Antoun Al Absi

Hannah Wurzer

Coralie Guerin

Celine Hoffmann

Antoun Flora Moreau

Xianqing Mao

Joshua Brown-Clay

Rémi Petrolli

Monika Dieterle

Jean-Paul Thiery

Salem Chouaib

Guy Berchem

Bassam Janji
email: bassam.janji@crp-sante.lu

Al Absi

Barbara Soussa

Fernando Schmitt

Thomas C 2016 Oncotarget

Viry E Al-Absi

Actin cytoskeleton remodeling drives breast cancer cell escape from natural killer cell lysis

-SMA -smooth muscle actin ABC ATP-binding cassette ABP Actin-binding proteins ADCC Antibody-dependent cellular cytotoxicity ADP Adenosine 5'-diphosphate AF Actin filament AML Acute myeloid leukemia Apaf-1 Apoptotic peptidase activating factor 1 aPKC Atypical protein kinase C Arl8b ADP-ribosylation factor-like 8b ARPC ARP complex component ATG Autophagy related genes ATP Adenosine 5'-triphosphate BAG-6 Bcl-2associated athanogene 6 BC Breast cancer Bcl-2 B-cell lymphoma-2 BCR B cell receptors bHLH Basic helix-loop-helix Bid BH3 interacting domain BMI1 B lymphoma Mo-MLV insertion region 1 homologue BTLA B

To the greatest family one could ever have, To Syria...

Acknowledgement

First, I would like to express my gratitude to all the members of the jury, Dr. Catherine-Laure Tomasetto, Dr. Juan J. Garcia Vallejo and Dr. Jacques

Antoun AL ABSI

Rôle du cytosquelette d'actine dans la résistance des cellules de cancer du sein à la lyse induite par les cellules « natural killer » Résumé L'évasion immunitaire tumorale joue un rôle central dans la progression tumorale et représente un obstacle majeur au succès des immunothérapies. Dans cette Thèse nous avons étudié le rôle du cytosquelette d'actine dans la résistance des cellules de cancer du sein à la lyse induite par les cellules "natural killers" (NKs). Nous avons trouvé que les cellules de cancer du sein résistantes échappent à l'attaques des cellules NKs par une accumulation importante et rapide d'actine près de la synapse immunologique, un processus que nous avons nommé "réponse actine". Nos analyses mécanistiques suggèrent que la réponse actine induit la polarisation d'autophagosomes vers la synapse immunologique et facilite ainsi la dégradation des molécules cytotoxiques sécrétées par les cellules NKs, tel que le ganzyme B, par autophagie. De plus, la réponse actine est associée au regroupement de ligands inhibiteurs à la synapse, suggérant qu'elle est au centre de plusieurs mécanismes de résistance. Dans leur ensemble, nos résultats constituent une base pour le développement d'approches thérapeutiques visant à interférer avec la réponse actine et à restaurer une réponse immunitaire anti tumorale efficace.

Mots-clés : Cancer du sein, cellules Natural Killer, cytosquelette d'actine, Cytotoxicité, évasion immunitaire, granzyme B.

Résumé en anglais

Tumor immune evasion plays a central role in cancer progression and is a major hurdle to effective immunotherapy. In this Thesis, we examine the role of the actin cytoskeleton in breast cancer cell resistance to natural killer (NK) cell-mediated cell lysis. We found that resistant breast cancer cells escape from NK-cell attack through a rapid and prominent accumulation of actin near the immunological synapse, a process we termed the "actin response". Our mechanistic investigations suggest that the actin response drives autophagosome polarization toward the immunological synapse and thereby facilitates the autophagy-mediated degradation of NK cell-derived cytotoxic molecules such as granzyme B. In addition, the actin response was associated with inhibitory ligand clustering at the immunological synapse, suggesting that it is a common driver of different immune evasion mechanisms. Taken together, our data lays the groundwork for therapeutic approaches aimed at interfering with the actin response and restoring an effective anti-tumor immune response.

Key words: Actin cytoskeleton, breast cancer, cytotoxicity, granzyme B, immune escape, natural killer.

2.1.

Inhibition of autophagy reverts breast cancer cell

Summary of the thesis in French

Objectifs et approches

Mon travail de thèse a consisté à atteindre deux objectifs principaux :

. The immune system

The immune system is composed of different and complementary networks of cells, tissues and organs that interact together for the purpose of protecting the host from pathogens and clearing infected and diseased cells such as cancer cells. In the absence of functional immune defenses, minimal infections can have dangerous effects on the organism. The innate and adaptive immune cells are commonly considered the two arms comprising the immune system 1 (Figure 1).

Adaptive immunity

The adaptive immune system is more specific to pathogens than the innate immune system and it needs more time to become protective and functional. Its specificity relies on the recognition of antigens present on target cells via antigen-specific receptors. The sophistication of the system derives from its ability to memorize and have an effective

Innate immunity

The first line of defense that responds immediately against invading tumors and pathogens is the non-specific innate immune system. It is composed of three main components: 1) anatomic barriers, which involve the skin, saliva, tears, mucus and cilia;

2) the humoral barriers, including a number of small proteins synthesized in the liver; and

3) cellular barriers that comprise different types of cells with distinct and complementary functions. Innate immune cells express pattern-recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs), which are motifs that are unique to microorganisms 11 .

Mononuclear cells, including monocytes and macrophages, are a type of phagocyte that finds and digests foreign microorganisms. Monocytes are derived from the bone marrow and circulate in the blood at a steady state. They can migrate to tissues to engulf cells and toxic molecules. During development, monocytes differentiate into dendritic cells (DCs) or macrophages, which are long-lived cells that can survive within the body for months and even years. Their function is essential to phagocytizing apoptotic erythrocyte cells and cellular debris as well as for the secretion of pro-inflammatory cytokines such as IL-6 and tumor necrosis factor (TNF). Macrophages also induce the proliferation of other immune cells 12 .

Dendritic cells are the granular antigen-presenting sentinels of the innate immune system. In the skin, DCs are called Langerhans cells, whereas immature DCs with phagocytic properties circulate in the blood. Upon infection, DCs migrate to the lymph nodes where they interact with adaptive immune cells to shape the immune response 13 .

Neutrophils along with eosinophils and basophils are known as granulocytes due to the presence of multiple granules in their cytoplasm. They are polymorphonuclear cells, having lobed nuclei. Neutrophils are the most abundant phagocytic cells, and they are usually the first to arrive at a site of infection to kill bacteria and fungi. When activated, neutrophil granules containing a variety of cytotoxic compounds are released. Basophils and eosinophils release histamine, an important mediator of allergic reactions and a defense against parasites. Mast cells are morphologically and functionally very similar to basophils, but they are more particularly involved in wound healing, immune tolerance and angiogenesis 14 .

Complement proteins are small polypeptides synthesized in the liver that circulate in the blood in an inactive state. When stimulated, proteases cleave the complement proteins into fragments 15 . The role of these proteins is to enhance the ability of antibodies and phagocytes to eliminate pathogens and damaged cells 16 . As I focused on this innate cell type during my research, Section 1.2 describes NK-cell properties and functions in more detail.

Figure 1 | The innate and adaptive immune systems

The innate immune response functions as the first line of defense against infection. It consists of soluble factors, such as complement proteins, and diverse cellular components including granulocytes (basophils, eosinophils and neutrophils), mast cells, macrophages, dendritic cells and natural killer cells. The adaptive immune response is slower to develop, but manifests as increased antigenic specificity and memory. It consists of antibodies, B cells, and CD4 + and CD8 + T lymphocytes. NKT cells and γδ T cells are cytotoxic lymphocytes that straddle the interface of innate and adaptive immunity. Adapted from Dranoff, 2004.

Innate immune system specificity: recent paradigm changes

Until recently, the innate system was considered as a non-specific arm of the immune system that detects and eliminates aberrant cells without education or specificity. The discovery of the specificity in some innate immune responses in the last 20 years has shifted our understanding of the of immune system classifications. These observations led researchers to re-think about the simplistic classification of immune cells into the innate and adaptive arms 17 . In this section, I summarize some of the recent discoveries about the adaptive arm of innate immunity.

Sensing Gram-negativity by a host through the detection of lipopolysaccharides and other components by the host was the first observation of a specific innate response against infectious agents. Further, the stress sensing pathway was found to induce innate immunity in some species such as nematodes [START_REF] Baldewijns | VHL and HIF signalling in renal cell carcinogenesis[END_REF] . Mammalian stress signaling was also reported in the induction of inflammatory responses involving heme, which is abundant in red blood cells. Heme functions to activate TLR4 on innate immune cells and in initiating an oxidative stress response. An adaptive response re-occurs following red blood cell damage 19 .

Studies have shed light on the non-apoptotic regulated cell death of certain innate immune cells. One of the best-described example is the formation of the neutrophil extracellular traps, a process known as NETosis. Neutrophils and other granulocytes can undergo a self-immune cell death and release their own chromatin as an anti-microbicidal response. It has been well established that bacteria, viruses and fungi can be trapped and killed by the released network of histones, DNA and granular proteins. The implication of this type of innate immune-cell death revealed a high specificity toward pathogen infections 20 . Moreover, the Fc receptors of basophils and mast cells show an adaptive functionality, as the generated signaling pathways have an adaptive response toward allergic reactions that can be regulated and tuned 21 .

Relevant to my work is the recent discovery of Innate Lymphoid Cells (ILCs), which include NK cells as well as the recently discovered subsets, ILC1, ILC2, and ILC3.

Whereas NK cells share the cytolytic molecules, IFN- and Eomes, with CD8 + T cells, ILCs share common transcription factors with T cells that give rise to strikingly similar effector functions, such as cytokine production. Profiling the subsets has revealed that ILC1 shares the expression of T-bet and IFN- with Th1 cells, ILC2 shares GATA-3, IL-5 and IL-13 with Th2 cells and ILC3 shares RORt, IL-17 and IL-22 with Th17/Th22 cells. The link between T cells and the ILC groups is still not fully understood, but recent studies support the importance of ILCs in protecting of epithelial and mucosal surfaces when T cells are inefficient or absent 22 (Figure 2). Upcoming studies will establish the precise biological functions of such responses in specifically regulating the host defense immune system.

Figure 2 | Parallels between innate and adaptive immune lineages

Innate lymphoid cells share a number of features with T cells including the cytokines and transcription factors necessary for differentiation and the effector cytokines produced by each subset. SLEC, short-lived effector CD8 + T cells; MPEC, memory precursor CD8 + T cells; GzmB, GzB. Adapted from Huntington et al., 2016.

NK cells: generalities

A fraction of effector lymphocytes was originally considered as typical, with high cytotoxicity and cytokine secretion capability but without education 23 . The intense focus on the role of these cells led to the identification of a new sub-population of cells defined as natural killer cells. Natural Killer cells are granular lymphocytes in the ILC group and a part of the innate immune system. They have the ability to lyse aberrant cells, including tumor and pathogen-infected cells, without prior sensitization. The NK cell employs sophisticated mechanisms to discriminate between abnormal and healthy cells 24 .

Moreover, NK cells provide a source of immunoregulatory cytokines. This regulatory function of the NK cells, in addition to their cytotoxicity, holds promise for improvements in immunotherapy, autoimmune and inflammatory diseases and organ transplantation 25 .

The NK-cell membrane expresses a variety of germline-encoded activating and inhibitory cell surface receptors whose engagement leads to target recognition. The activation of NK cells derives from the complex system that detects activation and inhibitory ligands on the target surface. Thus, the signals derived from the interaction of receptors on the NK surface and their corresponding ligands on neighboring cells governs the "dynamic" equilibrium regulating NK-cell activation and inhibition 26 .

In this chapter, the biology and features of NK cells will be described in detail, with particular emphasis on the critical roles played by NK-cell receptors and the different components of NK cell-mediated cell-death receptors.

NK cells: a historical perspective

From 1970 until the present, much progress has been made on elucidating NK-cell functionality, which is not limited to cell lysis and elimination, but also includes reciprocally regulating of the activity of other immune cells 27 . That NK cells represent a separate cell lineage took long to be established because their ability to kill tumor target cells was considered as an artifact. However, in the early 1970s, Rolf Kiessling, a PhD student from the Karolinska Institute in Stockholm, discovered a new type of lymphocytes in the spleen of BALB/c mice with the capacity to kill aberrant cells without prior stimulation. Theses remarkable cells were described as naturally occurring killer lymphocytes with a specificity to mouse Moloney leukemia and with unique surface and functional properities 23,28 . The lysis of tumor cells occurred within one to four hours after contact with the effector cells in vitro, a more rapid induction than that of other adaptive immune cells. "The killer cells involved would seem to be neither T nor B lymphocytes as classified by conventional markers but rather belong to yet a third group of lymphocytes", Kiessling stated. In 1976, Hugh Pross identified the same immune cell population in peripheral human blood 29 . Studies on NK cells began to attract growing interest, and in the same year, Ronald Herberman published data confirming the functionality of the new cell type to kill target cells without any previous sensitization [30][31][32] .

The number of studies continued to rise, and Thornthwaite eventually showed how the newly discovered cells kill P815 cells, a mastocytoma tumor model, under a microscope for the first time 33 . After the discovery of NK cells, experiments were conducted on whole peripheral blood mononuclear cells (PBMCs) because there were no efficient isolation methods. In 1980, however, Timonen and Saksela isolated NK cells with the use of a density gradient, allowing them to visualize the cells under a microscope and characterized them as large granular lymphocytes 34 . Human NK cells are defined by the positivity for the CD56 antigen, an isoform of the human neural-cell adhesion molecule (N-CAM), and by their negativity for CD3. In human peripheral blood, NK cells represent from 2% to 18% of the total lymphocytes.

The biology of NK cells

Studies on mice have shown that the percentage of NK cells defined as NK1.1 + CD32 -is about 2% in mice spleen, 5% of PBMCs, and about 8% to 10% in mice lung and liver 36,37 .

In humans, NK cells are divided into two subsets with different roles and functions based on the expression levels of the unique CD56 markers, CD56 bright and CD56 dim . The largest fraction of NK cells (~90% of PBMCs and spleen NK cells) is the highly cytotoxic CD56 dim subpopulation, which expresses high levels of the Fcγ receptor III (Fcγ RIII, CD16 + ) 38 . In contrast, NK cells are CD56 bright CD16 -in lymph nodes and tonsils. This subpopulation is known to be non-cytotoxic in comparison to the CD56 dim NK cells due to the absence of perforin (detailed in section 1. 6.3.2). However, the CD56 bright CD16 -subpopulation secretes high levels of cytokines after being activated by monocytes.

Interestingly, the recently discovered cell subpopulation of CD56 dim CD16 dim NK cells has been found to be present in higher in numbers than the bright subsets in more immature cells 39 .

The distribution and trafficking of NK cells in an organism are regulated by chemokine-receptor expression on the NK-cell membrane. The expression of some of these receptors is dependent on the maturation and the activation level of the cell 40 .

Immature NK cells express the chemokine receptors CXCR3, CXCR4 and CXCR6 until maturation, when they gain the ability to express CX3CR1 and sphingosine-1-phosphate receptor 5 (S1P5), mainly responsible for NK cell egress from the bone marrow and lymph nodes. Once NK cells enter the periphery, they are directed to the site of action via chemotaxis 41 . All NK cells express high levels of CXCR4, a receptor for CXCL12, and SDF-1/, a receptor for CXCL12. CD16 + cells express high levels of CXCR1 and CX3CR1, low levels of CXCR2 and CXCR3 and no CXCR5. These cells migrate in response to CXCL8 and CX3CL1 42 . By contrast, CD56 bright CD16 -NK cells express high levels of CXCR3, CCR5 and CCR7, but have no CXCR1, CXCR2, or CXCR5 expression. Accordingly, they migrate in response to CCL19, CCL21, CXCL10, and CXCL11 43 .

The NK cell membrane expresses a wide variety of activating and inhibitory receptors that determine the NK cell's killing activity and interactions with other immune cells. The receptors are divided into several groups based on their corresponding ligands. Healthy 44 (Figure 4). Immune-checkpoint receptors are described in the chapter on immunotherapy.

Killer cell immunoglobulin-like receptors (KIRs)

The KIR receptor class (also known as CD158) comprises 7 activating and 7 inhibitory receptors. KIRs are monomeric type I glycoproteins whit two or three Ig-like domains (KIR2D or KIR3D) in the extracellular region 45 . All inhibitory KIR receptors contain one or more an immune-receptor tyrosine-based inhibitory motifs (ITIM) that recognizes MHC class 1 ligands 44 . Upon the engagement of KIRs with HLA-A, B or C, the ITIM domain is phosphorylated by Src family protein kinases. This phosphorylation promotes the formation of a docking space and the recruitment of the SHP-1/2 protein kinase phosphatases, which in turn, leads to the suppression of the activating receptor signals.

All activating KIR receptors lack ITIMs and instead, express the DAP12 protein that delivers activating signals through the immune-receptor tyrosine-based activation motif (ITAM) 46,47 .

Natural killer group 2 (NKG2) receptors

NKG2 receptors are a group in the C-type lectin family that contains six heterodimers (NKG2A, B, C, E, F and H) coupled to CD94 and a non-coupled NKG2D. They are predominantly expressed and divided into activating and inhibitory receptors. Both activating and inhibitory NKG2/CD94 receptors bind to the corresponding non-classical MHC HLA-E ligand expressed on target cells 44 . The receptor subtypes NKG2A and B are classified as inhibitory receptors as they contain 2 ITIM domains that drive inhibitory signal cascades. NKG2C, E and H associate with DAP-12 and accordingly generate activating signals. NKG2F is classified as an orphan receptor because its expression and function on NK cells is not well established. Nevertheless, it is usually accepted that NKG2F can associate with DAP-12 and therefore functions as an activating receptor [START_REF] Borrego | The CD94/NKG2 family of receptors: from molecules and cells to clinical relevance[END_REF] .

The most critical member of the NKG2 class is the NKG2D receptor, which is expressed on a wide range of cytotoxic cells, including NK cells, and γδ + and CD8 + Tcells. NKG2D is encoded by KLRK1 gene, located on the chromosome 12 in humans [START_REF] Spear | NKG2D ligands as therapeutic targets[END_REF] .

In contrast to the other NKG2 class members, NKG2D does not form a heterodimer with CD94, but instead is expressed as a homodimeric receptor. NKG2D monomers associate with DAP-10 dimers, which transduce activation signals [START_REF] Mistry | Regulation of ligands for the activating receptor NKG2D[END_REF] . Due to their short binding site, NKG2D receptors recognize many different types of ligands. Importantly, the MHC class I-related chain A and B (MICA/B) and the family of six UL16-binding protein (ULBP1-6) [START_REF] Sutherland | human ligands of the NKG2D receptor, stimulate tumor immunity with enhancement by IL-15[END_REF][START_REF] Zhang | NKG2D Ligands in Tumor Immunity: Two Sides of a Coin[END_REF][START_REF] Andre | Comparative analysis of human NK cell activation induced by NKG2D and natural cytotoxicity receptors[END_REF] . The ligation of NKG2D with MICA/B or ULBPs and the subsequent activating signals represent an important regulatory axis of cancer immune surveillance. Cellular stress ligands expression is regulated by DNA damage and heat shock response pathways, which are increased accordingly in cancer cells [START_REF] Nausch | NKG2D ligands in tumor immunity[END_REF][START_REF] Waldhauer | NK cells and cancer immunosurveillance[END_REF] . Nevertheless, healthy cells also express NKG2D ligands but at a baseline level [START_REF] Molfetta | Regulation of NKG2D Expression and Signaling by Endocytosis[END_REF] . Experiments on E-myc mice, which lack NKG2D receptors, have shown the importance of these receptors in the immune response. 44 The LILRs subfamily B or CD85 is widely expressed on NK cell membranes. These receptors are type I transmembrane glycoproteins with an extracellular Ig-like domain.

Leukocyte immunoglobulin-like receptors (LILRs)

Upon binding to MHC class I molecules, and more particularly to the HLA-G ligands from tumor cells, the 5 isoforms of the receptors (LILRB1-5) transduce signals via ITIMs that recruit the SHP-1/2 protein kinase, leading to the inhibition of NK-cell activity [START_REF] Hirayasu | Functional and genetic diversity of leukocyte immunoglobulin-like receptor and implication for disease associations[END_REF] . This interaction reportedly serves as an immune checkpoint, although its impact on tumor immune escape is less documented than that of CTLA4 or PD-1 [START_REF] Carosella | HLA-G: An Immune Checkpoint Molecule[END_REF] .

Natural cytotoxicity receptors (NCRs)

Another important class of activating receptors on NK-cell membranes is the Igsuperfamily called natural cytotoxicity receptors (NCRs) [START_REF] Shemesh | Splice variants of human natural cytotoxicity receptors: novel innate immune checkpoints[END_REF] . Their role in promoting NK cell mediated-tumor cell lysis was discovered following the combined knockout of each receptor in mice or through the use of specific blocking monoclonal antibodies 44 . NKp30, NKp44 and NKp46 are the identified activating receptors that were identified and shown to bind to viral-, parasite-, bacterial-and tumor-derived ligands [START_REF] Bozzano | Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment[END_REF] . NKp30 and NKp46 are constitutively expressed on the membrane of both resting and activated NK cells whereas NKp44 is only expressed upon IL-2 stimulation 61 .

NKp30 is a major receptor that mediates NK cell-meditated DC maturation as well as virus-infected cell and tumor lysis [START_REF] Alvarez-Breckenridge | NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors[END_REF] . Studies are still ongoing to uncover the precise role of NKp30 in the anti-tumor immune response [START_REF] Herrmann | Homo-oligomerization of the activating natural killer cell receptor NKp30 ectodomain increases its binding affinity for cellular ligands[END_REF] . Recent studies have shown that the nuclear factor HLA-B-associated transcript 3, tumor antigen B7-H6, Bcl-2-associated athanogene 6 (BAG-6) and heparin sulfate proteoglycans can bind to NKp30 resulting in cell lysis [START_REF] Matta | Induction of B7-H6, a ligand for the natural killer cell-activating receptor NKp30, in inflammatory conditions[END_REF][START_REF] Li | Structure of the human activating natural cytotoxicity receptor NKp30 bound to its tumor cell ligand B7-H6[END_REF] . To date, only a few studies have shed the light on the NKp46 receptors, their ligands, and their implications in tumor recognition and elimination [START_REF] Bozzano | Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment[END_REF] . Viral ligands such as the influenza virus's hummagglutinin were the first ligands identified to bind to NKp46 66 . Viral, parasite and bacterial ligands were later shown to have the capacity for signal induction after NKp46 binding [START_REF] Hadad | NKp46 Clusters at the Immune Synapse and Regulates NK Cell Polarization[END_REF] . Although the ligands of NKp44 on tumor cells remain to be identified, in vivo studies in NKp44 KO mice provide evidence that NKp44 is implicated in tumor immune surveillance [START_REF] Cantoni | The three-dimensional structure of the human NK cell receptor NKp44, a triggering partner in natural cytotoxicity[END_REF] . A cellular ligand of NKp44 has been recently identified as NKp44L. This ligand is a novel isoform of the mixed-lineage leukemia-5 protein (MLL5) and has been shown to control NK cell-mediated cell lysis [START_REF] Baychelier | Identification of a cellular ligand for the natural cytotoxicity receptor NKp44[END_REF][START_REF] Bialoszewska | Constitutive expression of ligand for natural killer cell NKp44 receptor (NKp44L) by normal human articular chondrocytes[END_REF] .

2B4 receptors

2B4 or CD244 is a non-MHC multifunctional binding receptor that is abundant on NK cells, monocytes, basophils and a subpopulation of T cells. There are two isoforms, 2B4-A and 2B4-B, which differ in their affinity for CD48, a ligand expressed on all hematopoietic cells. Depending on the maturation stage of the NK cell, activating or inhibitory signals can derive from these receptors upon ligation with CD48. A recent study has reported that the interaction between 2B4-A and CD48 leads to increased intracellular calcium release, induces IFN- secretion, and contributes to initiating of the cytotoxicity process 70 .

Killer cell lectin-like receptor G1 (KLRG1)

Killer cell lectin-like receptor G1 (KLRG1) is an abundant receptor on both NK cells and T cells. They were originally defined as inhibitory receptors that induce inhibitory signals through an ITIM domain. The ligands for KLRG1 receptors include the cadherin family members (E-, N-, and R-cadherins), which are abundant on healthy cells and are downregulated on malignant mesenchymal tumors. Thus, NK cells implicate KLRG1 receptors in playing an important role in preventing healthy cell damage and lysis [START_REF] Ito | Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity[END_REF] .

Co-stimulatory receptors

The co-stimulatory DNAM-1 (also known as CD226) receptors are expressed on 50% of NK cells and they bind to CD155 (PVR) and CD112 (PVRL2, nectin-2), which are expressed on tumor cells [START_REF] Bozzano | Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment[END_REF] . Beside their role in lymphocyte migration, DNAM-1 is involved in tumor cell recognition and adhesion [START_REF] Shibuya | DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes[END_REF] .

T-cell immunoglobulin and the ITIM domain (TIGIT) are co-inhibitory receptors of the Ig superfamily expressed on NK cells, and they bind to CD155 with a higher affinity than to DNAM-1. Remarkably, the interaction between DNAM-1 and CD155 is inhibited by TIGIT [START_REF] Wang | TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals[END_REF] . Another group of co-stimulatory receptor is the NKR-P1 receptors (NK1.1 in mice), which are classified into 5 isoforms. NKR-P1 B/D inhibits NK-cell activation through the ITIM domain, while the NKR-P1 C has an ITAM domain [START_REF] Kirkham | Complexity and Diversity of the NKR-P1:Clr (Klrb1:Clec2) Recognition Systems[END_REF] . In addition, B and T lymphocyte attenuator (BTLA) and CD160 are immunoglobulin inhibitory receptors that bind to herpes virus entry mediator (HVEM) [START_REF] Sedy | A herpesvirus entry mediator mutein with selective agonist action for the inhibitory receptor B and T lymphocyte attenuator[END_REF] . The ligation between HVEM and the TNF suprafamily 14 (TNFSF14, also known as LIGHT) co-stmulatory receptors induces activating signals [START_REF] Ware | Targeting the LIGHT-HVEM pathway[END_REF] . Sialic acid-binding immunoglobulin-like lectin is another inhibitory receptor that binds to sialylated glycan to induce inhibitory signals [START_REF] Shao | Siglec-7 Defines a Highly Functional Natural Killer Cell Subset and Inhibits Cell-Mediated Activities[END_REF] .

Figure 4 | NK cell activating and inhibitory receptors

Major activating (green) and inhibitory (red) receptors expressed by human NK cells and their corresponding ligands are represented. Adapted from Chiossone et al., 2017.

NK cell-mediated-cell death

The main function of NK cells is as a defense against tumor and virally-infected cells via different and well-established lytic mechanisms 24 . These mechanisms include (1) death receptor pathways 31 (2) NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) [START_REF] Wang | Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy[END_REF] , and (3) the lytic granule-dependent pathway [START_REF] Vivier | Functions of natural killer cells[END_REF] . Along with these well-defined lysis mechanisms, NK cells release exosomes that are known to play a significant role in immune-surveillance [START_REF] Fais | NK cell-released exosomes: Natural nanobullets against tumors[END_REF] . NK cell-derived exosomes (NKEXOs) have been reported to have lytic properties when they enter tumor cells. They express the exosomal markers Rab5B and CD63 as well as the CD56, NKp30, NKp46, and NKp44, NK-cell markers [START_REF] Whiteside | Immune modulation of T-cell and NK (natural killer) cell activities by TEXs (tumour-derived exosomes)[END_REF] .

Importantly, NKEXOs contain cytolytic proteins such as Fas ligand and perforin [START_REF] Lugini | Immune surveillance properties of human NK cell-derived exosomes[END_REF] .

Death receptor pathways

NK cells contribute to the suppression of tumor growth and metastasis via deathreceptor pathways independently from the cytotoxic granule pathway [START_REF] Screpanti | A central role for death receptor-mediated apoptosis in the rejection of tumors by NK cells[END_REF] . This pathway is activated upon the ligation of death ligands with their corresponding death receptors, which promotes caspase-dependent death pathways 84 31,85 .

The Fas receptor-Fas ligand pathway

FasL (also known as CD95L, APO-1, or TNFRSF6) and its receptor Fas (also known as CD95, APO-1, or TNFRSF6) are members of the TNF and TNF-receptor family [START_REF] Vujanovic | Role of TNF superfamily ligands in innate immunity[END_REF] . Fas is a type I transmembrane protein containing a death domain in its cytoplasmic region that is responsible for inducing apoptosis [START_REF] Volpe | Fas-Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis[END_REF] . Not all tumor cells express the death receptor on their membrane. Nevertheless, NK cells can stimulate Fas expression on target cells via the secretion of IFN-. This 40 kDa Fas ligand is expressed at the surface of both NK and cytotoxic T-lymphocyte (CTL) cells and its ligation with Fas allows for recruitment of the protein Fas-associated death domain (FADD). FADD promotes the formation of the death-inducing signaling complex (DISC) and the activation of caspases 8 and 3.

Moreover, the anti-apoptotic FLICE/ caspase-8-like inhibitory protein (FLIP) blocks Fas pathway-mediated cell lysis by disturbing the formation of the DISC [START_REF] Salvesen | Structure of the Fas/FADD complex: a conditional death domain complex mediating signaling by receptor clustering[END_REF] . Of note, metalloproteinases can split FasL into a soluble inactive form that competes with the FasL on the membrane [START_REF] Kayagaki | Metalloproteinase-mediated release of human fas ligand[END_REF][START_REF] Tsutsui | Caspase-1-independent, Fas/Fas ligand-mediated IL-18 secretion from macrophages causes acute liver injury in mice[END_REF] .

The TNF-related apoptosis-inducing ligand (TRAIL) pathway

TRAIL (also known as APO-2 ligand) is a type II transmembrane protein that belongs to the TNF superfamily. Five TRAIL receptors have been identified in human cells, but only two of them, DR4 and 5, are capable of inducting apoptotic signals due their death domain 31 . The high expression of TRAIL on NK-cell membranes is stimulated by with IL-2, IL-15, and IFNs. Like Fas receptors, the activation of DR 4 or 5 induces DISC assembly. FADD then translocates to the DISC, where it interacts with the death domain of the receptor [START_REF] Falschlehner | Following TRAIL's path in the immune system[END_REF] . FADD recruits pro-caspases 8 and 9 to the DISC where they are activated. In some types of cells, DISC also activates the mitochondrial pathway. The BH3 interacting-domain death agonist (BID) is then cleaved to tBid by caspases 8 and 10 and translocated to the mitochondrial. tBID activates Bax and Bak resulting in cytochrome-C release and the activation of caspases 9 and 3 [START_REF] Falschlehner | Following TRAIL's path in the immune system[END_REF][START_REF] Walczak | Death receptor-ligand systems in cancer, cell death, and inflammation[END_REF][START_REF] Dang | Fasting enhances TRAIL-mediated liver natural killer cell activity via HSP70 upregulation[END_REF] .

Antibody-dependent cellular cytotoxicity (ADCC)

ADCC is a mechanism of the cell-mediated immune response whereby an NK cell detects and lyses a target cell whose membrane antigens are bound to specific antibodies. NK cells and hematopoietic cells (except most T cells) express three types of Fcγ receptors, which bind to the Fc part of the IgG antibodies. FcγRI (CD64) is expressed on the membranes of neutrophils and macrophages and binds to human IgG1 and 3 [START_REF] Yamashita | A novel method for evaluating antibody-dependent cellmediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells[END_REF] . The second type is FcγRII (CD32), which comprises two isoforms that function as activating or inhibitory receptors. The third type is FcγRIII, which has two isoforms, FcγRIIIa with an ITAM domain and FcγRIIIb with an ITIM domain [START_REF] Clynes | Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets[END_REF] . The balance between the activating and inhibitory signals of FcγRs and their antibody-affinity thresholds control the efficacy of ADCC.

NK cells represent an important model of ADCC as the activating FcγRIIIa is highly expressed on CD56 dim CD16 + NK cells, while inhibitory FcγRIIIb is absent. The binding of FcγRIIIa to the IgG1 and IgG3 subclasses induces a potent activating signal. NK-cell activation triggers cytotoxic granule polarization and secretion. ADCC has been implicated in tumor elimination in several in vivo studies [START_REF] Wang | Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy[END_REF] . Antibodies specifically engineered to prevent Fc binding to FcγRIIIa and human epidermal growth factor receptor 2/neu (Her2/neu, CD240) induce a potent inhibition of the ADCC pathway and lead to increased tumor size in BC xenograft-mouse models. It is worth mentioning that vaccination and monoclonal antibody-based immunotherapies takes advantage of the ADCC mechanism [START_REF] Wang | NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement[END_REF][START_REF] Seidel | Natural killer cell mediated antibodydependent cellular cytotoxicity in tumor immunotherapy with therapeutic antibodies[END_REF] .

The lytic granule-dependent death pathway

Upon target recognition, the main elimination pathway is the secretion of the cytolytic granules into the target cells and the subsequent induction of apoptosis [START_REF] Voskoboinik | Perforin and granzymes: function, dysfunction and human pathology[END_REF] . Whereas CTLs have TCRs that recognize MHC class I, NK cells mainly recognize their targets via a set of activating and inhibitory receptors. Despite NK cells and CTLs having different target recognition mechanisms, the following lytic granule-dependent killing mechanism is conserved in both cell types. The lytic granules are electron-dense organelles that contain the pore-forming protein perforin, the serine protease proteins called granzymes, and the lipid-disrupter granulysin [START_REF] Chamkha | Perforin, granzyme and granulysin association to kill intracellular parasites[END_REF] . Once NK cells are activated, Ca +2 concentration increases in the cytosol, the microtubule organizing center (MTOC) moves toward the target cell and the cytotoxic granules are polarized along with the MTOC toward the immunological synapse (IS) 100 . Perforin and granzymes synergize to induce apoptosis in the target cells 101 (Figure 5).

Granzymes

Granzymes constitute a family of serine proteases whose members differ in their substrate specificity and functionality. Five granzymes have been identified in humans, granzymes A, B, H, K and M 102,103 . Granzymes reportedly to cleave both intracellular and extracellular proteins. However, there is a gap in our knowledge regarding their extracellular functions. Granzyme A-mediated cell death is not related to the caspaseactivation pathways, but rather involves a loss of the mitochondrial inner membrane, the release of reactive oxygen species (ROS), and nuclear histone cleavage 104,105 . The mode of action of granzymes H and K are not well characterized, but recent studies support that granzyme H-induced cell death is also independent of caspase activation [START_REF] Fellows | Natural killer cellderived human granzyme H induces an alternative, caspase-independent celldeath program[END_REF] . In contrast to granzyme H, granzymes K and M mediate cell death through caspase pathway activation and ROS production 106 . Granzyme B (GzB) is the most abundant protein and the best functionally characterized among the other forms of cytolytic granules. GzB is responsible for the rapid induction of caspase-dependent apoptosis in target cells promoting directly or by indirectly caspase activation. Although its relative importance to tumor cell elimination remains unclear, caspase-independent cell death has also been reported after GzB is internalized 107 . 1.6.3.1.1.

Granzyme B structure and synthesis

GzB was discovered in 1987 by Schmid and Weissmann as an abundant serine protease in human leukocytes 108 . In 1991, Poe et al., showed that the 32 kDa GzB could be purified from the granules in human cytolytic lymphocytes, and that they established its enzymatic activity [109][110][111] . The protein is composed of two 6-stranded β-sheets which are connected by three trans domains. In the first step, GzB is first synthesized as a preproenzyme before being transported into the endoplasmic reticulum. The subsequent pro-GzB is then transported in specific vesicles into the Golgi apparatus, where it is modified by mannose-6-phosphate 112 . Once modified, the pro-GzB is ready to be activated by dipeptidyl peptidase I or cathepsins C and H in secretory granules, which are stored in association with the proteoglycan serglycin-containing chondroitin sulphate 113 .

Granzyme B delivery to and internalization in the target cell

At steady state and in the absence of target cells, CTLs and NK cells constitutively synthesize GzB. A major difference between the NK cells and CTLs is that NK cells secrete and release GzB in its active form while CTLs secrete the inactive form. The regulators of cytotoxic granules such Rab-27A, Munc13-4, and synaptotagmin VII play an essential role in granzyme maturation and the fusion with the plasma membrane 114,115 .

Additionally, NK cells can recycle a fraction of the secreted GzB via clatherin-dependent endocytosis. The entry of GzB into the target cells can be achieved either by co-entry into the cytoplasm with perforin or via the mannose-6-phosphate receptor 116 .

Granzyme B-induced apoptosis

The exposure of target cells to nanomolar concentration of GzB is rapidly followed by apoptosis (within 5-8 minutes). Granzyme B's entrance into the target cell's cytoplasm is followed by the recognition of a broad range of intracellular signaling components that directly or indirectly promote cell death 107 . These pathways are briefly described below.

BID is an important molecular target of GzB. The proteolysis of Bid leads to the translocation of its C terminal into mitochondria and the subsequent permeabilization of the mitochondrial membrane via Bak or Bax protein oligomerization 117 . In addition, GzB is responsible for the cleavage of anti-apoptotic B-cell lymphoma-2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1) 118 . Both mechanisms lead to the release of the mitochondrial cytochrome C and diablo homolog (Smac/diablo) into the cytosol 119 .

Cytochrome C, together with apoptotic peptidase activating factor 1 (Apaf-1), directly activates caspases 9 and 3. Additionally, GzB is capable of directly cleaving caspases 8, 10, 3, and7 andindirectly cleaving caspases 2, 6 and9 120 . The indirect induction of target cell death involves the activation of Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) and results in membrane blebbing 121 . In addition, GzB cleaves -tubulin, leading to cytoskeleton collapse and microtubule polymerization 122 . GzB also directly cleaves nuclear components such as lamin A/C, lamin B, and nuclear mitotic apparatus protein A (NUMA1), which results in lamina disruption and a loss of nuclear integrity 123,124 . Moreover, GzB-dependent targeting of the poly (ADP-ribose) polymerase 1 (PARP-1) or the DFF45/ICAD (cytoplasmic DNA fragmentation factor 45) alpha peptide promotes apoptosis via DNA fragmentation.

Finally, GzB can negatively affect fibroblast growth factor receptor 1 (FGFR1) and notch homolog 1 translocation-associated (Notch/NEXT) to enhance apoptosis 125 .

Perforin

The 67 kDa multi-domain protein perforin was discovered in 1980 by 3 groups of scientists: Henkart et al.,Okumura et al.,and Podack et al 126 . Perforin is a poreforming member of the membrane-attack-complex/perforin (MACPF) protein family that induces transmembrane tubules 127 . Additionally, it can oligomerize into membrane-spanning pores at neutral pH and when Ca + is present 2128 . Such pores allow the passive diffusion of the GzB into the target cells 129 . The amino-terminal MACPF domain is followed by an epidermal growth factor (EGF) domain. The C-terminal domain of perforin is related to the Ca +2 -dependent C2 domain family and is extremely important to its activity. Perforin is an essential key for granzyme-dependent apoptosis, as noted by the loss of perforin in mice abolishing targeted cell death 130 . It is also associated with various human diseases such familial haemophagocytic lymphohistiocytosis (FHL; a congenital perforin deficiency), hematological malignancies, and protracted viral infections 131 .

Granulysin

The saponin-like protein family contains the 15 kDa member granulysin. In addition to perforin and granzymes, secretory granules also contain the granulysin, which is responsible for the lysis of microbes and tumor cells 132 . Interestingly, there is no homolog of granulysin in mice, and no in vivo studies have been conducted to elucidate its functionality. However, it has shown in vitro that granulysin kills a spectrum of parasites, fungi, and bacteria, particularly Mycobacterium tuberculosis 133 .

Structure and function of the immunological synapse (IS)

Target cell lysis by NK cells depends mainly on a lytic granule-dependent apoptotic pathway. This process relies on strict and well defined biological steps to ensure the precise targeting of the lytic molecules 134 . A key element governing NK cell-mediated cell lysis is the formation of the IS, which is a specialized cell-cell interface characterized by the tight apposition between the membrane of an immune cell and its target. Originally the IS was defined between a T cell and an antigen-presenting cell, but studies have shown the critical role of NK-IS upon target recognition 135 . For the first time, in 1983, Abraham Kupfer showed the polarization of the Golgi apparatus and MTOC in an NK cell towards its target. Originally the interface was named after the neuronal synapse due to their having the function of connecting two cells in common 136 . Studies on the NK-IS have improved our understanding about its multiple functions. The IS serves first as a region of contact between the membranes of the two cells, creating a critical zone for ligand-receptor ligation 137 . Upon target recognition, the purpose of the IS is first to ensure the adhesion and stability of the connection between the two cells. Subsequently, the direct secretion of cytolytic granules into the target cells guarantees their elimination and lysis. Highly regulated steps, components, and pathways participate in the formation of a functional IS 138 . Importantly, the sizes of the activating and inhibitory clefts are similar, between 10 and 30 nm. The lytic clefts between the effector and opposing cells play a major role in preventing the leakage of lytic granules into the surrounding tissue and ensure targeted secretion.

Stepwise stages of IS formation and activity

Defining the steps required to establish an IS is crucial for our understanding of the NK cell-mediated cell lysis. Of note, any dysregulation of one of the main stages or detailed steps is critical and may present a high risk of immune deficiency and malignancy 139,140 (Figure 6).

Target recognition and adhesion stage

Resting or primed NK cells are able to recognize their targets via either tethering or a more potent adhesion component on their cell membrane, but before the adhesion receptors engage in the process, heterogeneous structures called nanotubes are rapidly formed to guide the NK cell toward its target and to transmit signals over long distances 141 . Subsequently, selectin family members on the NK membrane such as Lselectin and P-selectin glycoprotein ligand-1 (PSGL-1), initiate the adhesion with the target. Lymphocyte function associated antigen-1 (LFA-1/CD11B/CD18) and CD2 are also implicated in the creation of a firm junction 142 . LFA-1 ligation with the intercellular adhesion molecule-1 (ICAM-1) on the surface of a target cell leads to a tight junction. This adhesion step induces downstream signals via the phosphorylation of the guanine nucleotide exchange factor Vav1 by an Src-family kinase, Rac1 activation, and cytoskeletal rearrangement 143,144 . Ligand-receptor ligation is strengthened and activated upon engagement of the activating receptors DNAM-1, NKG2D, and NCRs along with their corresponding ligands, giving rise to the initiation of the cytotoxic process [START_REF] Shibuya | DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes[END_REF] .

Afterwards, the lytic granules move along microtubules in a dynein-dependent manner and converge upon the MTOC polarization 145 .

Effector stage

The most important stage for the lytic process is the effector stage when the degranulation of the cytotoxic granules occurs. This stage is marked by a rearrangement of the cytoskeleton, MTOC polarization and lytic protein secretion 146 . The mature activating IS contains a region called the supramolecular activation cluster (SMAC) that is divided into a central (cSMAC) and a peripheral (pSMAC) forms. The SMAC serves as a region for surface receptors and actin filaments (AF) to cluster 147 . The 2 integrin LFA-1 receptors and AF cluster at the pSMAC, while perforin polarizes to at the cSMAC 148 . Dynamic AF polymerization is crucial for the establishment of a lytic IS through MTOC polarization and the delivery of cytotoxic granules 149 . Importantly, AF polymerization drives the maintenance of adhesion molecules at synaptic region during the effector stage. Pharmacological-or gene-targeting of the actin cytoskeleton at the NK-IS has been found to impair IS development, cleft size, receptor distribution, and MTOC polarization 150 . Actin filament dynamics at the synapse are discussed in detail in Section 5.4.

Activating and inhibitory receptors also cluster at the SMAC. One key to an inhibitory synapse is the domination of signals derived from inhibitory receptors and the functionality of phosphatases, especially SHP-1 45,151 . These inhibitory signals swiftly act to disrupt multiple IS checkpoints such as target-cell adhesion, LFA-1 stimulation, the phosphorylation state of Vav1, activating receptor clustering, and Ca+2 flux 152,153 .

It is well established that activating receptors, such as NKG2D and 2B4, cluster at the periphery of the synapse suggesting a more potent activating signal transmission.

Interestingly, the inhibitory receptor KIR2DL1 also clusters at the synapse but its concentration is highly reduced when NKG2D signaling is dominant. Two main axes of activating signaling occur after NK cell activation: the phosphatidylinositol 3-kinase/extracellular signal-regulated kinases (PI3K/ERK) pathway and the phosphoinositide phospholipase C-/c-Jun NH(2)-terminal kinase (PLC -JNK)

pathways. An early marker of NK cell activation is the calcium flux from the endoplasmic reticulum (ER), which is mediated by an IP3 channel downstream of PLCy signaling. The ER likewise polarizes toward the synapse to ensure a sustained Ca +2 flux during the lytic process [START_REF] Nausch | NKG2D ligands in tumor immunity[END_REF]154 .

Following activation and actin rearrangement, the MTOC is reoriented toward the synapse accompanied by the lytic granules. The movement of MTOC depends on either microtubule growth (push) or on microtubule shrinkage (pulling). Signals that direct MTOC polarization are derived from the LFA-1-derived Vav1 155 . Moreover, the actin cytoskeleton is linked to the microtubule network via Cdc42-interacting protein-4 (CIP4), the formin hDia, and the IQ-containing GTPase-activating protein 1 (IQGAP1) to ensure MTOC polarization 156,157 . Previous studies have also shown that the ADP-ribosylation factor-like 8b (Arl8b) interacts with kinesin-1 and regulates MTOC polarization 158,159 . This process is directed and regulated by the factor Munc13-4 and its ligation to Rab27a, a Rab GTPases member that regulates subcellular compartment trafficking 114 . Myosin IIA is likewise associated with lytic granules movement to the plasma membrane 160 . The lytic granules must traverse an actin meshwork by identifying actin hypo-density areas where the actin is remodeled again to provide the way 161 . Afterwards, Munc13-4 and rab27a mediates granule docking and fusion with the plasma membrane as directed by the targetsoluble NSF attachment protein receptor (SANRE) 162 .

NK cell mediated cell lysis is achieved by granule exocytosis in the cleft of the target 163 . Notably, proteinase inhibitor and cathepsin B are upregulated at the synapse to protect the NK cells from self-suicide or self-destruction from the released lytic proteins in the cleft 164 .

Lysis and elimination stage

The final stage of the cytotoxicity process is target cell lysis. Upon lytic granule delivery into the target, effector cells detach and either reinitiate the lysis of another target with granules still converged at the MTOC or they return to a resting state.

Phosphatidylserine is exposed on the target-cell surface after perforin protrusion and is responsible for the signal transmitted to the inhibitory CD300a on the NK cell membrane that stops the process 127,165 . Detachment from the target cell is faster when the target cell lysis is the result of this process rather than a non-lytic IS, but it can leave the NK cells in a state of exhaustion state 40 . However, the exposure of NK cells to an activator and stimulator such as IL-2 could reinitiate their activity 166 .

Figure 6 | Stepwise molecular progression through NK cell cytotoxicity

NK cell cytotoxicity can be broken down into three main stages: recognition (a-d), effector (d-p) and termination (q, r). During the recognition stage, the NK cell is particularly sensitive to inhibitory signaling (d). Key events include lytic granule convergence (c), the actin-dependent firm adhesion of the NK cell (e) and subsequent F-actin conduit formation (h), MTOC polarization (j) and LG fusion and exocytosis (o). This is followed by vesicle recycling (p), a period of relative inactivity (q) and detachment and subsequent serial killing (r). Adapted from Mace, 2014.

NK-cell regulation and cross-talk with immune cells

It is well established that the immune function of NK cells is defined by cytokine secretion and the elimination of aberrant cells. However, a large number of events occur between NK cell trafficking to the site of action and their activation. The maturity and functionality of the NK-cell response generally depends on constant signals from their microenvironment, including cytokine secretion and interactions with other immune cells 167 .

Cytokine regulation of NK cells

The main activators of NK activity are type I IFN, IL-12, IL-18, and IL-15. IL-15 ligates with a high affinity to the IL-15R receptor, and the derived signal plays an important role in NK survival through regulation of the anti-apoptotic family Bcl2 168 . It is also a key element in priming NK cells to react more rapidly and efficiently by establishing a stock of lytic granules. Furthermore, IL-15 is required for IFN- secretion and NK-cell maturation and proliferation 169 .

In vitro, IL-2 is a powerful activator for NK-cell proliferation and functionality 170 . The receptor IL-2R on NK-cell membranes is triggered upon ligation with IL-2, which activates the downstream signals JAK1 and STAT4. The role of IL-2 is well established as promoting NK cell proliferation, cytokine secretion, and cytotoxicity 171 . Together, IL-12/IL-18 principally affect IFN- secretion, while IL-18 plays a role in the expression of CCR7 and the secretion of CCL3 172,173 . However, some cytokines such as IL-21 act negatively to inhibit the proliferation of NK cells while increasing their cytotoxic effect 174 . IL-4 also inhibits NKG2D expression and the production of TNF, IFN-, and granulocyte-macrophage colony-stimulating factor (GM-CSF) 175

NK cells: beyond natural killing

An effective immune response relies on rapid and flexible coordination between the innate and adaptive immune systems. Cytokine secretion and direct interactions between immune cells control the maturation, development, and activity of each cell population.

Besides the cytolytic function of NK cells, their functions go beyond killing. Indeed, NK cells play a critical role in shaping other immune cells by mediating a refined and developed activity that impacts innate and adaptive immune cell function 176 .

One of their primary important effects is regulating the function of the antigenpresenting DC 177 . The interaction between the two cell types is considered reciprocal as the cells regulate one another, and the generated results are required for an appropriate immune response 178

Immune-based cancer treatment

Immunotherapy is a form of cancer treatment with the intent of empowering the immune system against tumor cells, either by energizing the immune cells themselves or by ending break-off and resistance against the immune system. Cancer immunotherapy is favorably evolving and can now be considered a primary choice of therapy, joining such classical therapies as surgery, radiation, chemotherapy, and targeted therapy. With the development of immune-based therapy, attention is not only on targeting the tumor itself, but on targeting immune cells in the tumor microenvironment (TME) to reinforce their intrinsic immunological killing 183 . Thus, immunotherapy relies on the insight that the innate and adaptive immune systems recognize and eradicate tumor cells. Interestingly, some types of tumors show a dramatic and efficient response, while others do not respond at all 184 . Cancer immunotherapy seeks to reinforce the patient's immune mechanisms against tumor cells with an aim of having fewer side effects by not targeting healthy cells.

Unfortunately, therapy strategies such as chemotherapy and radiation are unable to distinguish between healthy and malignant cells when they kill 184 .

2.1.Types of immunotherapy

Approaches to stimulate immune cells involve vaccination with tumor antigen or increasing the number of antigen presentations. Adoptive cell therapy (ACT) is an additional strategy that involves the direct uptake of activated immune cells. Moreover, administering oncolytic viruses has proven to be highly efficacious in initiating antitumor immunity. Recombinant proteins that either block immune checkpoints or stimulate immune cells are also used 185 .

Cancer vaccination

For decades, the idea of developing therapeutic cancer vaccines similar to those used against infectious agents has been of interest to researchers 186 . The objective of cancer vaccination is to stimulate immune cells to elicit an immune response against tumors. Although many vaccines have shown the capacity to activate immunity, their clinical efficiency has remained less than expected 187 . Activating host T cells against tumors was one the first targeted approaches. Vaccines are composed of antigens and adjuvants that elicit the activation of the antigen-presenting cells responsible for shaping the immune cells. Whole cells, cell lysates, and purified antigens are sources for tumor antigens.

However, the main obstacle is finding the optimal and suitable antigen that can stimulate immunity. The first cancer vaccine to be approved by the FDA was in 2010 for sipuleucel-T, a treatment for metastatic castration-resistant prostate cancer. The vaccine is made from a patient's PBMCs and enriched ex vivo. The cells are subsequently fused with prostatic acid phosphatase (PAP) and GM-CSF 188 .

DCs are the most effective antigen-expressing cells that bridge the innate and adaptive immune systems 183 . The strategy is to provide a high quantity of appropriate antigens to load onto the DCs, and accordingly, allow the tumor cells to break the tolerance to immune cells. A DC-based vaccine strategy relies on isolating DCs from a patient's PBMCs and activating them with tumor antigens before re-infusing them into the patient 189 .

Oncolytic virus

The therapeutic outcomes for oncolytic viruses derive either from the ability of some viruses to induce tumor killing or by their stimulation of immune-based lysis 190 . The viruses are engineered to limit their lytic activity exclusively to cancer cells 191 .

Talimogene laherparepvec (T-VEC), a modified herpes simplex virus type 1 and the first oncolytic virus approved by the FDA (in 2015), is for the treatment of advanced myeloma 192 .

Adoptive cell therapy (ACT)

Immunotherapy's exploitation of the cytotoxic granule activity in immune cells has revolutionized immune-based cancer treatment. For instance, ACT is taking advantage of the perforin and granzyme pathways. The concept of ACT is to isolate a patient's PBMCs, expand them ex-vivo, and re-infuse them back into the patient 193 . The lymphocytes can be isolated from peripheral blood, lymph nodes, or tumor tissue. In melanoma patients, depleting immunosuppressive cells such as Treg cells or co-injecting IL-2 has improved the efficiency and clinical outcome of ACT for tumor infiltrating lymphovytes 194 . Another strategy is to transfer T cells engineered and generated ex vivo.

One example involves the expression of the TCR  and  chains that ligate with HLA ligands 195 . Another approach is the transfer of chimeric antigen-receptor (CAR) cells, which are an antibody-recognition domain fused to a TCR-constant domain. This new FDA-approved strategy has shown promise in treating patients with hematological malignancies such as acute B-cell lymphoblastic leukemia and B-cell lymphoma [196][197][198] .

Immune checkpoint blockade

Immune checkpoints play a critical role in controlling the immune response against pathogens and cancers 199 . A plethora of immune inhibitory pathways are generated upon contact with cancer cells. Any blockage to inhibitory checkpoints, and therefore their derived inhibitory signals, is a promising approach for triggering an anti-tumor immune response 200 . Here we focus on the main approved checkpoint inhibitors for cancer immunotherapy 201 .

The first inhibitory checkpoint to be targeted was cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152, an inhibitory receptor and negative regulator expressed on regulatory T cells 202 . CTLA-4 is a homolog of the co-stimulatory receptor CD28, but with a high affinity to CD80 (B7-1) or CD86 (B7-2) ligands on the membranes of antigen-presenting cells. Ligation with B7 molecules leads to a net negative signal, which limits IL-2 production and T-cell survival 200 Pembrolizumab and nivolumab antibodies target PD1 and are approved by the FDA to treat melanoma, head and neck squamous cell carcinoma, and NSCLC 205 . Additionally, due to the high clinical response it has shown, nivolumab was also approved for Hodgkin lymphoma and renal cell carcinoma. Moreover, atezolizumab, avelumab, and durvalumab are antibodies against PD-L1. Atezolizumab produces a therapeutic response in many types of cancers such as melanoma and colon, lung, gastric, and head cancers. It was approved by the FDA in 2016 for the treatment of NSCLC and urothelial carcinoma 184 .

V-domain immunoglobulin suppressor of T cell activation (VISTA) is an inhibitory immune checkpoint that downregulates CD4 + and CD8 + T-cell activation 206,207 . It is expressed on naïve CD4 + and CD8 + cells, Foxp3 + CD4 + regulatory cells, and DCs. VISTA blockage could soon be a target for novel therapies 200,208 . Lymphocyte activation gene 3 The blockade of TIGIT in combination with a PD1 mAb resulted in the improvement of CD8+ T-cell functionality and tumor rejection. Anti-TIGIT can also be combined with anti-LAG3 or anti-TIM3 to confer anti-tumor immunity [START_REF] Wang | TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals[END_REF] .

2.2.NK cell-based immune editing

Although immunotherapy studies have focused more on T cell-based therapy, recent studies have shed light on the potential of NK cells as a powerful directed immunotherapy approach. Overcoming the immunosuppressive microenvironment and inducing antitumor activity in NK cells have been exploited to cure cancers in different approches 211 .

Adoptive NK-cell therapy

The first approach is adoptive NK-cell therapy whereby NK cells from healthy donors (allogenic NK cells) or the patient (autologous NK cells) are isolated and re-infused into the patient after ex vivo expansion and activation with cytokines (IL-2, IL-12, IL-15, and IL-18) or stimulatory molecules. Cytokines, and especially IL-2, are administrated after the re-infusion in repeated injections at low doses. Allogenic NK-cell transfer shows a higher efficacy and optimal anti-tumor effect than autologous NK-cell transfer due to the KIR mismatch (haploidentical). Furthermore, NK cells don't exert graft-versus-hostdisease (GvHD) 212 . The disadvantage of this method is the poor proliferation and persistence of NK cells in the patient. Given the donor-dependent variability of NK cells, studies have shown the high efficiency of infusing NK-cell lines such as NK-92, which lacks the KIR inhibitory receptor and therefore is highly cytotoxic 213,214 . Genetic modifications to NK cells have also proven to enhance NK-cell anti-tumor activity. The infusion of CAR T, CAR-NK, or CAR-NK-92 cells is a promising approach at the preclinical stage that involves increasing tumor specificity in NK cells for the treatment of B-lineage acute lymphoblastic leukemia 215 (Figure 7).

Tumor antigen-specific antibody administration

Targeting tumor-associated antigens with therapeutic antibodies plays a role in inducting NK cell-mediated ADCC. The chimeric monoclonal antibody (mAb) anti-CD20 (rituximab) is indicated for the treatment of chronic lymphocytic leukemia and non-Hodgkin lymphoma. The Fc portion of rituximab mediates ADCC as well as CDC, and thus enhances NK-cell cytotoxicity [START_REF] Wang | NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement[END_REF]216 . Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb, is used for colorectal cancer, NSCLC, and head and neck

NK cell genetic modifications

Clinical studies with NKG2D-or TRAIL-overexpressing NK cells showed they have improved tumor recognition and lysis capability after re-infusion in the patient.

Immunosuppressive cytokines such TGF- are secreted into the TME to downregulate the activity of NK cells. Studies show that NK cells engineered with TGF- dominantnegative receptor II protect NK cells and improve their survival. In addition to the genetic modification of NK cells, clinical trials with TGF- inhibitors and adenosine A2A agonists are ongoing 215 .

Supplement cytokine uptake

IL-2, IL-12, and IL-15 are activator cytokines for NK cells, and their uptake could enhance the anti-tumor response. In melanoma and metastatic renal cell carcinoma, the combined administration of IL-15 or IL-2 with lymphokine-activated killer cells has a 20% response rate 215 .

NK-cell immune checkpoint targeting

Immunomodulatory drugs

Immunomodulatory drugs can regulate NK-cell function and anti-tumor activity.

Thalidomide and its derivate lenalidomide have proven to be efficient in multiple myeloma by increasing IL-2 secretion 223 . Equally, tyrosine-kinase inhibitors, proteasome inhibitors, and serine-threonine kinase GSK3 have direct effects on NK cell-mediated cell activity 224 and thus their combination with anti-cancer drugs could be a promising way to enhance NK-cell function 222 (Figure 8).

Figure 8 | Targeting the tumor microenvironment to improve NK cell responses

Various strategies can be adopted to create a microenvironment favorable to NK cells to improve the efficacy of NK cell-based therapies including the administration of activating cytokines, blocking antibodies, chemotherapeutics and small-molecule inhibitors. Adapted from Guillerey et al., 2016. Mammary glands are made of individual lobes that are regulated upon puberty in response to the endocrine system and secreted hormones. Each gland consists of a central duct connected to many branched ducts. These ducts and lobules are connected and surrounded by the stroma, which is made up of fatty connective tissues as well as blood and lymphatic vessels. Breast cancer that starts in the epithelial lining of milk ducts is termed ductal carcinoma. However, in some cases, it can also start in the milk-producing glands (lobules). Two forms of ductal carcinoma exist: ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). The latter tends to spread to other parts of the breast and body via neighboring lymph nodes. Less common types of BC also occur such as inflammatory BC, medullary carcinoma, mucinous carcinoma, and tubular carcinoma 227,228 .

Breast cancer and immune evasion

Mammary glands develop due to ovarian hormones as well as pituitary hormones and growth factors. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are secreted from the pituitary gland and stimulate the ovaries to secrete the female sex steroid hormones estrogen (estradiol) and progestin (progesterone). These hormones are a key element in the development of mammary glands and tumorigenesis 229 .

Types of breast cancer

Breast cancer shows a high level of heterogeneity in terms of biomarkers, histological grades, and morphology. The classification of BC is defined by the expression or lack of expression of three distinct protein receptors: estrogen receptor (ER), progesterone receptor (PR), and HER2. Breast cancer is also classified based on the tumor size, lymph node implications, and age.

The ER + group represents the most varied group with regard to genetic diversity, but it responds well to treatment. This group is also categorized as luminal A, which is ER- positive with a low histological grade, or luminal B, which is low ER- positive with a high histological grade. The luminal A phenotype responds to anti-estrogen therapy better than the luminal B phenotype. The second group is the HER2 + group, which is characterized by the high expression of HER2 receptors and accounts for 30% of all BCs 230 . Compared to the ER + group, HER2 + has shown better prognoses and clinical outcomes. The basal-like phenotype of cells lacking the three receptors is defined as a triple-negative breast cancer (TNBC). TNBCs tend to be the more aggressive form, as generally this group does not respond to targeted treatments such as anti-estrogen or anti-HER2 antibodies 231 . Nevertheless, the complicity and heterogeneity of BC groups limit the possibility of a common therapeutic effect for treatment 232 .

Breast cancer therapeutic strategies

Breast cancer is becoming a major area of interest for researchers due to its high malignancy in women. Traditional therapies such as radiation and chemotherapy are becoming less effective, as they harm the healthy tissues of the body. Surgery is also considered to have a limited effect due to the rapid metastasis of BC after prognosis.

Alternative therapies, especially immune-based therapies, are emerging as promising therapeutic strategies 233 .

General treatments

Owing to the heterogeneity of BC, the choice of an appropriate and optimal therapy option relies on many factors such as the type and stage of the tumor, genomic markers, patient age, menopausal status, and the presence of mutations. Likewise, the biology of the cancer and its size affect the decision on a strategy. Several therapeutic approaches can be used such as surgery, chemotherapy, radiation, hormone therapy, targeted therapy, and immunotherapy. Surgery is the choice for DCIS and early stage invasive BC. Lumpectomy is the removal of the tumor and the tissue surrounding it, whereas mastectomy is the removal of the entire breast. The removal of lymph nodes is also one of the surgical choices. Usually surgery is followed by radiation therapy (ionizing irradiation) as an adjuvent 234,235 . Systemic chemotherapy is one powerful treatment option because it destroys cancer cells and their ability to grow and divide. There are many common drugs used to treat BC, alone or in combination with other adjuvant therapies. Methotrexate, cisplatin, fluorouracil, cyclophosphamide, and doxorubicin are commonly used chemotherapeutic agents 227 .

Endocrine or hormonal therapy is one of the most effective treatments for the corresponding cancer type that can be used alone or as an adjuvant therapy. Blocking hormones can lead to the prevention of cancer growth and recurrence. Tamoxifen acts as an antagonist and blocks the effect of estrogen by binding to estrogen receptors. It is the drug of choice for ER + patients and has proven to lower the growth, recurrence, and risk of developing cancer 236 . Aromatase inhibitors are used to decrease the amount of estrogen by blocking the enzymatic activity of aromatase, which is responsible for converting the male hormone androgen into estrogen 229 .

Breast cancer immune-based treatment

Although BC has been considered a non-immunogenic tumor type, studies have shown that certain types of BC are highly infiltrated by immune cells and have a high response rate to immune checkpoint inhibitors. These are the TNBC and the HER2 -BCs. The next section will provide insight into the various processes with a focus on the implications of the EMT and autophagy to better understand how cancer cells escape immune surveillance and resist T and NK cell attacks.

Mechanisms of immune evasion in breast cancer

Intrinsic tumor resistance

Immune cells recognize tumors via antigens and ligands presented on the surface of the tumor. Breast cancers express surface antigens distinct from normal cells, triggering immune cells to recognize them. Melanoma-associated antigens (MAGEs) are a family of cancer/testis antigens expressed on the surface of BC cells, but not on normal cells (only in testis and placenta). MAGE-A9 is highly expressed in ductal BC and is associated with negative consequences. However, MAGE-A9 is expressed less in ER -and HER2 - BC 244,245 . Another antigen is mesothelin, a cell-surface glycoprotein highly expressed on the surface of TNBC. It is considered a tumor marker and can modify TCRs. Moreover, HER2/neu is known to be overexpressed in HER2 + BC, and higher levels of HER2/neu were found in patients with BCs possessing a more aggressive phenotype 246

Immune cell suppression

Tumor progression and metastasis is not only restricted to modifications in tumor cells, (Figure 9).

Figure 9 | Mechanisms of immune evasion in breast cancer

A summarized network of immune evasion mechanisms and their interactions. Adapted from Wang et al., 2017.

Epithelial-to-mesenchymal transition

A key element mediating tumor metastasis and invasion is the transitional state between the epithelial and mesenchymal phenotypes.

EMT biomarkers

E-cadherin is a calcium-dependent cell-surface protein responsible for adhesion in epithelial cells through the cytoplasmic and extracellular domains. Its expression is a hallmark of the epithelial phenotype, and any suppression of its expression can lead to a mesenchymal phenotype. In BC cells, the loss of E-cadherin and the acquisition of Ncadherin are correlated with the acquisition of invasiveness and metastatic behavior, as well as a poor prognosis 266,267 .

Vimentin is a hallmark of the mesenchymal invasive phenotype. It is an intermediate filament expressed at the site of cellular elongation and spikes. In BC cells, vimentin expression is regulated independently by Smad-interacting protein 1 (SIP1) in epithelial cells 268 .

In the BC basal phenotype, -smooth muscle actin (-SMA) is abundant in metastatic cells in comparison to non-metastatic one 269 . -Catenin is a protein localized to the membrane of epithelial cells that translocates to the cytoplasm and the nucleus upon EMT

Gene expression and transcription factor regulation in EMT

The downregulation of E-cadherin and loss of the epithelial barrier function is controlled by the repression of genes encoding cell-junction proteins and the activation of genes generating mesenchymal proteins such as N-cadherin. The latter connects to the cytoskeleton and interacts with receptor tyrosine kinases (RTKs), comprising plateletderived growth factor (PDGF) and fibroblast growth factor receptors (FGFRs). In mesenchymal cells, the overexpression of vimentin regulates the trafficking of organelles and membrane associated proteins, and it interacts with motor proteins that enable cell

Mesenchymal cell resistance to T cells and NK cells

The EMT is defined as a tumor-related immune evasion and cancer cell intrinsic

EMT implication in resistance mechanisms

In vitro and in vivo studies showed that induction of the EMT is associated with

Autophagy

Cancer cells respond to the hypoxic TME by activating autophagy to increase their anaerobic-dependent metabolism. Autophagy (also known as macroautophagy) is a highly conserved cytoplasmic degradation system and catabolic process. It is based on the formation of autophagosomes, the double-membrane vesicles responsible for surrounding cellular debris such as organelles and proteins. Autophagy has different cellular functions such as adaptation to starvation, protein and organelle degradation, cell death, anti-aging, development, and tumorsuppression 302 .

The discovery of the autophagy process began in 1963 in Christian de Duve's group.

Soon after, the anti-malarial drug chloroquine (CQ) proved to have anti-tumor activity. It took from then until 1998 to discover that CQ inhibits the autophagic process. Since then, a large number of findings have highlighted the biological functions of autophagy within healthy and aberrant cells. The Nobel prize for physiology and medicine in 2016 was awarded to Yoshinori Ohsumi for his contribution in shaping our knowledge of autophagy 303 . The autophagy process was known to be a key inhibitor for tumors, but studies have now shown that an increase in autophagic flux promotes tumor growth, resistance, and survival. The important question in cancer therapy is whether the activation or inhibition of autophagy should be the objective when targeting tumors.

Understanding the implications of autophagy and related pathways in tumor cells has become a major area of interest for researchers in recent years.

5.1.Types of autophagy

Three different types of autophagy have been described thus far, including macroautophagy (here referred to as autophagy), microautophagy, and chaperonemediated autophagy (CMA). Microautophagy is a process by which lysosomes degrade cytoplasmic components that enter through a metabolite transporter or a deformation in the lysosomal membrane. In contrast to both micro-and macro-autophagy, which nonspecifically degrade cellular components, CMA is a specific autophagy in which the engulfed proteins are translocated to the lysosomal membrane via cytosolic chaperones 302 .

The CMA process destroys proteins, transcription factors, glycolytic enzymes, and proteasome subunits. During starvation, CMA is responsible for oxidized-protein elimination and amino-acid delivery. The cytosolic substrate includes the KFERQ-related motif, which is recognized by the heat shock cognate of Hsp70, a cytosolic chaperone.

Lysosome-associated membrane protein 2A (LAMP-2A) translocates the complex throughout the lysosomal membrane 304 (Figure 13).

The next section will focus on and provide an overview of the main autophagic processes and signaling pathways in cancer and their implication for tumor evasion from the immune system.

Functions of autophagy

Autophagy is considered a survival mechanism used by cells under specific conditions. A principal role of the autophagic process is its contribution to basal cellular and tissue homeostasis by the elimination of cytoplasmic contents, including proteins and organelles. The best evidence for this function is the accumulation of aberrant molecules such as ubiquitinated proteins and deformed organelles in autophagy-deficient neurons and hepatocytes. Autophagy is also implicated in lipophagy for regulating lipid

Figure 14 | Stages of autophagy

The process of autophagy is divided into five distinct stages: initiation, vesicle nucleation, vesicle elongation, vesicle fusion and cargo degradation. Adapted from Mulcahy Levy et al., 2017.

5.3.The autophagy link to the cytoskeleton

The interaction between autophagy and the cytoskeleton is still not well studied. Few reports describe the implication of the actin cytoskeleton in the biogenesis of autophagosomes from the ER or cargo transport. Actomyosin plays a role in integrating different cellular debris types into the growing cargo before detachment from the ER membrane 317 . The first evidence implicating the actin cytoskeleton in the autophagy process was the failure of autophagosome formation in cells treated with actin-disrupting drugs such as cytochalasin D and latrunculin B 318 . Other studies further established the colocalization of actin filaments and autophagy markers during the formation and transport of autophagosomes 319 (Figure 15).

The first actin element found to interfere with autophagosome formation was the nucleation factor ARP2/3. Pharmacological or genetic targeting of ARP2/3 produces decreased LC3-II and autophagosome levels and inhibits the formation of omegasomes, an early structural stage of phagophors 320 . Furthermore, ARP2/3 is localized to the sides of autophagosomes at the points of actin comet tails, which are responsible for transporting autophagosomes after detachment from the ER 321 . ARP2/3-dependent actin filaments are important in phagophore formation, as are cofilin and CapZ 322 . Cortactin, which stabilizes actin branches formed by the ARP2/3 complex, is recruited to the autophagosome and lysosome fusion lysosomes 323 .

Besides the evident role of ARP2/3, its activator also seems to play a crucial role in the autophagic process. The WAS protein family homologue (WASH) complex has been found to co-localize with phagosome markers, and its inhibition leads to autophagy being inhibited due to Beclin-1 suppression and defective ATG9 transportation 324 . The nucleation-promoting factors JMY (junction-mediating and regulatory protein) and WHAMM (WAS protein homolog associated with actin, Golgi membranes, and microtubules) are involved in the early stages of autophagosome formation and maturation 321 .

The Rho-family GTPases control a wide variety of actin-related processes, but their precise role in autophagy is still not clear. RhoA has been shown to have a stimulatory effect on autophagosomes via the regulation of Rho-associated protein kinase (ROCK),

while Rac depletion appears to enhance the autophagic pathway 325 . Recent data have shown that Cdc42 is as an important regulator of the autophagy process, as knocking it down produces reductions in LC3-I and LC3-II levels. These results suggest a crucial role for Cdc42 and its downstream proteins in the autophagic pathway.

The role of autophagy in cancer immune evasion

Studies in recent years have focused on the implications of the autophagic process in various cancer types. However, the process can either inhibit or promote tumorigenesis, suggesting its role is context dependent. Some results have shown that autophagy has an important anti-tumor function and influences the initiation and progression of tumors 304 .

In a large number of cases, patients with ovarian, breast, and prostate cancers with aggressive phenotypes have had monoallelic disruptions of BECN1 on chromosome 17q21. Indeed, many autophagy-associated protein deficiencies in mice are associated with tumorigenesis, while the deletion of Beclin 1 results in embryonic lethality 311 .

Moreover, cytoplasmic LC3 has been found to be elevated in tumor tissues after chemotherapy.

It is, however, well established that autophagy is highly associated with the survival of tumor cells under stress related to hypoxia and inadequate angiogenesis. Tumor cells depend on autophagy due to deficiencies in the TME and the resulting increased metabolic demands. Remarkably, there is a logical association between autophagy and apoptosis given that one role of autophagy is in protecting cells from undergoing programmed cell death. In a recent study on BC cell lines, shRNAs targeting more than 100 autophagy regulators were applied to detect the level of survival and dependence on the autophagic process. The results showed that some cell lines experienced no global effects from inhibiting autophagy, while other cell lines were dependent on it. These outcomes were related to STAT3-mediated autophagy dependence 326 .

Autophagy has been implicated in the resistance of several cancer types such as NSCLC, bladder, thyroid, melanoma, and BRAF-mutated central nervous system cancers.

A recent study in B16 mouse-melanoma cells and the 4T1 human mammary carcinoma cell mouse model showed that T-cell responses to autophagy-competent and autophagydeficient tumors (either genetic deletion of autophagy genes or CQ treatment) are similar 327 . Moreover, another study showed an enhanced immune response against tumors upon autophagy activation 328 . Countering the studies that showed no effect from autophagy inhibition, other studies have reported that autophagy inhibition resulted in an increase in immune anti-tumor activity and tumor regression in breast and melanoma cancer models in mice 288,329 . One struggle in targeting autophagy during tumor treatment Overall, targeting autophagy in tumors promises to be a novel therapeutic strategy by enhancing the immune response, disrupting the immunosuppressive TME, and supporting anti-tumor therapies.

The actin cytoskeleton

Cellular functions, including cell division, migration, and shape, are regulated by a dynamic cytoskeleton. The three filament networks (actin, microtubules and intermediate filaments) that constitute the cellular skeleton are present in all types of cells, from plants to prokaryotes and eukaryotes. Contrary to older research that described a rigid cytoskeleton, recent decades have shown the cytoskeleton to be a dynamic structure with many essential biological functions. Along with its role in shaping the cell, the cytoskeleton participates in cell migration, division, intracellular trafficking, and many diverse processes.

Since the main goal of my PhD thesis is to reveal how actin cytoskeleton dynamics in tumor cells control immune evasion, this chapter will provide an overview of the actin cytoskeleton and its regulation.

A historical overview of actin

Structure of the actin molecule

The actin monomer is a 43 kDa globular protein with dimensions of 55 Å × 55 Å × 35 Å.

It is the most abundant protein in most eukaryotic cells and the most conserved, with 90% identity between yeast, plants, and mammals. Studies in recent decades have also shown actin homologues in archae and bacteria (15% identity). Three main actin isoforms are described: the -isoforms of cardiac, skeletal, and smooth muscles and the -and isoforms of muscle and non-muscle cells, respectively. Each isoform has a different function attributed to differences in a few amino acids at the N-terminus. -Actin is implicated in contractile structures and functions, while -and γ-actin provide mechanical support to maintain cell shape via actin polymerization in addition to their involvement in cell movement and intracellular transport.

Since the first atomic structure was revealed in 1990, eighty crystal structures have been reported for G-actin by co-crystallizing it with DNase I or profilin. An actin polypeptide is composed of 375 residues folded together into a flat protein. G-actin is composed of two primary domains ( and -domains, also called the small and large domains) that are separated by two clefts. Four subdomains have been identified. Subdomains 1 and 3 are small and structurally related, whereas subdomains 2 and 4 are large and insert into subdomains 1 and 3, respectively. The upper cleft binds the nucleotide (ATP or ADP) and is associated with divalent cations (Mg +2 or Ca +2 ), while the lower cleft mediates contact between the subunits in a filament and is a major binding site for ADP. The strength of subunit interactions and the binding affinity of actin-binding proteins (ABPs) are controlled by nucleotide-dependent conformational changes, which essentially relies on interactions between the two clefts. Remarkably, monomeric actin exhibits different conformations when bound to ATP and ADP.

The actin cytoskeleton is composed of a network of assembled double-stranded AFs with different architectural types. The first organization is the cell cortex, a cross-linked network that creates a thin layer, which is important for cell shape and motility.

Lamellipodia, localized at the front of the cell as branched and cross-linked networks, are implicated in cell motility and movement. Another structure is the aligned and parallel bundles of filopodia, which are thin protrusions at the front of the cell and important for directional cell migration and adhesion. The fourth architectural form is stress fibers, composed of an anti-parallel contractile 3D network that is abundant throughout the body of the cell and provides force for many cellular processes (Figure 17).

Monomeric G-actin is gathered in a highly regulated process by numerous ABPs to form an AF. Each filament is approximately 7 to 10 nm long with a right-handed helical twist, with each twist composed of 13 subunits. Remarkably, the reconstruction of electron micrographs of frozen-hydrated filaments provided the best structure of an AF.

Principles of actin polymerization

Actin monomers continuously cycle through the process of polymerization and depolymerization. The polymerization process happens spontaneously under physiological salt conditions (in vitro) in the presence of either monovalent or divalent cations.

Importantly, the process relies strongly on the concentration of G-actin. Two steps shape the polymerization process: a rate-limiting step defined by the nucleation of short oligomers and the elongation step. Polymerization begins with the formation of a dimer, and subsequently, the addition of a third monomer to form a trimer 335 . This association reaction is unstable and rapid; however, it becomes more stable with the addition of a forth monomer to produce a large oligomer. Remarkably, the concentration of dimers and trimers is low during the polymerization reaction 337 .

Once trimers are formed, the elongation step begins. This involves the addition of

Actin binding proteins

It is well established that the dynamics of the cytoskeleton are governed by a vast array of ABPs. Actin filaments employ diverse mechanisms through ABPs to cope with the cellular condition, migration and invasion, and responding to intra-and extracellular signals. These proteins function to provide a large pool of actin monomers for the polymerization process. The ABPs are listed below and classified by their proper group function. Given the time and space, among the more than 100 ABPs, I will highlight the most effective proteins that regulate the cytoskeleton 338 .

Monomer-binding proteins

Monomer-binding proteins are essential in regulating the actin monomer concentration within the cell. Under specific conditions and requirements, they either accelerate or prevent actin polymerization. Spontaneous actin assembly begins with one oligomer and depends on one critical concentration, that of the actin monomers 339 .

Profilin is a small protein (~15 kDa) that is highly expressed in eukaryotes and abundant in the cytoplasm. It competes with thymosin-4 in maintaining large amounts of non-polymerized actin. Several functions have been established for profilin. First, it promotes polymerization by transporting actin monomers to the barbed end 340 . This transport is mediated by binding to the actin nucleator formin at its proline-rich forminhomology 1 (FH1) domain, which leads to an increase in the elongation rate. Remarkably, profilin prevents the nucleation of new branches or the addition of new subunits to the pointed end by binding to ATP-G-actin. Moreover, profilin catalyzes nucleotide exchange by binding ATP to recently depolymerized monomers after ADP severance 341 . Thymosin-4 is abundant in the cytoplasm and is a highly conserved polypeptide containing 43 amino acids with a high similarity to the WH2 domain. It not only sequesters actin monomers, but it is also a key player in determining the actin critical concentration by its formation of a complex with profilin and actin. It has a high affinity for Mg-ATP-actin, and therefore maintains the pool of non-polymerized actin monomers.

Nucleating proteins

Monomer-binding proteins can prevent AF polymerization. As a consequence, other mechanisms have evolved in cells to substitute for spontaneous actin assembly. Actin nucleator proteins are required at the initial step to promote the de novo polymerization of AF. Formin and actin-related protein 2/3 (ARP2/3) complex are the main classes of nucleating proteins in eukaryotes. As these proteins are of major concern in my study, this section will describe their structure, function, and regulations 342 .

ARP2/3 and its related regulators

The first nucleating factor to be identified was the actin-related protein ARP2/3, which is a highly conserved large 220 kDa complex. ARP2/3 is composed of seven evolutionarily related subunits: ARP2, ARP3, and 5 isoforms of ARP complex component (ARPC1-5). Upon binding to the side of an existing AF, the complex functions to nucleate filaments and organize them into a Y-branched structure (~70°). The newly formed branch has a capped pointed-end and a polymerization-competent barbedend. ARP2 and ARP3 are known to bind ATP, and this interaction is crucial for the activity of the complex. The hydrolysis of ATP leads to the detachment and release of the complex. It has been shown that ARP2 and ARP3 localize at the pointed-end of the new branch, while APRC2 and APRC4 associate with the parental filament. This interaction with the filament gives ARP2/3 its activity. Moreover, the phosphorylation of Thr and Tyr in ARP2 and interactions with nucleation-promoting factors (NPFs) are essential for a potent nucleation process (Figure 19).

Figure 19 | ARP2/3 structure and function

a) The morphology of a Y-branched actin filament and the actin-related protein 2/3 (ARP2/3) complex is shown in an electron micrograph and in structural models based on electron tomography. The ARP2/3 complex consists of ARP2 and ARP3 plus the additional subunits ARPC1-ARPC5. In this model, all seven subunits participate in binding to the existing filament and ARP2 and ARP3 act as the first two subunits of the nascent filament. b) ARP2/3 is recruited by the WCA domains of class I nucleation-promoting factors (NPFs) in proximity to cellular membranes (1). The collective activities of WASP homology 2 (WH2; W), connector (C) and acidic (A) segments serve the basic purpose of bringing ARP2/3 together with the first actin subunit in the new filament to generate a branch (2). ARP2/3 branch points can be stabilized by filamentous actin (F-actin)-binding class II NPFs, such as cortactin (3). Coronin family proteins interact with ARP2/3 and F-actin to prevent cofilin-mediated disassembly of newly formed filaments (4, top).

Coronin can also replace ARP2/3 and synergize with cofilin to trigger debranching and disassembly of older ADP-actin filaments (4, middle). Disassembly of older branches can also occur spontaneously, following phosphate release from ARP2 and actin ( 4

Formins

Formins are homodimeric proteins with formin-homology domains 1 and 2 (FH1 and 2) that bind to the barbed end of an AF. They have the role of nucleating and elongating unbranched actin complex and bundling new filaments. The formin family contains seven members that share FH domains 345 . When a formin protein encounters an actin subunit, the -helical linker of FH2 stretches, leading to it wrapping around the AF. The FH1 domain is enriched with proline-rich sequences that enable the formin protein to interact with profilin. Another important domain is the GBD. Due to the presence of the diaphanous auto-inhibitory domain (DAD) and the DAD-interacting domain (DID), formin functions can be auto-inhibited. Nevertheless, instead of using the WH2 domain, formins use the FH2 domain for interaction with AFs to promote elongation 346 .

Severing and depolymerizing proteins

The actin-depolymerizing factor/cofilin family is an essential member of the ABPs In addition, the gelsolin family contains 8 members that act to sever AFs. The principal gelsolin member is a potent Ca +2 -dependent protein that acts mechanically by changing the actin conformation and weakening the non-covalent bonds between actin molecules. After rapidly binding to the actin subunit, gelsolin acts slowly to dissociate the filament and actin as it caps the disassembled part. The uncapping of these small filaments enriches the cytoplasm with numerous polymerization-competent ends 350 .

Capping proteins

Eukaryote cells express proteins dedicated to capping the end of AFs and therefore inhibit continuous elongation. Their function is synchronized with profilin to ensure a limit on the number of polymerization-competent barbed ends. The capping process generally occurs in seconds and can last for 30 minutes. To end the capping process, polyphosphoinositides bind to the protein and inhibit its ability to form connections; V-1/ myotrophin is responsible for dissociating the capping proteins from the filament.

Moreover, capping protein Arp2/3 myosin I linker (CARMIL) contains a capping protein interaction motif that regulates the activity of capping proteins.

Tropomodulin is the main capping protein oriented to specifically cap the pointed ends of AFs to block new subunit addition or loss. -Actinin, CapZ, and Cap32/34 are also implicated in protecting the cytoskeleton from AF loss by binding to the barbed end., Some members of the gelsolin family also act as capping proteins.

Cross-linking and bundling proteins

An intact cytoskeleton is crucial for all processes that require spatial and temporal variation such as cell migration, invasion, division, and trafficking 351 . To meet these requirements, AFs are linked to each other to construct networks using actin cross-linking and bundling proteins 352 . One of the central functions of these proteins is to support the cytoplasm and cellular protrusions. Specialized and different polymorphisms can be obtained depending on the unique properties of the protein (e.g., its type and concentration) used to connect the network 353 . The importance of bundling proteins arises from their crucial role in the formation and function of actin structures such as lamellipodia, filopodia, stress fibers, and invadopodia. These organelles often contain more than one type of bundling protein, which coordinates cell dynamics. For instance, fascin, L-plastin, tensin, and -actinin form the parallel bundles and orthogonal networks of stress fibers and lamellipodia. -Actinin, talin, plectin, fimbrin, and vinculin form the protrusion's core. Dystophin is known to connect the cytoskeleton to the plasma membrane 351 .

The 55 kDa fascin polypeptides are the most abundant bundling proteins in all cytoskeletal organelles. Fascin consists of four -trefoil domains, including two major actin-binding sites on domains 1 and 4. It is well established that the expression of fascin is upregulated in many invasive and metastatic cancers. Therefore, fascin has emerged as a potent marker for cancer prognosis and a target for many therapeutic drugs 354,355 .

Another interesting member of the actin bundling proteins is L-plastin (also known as fimbrin). The expression of L-plastin was originally believed to only be in hematopoietic cells, but it was later shown that it is highly regulated in many types of cancer, and therefore, it is a vigorous marker for diagnosis 285 . The protein consists of two actinbinding domains that include two calponin-homology domains, and it is highly expressed in invadopodia and filopodia 356 .

6.5.Rho GTPases in the actin cytoskeleton regulation

Various extracellular stimuli can induce changes in the actin cytoskeleton through the Rho-family of p21 GTPases and their downregulation proteins. Among the 20 discovered Rho GTPases, Rho, Rac, and Cdc42 are the key regulators of AF dynamics. The abnormal regulation of Rho GTPase activation affects all cellular processes, including motility, invasion, division, and tumorigenesis 357 . Upon stimulation, Rho GTPases regulate the polymerization and organization of AFs by binding to ABPs. This process is mediated by the cyclic interaction of GTPases with GTPase activating proteins (GAPs) and guanine-nucleotide-exchange factors (GEPs), which are responsible for cycling GTP hydrolysis. Guanine nucleotide-dissociation inhibitors (GDIs) act to sequester the activation of GTPases 358 .

The implication of GTPases in actin cytoskeleton regulation was observed by Hall in 1993 after the injection of activated mutants of Rho GTPases into Swiss3T3 cells 359 . The first observations showed roles for RhoA in stress-fiber formation, Rac1 in forming lamellipodial protrusions, and Cdc42 in forming filopodial protrusions. All later studies have shown that the GTPases are tightly coupled and coordinate together to regulate the cytoskeleton through complex activation cascades 360 .

The stimulation of tyrosine kinase receptors on PI3K produces PIP3, which is a regulator of GEF, a promoter of GTP-nucleotide exchange. The downstream targets of Rho GTPases consist primarily of formins, WASP, and kinases. Rho-associated coiledcoil kinase 1/2 (ROCK), p21-activated kinase (PAK), and mammalian diaphanous formin (mDia) are the major downstream effector molecules for Rho GTPases 359 (Figure 21).

RhoA

RhoA is a ubiquitously expressed member of the Rho GTPase family with a crucial role in regulating the actin cytoskeleton and stress-fiber formation. GDIs maintain RhoA in an inactive GDP-bound state in the cytosol 361 . The dissociation of GDIs after activation leads to the translocation of RhoA to the plasma membrane, and hence, interaction with downstream molecules 362 . RhoA triggers ROCK-mediated actin cytoskeleton regulators such as myosin light-chain phosphatases, an important substrate for cell contractility.

Moreover, ROCK activates LIM kinase (LIMK), which is responsible for ADF/cofilin phosphorylation and stimulation. Furthermore, RhoA is responsible for the activation of formin and its isoform mDia1, which is involved in cytoskeletal and microtubule rearrangement 363 .

Rac1

Rac1 is an intracellular signal transducer implicated in the regulation of the actin cytoskeleton. It has been established that Rac1 plays an important role in lamellipodia induction and cell adhesion and migration. The downstream molecular signaling underlying actin regulation has also been well established. Rac1 is responsible for LIMK activation via PAK1. Additionally, Rac1 activates WAVE, whose structure lacks the GBD domain. This activation is mediated by binding to the adapter molecules IRSp53

and Abl. In addition, Rac1 regulates PI3K as well as the ERK pathway 364 .

Cdc42

Cdc42 is a ubiquitously expressed small GTP-binding protein in the Rho family of and their binding with Cdc42 is an important pathway in tumors. The increased expression of both proteins tends to be oncogenic and related to metastasis 358,366 .

Microinjecting constitutively active Cdc42 increases GTP-GDP cycling and hence, regulates the cytoskeleton and formation of filopodia. Some activating mutants lead to the induction of cellular transformation and foci formation. In the case of BC, Cdc42 deletion is key to the degradation of EGFR and consequently the reduction of cell growth and metastasis, whereas in melanoma and lung cancer, cdc42 overexpression serves as a prognosis marker 367,368 .

Cdc42 represents a potent therapeutic target for many types of cancer. Due to the difficulties in blocking only one of the GTPases without affecting the whole process, inhibitors of the downstream effectors are used. ZCL278 acts against interactions between Cdc42 and the GEFs, while secramine blocks the recruitment of GDIs. Another potent molecule is CID2950007, which binds to the guanine nucleotide site on Cdc42, inhibiting the GTP-GDP cycle. Moreover, the PAK inhibitors PF-3758309 and FRAX597 have been used in many types of cancer models to block Cdc42 downstream pathway 369 .

Actin cytoskeleton dynamics at the IS

Actin remodeling of the IS has been a focus of interest for many studies. Clarifying the actin filament and microtubule dynamics has led to a better understanding of the basics of immune cell-mediated cell lysis., Actin remodeling is a marker of immune cell activation in both T and NK cells and is a driver for cytotoxic granules 139 . The ISs of both T and NK cells show similarities in function and organization when considering the cytoskeletal rearrangement. This section will detail the AF dynamics 134 .

WASP, WAVE, and HS1 are key regulators of ARP2/3, which is responsible for AF polymerization. WAVE is known to regulate actin polymerization in T cells, but it does not play a role in NK cells. Interestingly, the IL-2-dependent activation of WAVE can compensate for the absence of WASP in patients with Wiskott-Aldrich syndrome. This result would explain the cooperation between immune cells to induce a functional NKcell lytic response. ARP2/3 complex activation can also be achieved by HS1, the hematopoietic homologue of cortactin. HS1 tends to cluster at the synaptic region where it is essential for connecting with LFA-1 signaling 370 . Interestingly, formin is implicated in the actin polymerization at T-cell synapses, but not in NK cells, where it functions only in assisting microtubule polarization 371,372 .

As discussed in Section 2. A further ligation of 2B4 receptors participates in destabilizing LFA-1 and the actin connection, and thus NK-cell detachment 375 .

The implication of actin at the synapse is not limited to adhesion receptor clustering, but also to activating and inhibitory receptor polarization. In T cells, the movement of activating receptors occurs in an actin and myosin IIA-dependent manner. Comparably, the NK-cell synapse shows a clustering of activating and inhibitory receptors (i.e., NKG2D and KIR2D) coupled to actin accumulation. Of note, NKG2D clustering at the IS has been observed to reduce KIR2D clusters in the same region 376 .

As actin remodeling is a dynamic process, AFs undergo continual re-organization after the adhesion step. Actin hypodensity is observed in the same region where activation receptor clustering functions to clear the way for MTOC and lytic granules. This specific remodeling is governed by cofilin and coronin 1A, which promote the turnover of actin and ARP2/3 377,378 . At the termination stage, actin is remodeled to its basic state with no further secretion observed 379 .

PhD project objectives

An effective NK cell anti-tumor response relies mainly on the establishment of an

Main article

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Supplementary Materials and Methods

Western blot analysis

Protein extracts were isolated in RIPA lysis buffer (Millipore) supplemented with a protease and phosphatase inhibitor mixture (Roche). The Bradford protein assay was used to determine the protein concentration (5000006; Bio-Rad). Next, 30 μg of protein lysate was separated by SDS-PAGE on a 12% SDS agarose gel and transferred to a PVDF membrane (Millipore). Membranes were immunoblotted with monoclonal rabbit anti-N-WASP (#4848, Cell Signaling), polyclonal rabbit anti-CDC42 (#2462, Cell Signaling), monoclonal mouse anti-Ecadherin (#610182, BD biosciences), monoclonal mouse anti-vimentin (#SC-6260), monoclonal rabbit anti-GAPDH (#5174S, Cell Signaling), polyclonal rabbit anti-actin (#A2066, SIGMA), polyclonal rabbit anti-SNAI1 (#sc-28199, Santa Cruz), or polyclonal rabbit anti-GFP (#A11122, Invitrogen) antibodies overnight.

Membranes were washed and incubated with HRP-conjugated goat anti-rabbit IgG. Protein bands were detected using the SuperSignal™ West Femto Chemiluminescent Substrate (Thermo Scientific) and visualized by ImageQuant LAS 4000 (GE Healthcare Life Science).

Cell labelling and confocal microscopy

For the conjugate formation assay, NK-92MI cells were counted, re-suspended and stained with PKH26 dye (PKH26 red fluorescent cell linker kit, Sigma-Aldrich) for 5 minutes at room temperature. NK-92MI cells were washed twice with RPMI medium containing FBS and horse serum and subsequently incubated with 1 x 10 5 target cells (E:T ratio of 2:1) per 200 μl of medium. Conjugates were plated on poly-L-lysine-coated chambered coverslips (μ-Slide 8 Well, Ibidi) for 45 minutes prior to fixation with 2% paraformaldehyde (PFA, Thermo Fisher Scientific) for 10 minutes. Cells were washed with PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich), and counterstained with DAPI (0.3 μg/ml, Sigma-Aldrich). For target cell staining, 1 x 10 5 target cells were seeded into chambered coverslips. The next day, cells were washed twice, fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and immunostained with anti-vimentin (Santa Cruz) or anti-Ecadherin (BD biosciences) primary antibodies, followed by secondary goat antimouse Alexa Fluor 488 antibodies for detection. For AF visualisation, cells were co-stained with Acti-stain 488 Phalloidin (Cytoskeleton) and DAPI. For assays with cytochalasin D, cells were stained with Acti-stain 555 Phalloidin (Cytoskeleton). For live-cell imaging, target cells were seeded into chambered coverslips (Ibidi) at a concentration of 1 x 10 5 cells per well. Slides were mounted within a temperature-controlled chamber maintained at 37 C under a constant atmosphere of 5% CO 2 . NK cells were labelled with the PKH26 Fluorescent Cell linker (Red) for 5 minutes at RT and washed three times. At the beginning of recording, 2 x 10 5 NK cells were added to each slide. Images of conjugates were acquired on a laser scanning confocal microscope (LSM880 FastAiry, Carl Zeiss) with an oil immersion Plan-Apochromat 63x objective. 3D projections and time lapse movies were assembled using Zen imaging software (ZEISS) and NIH ImageJ software (49), respectively.

Actin quantification

For each cell line, 25 conjugates from three independent experiments were analyzed for actin cytoskeleton accumulation. To quantify actin filament enrichment at the target side of the IS, the target cell image was divided into two equal regions of interest (one at the synapse and one at the opposite side). Florescence intensity of LifeAct-mEGFP was measured using the ROI tool of ImageJ software. Results were reported as the fold increase in the intensity of LifeAct-mEGFP at the synaptic region divided by the same intensity at the distal region.

Cytotoxicity analysis

Target cells expressing LifeAct-mEGFP were incubated with effector cells at an E:T ratio of 1:1 or 5:1 for 4 hours at 37° C. Before analysis, TO-PRO®-3 (Invitrogen) was added to assess the percentage of death target and then samples were immediately analyzed by flow cytometry (FACSCanto; BD Biosciences).

Degranulation assay

NK-92MI cells were incubated for 4 hours at 37°C alone or with LifeAct-mEGFPexpressing target cells (5:1). NK cells incubated without target cells were used as a negative control. The NK cell-susceptible K-562 cell line (kindly provided by the NORLUX Neuro-Oncology Laboratory from LIH; ATCC) was used as a positive control for the assay. To block intracellular protein transport and CD107 degradation, monensin-containing GolgiStop reagent (BD Biosciences) was added to samples after 1 hour of incubation at a concentration of 6 μg/ml. Two hours later, cells were stained with anti-human CD107a-APC (BD bioscience, clone: H4A3) and samples were immediately analyzed by flow cytometry (FACSCanto; BD Biosciences).

Discussion of the article in French

The actin response facilitates autophagy-mediated degradation of

Granzyme B in tumor cells (results and discussion)

In the article presented in section 1.2 of the results, we provide evidence that the actin response is associated with, and is actually required for, GzB level reduction in resistant target cells. Very interestingly, autophagy was recently proposed to promote NK cellderived GzB degradation in hypoxic MCF-7 cells, and thereby reduce target cell susceptibility to NK cell-mediated lysis 288,409 . In keeping with this, inhibition of autophagy in target cells improves tumor elimination by NK cells in in vivo mouse models of BC and melanoma 303 . Considering the multiple and critical roles of the actin cytoskeleton in autophagy 317 , I decided to investigate the link between the actin response and autophagymediated GzB degradation in tumor cells. The section below directly follows-up the article presented in section 1.2 and addresses the mechanism by which the actin response leads to a decrease in GzB levels in resistant breast cancer cells. The results and discussion have been merged and there is no separate discussion section.

Inhibition of autophagy reverts breast cancer cell resistance to NK cell-mediated cell lysis

First, we examined if autophagy is implicated in the resistance of normoxic breast cancer cells to NK cell-mediated cell death. To do so, MCF-7, 1001 and MBA-MB-231 cells were transfected with 2 different siRNAs targeting Beclin-1 transcripts. Beclin-1 is a key regulator of autophagy that is required for autophagic vesicle formation 302 . Beclin-1 knockdown has been previously shown to effectively inhibit autophagy in various types of cells, including cancer cells 303,313 . As shown in figure 22, western blot analysis confirmed that Beclin-1 was significantly knocked down in the three breast cancer cell lines after the transfection with the two siRNAs. We then analyzed the susceptibility of autophagy-deficient breast cancer cells to NK cell-mediated cell lysis after 4 hours incubation with NK-92MI effector cells at an effector-to-target ratio (E:T) 5:1. Cytotoxic assays revealed that the two highly resistant cell lines 1001 and MDA-MD-231 became significantly (2 to 3 times) more susceptible to NK cell-mediated lysis upon Beclin-1 knockdown. These data support that autophagy is an important mediator of breast cancer cell resistance to NK cell-mediated lysis under normoxia. In contrast, the highly susceptible phenotype of the MCF-7 cell line was not significantly modified by Beclin-1 depletion. This result is consistent with the low basal level of autophagy reported in MCF-7 cells cultured in standard, normoxic, conditions 410

Autophagy promotes Granzyme B degradation in resistant breast cancer cells

As previously stated, autophagy was reported to promote NK cell-derived GzB degradation in hypoxic MCF-7 cells 288,409 . We hypothesized that autophagy may have a (Figure 22). In addition, they extend the previous findings on hypoxia 288,409 and generalize autophagy-mediated GzB degradation as a general resistance mechanism against NK cell-mediated cell death. This also raises the possibility that hypoxia may increase tumour cell competency for the actin response. Although this remains speculative, it would be consistent with the fact that hypoxia is potent inducer of EMT 411 and that EMT, in turn, strongly increases the actin response rate in breast cancer cells (see Figure 3 from the article, p.129). If this turns to be right (experimental work in progress), then our data would provide a mechanistic insight into how hypoxia promotes autophagymediated GzB degradation (see below).

The actin response drives autophagosome polarization toward the synaptic region (preliminary results)

The actin cytoskeleton plays central roles in intracellular trafficking by promoting the formation and transport of various types of vesicles 336 . Furthermore, evidence has emerged over the recent years that actin polymerization is critically required for the formation and transport of autophagosomes 317 . To investigate the possible link between the actin response and autophagosomes in the resistance of breast cancer cells to NK cellmediated cell death, we analyzed autophagosome distribution in NK cell-conjugated breast cancer cells with respect to the presence or absence of an actin response. To do so, we used the autophagosomal marker LC3, imaging flow cytometry and specific masks and features in the IDEAS software for analysis. The peak mask allowed us to distinguish between cells with unspecific diffuse signal versus specific and highly localized LC3 puncta. The BDI R7 feature was used as it computes the intensity of bright spots at the selected region of interest that are less than 7 pixels in radius. In addition to LC3 intensity-based measurements, the spot count feature was used as a complementary method as it was shown to be a potent tool to assess the activation of autophagy 316 (Figure 24). The above data were validated using primary NK cells isolated from five healthy donors (Fig. 26). Both the BDI and spot count quantification approaches confirmed a higher level of autophagosome in the synaptic region of NK cell-conjugated 1001 cells exhibiting an actin response, as compared to that of target cells without actin response.

Preliminary data regarding the mechanism underlying actin response initiation (results and discussion)

This last result section presents some preliminary data regarding the mechanism(s) by which the actin response is induced in breast cancer cells, with a focus on NK cell adhesion to its target and NK cell activation.

NK cell adhesion and activation in actin response initiation

A critical question that remains to be answered is: what is (are) the signal(s) triggering actin response initiation in competent cancer cells? We hypothesized that either effector cell adhesion or effector cell activation could be key event in directing actin response initiation in the target cell. To evaluate such possibility, we analyzed the effects of pharmacological blocking of the adhesion molecule ICAM-1 416 and of MICA/B, a highly polymorphic ligand of the NK cell activating receptor [START_REF] Andre | Comparative analysis of human NK cell activation induced by NKG2D and natural cytotoxicity receptors[END_REF] . We conducted these analyses on MDA-MB-231 cells since they contain a large subpopulation of cells competent for the actin response.

First, target cells were incubated for one hour with purified mouse anti-human ICAM-1 (CD54) antibodies prior to washing and presentation to NK cells. Control cells were similarly treated using purified mouse IgG1, κB, Wnt, Isotype. As expected, blocking ICAM-1 reduced the number of conjugates (by about 32%) between MDA-MB-231 cells and NK cells (Figure 23a). Accordingly, it also decreased the overall cell line susceptibility to NK cell-mediated cell death in cytotoxic assays (Figure 23b). However, within the cell-cell conjugates that still formed upon ICAM-1 blocking, the rate of actin response was unchanged as compared to the control cells (Figure 23 c). This suggests that, although the adhesion molecule ICAM-1 is critical for conjugate formation (and accordingly efficient target lysis), it is not specifically involved in actin response induction.

Second, MDA-MB-231 cells were treated with purified mouse anti-human MICA/B antibodies or control mouse IgG2a, κB, Wnt, Isotype. As shown in Figure 23d, blocking of the activating ligand resulted in a statistically-significant increase of target cell resistance to NK cell-mediated lysis. Very interestingly, we found that MICA/B antibodies induced a significant reduction of the rate of actin response in conjugated target cells. This result suggests that NK cell activation is a prerequisite for the induction of the actin response in competent tumor cells. In other terms, the actin response would be triggered by a signaling pathway downstream of NK cell activation. and Spot Count features were applied on the mask of mEGFP-LifeAct to measure LC3 in whole target cell.

Statistical analysis

The During step 1, the effector cells recognize and adheres to its target. Physical contact between the two cells appears to be necessary but not sufficient to drive the actin response. During step 2, the NK cell cytotoxicity is activated following the recognition of upregulated activating ligands and/or deficient self-molecules on the target cell surface.

Our preliminary data indicate that blocking activating ligands, such as MICA/B, significantly lowers the rate of actin response observed upon NK cell attack, suggesting that NK cell activation generates a feedback signal towards the target cell that is responsible for actin response initiation. Such mechanism might serve as a safeguard against unnecessary induction of the actin response upon contact with non-killer cells.

Although the signaling molecules involved in actin response initiation remain to be identified, our data support that they converge towards Cdc42 and N-WASP, two key regulators of the ARP2/3 actin nucleation machinery 368 . During step 3, the massive accumulation of F-actin in the synaptic region of the target cell mediates (at least) two processes leading to cancer cell immune resistance. In process 1, the actin response induces a significant reduction in NK cell-derived GzB levels within the target cells. We assume that the quantity of GzB in target cells exhibiting an actin response is kept below the critical threshold required for effective apoptosis. Our mechanistic investigations show that the actin response promotes autophagosome accumulation near immunological synapse. This presumably locally increases autophagy at the main site of GzB entry, and thereby facilitates GzB degradation. Although actin polymerization plays several central roles in autophagosome biogenesis and transport 318 , our preliminary data support that the actin response induces polarization of pre-existing autophagosomes towards the synaptic region without modifying the overall quantify of autophagosome in the target cell. The exact mechanism by which the actin response mediates autophagosome recruitment at the synapse should be investigated. In process 2, the actin response stimulates the recruitment of inhibitory ligands, such as HLA-A, -B, -C in the synaptic region. This process is likely to alter "long term" signaling towards the effector cell and may turn the lytic synapse into an inhibitory synapse 417 . Although the mechanism underlying actindependent recruitment is unknown it is tempting to draw a parallel with the actin cytoskeleton-dependent recruitment of activation receptors in the NK cell 44,140,418 . A limitation of our work is that the NK92MI cells we use as effector cells do not express

Figure 27 | Model for actin response-mediated breast cancer cell resistance to NK cells

Step1: the NK cell recognizes and adheres to the target tumor cell.

Step 2: the NK cell is activated and this is, in turn, generates a feedback signal towards the target cell directing local and sustained actin polymerization. Downstream components of the related signaling pathway likely involve CDC42 activation and subsequent N-WASP-mediated induction of the ARP2/3 complex. Step3: The massive accumulation of F-actin in the synaptic region of the target cell drives two important processes. First, it drives autophagosomes recruitment at the synaptic region, and thereby facilitates autophagy-mediated GzB degradation (process 1). Second, it increases inhibitory ligand density (and possibly, decreases activating ligand density) at the synaptic region, leading to a shift of the balance of inhibitory/activating signals and attenuation/inhibition of NK cell cytolytic activity (process 2).

Step 4: The NK cell detaches from the surviving tumor cell, the feedback signal directing actin polymerization stops, and the actin response rapidly dissipates upon actin filament depolymerization and, possibly, severing.

An important finding of my work is that all the BC cell lines we examined contain two cell subpopulations differing in their capacity to generate an AR in response to NK attack and to survive this attack. A direct implication of this novel layer of intra-cancer cell line heterogeneity is that the apparent (overall) high susceptibility exhibited by some cell lines, such as MCF-7, actually masks the existence of a minor, AR-competent and highly resistant, cell subpopulation. We hypothesize that a similar cell subpopulation exists in tumors and contributes to cancer progression, metastasis and relapse. Future work should examine this hypothesis. First, highly infiltrated clinical breast tumor specimens can be searched for the presence of synaptic actin accumulation using electron microscopy. In addition, a relevant (syngeneic and immunocompetent) mouse model should be used to confirm the role of the AR in tumor immune evasion in vivo, and to provide a rationale for targeting this process as a therapeutic approach to improve the anti-tumor immune response. In that attempt, we already verified inter species conservation of the actin response in mouse 4T1 cells modified to stably express LifeAct-mEGFP.

Another important question to be evaluated in follow-up studies is the possibility that other cytotoxic immune cells, such as CTLs, also induce an actin response in breast cancer cells. Although the mechanism of CTL activation differs from that of NK cells, both types of immune cells induce cancer cell death through highly similar mechanisms, including the secretion of cytotoxic molecules, such as GzB 373 . It is accordingly conceivable that the actin response protects cancer cells against different types of cytotoxic cells.

Our in vitro data indicate that Cdc42 is an effective point of intervention to block the actin response and its protective effects against NK cells. However, from a clinical standpoint, direct targeting of Rho GTPase, such as Cdc42, has proven to be challenging [366][367][368][369]420 . Although direct small-molecule inhibitors of Cdc42 have been developed and found to have anti-tumor activity in preclinical studies 369,421 , none of those has been clinically approved or entered clinical trials to our best knowledge. Importantly, Cdc42 has been reported to have critical roles in NK cell-mediated lysis 422,423 .

Accordingly, the use of direct inhibitors of Cdc42 to inhibit cancer cell immune resistance can be expected to simultaneously harm NK cell activity. Thus, the translation of our findings into clinical applications requires the identification of clinically relevant targets.

For instance, some of Cdc42 upstream or downstream effectors may exhibit much higher specificity to the AR as compared to Cdc42 itself, which regulates multiple cellular pathways. The clinical efficacy of inhibitors of Cdc42 downstream effectors, such as p21 Activated Kinases (PAKs) is currently evaluated, and there are various additional druggable kinases further downstream of Cdc42 signalling and PAKs 424 . Follow-up studies should characterize the molecular signalling pathways involved in the actin response and identify clinically relevant targets to interfere with this process. Considering the that the actin response is a highly dynamic process, changes in gene expression are unlikely involved in actin response initiation. Such rapid cytoskeletal remodeling presumably involves posttranslational modifications like phosphorylation, a key component of the signaling pathways controlling actin dynamics [425][426][427] . We thus plan to conduct phosphoproteomic analyses aimed to identify proteins whose phosphorylation status is modified during the actin response.

Journal of Immune Research

Open Access

Abstract

Since many years, the tumor microenvironment is recognized as an important promoter of cancer initiation and progression. Although tumors primarily consist in cancer cells, various components/factors of the microenvironment and more specifically the immune landscape, dramatically impact cancer progression. Comprehensive analyses conducted on diverse tumors have identified and characterized the most relevant components/ factors of the tumor microenvironment that supports the malignant behavior of a growing primary tumor. Tumor hypoxia is a common characteristic of the tumor microenvironment that is associated with tumor progression, metastasis, treatment failure and escape from immune surveillance. Although immune cells are usually efficiently recruited into the tumor bed, hypoxic microenvironment was reported to compromise immune cell functions and, in some cases, switch immune cell activity towards a pro-tumorigenic profile. Mechanistic studies have highlighted that hypoxia acts as double-edged sword: it simultaneously impairs the function of immune cells in the tumor microenvironment and activates intrinsic cell resistance mechanisms in tumor cells. The present article aims at reviewing some recent findings on how hypoxia impairs the anti-tumor immune response by focusing on emerging mechanisms by which hypoxia damps the function of immune effectors cells and activates intrinsic immune resistance mechanisms in tumor cells including autophagy and actin cytoskeleton remodeling. Keywords: Cancer; Hypoxia; Immune response; Tumor microenvironment; Autophagy; Actin cytoskeleton cancer immune surveillance which predicts that the immune system can recognize precursors of cancer and, in most cases, destroy them or slow their growth before they become clinically apparent [1, 2]. Several types of immune cells are involved in tumor immune surveillance. Briefly, key cells of the adaptive immune system recognizing cancer cells are cytotoxic T lymphocytes (CTL) which are able to identify antigens via the T cell receptor (TCR) [3]. Some of these antigens are expressed exclusively by tumors and thus are called tumor-specific antigens [3]. Natural killer (NK) cells of the innate immune system also play an important role in tumor immune surveillance [3] by mechanisms called "missing-self" and "induced-self" recognitions [5]. In addition to CTL and NK cells, macrophages and neutrophil granulocytes are also involved in antitumor immunity [6]. Macrophages are antigen presenting cells (APCs) that display tumor antigens and stimulate other immune cells such as CTL, NK cells and other APCs [7]. While the molecular mechanism by which CTL and NK cells recognize their target tumor cells is fundamentally different, both immune cells kill their target following the establishment of immunological synapse (IS) [8]. The formation of IS requires cell polarization and extensive remodeling of the actin cytoskeleton at various stages [9]. It is now well established that CTL and NK cells recognize and kill target cells by two major pathways: either through the release of cytotoxic granules containing perforin and granzymes to the cytosol of target cells [10], or through tumor necrosis factor (TNF) super family-dependent killing [11].

Introduction

Many microenvironmental factors (e.g.hypoxia, composition/ organization of the extracellular matrix (ECM), inflammation, immune suppressive tumor-associated cells) contribute to various aspects of cancer progression, including immune evasion of tumor cells [12, 13]. Recently, it has been reported that the immune system also sculpts the immunogenic phenotype of an established tumor to favor the emergence of resistant tumor cell variants [14]. Accumulating experimental and clinical evidence indicates that multiple mechanisms suppressing the anti-tumor immune functions are directly evolved in the tumor microenvironment [15]. Recently, attention has been focused on the mechanisms by which hypoxic stress within the tumor microenvironment alters tumor transcriptional profiles to modulate glycolysis, proliferation, survival and invasion [16].

In this review, we focus on recent progresses in understanding the influence of hypoxic stress on the tumor survival mechanisms and tumor escape from immune surveillance. We discuss how hypoxia impairs tumor cell killing mediated by both innate and adaptive immune cells. We also review how hypoxia confers resistance to immune attack by activating intrinsic resistance mechanisms in tumor cells. Particular attention is given to hypoxia-induced immune escape mechanisms that rely on actin cytoskeleton remodeling in tumor or immune cells. Finally, we briefly address how hypoxia-targeted strategies may have potential clinical application for restoring an efficient anti-tumor immune response.

Hypoxia and Hypoxia-inducible Factors (HIF) Regulation

Hypoxic stress plays a major role in the acquisition of tumor resistance to immune cell-mediated apoptosis and in the impairment of immune cell activity. Tumor cells adapt to hypoxic microenvironment by inducing transcription factors of the hypoxia inducible factor (HIF) family [17]. The HIF family comprises three members, HIF-1,-2 and-3. HIF-1 is a hetero dimeric protein composed of a constitutively expressed HIF-1β subunit and an oxygen-regulated HIF-1α subunit. In the presence of oxygen, HIF-1α is hydroxylated on proline residue 402 and/or 564 by prolyl hydroxylase domain protein 2 (PHD2). Hydroxylated HIF-1α interacts with the Von Hippel-Lindau (VHL) tumor suppressor protein [18] and recruits an E3 ubiquitin-protein ligase to catalyze the polyubiquitination of HIF-1α and itssubsequent targeting to theubiquitin proteasome system for degradation [19] (Figure 1). Under hypoxic condition, the hydroxylation of HIF-1α is inhibited leading to HIF-1α accumulation. Subsequently, HIF-1α Trans locates to the nucleus, dimerizes with HIF-1βsubunit, binds to the hypoxia-response elements (HRE) present in the promoter of target genes, and recruits co-activators to activate gene transcription (Figure 1). Similar to HIF-1α, HIF-2α is regulated by oxygen-dependent hydroxylation [20]. Although they share structurally similar DNA-binding and dimerization domains, HIF-1α and -2α differ in their trans activation domains. Accordingly, HIF-1α and -2α share overlapping target genes, and each also regulates a set of specific targets [21]. HIF-3α lacks the trans activation domain and was proposed to function as an inhibitor of HIF-1α and HIF-2α. Interestingly, its expression is transcriptionally regulated by HIF-1 [22].

Influence of Hypoxia on Cancer Cell Sensitivity to CTL and NK-mediated Lysis

As previously mentioned, hypoxia modulates both the activity of immune effectors and the response of tumor cells to these effectors. It has been reported that hypoxia decreases the sensitivity of tumor cells to CTL-mediated killing by several mechanisms involving HIF-1. For example, Noman et al. showed that HIF-1α induces the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in tumor cells by a mechanism involving at least in part, vascular endothelial growth factor (VEGF) secretion. Following its hydroxylatesHIF-1α subunit which is bound to Von Hippel-Lindau protein (VHL), ubiquitinated and subsequently degraded by the ubiquitin-proteasome system. Under hypoxia, PHD2 protein is inactivated, and HIF-1α is therefore stabilized. Stabilized HIF-1α translocated to the nucleus and formed with HIF-1β, a heterodimeric complex responsible for transcriptional activation of HIF-target genes including those responsible for the activation of autophagy. In addition, HIF induced the phosphorylation of the signal transducer and activator of transcription (STAT) 3 by a mechanism which is not fully described. The activation of autophagy leads to the degradation of the cargo protein p62, responsible for the elimination of phospho-STAT3 by the ubiquitin proteasome system. The degradation of phospho-STAT3 inhibits CTL-mediated lysis of tumor cells. Furthermore, excessive autophagy in hypoxic target cells leads to the selective degradation of granzyme B (GZMB), a serine protease contained into the cytotoxic granules released by natural killer (NK) cells, thereby inhibiting NK-mediated lysis.

J Immun Res 1(2): id1010 (2014) -Page -03

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Austin Publishing Group Submit your Manuscript | www.austinpublishinggroup.com translocation to the nucleus, HIF-1α cooperates with pSTAT3 to alter the susceptibility of non-small cell lung carcinoma to CTL-mediated killing [23] (Figure 1). A follow-up study has reported that the level of pSTAT3 is tightly controlled by the activation of autophagy in hypoxic cells. Indeed, the accumulation of pSTAT3 was no longer observed when the autophagic flux was inhibited in hypoxic tumor cells [24]. Briefly, autophagy is a catabolic process where a cell self-digests its own components. It can be activated in response to multiple stresses including hypoxia, nutrient starvation, growth factor withdrawal and endoplasmic reticulum stress. Autophagy primarily serves as an adaptive metabolic response providing nutrients, preventing accumulation of altered cell components [25], shaping the anti-tumor immune response [26] and the acquisition of resistance to TNF α [27]. Studies aimed at investigating the mechanisms by which autophagy controls pSTAT3 revealed that targeting autophagy decreases the level of pSTAT3, and that this decrease was mediated by the ubiquitin proteasome system [24, 28].

Another study showed that the transcription factor NANOG, which is involved in stem cell self-renewal, is a new mediator of hypoxia-induced resistance to CTL-lysis [29, 30]. NANOG is induced at both transcriptional and translational levels under hypoxic stress and its targeting leads to significant attenuation of hypoxia-induced tumor resistance to CTL-dependent killing. NANOG depletion results in inhibition of STAT3 phosphorylation and nuclear translocation. Furthermore, it has been shown that HIF-1 induces hypoxia-inducible micro RNA (miR)-210. Over expression of miR-210 in tumor cell stargets protein tyrosine phosphatase non-receptor type 1 (PTPN1), homeobox A1 (HOXA1), and tumor protein p53-inducible protein 11 (TP53I11), and thereby decreases tumor cell susceptibility to CTL [31]. Hypoxia has also been reported to increase the shedding of the major histocompatibility complex (MHC) class I polypeptide-related sequence A (MICA), a ligand for the activating receptor natural killer group 2 member D (NKG2D), at the surface of prostate cancer cells. This shedding is related to the impairment of NO signaling [32] and leads to tumor escape from NK and CTL cells. Furthermore, HIF-1 is able to down regulate MICA expression in osteosarcoma cells resulting in tumor resistance to NKmediated lysis [33]. Recently, we showed that targeting autophagy under hypoxia restores NK-mediated lysis in breast cancer cells. We provided mechanistic evidence that activation of autophagy under hypoxia leads to the degradation of NK-derived granzyme B making hypoxic tumor cells less sensitive to NK-mediated killing [34, 35] (Figure 1). Taken together, it is now well documented that hypoxic microenvironment is an important determinant involved in the control of anti-tumor immune response. Indeed, its immune suppressive effect contributes not only in damping the immune cell function but also in conferring resistance mechanisms to tumor cells to resist immune cells attack [36].

The Actin Cytoskeleton: A Point of Convergence for Various Processes Involved in Tumor Immune Evasion

To fulfill their biological functions, immune cells require exceptional capabilities to proliferate, move through the entire body, infiltrate tissues, establish cell-cell contacts and exchange various types of compounds/signals with both other immune cells and their targets. The related cellular processes, including cell division, cell migration and invasion, cell shape plasticity, intracellular transport, endo and exocytosis, membrane trafficking and phagocytosis, largely rely on dynamic re-arrangements of the actin cytoskeleton. Accordingly, dysregulation of the actin cytoskeleton organization or dynamics can compromise immune cell function and lead to severe diseases, including cancer [37]. A typical illustration is provided by the Wiskott-Aldrich syndrome (WAS) which is caused by alterations of WAS protein (WASp) activity. One major function of WASp is to stimulate actin polymerization through the actin-related proteins (ARP) 2/3 nucleation complex in response to signals from the cell surface [38, 39]. Although some signaling activities of WASp are independent of actin remodeling, most immune cell defects induced by the loss of WASp are at least to some extent, related to defective actin responses, including cell migration, activation, IS formation, and phagocytosis [40-43].

One important aspect of the anti-tumor immune response is the establishment of the IS between cytotoxic immune cells, such as CTL and NK cells, and their targets. Although the signaling pathways activating CTL and NK cell-mediated tumor cell death differ, the establishment of a functional IS and the directed secretion of cytolytic granules toward the tumor cell are relatively well conserved processes that require extensive cytoskeletal remodeling. Following immune cell activation, actin polymerization is activated [44, 45] and granules polarize toward the cell-cell contact region in a microtubule network and microtubule motor activity-dependent manner [46-48]. A critical feature of this process is the translocation of the microtubuleorganizing center (upon which granules converge) from the rear of the immune cell to the leading edge where the immunological synapse is established. The presence of pre-docked granules at the cell cortex of CTL and NK cells, that can be exocytosed before centrosome polarization has been reported [49-51]. Both pharmacological and genetic studies indicate that immune cell activation, IS formation, and the steps following granule polarization largely rely on actin cytoskeleton remodeling [46, 48, 52-54]. A substantial remodeling event occurring simultaneously with centrosome translocation is the accumulation of actin toward the edge of the cell-cell contact region in the so-called distal supra molecular activation cluster where integrin proteins concentrate to mediate cell adhesion [55-57]. The central region of mature synapses was initially proposed to function as an actin-free clearance through which granules are passively released [47, 56]. Such a model was however challenged by studies supporting that granules associate with actin in a myosin IIA dependent manner prior to their delivery to the precise de-granulation sites [58, 59]. Recently, high-resolution microscopy has provided compelling evidence that an actin meshwork is present at the center of the IS from NK cells and that it functions as a facilitator for granule secretion rather than as a barrier [46, 60-62].

In contrast to the well-characterized configurations and roles of actin filaments (AFs) in NK and CTL cells during the cytolytic process, there is a relative gap of knowledge about AF organization and dynamics in target tumor cells. However growing evidence indicate that AF reorganization in tumor cells can promote resistance to anti-tumor immunity (Figure 2). Thus, knock-down of two actin-regulatory proteins, namely the actin-severing factor scinderin [63, 64] and the signaling molecule ephrin-A1 [65] that J Immun Res 1(2): id1010 (2014) -Page -04

Bassam Janji

Austin Publishing Group Submit your Manuscript | www.austinpublishinggroup.com are over expressed in resistant lung tumor cells, was sufficient to restore normal cell morphology and to increase cell sensitivity to CTL-mediated cell death [66]. A follow-up study established that the focal adhesion kinase (FAK) signaling pathway, which controls actin organization and modulates cell adhesion and proliferation was defective in tumor resistant cells [67]. Remarkably, the suppression of FAK in tumor cell was sufficient to promote their resistance to CTL by blocking IS formation. Furthermore, increased AF rearrangement and adhesion following pharmacological activation of Ras homolog (Rho) GTP ases has been reported to induce tumor cell susceptibility to CTL mediated killing. These data further support the role of actin remodeling as a switch in the control of tumor cell susceptibility to cytotoxic immune cells. In addition, depolymerization of AFs in NKsensitive target cells abrogates the conjugate formation mediated by lymphocyte associated antigen (LFA)-1 and subsequently affects granule polarization [68]. Experimental data point to a mechanism where actin cytoskeleton-dependent tethering of LFA-1 ligand such as intercellular adhesion molecules (ICAM)-1 and -2, is required for proper integrin signaling in NK cells [68]. Thus, the modulation of the lateral diffusion of integrin ligand by actin remodeling at the tumor cell cortex represents a potential facet of the immune evasion.

Tumor cells do not only remodel their own actin cytoskeleton to escape from the immune system but are also capable of promoting cytoskeletal modifications in immune cells to impair their activity (Figure 2). Gene-expression profiling study revealed that genes involved in actin cytoskeleton formation and organization in peripheral blood CTL are considerably dysregulated in chronic lymphocytic leukemia patients [69]. Thus, leukemia cells were able to induce similar cytoskeletal defects in CTL from healthy allogeneic donors. Interestingly, the underlying process required cell-cell contact and was independent on cytokine release. Follow-up studies established that malignant cells up-regulate inhibitory ligands such as the B7 super family-related ligands CD200, CD274 and CD276, and TNF-receptor super family member CD270, to compromise the activation of key regulators of T cell synapse actin dynamics, including the Rho GTPases RhoA, Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein 42 (Cdc42) and eventually compromise the establishment of a functional IS [70, 71]. Remarkably, similar inhibitory ligand signaling axes were found in a range of other hematologic and solid carcinoma cells, suggesting that tumor cell-induced T cell actin synapse dysfunction is a common immunosuppressive strategy. Recently, elegant work by Ramsay et al demonstrated that leukemia cells from patients impair T cell LFA-1-mediated migration by altering Rho GTPase activation signaling. Importantly, the immune modulating drug lenalidomide was shown to restore T cell Rho GTPase activity and LFA-1 activation, laying the ground for immunotherapy approaches promoting T cell motility in cancer patients [72].

Tumor Microenvironment-induced Cancer Progression and Immune Evasion Involve Actin Cytoskeleton Remodeling

It has been reported that the physical properties of tumor stroma considerably differ from those of corresponding healthy tissues. Hypoxic stress within the tumor microenvironment induces an increase in ECM stiffness [73] and this increase is largely associated with the ability of hypoxia to induce the expression of ECM genes in cancer-associated fibroblasts, such as those involved in collagen fiber deposition and organization [74-76]. The perception of matrix stiffening by actin cytoskeleton-linked integrins activates signaling pathways that promote Rho GTPase-dependent cytoskeletal tension, focal adhesion assembly and cell migration [73, 77]. Interestingly, a recent study showed that an increase in matrix stiffness and actin cytoskeleton contractibility activates the transcription factor Yesassociated protein(YAP) in cancer-associated fibroblasts and that this activation is required for these fibroblasts to further increase matrix stiffening, cell invasion and angiogenesis [78]. Thus, by inducing a stiff microenvironment, hypoxia promotes actin remodeling in both tumor and cancer-associated fibroblasts (CAFs) leading to increased fibrillar collagen deposition and promoting tumorigenic functions of CAFs (Figure 2). Hypoxic stress has been described to modify the expression and/ or activity of Rho GTPases and/or of their downstream targets in both healthy and cancer cells. Recently, HIF-1 and -2 were shown to directly activate transcription of RhoA and Rho-associated

Figure 1 | 27 Figure 2 | 29 Figure 3 | 33 Figure 4 | 38 Figure 5 | 42 Figure 6 | 49 Figure 7 | 56 Figure 8 | 59 Figure 9 | 66 Figure 10 | 72 Figure 13 | 78 Figure 14 | 82 Figure 15 | 84 Figure 16 | 88 Figure 17 | 90 Figure 18 | 91 Figure 19 | 94 Figure 20 | 96 Figure 21 |Figure 25 |

 1272293334385426497568599661072137814821584168817901891199420962125 Figure 1 | The innate and adaptive immune systems....................................................27 Figure 2 | Parallels between innate and adaptive immune lineages............................29 Figure 3 | NK cell recognition of tumor cells.................................................................33 Figure 4 | NK cell activating and inhibitory receptors.................................................38 Figure 5 | Stepwise stages during NK cell-mediated cytotoxicity................................42 Figure 6 | Stepwise molecular progression through NK cell cytotoxicity...................49 Figure 7 | NK cell transfer...............................................................................................56 Figure 8 | Targeting the tumor microenvironment to improve NK cell responses....59 Figure 9 | Mechanisms of immune evasion in breast cancer........................................66 Figure 10 | EMT scores in different cancers types........................................................70 Figure 11 | EMT process..................................................................................................71 Figure 12 | EMT transcription factors...........................................................................72 Figure 13 | Types of autophagy.......................................................................................78 Figure 14 | Stages of autophagy......................................................................................82 Figure 15 | The cytoskeleton-autophagy connection.....................................................84 Figure 16 | First visualization of actin............................................................................88 Figure 17 | Overlay of actin architecture and mechanics in the moving cell..............90 Figure 18 | Structures of the actin molecule and AF....................................................91 Figure 19 | ARP2/3 structure and function....................................................................94 Figure 20 | Nucleating protein function.........................................................................96 Figure 21 | Rho GTPases and downstream effectors..................................................100 Figure 22 | Beclin-1 knockdown reverts breast cancer cell resistant to NK cellmediated cell lysis...........................................................................................................142 Figure 23 | Granzyme B levels in Beclin-depleted and control breast cancer cells..144 Figure 24 | LC3 staining in breast cancer cells and the Peak mask..........................145 Figure 25 | The actin response is associated with autophagosome polarization towards the synaptic region in target cells...................................................................146 Figure 26 | Relative autophagosome concentration at the synaptic region of 1001 cells challenged by primary NK cells...........................................................................147 Figure 29 | Effects of ICAM-1 and MICA/B blocking on the actin response...........149 Figure 30 | Model for actin response-mediated breast cancer cell resistance to NK cells..................................................................................................................................157

  Les cellules natural killers (NKs) sont des sentinelles du système immunitaire inné qui patrouillent à travers l'organisme pour détecter et détruire les cellules tumorales ainsi que les cellules infectées par des agents pathogènes. Contrairement aux cellules cytotoxiques du système immunitaire adaptatif, telles que les lymphocytes T cytotoxiques CD8 + , les cellules NKs détruisent leurs cibles de manière rapide et sans nécessiter de pré-activation par exposition préalable aux antigènes. Ainsi les cellules NKs sont souvent considérées comme la première ligne de défense immunitaire anti-tumorale. Les cellules NKs sont équipées de récepteurs activateurs et inhibiteurs qui peuvent reconnaître leurs ligands au niveau de la membrane des cellules cancéreuses. La balance entre les signaux inhibiteurs et activateurs régule l'activation de la cytotoxicité des cellules NKs. Suite à la reconnaissance d'une cellule cible, les cellules NKs s'engagent dans une série d'étapes aboutissant à l'exocytose de granules cytolytiques contenant des agents lytiques tels que la perforine et les granzymes. Une étape clé de ce processus est la formation d'une interface entre la cellule NK et sa cible nommée "synapse immunologique". L'établissement d'une synapse lytique est un processus hautement contrôlé qui implique plusieurs phases. Lors de la phase de reconnaissance, la cellule NK établie un conjugué avec sa cible via l'interaction de récepteurs et molécules d'adhésion. La formation du conjugué « cellule NK-cellule cible » requiert une réorganisation importante du cytosquelette d'actine dans la cellule NK, ainsi que la polarisation du centre d'organisation des microtubules (MTOC) et des granules cytolytiques vers la synapse. Lors de l'étape effectrice, les granules cytolytiques sont secrétés vers la cellule cible et induisent l'apoptose. Enfin, lors de l'étape de terminaison, la cellule NK se détache de sa cible morte et régénère son potentiel cytolytique. Certaines cellules tumorales parviennent à échapper à la lyse cellulaire induite par les cellules NKs et contribuent ainsi la progression de la maladie. Par ailleurs, les mécanismes d'évasion immunitaire limitent l'efficacité des immunothérapies, tels que le transfert adoptif de cellules NKs ou les inhibiteurs de checkpoints immunitaires. Il est donc primordial de mieux caractériser les mécanismes moléculaires de la résistance au système immunitaire, notamment aux cellules NKs, afin d'élaborer de nouvelles approches thérapeutiques visant à interférer avec ces mécanismes et à restaurer une réponse immunitaire anti-tumorale efficace. De précédents travaux ont permis de caractériser en détail le rôle clé du cytosquelette d'actine des cellules NKs dans la formation et l'activité de la synapse immunologique. Par exemple, des études d'imagerie à haute résolution ont montré qu'une accumulation d'actine filamenteuse près de la synapse est nécessaire à la polarisation du MTOC et des granules cytolytiques vers la cellule cible. Contrairement à notre connaissance approfondie de l'organisation et des fonctions du cytosquelette d'actine des cellules NKs lors de la reconnaissance et la lyse des cellules cibles, le rôle du cytosquelette d'actine dans les cellules tumorales au cours de ces processus reste énigmatique.

  1) documenter l'organisation et la dynamique du cytosquelette d'actine dans les cellules tumorales au cours de leur attaque par les cellules NKs et 2) évaluer le rôle du cytosquelette d'actine des cellules tumorales dans la résistance à la lyse induite par les cellules NKs. Pour atteindre ces objectifs, j'ai focalisé mes travaux sur le cancer du sein dont plusieurs lignées cellulaires différant par leur susceptibilité aux cellules NKs étaient disponibles au laboratoire. J'ai introduit dans chacune de ces lignées un marqueur du cytosquelette d'actine constitué d'un petit peptide (17 acides aminés) dérivé d'une protéine de Saccharomyces cerevisiae et fusionné à la GFP (LifeAct-mEGFP). L'utilisation combinée de la microscopie confocale de dernière génération et de la cytométrie de flux en image m'a permis de révéler, pour la première fois, les changements de configuration du cytosquelette d'actine dans la cellule tumorale au cours de l'attaque par les cellules NKs avec une haute résolution spatiale and temporelle, et de générer des résultats statistiquement significatifs. Résultats Les données que j'ai obtenues au cours de ma thèse indiquent que le remodelage du cytosquelette d'actine joue un rôle majeur dans le processus de résistance des cellules de cancer du sein à la lyse induite par les cellules NKs. Les principaux résultats de cette étude sont énumérés ci-dessous : Certaines cellules de cancer du sein répondent à l'attaque des cellules NKs par une rapide et massive accumulation d'actine filamenteuse dans la région de la synapse immunologique, un processus que nous avons nommé « réponse actine ». Toutes les lignées cellulaires de cancer du sein que nous avons analysées comportent une souspopulation de cellules capables de répondre à l'attaque des cellules NKs par une réponse actine. En outre, l'abondance relative de cette sous-population dans une lignée donnée est directement liée à la susceptibilité globale de cette lignée à la lyse induite par les cellules NKs. Les lignées cellulaires avec une sous-population abondante de cellules compétentes pour la réponse actine présentent une faible susceptibilité aux cellules NKs. Les lignées cellulaires avec une sous-population réduite de cellules compétentes pour la réponse actine présentent une susceptibilité élevée aux cellules NKs. Les lignées cellulaires de type mésenchymal contiennent une plus large souspopulation de cellules compétentes pour la réponse d'actine et sont plus résistantes à la lyse induite par les cellules NKs que les lignées cellulaires de type épithélial. Une induction expérimentale de l'EMT augmente simultanément la sous-population des cellules compétentes pour la réponse d'actine et la résistance à la lyse induite par les cellules NKs. La réponse d'actine est responsable de la résistance des cellules tumorales à la cytotoxicité induite par les cellules NKs. L'analyse de cellules au microscope confocal procure une preuve directe que les cellules tumorales avec une réponse d'actine survivent à l'attaque des cellules NKs (et ce, plusieurs heures après le détachement des cellules effectrices), alors que les cellules tumorales sans réponse d'actine sont efficacement lysées. En accord avec ces résultats, nous avons établi que le taux d'apoptose est significativement réduit dans les cellules tumorales présentant une réponse d'actine, par rapport aux cellules sans réponse d'actine. Enfin, l'inhibition de la réponse d'actine induite par la déplétion de régulateurs clés de la polymérisation d'actine est suffisante pour convertir des lignées cellulaires fortement résistantes en lignées très sensibles. La réponse d'actine est associée à une réduction de la quantité de Granzyme B (GzB) dans la cellule cible. Le blocage de la dynamique de l'actine (par traitement pharmacologique ou ablation génétique de Cdc42 ou N-WASP) suffit à restaurer une quantité élevée de GzB dans les cellules cibles. La réponse d'actine entraîne la polarisation d'autophagosomes au niveau de la région synaptique dans les cellules cibles. Ce processus pourrait être à l'origine d'une dégradation facilitée du GzB dérivé des cellules NKs dans les cellules tumorales. En accord avec cette hypothèse, l'inhibition de la formation d'autophagosomes augmente simultanément la quantité de GzB dans les cellules tumorales et la susceptibilité cellulaire à la lyse induite par les cellules NKs. La réponse à l'actine et ses effets protecteurs ont pu être validés à l'aide de cellules NKs primaires dérivées de donneurs sains Conclusions Mes travaux de thèse ont permis d'établir le rôle clé du cytosquelette d'actine dans la résistance des cellules de cancer du sein à la lyse par les cellules NKs. Au-delà d'une meilleure compréhension des interactions entre les cellules du système immunitaire et les cellules tumorales, nos résultats ouvrent des perspectives pour le développement de stratégies innovantes visant à cibler la réponse actine afin de restaurer une réponse immunitaire anti-tumorale efficace et/ou améliorer l'efficacité des immunothérapies. Dans cette optique, il serait intéressant de caractériser le mécanisme par lequel la réponse actine induit le recrutement d'autophagosomes à la synapse et d'identifier les principaux régulateurs de la réponse d'actine.Compte tenu de la rapidité de l'établissement de la réponse d'actine lors de l'attaque des cellules NKs, nous anticipons que des modifications post-traductionnelles, telles que la de/phosphorylation, sont à l'origine de l'activation de protéines du cytosquelette responsables du « burst » local de polymérisation d'actine à la synapse. Ainsi, il est envisagé au laboratoire de conduire des analyses phospho-protéomiques visant à identifier les protéines du cytosquelette d'actine et de signalisation dont le statut de phosphorylation est modifié au cours de l'établissement de la réponse actine. Les candidats les plus prometteurs, notamment ceux pour lesquels il existe des inhibiteurs cliniquement approuvés ou en phase d'évaluation, seront validés dans des tests in vitro puis dans des modèles murins de cancer du sein.

  immune response upon re-infection with the same antigen[START_REF] Denardo | Inflammation and breast cancer. Balancing immune response: crosstalk between adaptive and innate immune cells during breast cancer progression[END_REF] . The two major effectoradaptive cells types are the T cells expressing T-cell receptors (TCRs), and B cells, which express B cell receptors (BCRs).T cells are derived from bone marrow, migrate as immature thymocytes and mature in the thymus where they undergo gene rearrangements that lead to the development of TCRs. They recognize the major histocompatibility complex (MHC) molecules referred to as human leukocyte antigens (HLAs). HLA class I includes HLA-A, B and C while HLA class II contains HLA-DP, DQ and DR[START_REF] Den Haan | The activation of the adaptive immune system: cross-talk between antigen-presenting cells, T cells and B cells[END_REF] . The TCR is a disulfide-linked heterodimeric membrane protein composed of a constant region and a variable region with two polypeptide chains,  and , responsible of antigen recognition. T cells are CD3 + as well as CD4 + or CD8 + . Additionally, a fraction of T cells is composed of TCRs with  and  chains and are referred to as  T cells. They are CD4 -and CD8 -and share innate and adaptive features. CD4 + T cytotoxic cells represent a low frequency subpopulation of T cells in the circulation, which increase upon chronic inflammation. They have a lytic function against MHC class II-presenting cells. CD8 + cells are the most cytotoxic T cell subpopulation, able to eliminate pathogens and aberrant cells. T cell-mediated cell death occurs upon contact with antigen-presenting cells, which leads to the secretion of lytic granules 4 . Naïve CD4 + T cells differentiate into four types of CD4 + T-helper cells Th1, Th2, Th17, and regulatory T cells (Treg). Th1 cells are characterized by the expression of the transcription factor Tbet as well as by the production of IFN- and IL-2. They participate in the elimination of intracellular pathogens, regulation of inflammation, and healing tissue injuries. Th2 cells express the transcription factor GATA3 and secrete cytokines, including IL-4, IL-5, IL-6, IL-10, and IL-13. They are involved in the clearance of extracellular pathogens as well as in mediating humoral immunity 5 . Th17 cells develope in response to TGF, IL-6, and IL-21, and they are responsible for IL-17 secretion. They are characterized by their expression of the transcription factor RORt and provide protection against bacteria and fungi. Moreover, Th17 cells recruit neutrophils and macrophages to the site of infection, stimulate cytokine production and regulate T-cell proliferation 6 . Tregs are a subset of T cells expressing CD25, cytotoxic T lymphocyteassociated antigen 4 (CTLA-4), glucocorticoid-induced TNF receptor family-related gene (GITR) and the X-linked forkhead/winged-helix transcription factor box P3 (FoxP3). Treg cells can secrete inhibitory cytokines and can suppress the expansion and activation of immune cells 7 . Natural killer T (NKTs) cells are a T-cell subset that bridges the innate and adaptive immune system. They express CD1d and NK cell-surface marker NK1.1 (CD161) as well as the T-cells marker CD3 and the  TCR 8 . They are defined as CD1d-restricted T cells that express an invariant TCR with a variable (V) and joining (J) V24-J18 repertoire combined with a limited TCR- chain repertoire. They modulate immunity against bacteria and viruses as well as in allergy and autoimmunity by producing cytokines within minutes 9 . B cells are derived from bone marrow where they remain until maturation with a unique BCR expressing the IgM and IgD isotypes. Activation promotes isotype substituting, resulting in the development of antibodies with different heavy chains. Most of B cells are activated via the binding of BCRs and an antigen followed by ligation between CD40 on the B cells and the CD40-ligand on T-helper cells. Upon activation, B cells differentiate into the short-lived plasma cells responsible for producing specific antibodies. Microbes are attacked by B cell-derived antibodies making them accessible for phagocytosis by macrophages. These antibodies can also neutralize toxins and viruses. In addition, B cells can differentiate into a small fraction of memory cells that can become active in the case of re-infection 10 .

  Consistent with their innate immune function as the first line of defense against aberrant and pathogen-infected cells, NK cells are widespread throughout lymphoid and non-lymphoid tissues. The NK cell lineage is generated from the common lymphoid progenitor (CLP), and they further develop in the bone marrow as well as in the liver and the thymus. The lineage's development fundamentally relies on the secretion of cytokines such as interleukin (IL)-15 or IL-12 and on the expression of transcription factors such as Nfil3, PU.1 and Tox. During the maturation process, NK cells acquire the ability to recognize cells lacking self-recognizing MHC class I molecules 35 .

  cells are not affected by NK-cell activity as they express the inhibitory ligands MHC class I molecules while downregulating the activating ligands. Conversely, NK cells can detect the overexpression of activating ligands and the absence of MHC class I on the surface of target cells. In addition, the NK cell membrane expresses the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or CD95-L, which can ligate with the corresponding receptor on a target cell and initiate target cell apoptosis 44 (Figure 3).

5 .

 5 Figure 3 | NK cell

Figure 5 |

 5 Figure 5 | Stepwise stages during NK cell-mediated cytotoxicity YTS GFP-actin (green) cells were labeled with Lysotracker Red (red) to selectively label lytic granules and co-incubated with CellMask-labeled (Life Technologies, Carlsbad, CA, USA) (yellow) 721.221 target cells in the presence of SYTOX Blue (Life Technologies) (blue) for detection of cell death. Conjugates were imaged by spinning disk confocal microscopy for a total of 90 min. The images shown are frames acquired approximately every 15 min. a) The NK cell is conjugated to the target cell and lytic granules are dispersed. b) Granules are converged at the MTOC. c) The MTOC along with converged lytic granules are polarized at the immune synapse. F-actin is evidently reorganized from a to c. d) More F-actin reorganization is observed and lytic granules are docked at the plasma membrane. e) Target cell death begins as SYTOX Blue enters the cell. f) Target cell undergoes apoptosis and NK prepares for detachment as shown by reversal of shape change. Scale bar=4 μm. Adapted from Mace, 2014.

  . Type I IFN induces the proliferation and cytotoxicity of NK cells. IFNs ligate with their corresponding receptor IFNAR, and the absence of that receptor provokes an activation failure. The role of TGF- in decreasing the activation and proliferation of NK cells by affecting NKG2D expression has also been established. In contrast, inhibiting TGF- signaling increases the capacity of NK cells to secrete cytokines. The majority of cytokine receptors activate JAK/STAT signaling pathways, where the Janus kinase (JAK) allows for the recruitment and phosphorylation of signal transducer and activator of transcription (STAT), which migrates to the nucleus to regulate transcription factors 175 .

  . Studies have shown that the depletion of NK cells affects the functionality of DCs and that NK cells kill immature DCs lacking MHC class I via the lytic or death-receptor pathway. The NK cell-mediated maturation of DCs depends on TNF- and IFN- secretion upon direct contact 179 . In lymph nodes, CD4 + T cell-derived IL-2 activates NK cells, while CD4 + -derived TGF- inhibits NK-cell functions and suppresses their cytotoxic effect 180 . CD4+ T-helper type 1 (T H 1) cells are primed by NK cell-derived IFN- in the lymph nodes 181 . Indirectly, T H 1 polarization is enhanced via the maturation of DCs. However, NK cells induce the CD8+ T-cell response via DC activation. The role of CD4 + CD25 + Treg in suppressing immune cells is well studied. Interestingly, Treg can negatively affect various aspects of NK-cell functions such as the cytolytic process, proliferation, and cytokine production 182 . NK cells also induce the differentiation of CD14 + monocytes into DCs through direct contact with CD56 bright NK cells and the production of GM-CSF. Moreover, macrophage cross-talk with NK cells has proven to be inducible, as cytokine secretion increases upon interaction 178 .

  . In 2011, the anti-CTLA-4 monoclonal antibody ipilimumab was approved by the FDA for treating metastatic melanoma. Clinical trials for CTLA-4 with prostate, bladder, and non-small-cell lung carcinoma (NSCLC) are ongoing. Results have shown T-cell compartment activation, tumor regression, and overall survival improvement 203 . The programmed cell death 1 (PD1) receptor expressed on activated T-cell membranes is among the important inhibitory checkpoints. Activation of PD1 (also known as CD279) after it interacts with its ligands regulates the antigen response threshold of T and B cells and thus, inhibits cell proliferation, cytokine release, and cytotoxicity 204 . PD-L1 and PD-L2, expressed on tumor cells, are ligands that bind to PD1 receptors. Blocking this interaction can enhance T-cell anti-tumor activity 202 .

(

  LAG3) is a selective marker and cell surface molecule on the membrane of T, B and NK cells. It has been reported that this ligand downregulates the proliferation and activation of T cells and plays a role in the suppressive function of Tregs. Targeting LAG3 reverses T-cell exhaustion and thus, enhances anti-tumor activity 209 . T-cell immunoglobulin and mucin domain-containing 3 (TIM3) is another immune checkpoint expressed on NK cells, DCs, and various T-cell populations. Its co-expression with PD1 in CD8+ cells makes it a potent therapeutic target 210 . The TIGIT receptor is strongly expressed on lymphocytes.

Figure 7 |

 7 Figure 7 | NK cell transfer a) In autologous transfer, NK cells from the patient are activated and expanded in vitro in the presence of cytokines. Combination of IL-12, IL-15 and IL-18 might generate NK cells and feeder cells such as K562 can be added to the culture. The expanded and activated NK cells are then transferred back into the patient, who generally receives cytokine administration to sustain the expansion and function of the infused NK cells. b) In allogeneic transfer, NK cells can be obtained from HLA-matched or haploidentical (partially matched) donors. In this setting, the best responses are obtained when haploidentical donors do not express KIRs that recognize the patient's HLA molecules, because donor NK cells do not receive an inhibitory signal from the patient's cancer cells. c) CARs can be engineered in autologous or allogeneic NK cells or in NK cell lines such as NK-92. Adapted from Guillerey et al., 2016.

  cancer 217 . Besides inhibiting EGFR, one of the mechanisms of action is considered to be the ADCC triggering by Fc-R on the NK cells 218 . Moreover, recent studies have focused on increasing the activity of ADCC by increasing the affinity of the activating receptor FcRIIIa. Mogamulizumab, which is a mAb that binds to CC chemokine receptor 4 (CCR4) is such a treatment 219 . The drug is used for T-cell leukemia-lymphoma, and research has shown it enhances anti-tumor activity via ADCC by removing fucose residues from the Fc region 220 .

  An important approach to treating cancer is to empower the lytic mechanisms of endogenous NK cells by blocking inhibitory receptors with specific mAbs, thus banning the inhibitory signals. Indeed, a few inhibitory receptors are currently being targeted in clinical or pre-clinical development. Furthermore, classical immune checkpoints are present on the NK cell membrane, and their blockade reverts the NK inhibitory state 211,221 . The inhibitory pathway of NK cells depends largely on KIR receptors and their interactions with HLA class I on target cells. The negative effects of this ligation influence NK-cell activity, cytokine production, and cytotoxicity. Studies have shown that blocking KIR receptors with specific antibodies induces NK-cell cytotoxicity to acute myeloid leukemia (AML) cells. This promising result led to the development of lirilumab, a recombinant IgG4 mAb against KIR2DL1, KIR2DL2, and KIR2DL3. Clinical trials have been conducted for lirilumab as a monotherapy for AML patients and in combination with elotuzumab for multiple myeloma, with rituximab for chronic lymphocytic leukemia, and with nivolumab for head and neck carcinoma 211 . An important receptor of the NKG2 family is the A form that binds to HLA-E to generate inhibitory signals. NKG2A expression on effector cells is constant and unaffected by the microenvironment. Blocking NKG2A receptors with the mAb monalizumab led to an increased proportion of lymphocytes that infiltrated to the tumor site and enhanced cytotoxicity in oral squamous cell carcinoma and gynecological malignancies. Clinical trials are now being conducted for monalizumab in combination with cetuximab and durvalumab 222 . PD1, the most promising immune checkpoint receptor, is expressed on the NK-cell membrane and is associated with the inhibition of NK-cell lysis. In vitro, blocking PD1 on NK cells with mAbs relieves this inhibition. Clinical responses to nivolumab have been observed by rescuing the anti-tumor NK-cell response 199 . The expression of LAG3 on NK cells is crucial for NK-cell function. Nevertheless, targeting LAG3 with mAbs enhanced Fas-mediated apoptosis in NK and melanoma cells. Similar to LAG3, targeting TIM3 shows a promising result in increasing NK activity. In lung adenocarcinoma, TIM3 mAbs act to increases IFN- production and the cytotoxicity of NK cells. Clinical trials with LAG3 and TIM3 mAbs are ongoing. Of note, the administration of a TIGIT mAb in combination with other immune checkpoints is being tested in clinical trials on various solid tumors 222 .

  Clinical trials using immunotherapy have resulted in good therapeutic outcomes in TNBC patients, who have no targeted-therapy options231 . Targeting PD1/PD-L1 to treat BC is also showing promising results with tumor regression and the improvement of patient outcomes. Pembrolizumab (a PD1 inhibitor), atezolizumab, and avelumab (PD-L1 inhibitors) enhance the local anti-tumor response. They can also be combined with MAP2K and GITR in TNBC cases to provide a more therapeutic outcome237 . In a preclinical study, co-treatment with mAbs against TIM3/PD-L1 showed complete BC regression in the majority of treated mice 210 . The new fusion protein M7824 is an innovative drug that blocks both PD-L1 and TGF-, and thus, activated CD8 + T cells and NK cells. Preclinical studies in a BC model have shown enhanced anti-tumor activity and reduced metastasis. Moreover, an increase in the buildup of hyaluronan, a glycosaminoglycan, was observed in the breast cancer TME, leading to a delay in immune cell and antibody infiltration to the tumor. In a preclinical study with a BC model, the depletion of hyaluronan by PEGylated recombinant hyaluronidase 20 (PEGPH20) enhanced the delivery of PD-1 and PD-L1238,239 .It has also been shown that tamoxifen enhances NK-cell ADCC in HER2/neu BC cells 240 . The use of trastuzumab and pertuzumab, anti-HER2 mAbs, in HER2 + BC cases improves the overall survival of patients via the recruitment of NK cells and the induction of NK cell ADCC 241 .Another new approach that shows a clinical outcome in BC studies is the use of stimulator of interferon genes (STING). STING induces interferon production and DC activation, promoting the priming of cytotoxic CD8 + T cells. The injection of ADU-S100, a STING agonist, in combination with PD1 mAb administration produced a delay in tumor progression and tumor clearance in TNBC and HER2 + BC 232 .The epigenetic modification of immune recognition is one of the mechanisms of tumor evasion. Inhibitors against histone-deacetylase (HDAC) have proven to be effective by priming the anti-tumor immune response in a variety of BCs, especially in the ER + group. Etinostat, a selective HDAC-1 inhibitor, has also been shown to increase the T cell-mediated cell death of BC cell lines in vitro 239 .

  While an immune response is directed to eradicate cancer, tumor cells can induce resistance among the immune cells and evade the elimination process. To withstand the growth of a tumor, a suppressive TME is necessary to neutralize the activation of immune cells and stop the elimination process. The majority of immune-cell types can infiltrate into or to the edge of a tumor, with the infiltration rate varying depending on the type and stage of the tumor. Remarkably, the hypoxic milieu and the stabilization of hypoxiainducible factor 1- (HIF1-) proteins in tumor cells mediate the adjustment of cells to the TME through phenotypic, genetic, or metabolic transformations 242 . The immunoediting of BCs and other solid tumors is a dynamic process in which immune cells lyse the tumor cells. It comprises three distinct stages: elimination, equilibrium, and escape. During the elimination stage, immune cells destroy tumor cells after infiltration and activation. Cells that manage to resist and survive this killing enter into the equilibrium phase. The escape stage represents the final stage of the process, when tumor cells keep progressing, spreading, and becoming clinically detectable. The microenvironment of the tumor at this stage tends to be suppressive, and tumors have diverse mechanisms of resistance 243 . In BC, tumor cells escape from the immunosurveillance of the innate and adaptive immune cells. Immunotherapy approaches have significantly increased remission and overall survival in patients. Nevertheless, the high proportion of non-responders represents a tough and serious challenge in the cancer field. Remarkably, many biological processes are implicated in tumor immune evasion as well as in resistance to various therapies. The mechanisms used by tumor cells for immune evasion fall under many categories and include tumor-related immune evasion and microenvironment alteration as well as morphological and cellular changes 242 . Recent findings have well established that cellular heterogeneity and plasticity, e.g., the induction of autophagy or the epithelial-tomesenchymal transition (EMT), have a serious impact on tumor evasion from T and NK cells.

  . Moreover, 2 microglobulin, calnexin, and transporter-associated antigen processing 1 are downregulated in metastatic BC and negatively affect CTL infiltration. The MHC class I expression level is also significantly downregulated in BC cells, leading to evasion of the T cell-dependent immune response, but also the simultaneous activation of NK cellmediated cell lysis by the "missing self" signal 247 . To avoid T cell-and NK cell-mediated killing, BC cells express HLA-G on the cell surface. HLA-G is considered a biomarker for identifying metastatic BC and hence, for targeting. It is a potent immunotherapeutic strategy. Controlling the apoptotic pathway is considered an important mechanism for tumor cells to evade the immune response. Any disruption of one of the components in the pathway can block the apoptotic process, leading to endless proliferation. Breast cancer cells can either downregulate the Fas receptor on the surface of their membrane or stimulate the immune cell apoptotic pathway by overexpressing FasL, which binds to Fas receptors on the immune cell surface 248 . In addition to the Fas pathway, high levels of Bcl-2 in BC cells inhibit the TRAIL-dependent pathway, leading to a prolonged life span for the tumor cells 249 . The overexpression of downstream proteins such as survivin in the apoptotic pathway plays a significant role in BC resistance in advanced grades 250 . BC cells express both PD1 and PD-L1 on their membranes. The interaction between PD-L1 on the BC-cell membrane and the receptor on immune cells leads to downregulation of the immune response due to the induction of T-cell and NK-cell apoptosis. The N-glycosylation and ubiquitination of the PD-L1 ligand on BC cells have direct positive effects on its suppressive activity 251 . Blocking PD-L1 or destabilizing it can significantly attenuate T-cell apoptosis. Moreover, a high correlation has been found between PD-L1 expression and increased FoxP3 + Treg infiltration in the high histological grade BCs (ER -, PR -, and TNBC). They work synergistically to promote BC immune evasion 252 . Additionally, the overexpression of CD80 and CD86 on TNBCs leads to the inactivation of T cell-mediated cell death, immunosuppressive-cell infiltration, and a reduction in cytokine production via interaction with the inhibitory CTLA-4 receptors 253 .

  but also to the TME. Recent studies have shown a tight association between the BC microenvironment and tumor evolution. The activation of infiltrated immune cells depends largely on chemokines and cytokines produced by the tumor 254 . Due to the impact of the inhibitory environment, T cells could ignore tumor antigens presented on BC cells. A high rate of Treg infiltration and proliferation has been observed in TNBCs. This recruitment is dependent on CCR4, CCL22, and CCL17 and modulated by compounds secreted from the BC cells and DCs 255 . Using MAPK/ERK inhibitors leads to blocking the TGF--dependent induction of Treg 256 . Tumor cells can also suppress the activity of NK cells that have infiltrated to the BC microenvironment. One immune-evasion strategy consists of shedding ligands that bind to NKG2D receptors or the secretion of transforming growth factor- (TGF-), which downregulates NKG2D-receptor expression. Additionally, tumor secretion of NKG2D ligands can saturate and abolish the activity of NK-cell receptors 257,258 . Furthermore, BC can produce soluble factors that induce the expression of NKG2D ligands on the immune-cell surface, thereby evading immunity 154 . BC cells produce numerous cytokines during the elimination stage to evade anti-tumor responses. The IL-6 secreted by BC cells affects NF-B, leading to EMT induction and more resistant and invasive tumor cells. Secreted IL-33 activates the production of GM-CSF and myeloid-derived suppressor cells. IL-19 and IL-20 enhance the proliferation and metastasis of tumor cells, while IL-23 affects CD8+ T-cell infiltration and activation 242

4 . 1 .

 41 The process is essential to development, stem cell behavior, wound healing, and cancer progression. The transition respects a conserved program with particular hallmarks and results in the downregulation of epithelial cells and the induction of mesenchymal cell characteristic 259 . Recent studies have shown a tight association between the EMT and invasiveness and resistance to apoptosis, chemotherapy, and radiation. Moreover, mesenchymal cells tend to have cancer stem-cell behavior and have an influence on TME components, possibly regulating anti-tumor immune editing 260 . Basic principles of the EMT Plasticity in cellular organization is a hallmark of early embryonic developmental stages. The EMT was first observed by Elizabeth Hay in the 1980s in the primitive streak of chick embryos 261 . Cells expand their intracellular contact immediately after cleavage formation and create the epithelium, a polarized layer. Subsequently, cells develop apicobasal cell surface polarity, with actin microfilaments concentrated at the basolateral membranes and microtubules positioned at the basoapical axis positioning the cytoplasmic organelles. In epithelial cells, integrin receptors are localized on the basal domain, forming a basal lamina that interacts with the extracellular matrix, while the lateral domain comprises the junctional complexes, including E-cadherin 260 . The acquisition of the mesenchymal motile phenotype is characterized by the loss of their apico-basal polarity, epithelial cell-cell junction, and E-cadherin. Simultaneously, the cells acquire vimentin and N-cadherin, redistribute the actin cytoskeleton, and reprogram some gene expression, giving rise to the initiation of a front-rear polarity and migratory features 262 . The formation of mesenchymal cells is initiated during gastrulation, an early phase in embryonic development when a single-layer blastula is reorganized into a multilayered structure called the gastrula. Mesenchymal endodermal and mesodermal cells detach from the epiblast (the epithelial layer) and ingress through the primitive streak 263 . Mesenchymal cells can convert back to the epithelial state, a reverse process called the mesenchymal-to-epithelial transition (MET), to establish paraxial and lateral mesodermal structures. The shifts between the EMT and the MET (crucial in some organogenesis such as heart development), gene expression, and non-transcriptional changes are regulated by TGF-, canonical Wnt, and the RTK pathways 264 . Upon initiating the EMT, the epithelial cell-cell junctions, including E-cadherin, the tight junction, adherens junctions, desmosomes, and gap junctions such as connexins, are deconstructed and degraded. This process is associated with decreased claudin and occluding expression and the diffusion of zonula occludens 1 (ZO1). In addition, catenin and p120 catenin are protected from degradation and act only in transcription following E-cadherin degradation 265 . Apical-basal polarity is associated with three important polarity complexes linked to the cell junctions. Upon E-cadherin suppression, complexes are either repressed or inhibited from interaction with the membrane. Partitioning-defective proteins include PAR3, PAR6, and atypical protein kinase C (aPKC). Crumbs complexes, which are mainly associated with tight junctions and cortical actin, contain Crumbs, protein associated with Lin-7 1 (PALS1), and PALS1-associated tight junction protein (PATJ). The Scribble complexes associate with adherens junctions and comprise discs large (DLG) and lethal giant larvae (LGL). The repression of Crumbs and Scribble in the mesenchymal phenotype allows for directional motility 262 .

  induction. It is later responsible for promoting gene transcription. The expression of catenin is higher in invasive carcinomas than in DCISs. It has been shown that Wnt/catenin signaling is upregulated in invasive ductal carcinoma 270 . Discoidin domain receptor 2 (DDR2) is an abundant receptor for tyrosine kinase in BC. It stabilizes SNAIL and plays a role in adhesion to collagen in the extracellular matrix, promoting invasiveness 271 .Cancers can be classified based on their transcriptomic signatures, a method called EMT scoring. The score can help to semi-quantitatively assess the position of certain tumor types over a wide range of the EMT spectrum, with values ranging from -0.8 for the most epithelial phenotype to +0.6 for the most mesenchymal phenotype. As an example, melanomas and renal carcinomas have a majority of mesenchymal features while colon carcinomas show epithelial features. Others, such as BC, exhibit a wide range on the EMT spectrum. In general, tumor cell lines protect the initial EMT features from their original tumor. In BC, luminal and HER2/neu tumors exhibit an epithelial-like phenotype, whereas basal-like and claudin-low tumors are mesenchymal 272 (Figure10).

Figure 10 | 4 . 3 .

 1043 Figure 10 | EMT scores in different cancers typesScatter plot of EMT scores (mean ± SEM;y-axis) of various cancers in clinical samples (upper panel) and cell lines (lower panel) sorted by cancer type and mean EMT score. EMT score nearer to +1.0 is more mesenchymal-like (Mes), whereas EMT score nearer to -1.0 is more epithelial-like (Epi). EMT scores of overlapping cell lines in Cancer Cell Line Encyclopedia (CCLE) and SANGER/COSMIC collections were averaged. Epithelial-mesenchymal transition spectrum quantification and its efficacy in deciphering survival and drug responses of cancer patients. Adapted from ZeaTan et al., 2014.

Figure 11 |

 11 Figure 11 | EMT process a) The first steps of EMT are the disassembly of epithelial cell-cell contacts that is, tight junctions, adherens junctions, desmosomes and gap junctions and the loss of cell polarity through the disruption of the Crumbs, PAR and Scribble complexes. The expression of epithelial genes is repressed, concomitantly with the activation of mesenchymal gene expression. b) Next, the epithelial actin architecture reorganizes, and cells acquire motility and invasive capacities by forming lamellipodia, filopodia and invadopodia, and by expressing matrix metalloproteinases

  motility275 . The main transcription factors involved in the EMT are SNAIL1/2, TWIST, and Zinc-finger E-box-binding 1 and 2. Other transcriptional factors such as Grainyheadlike-2 (GHL2), FOCX2, E47 (TFC3), KLF8, and PRRX1 are also implicated in inducing the EMT by targeting other genes and signaling pathways 276 (Figure12).

Figure 12 |

 12 Figure 12 | EMT transcription factorsEMT is driven by SNAIL, ZEB and bHLH transcription factors that repress epithelial marker genes and activate genes associated with the mesenchymal phenotype. Post-translational modifications regulate their activities, subcellular localization and stability. a | GSK3β phosphorylates (P) SNAIL1 at two motifs; phosphorylation of the first motif facilitates the nuclear export of SNAIL1, and phosphorylation of the second motif enables the Ub-mediated degradation of SNAIL1. Phosphorylation of SNAIL1 by PKD1 also leads to its nuclear export. Conversely, phosphorylation of SNAIL1 by PAK1 or LATS2, or dephosphorylation of SNAIL1 by small C-terminal domain phosphatase1 (SCP1) promotes the nuclear retention of SNAIL1 and enhances its activity. SNAIL2 is degraded as a result of its p53-mediated recruitment to the p53-mouse double minute 2 (MDM2) complex. b | TWIST is phosphorylated by the MAPK p38, JNK and ERK, which protects it from degradation, and thus promotes its nuclear import and

  mechanism, as tumor cells undergo the transition to a mesenchymal phenotype not only to invade other sites, but also to resist the TME and therapies. The most well-established model for studying the correlation between the EMT and tumor immune evasion is the human mammary carcinoma model MCF-7. MCF-7 cell lines begin as epithelial cells with an abundant expression of E-cadherin and no vimentin or N-cadherin expression.The EMT can be initiated in MCF-7 cells by inducing stable SNAIL expression or via prolonged exposure to TNF-. The MCF-7 cell line's EMT score places it in the epithelial state, while the derivatives display a mesenchymal state. The resulting cells are mesenchymal, with high vimentin and E-cadherin expression and a total loss of Ecadherin. Additionally, the EMTed MCF-7 cells lose their cell-cell junction with a spindle-like shape and acquire the invasive phenotype[285][286][287][288] .Mesenchymal cell resistance to CTL-mediated cell lysis can engage many pathways.Akalay et al. showed that EMTed MCF-7 cell lines are resistant to CTL-mediated cell lysis. This resistance can be linked to the activation of autophagy 287 Interestingly, EMTed MCF-7 cells (cells after silencing WISP2, or Wnt-inducible signaling pathway protein 2) are similarly resistant to CTLs. The cross-talk between Wnt and TGF- signaling promotes the reversion of CTL resistance in MCF-7-shWISP2 cells after treatment with TGF- blockers 289 . Moreover, PD-L1 expression is high in MCF-7-derived cells compared to the parental cell line.In the non-metastatic lung adenocarcinoma model IGR-Heu, a correlation has been observed between the hypoxic environment and the EMT spectrum. Remarkably, cells differed in their ability to shift to a mesenchymal phenotype under hypoxic conditions, and it was evident that the cells that underwent the EMT were more resistant to CTLmediated cell lysis. The loss of E-cadherin in mesenchymal IGR-Heu explains the resistance, as CTL function relies on CD103 (αΕβ7 integrin), an E-cadherin ligand290,291 .Mesenchymal NSCLC tumor sub-clones show a significant resistance to NK cellmediated cell lysis (compared with epithelial clones) due to defective IS signaling. A recent study suggested that high levels of the EMT-related factor Brachyury increases resistance to CTLs, NK cells, and lymphokine-activated killer cells. Nevertheless, SNAIL overexpression in colorectal cancer cells and the secretion of TGF- made the cells more susceptible to NK cells due to an increase in the expression of MICA/B and ULBP1-3, NKG2D ligands 292 .

  stemness pathways and resistance to chemotherapy and radiotherapy. It has been established that the latter therapies can activate TGF-β, NF-κB, Wnt, B, Wnt, FGF, and EGF/HER2 pathways, and thus stimulate EMT-transcription factors. From another aspect, miR-200/ZEB1 axis downregulation mediates the docetaxel resistance of prostate cancer 293 . MiR-21 has been implicated in the EMT and in trastuzumab and paxlitaxel or doxorubicin resistance in HER2 + BC cells 294,295 . Chemotherapy resistance occurs mainly through degradation of the drug by increasing the efflux of hydrophobic drugs regulated by the ATP-binding cassette (ABC). EMT cells have been shown to bind to the E-box in the ABC. Radiation therapy, as well as hypoxia, induces the generation of ROS that play a role in initiating the EMT 296 . TGF-, the main inducer of the EMT, is known to have a negative effect on adaptive and innate immune cells. It can impair their maturation, differentiation, cytotoxicity, or ligand expression. In contrast, it also induces inhibitory Treg activation. Thus, TGF- blockers can be a therapeutic strategy for immunotherapy, and they are now under clinical study 297 . Moreover, PD-L1 expression is related to the mesenchymal state via the miR-200/ZEB axis 298,299 . PD-L2, PD1, TIM3, LAG3, and CTLA-4 expression are also correlated with the EMT state 238 . SNAIL overexpression in mouse melanoma B16F10 cells induces an EMT associated with the impairment of DC maturation, less T-cell infiltration, and enhanced Treg suppressive functions. In an MMTV-PyMT mouse model for BC, SNAIL-expressing cells showed high Treg infiltration compared to control cells.The same results were obtained from a study on hepatocellular carcinoma wherein hypoxia-induced EMT cells had an immunosuppressive TME accompanied by defective T-cell proliferation and increased Treg-cell infiltration 300 . Altogether, these studies highlight the implication that the EMT process is involved in cancer-therapy resistance and immune editing 272 . Erlotinib, an EGFR inhibitor, and fulvestrant, an ER antagonist, may be efficient at improving the immune cell-mediated cell lysis of lung cancer cells by targeting mesenchymal cells 301 .

Figure 13 |

 13 Figure 13 | Types of autophagya) Macroautophagy is characterized by the sequestration of structures targeted for destruction into doublemembrane vesicles called autophagosomes. Complete autophagosomes first fuse with endosomes before finally exposing their content to the hydrolytic interior of lysosomes. The resulting metabolites are transported into the cytoplasm and used either for the synthesis of new macromolecules or as a source of energy. b) During chaperone-mediated autophagy, proteins carrying the pentapeptide KFERQ-like sequence are recognized by the Hsc70 chaperone, which then associates with the integral lysosome membrane protein LAMP-2A, triggering its oligomerization. This event leads the translocation of the bound protein into the lysosome interior through a process that requires Hsc70. c) Microautophagy entails the recruitment of

  metabolism302 . Starvation-induced autophagy is an essential process to ensure cell survival. Blood glucose maintenance is a result of the anabolism and catabolism of amino acids released by liver autophagosomes. Moreover, autophagy-derived amino acids are a source of energy through the tricarboxylic acid cycle, as well as a substrate for protein synthesis 305 306 . Furthermore, autophagy deficiency causes cardiac dysfunction and cardiomyopathy. Hypoxia-induced autophagy occurs in an ischemic-myocardium model307 .The contribution of autophagy to protecting host cells is crucial, as autophagosomes can degrade intracellular bacteria and viruses. Autophagy is also implicated in the suppression of inflammation and downregulation of interferons and pro-inflammatory cytokines (e.g., IL-18)308,309 . Furthermore, autophagosomes fuse with MHC class II loading compartments. The high expression of autophagy in MHC Class II-positive cells leads to the delivery of endogenous cytosolic proteins to MHC class II molecules and therefore contributes to CD4+ T-cell stimulation. Evidence for this function includes the fusion between ATG8/LC3 and influenza matrix protein 1, which leads to enhanced MHC class II presentation to CD4+ T cell 310 .Autophagosomes circulate randomly in the cytoplasm along microtubules, but their movement is influenced by the MTOC where lysosomes are concentrated. Subsequently, they fuse with the lysosomes to create the autophagolysosomes responsible for degrading their components. The process is controlled through intracellular signaling and extracellular induction and is divided into five distinct stages: vesicle initiation, vesicle nucleation, vesicle expansion, vesicle closure and fusion with lysosomes, and the degradation stage 302 . Autophagy-related genes (ATGs) control all these stages (Figure14). Diverse signals control the dynamics of the autophagic process and extra-or intracellular signaling initiates the first step of autophagosome formation. Cell starvation, hypoxia, and insulin/growth factor signals mediate a decrease in glucose transport, the end of mTOR-dependent inhibition, and Jun N-terminal kinase (JNK) activation. Energy loss can also induce autophagy by the activation of AMP kinase (AMPK). Calcium, ROS, cyclic AMP, inositol triphosphate, and calpain receptors control the process. Cell death can be associated with an increase in autophagosomes as death receptors such as TRAIL can induce autophagic cell death 302 .The first step in autophagosome formation is the constitution of the phagophore.Membrane nucleation and phagophore formation begin with activation of the ATG1 complex, which includes Unc-51-like kinase 1 or 2 (ULK1/2), ATG13, ATG101, ATG9A, and FIP200 (focal adhesion kinase (FAK)-family interacting protein of 200 kDa). Further, the phosphorylation of Beclin-1 by ULK1 supports the class III PI3K complex synthesis of PIP3 and facilitates its translocation to the membrane. The class III PI3K complex contains Vps34, ATG14, UV radiation resistance-associated gene protein (UVRAG or p63), and BECN1-regulated autophagy protein 1 (AMBRA1)306 .In 1998, Beclin-1 (Bcl-2 interacting myosin-like coiled-coil protein; or ATG6), a member of the ATG family, was found to interact with the Bcl-2 protein. It is highly conserved in eukaryotes and implicated in additional cellular processes such as phagocytosis, trafficking, and the control of cytokinesis. Beclin-1 is a 60 kDa protein containing three important domains: the coiled-coil domain (CCD), the evolutionarily conserved domain (ECD), and a Bcl-2 homology domain 311 . The ECD is necessary for binding to the class III PIK3 complex. Together, CCD and BH3 form the large homooligomer responsible for the recruitment of ATGs. Bcl-2 can bind to the BH3 domain, and therefore constrains Beclin-1-dependent autophagy at the endoplasmic reticulum and mitochondria. Starvation acts to dissociate this interaction and activates Beclin-1 via Bcl-2 phosphorylation by JNK1[311][312][313] .Membrane elongation is controlled by two ubiquitination-like reactions. First, ATG7 and ATG10 conjugate ATG5 with ATG12. The membrane continues to expand due to the non-covalent conjugation of the ATG5-ATG12 complex with ATG16L1. The second reaction involves an association of the ATG8 family with the lipid phosphatidylethanolamine (PE). The ATG8 family comprises the microtubule associated protein 1A/1B-light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP), and GATE-16. Moreover, ATG4B (associated with ATG7 and ATG3) conjugates cytoplasmic LC3I and PE to form lapidated LC3II, thus elongating the doubled membrane. Complete phagophore elongation results in the formation of a mature autophagosome 314 . Immediately after the autophagosome closes, the SNARE protein syntaxin 17 (STX17) is recruited to the LC3 + /ATG -membrane. The STX17 + membrane then fuses with the lysosomes (LAMP1/2 + ) to form the autophagolysosomes that degrade cargo components with hydrolases. The adaptor protein sequestosome 1/P62 is used as an autophagic-flux marker because it is degraded after vesicle recycling 314 .LC3 is a 17 kDa soluble protein abundant in mammalian tissues and cells. Upon LC3 conjugation with PE, LC3II is formed and localized on the inner and outer membrane of the autophagosomes. LC3II is often used as a marker to measure autophagic flux, as it remains associated with the autophagosomes until they fuse with lysosomes. It is the most used marker because the amount of LC3II represents the number of mature autophagososmes 315 . Nutrient starvation significantly increases the number of autophagosomes and correspondingly the quantity of LC3II. In some cases, an increased amount of LC3II could denote an inhibition of autophagy deprivation and autophagosome accumulation. Immunoblotting of LC3 results in LC3I and II bands. The LC3I band is not reliable for quantifying autophagy; comparing the amount of LC3II to a housekeeping protein is the best method. A decrease in the amount of p62 that binds to LC3 is a confirmation of autophagic flux as p62 gets degraded along with LC3. A precise quantification requires the addition of bafilomycin A, a lysosome inhibitor, or CQ, an inhibitor of fusion between the autophagosomes and lysosomes. A comparison between LC3II and p62 in the presence and absence of lysosome targeting provides the ideal representation of autophagic flux in a cell313,316 .

Figure 15 |

 15 Figure 15 | The cytoskeleton-autophagy connection Implication of the actin cytoskeleton in the overall autophagy process starting from (a) initiation, (b) omegasome formation, (c) expansion, (d-e) transport, and (f) ending with fusion with lysosomes and degradation. Timeline showing the arrival and departure of cytoskeleton assembly factors. Solid bars indicate the timing of appearance of autophagy and cytoskeletal proteins during the various stages of autophagy (gray arrow). Striped bars indicate the predicted time of arrival based on the known functions of proteins for which direct evidence is still lacking. Adapted from Kast et al., 2017.

  is its non-specificity to tumor cells. General autophagy inhibition by gene deletion shows high toxicity due to the disruption to homeostasis and neuronal toxicity. However, prolonged treatment with the lysosomal autophagy inhibitors CQ and HCQ have shown no serious adverse effects 330 . It has been established that targeting autophagy in tumor cells under hypoxia reestablishes CTL-mediated cell killing. Autophagy and HIF- control induction and stabilize the STAT3-survival pathway 331 . Interestingly, hypoxia impairs BC cells' susceptibility to NK cell-mediated cell lysis by activating the autophagic process responsible for GzB degradation. Targeting Beclin 1 restores GzB levels in target cells and increases NK cell-mediated cell death 288 . Additionally, targeting Beclin 1 in melanoma cells inhibits their growth via activated NK-cell infiltration into the tumor. It has been established that autophagy-defective cells release CCL5 cytokines into the TME upon MAPK8/JNK-JUN/c-Jun signaling pathway activation. Increased CCL5 overexpression and release leads to the high NK-cell infiltration associated with overall survival in melanoma patients with high CCL5 level 332 .

  This section will highlight key breakthroughs made since the discovery of the actin protein. The story behind the discovery of actin begins in 1864 with the discovery of myosin by Wilhem Kuhne. Later, the British physiologist William Dobinson Halliburton discovered a new protein extracted from muscle cells and called it "myosin-ferment". This finding led to no further investigations until 1942, when Albert Szent-Gyorgyi and Bruno Ferenc Straub showed that myosin was composed of two proteins. The first protein, myosin A, has a low viscosity and is extracted after 20 min of exposing muscle tissue to a high salt concentration, while the second protein, myosin B, results from overnight extraction. The addition of ATP led to a reduction in the viscosity of myosin A, but not B, and to a shortening of the myosin B threads, but not those of myosin A. After purification, Straub identified the protein responsible for myosin fiber contraction and called it actin. He also showed that actin exists in two forms: globular actin (G-actin) and actin filament (AF). Moreover, adenosine 5′-triphosphate (ATP) was shown to be a functional part of G-actin that is hydrolyzed to adenosine 5′-diphosphate (ADP) when polymerized into filaments.In 1954, two concurrent studies revealed that the force between actin and myosin is responsible for filament sliding and generating muscle fiber movement. Studies continued to uncover various elements of the cytoskeleton with the development of live imaging. However, in 1969, a surprise awaited when actin was observed in cells other than muscle cells. Later, in 1974, Lazarides and Weber were the first to visualize the spatial arrangement of the actin cytoskeleton 333 (Figure16). Typical electron microscopy and immunofluorescence techniques prevented the observation of actin dynamics. It was the revolution in reactive fluorescent dyes that opened up the study of actin dynamics. In 1978, Taylor and Wang first saw the actin cytoskeleton in vivo after injecting 5′-iodoacetamido-fluorescein (IAF)-labeled actin into living cells 334 . Discoveries continued to uncover many specific actin components and functions such as focal adhesion, microtubule dynamics, and motor proteins. The first atomic model of AFs was produced by Holmes in 1990 335 . By the early 1990s, signaling pathways governing the regulation of the actin cytoskeleton were discovered by Paterson. Rho GTPases were shown to be key components in the signal transduction connecting extracellular signals to the actin structures. Rho, Rac, and Cdc42 were shown to control cytoskeletal responses, and they are complicatedly inseparable as they guide one another.

Figure 16 |

 16 Figure 16 | First visualization of actinSDS gel electrophoresis of actin. SDS polyacrylamide slab gel electrophoresis was performed in 12.5% slabs. a) Highly purified chicken muscle actin (10g). b) A 30% ammonium sulfate cut of a low-salt extract from an ethanol powder of mouse fibroblasts (15g). A small amount of myosin copurifies with actin under the extraction conditions used. The arrow at the bottom of the figure shows the dye front of the gel. Indirect immunofluorescence of actin antibody with primary chick embryomyoblasts. The nuclear staining seen in this figure seems to be nonspecific. Purification of the antibody used in this experiment by 50%

G

  -actin differs from AFs by a relative rotation of the two major domains by about 20°. Additionally, the conformation of the DNase-I binding loop in S2 gives F-actin an openloop conformation 336 .

Figure 17 |

 17 Figure 17 | Overlay of actin architecture and mechanics in the moving cellSchematic representation of the cell with the different architectures indicated: the cell cortex; an example of a contractile fiber, the stress fiber; the lamellpodium; and filopodia. The zoom regions highlight architectural specificities of different regions of the cell.Blanchoin et al., 2014.

  ATP and Mg +2 -bound G-actin to both ends at different rates. The fast-growing end is the barbed-or plus-end, which usually grows 20-fold faster (k = 11.6 M -1 •s -1 ) than the slow-growing end, which is called the pointed-or minus-end. As a function of their concentration, actin monomers can assemble at the barbed end and disassemble at the pointed end simultaneously in a dynamic steady state called AF treadmilling. Upon incorporation into a growing filament, ATP is hydrolyzed into ADP, and ADP-actin subunits and phosphate (Pi) are generated 336 . However, treadmilling is not the only key player in AF assembly. The elongation process involves two well-established tools, monomer-binding protein and capping proteins. Both mechanisms are effective in creating a vast pool of non-polymerized actin, whereas separately, none of these is qualified to provide the necessary number of monomers required to initiate the elongation step. Additionally, numerous severing and nucleating proteins are implicated in the branching and regulation of AFs 336 (Figure18).

Figure 18 |

 18 Figure 18 | Structures of the actin molecule and AF

2 (

 2 , bottom). Adapted fromCampellone et al., 2010. Nucleation-promoting factorsMammalian NPFs are classified into 2 classes. Class I includes Wiskott-Aldrich syndrome protein (WASP), neural-WASP (N-WASP), WASP-family verprolinhomologous protein (WAVE1-WAVE3), WASP and SCAR homologue (WASH), WASP homologue associated with actin, Golgi membranes, and microtubules (WHAMM), and junction-mediating regulatory protein (JMY). Class II contains hematopoietic lineage cell-specific protein 1 (HS1) and cortactin. NPFs activate ARP2/3 to interact with actin through the carboxy-terminal WCA domains, which include a WASP homology 1 and WH1 and 2) motif 343 .WASP is highly expressed in hematopoietic cells, while N-WASP is expressed in most cells. The deletion of both isoforms is crucial for embryonic survival and is implicated in neurological and cardiac diseases. ARP2/3-complex activation by N-WASP leads to a functional actin nucleation. The structure of WASP has been shown to have the WH1 motif in addition to CDC42/Rac-interactive binding (CRIB) and auto-inhibitory motifs 344 . WASP, the main regulator of ARP2/3, can be activated by the direct binding of small GTPases such as Cdc42 to the GTPase binding domain (GBD), which frees the WCA domain. Additionally, proteins with the Src homology 2 and 3 (SH2 and SH3) domain can bind to the proline-rich domain (PRD) and activate N-WASP. Moreover, Tyr phosphorylation of the GBD can stabilize the activity of N-WASP. The structure of WASP has been shown to contain the WH1 motif in addition to CRIB and auto-inhibitory motifs. Interestingly, N-WASP has limited NPF activity due to inhibition of the GBD. This inhibition is enforced by WASP-interacting protein (WIP), a molecule that interacts with the WH1 motif. Class II NPFs, including cortactin, have weaker activity than class I NPFs in activating ARP2/3 and the nucleation process. Interaction with class I NPFs occurs through t PRD and SH3 domains at the C terminal. Their structure has shown a lack of the WH2 domain, but a specific ARP2/3-binding domain is present at the N-terminal (Figure 20).

Figure 20 |

 20 Figure 20 | Nucleating protein functionFilamentous actin (F-actin; red) is nucleated and organized into branched networks by the actin-related protein 2/3 (ARP2/3) complex and its nucleation-promoting factors or is generated in unbranched forms by formins and tandem WASP homology 2 (WH2) domain-containing nucleators. Functional roles for different nucleation factors in a generic mammalian cell are depicted during phagocytosis, cell junction assembly, endocytosis, membrane ruffling and lamellipodia dynamics, filopodia formation, Golgi and tubulovesicular membrane dynamics, and stress fibre formation. Question marks indicate that the precise role for the nucleation factor is unclear. Adapted fromCampellone et al., 2010.

  implicated in the recycling of actin monomers. The family includes destrin and twinfilin as actin depolymerizing factors (ADF), cofilin-1 in non-muscle cells, and cofilin-2 in muscle tissues. Both cofilin-1 and ADF have similar functions in regulating AFs by binding and severing ADP subunits, leading to cytoskeleton turnover 347 .Destrin is a small globular 19 kDa protein that regulates the actin cytoskeleton by rapidly depolymerizing AFs in a stoichiometric manner. It is widely expressed in eukaryotes and has a 71% similarity to cofilin. Destrin can directly bind to a filament and remove an actin monomer, thereby dissociating the AF 348 . Cofilin-1, hereafter called cofilin, is a small 15 kDa protein that is highly expressed in eukaryotes. Cofilin severs AFs by binding to the barbed-end, which leads to creating free ends. ATP hydrolysis and Pi dissociation trigger cofilin to bind to the ADP-actin subunit 349 . It is well established that cofilin promotes actin assembly or disassembly depending on its concentration in the cytoplasm and the presence of other ABPs. Moreover, it can bind to actin monomers and inhibit nucleotide exchange 347 .

Figure 21 |

 21 Figure 21 | Rho GTPases and downstream effectorsDownstream targets of Rho include the serine/threonine kinase ROCK which is mainly involved in the formation of stress fibers and focal adhesions. ROCK phosphorylates downstream myosin light chain (MLC) leading to actin-myosin contractility. At the same time, ROCK inhibits MLC dephosphorylation by inhibiting MLC phosphatases via their myosin binding subunit (MBS). The mammalian homolog of diaphanous (mDia) is another important Rho effector mediating actin nucleation. LIMK is also another downstream effector of Rho, which phosphorylates the actin severing protein cofilin inhibiting its severing activity and production of barbed ends. Downstream signaling of Cdc42 and Rac includes scaffold proteins belonging to the WASP/SCAR/WAVE family, key regulators of actin nucleation and polymerization. Active PAK phosphorylates MLCK thereby inactivating it and inhibiting MLC phosphorylation and contractility. PAK also phosphorylates and activates LIMK which potentially results in the phosphorylation of cofilin inhibiting its actin-severing function. Adapted fromHanna et al., 2013.

  GTPases. The role of Cdc42 in cells is crucial, and studies have shown that knocking out Cdc42 in mice causes embryonic death. The use of dominant-negative or constitutively active mutants showed the implications of Cdc42 in cellular processes despite the limitations such mutations place on other Rho GTPases 358 . The downstream effectors of Cdc42 are numerous (more than 20) and include actin proteins, kinases, and phospholipases. The most relevant pathways will be summarized. Cdc42 is the main and direct activator of the WASP family of proteins, including WASP, N-WASP, and WAVE, via binding to the CRIB motif. Cdc42 connects with the Toca (transducer of Cdc42dependent actin assembly) families, enhancers of Arp2/3 activation. Additionally, Cdc42 stimulates LIMK-dependent cofilin activation and PAK 365 .The regulation of Cdc42 also affects the expression and activation other oncoproteins such as Ras and EGFR via PAK. Ras modifications lead to the initiation and progression of tumors by the activation of anti-apoptotic pathways such as Raf-MEK-ERK. It has also been established that the activation of Cdc42 is associated with the prevention of EGFR degradation and thus tumor cell progression. The regulation of IQGAP1 and 2 proteins

1 ,

 1 MTOC polarizes to the leading-edge region upon targetcell recognition and adhesion. The MTOC and myosin IIA centered in the cSMAC are surrounded by protrusive actin polymerization in the dSMAC 161 . Actin remodeling at this stage is crucial for immune cell activation, as any disruption to the cytoskeleton leads to sequestering the activation state 156 . Remarkably, the pharmacological or genetic disruption of myosin IIA prevents TCR recruitment and activation. In T cells, AFs are responsible for TCR clustering at the IS. In NK cells, it is well established that the ligation of ICAM-1 on target cells and LFA-1 on NK cells initiates AF polymerization and accumulation 373 . At this stage, actin polymerization acts to maintain the LFA-1 clustering and thus ensures tight adhesion and a larger synapse diameter with the target 374 .

1 . 1 . 1 . 2 )

 1112 active and functional IS. Although the dynamic and function of the actin cytoskeleton at the NK side of the synapse is well established, there is a gap of knowledge about the role of tumor actin cytoskeleton upon immune cell attack. The two objectives of my PhD Thesis were to: (i) characterize actin cytoskeleton organization and dynamics in breast cancer cells during NK cell attack. (ii) examine the role of the tumor cell actin cytoskeleton in the resistance to NK cell-mediated cell killing. Most of the results obtained during my PhD were assembled into an article that was published in the journal of Cancer Research. In this article, we report, for the first time, actin cytoskeleton organization and dynamics in tumor cells during NK cell attack. In addition, we provide evidence that actin remodeling plays a central role in breast cancer cell escape from NK cell-mediated lysis. The article is provided as a main result section (1.2). The introduction and the discussion of the article are also provided in French to comply with the requirements from the University of Strasbourg (1.1 and 1.3, respectively). In follow-up studies, I investigated the mechanisms underlying actinmediated immune evasion. The corresponding results are presented as classical result sections (2 to 4). This result chapter is followed by a general discussion and perspectives as well as complementary materials and methods. Actin cytoskeleton remodeling drives breast cancer cell escape from natural killer-mediated cytotoxicity Introduction to the article in French Note : Les références de cette introduction se trouvent dans l'article qui suit (1.Les cellules NKs sont des lymphocytes du système immunitaire inné qui possèdent une activité cytotoxique et contribuent à débarrasser l'organisme des cellules cancéreuses et des infections induites par des agents pathogènes. Contrairement aux cellules cytotoxiques du système immunitaire adaptatif, tels que les lymphocytes T cytotoxiques CD8 + , les cellules NKs tuent leurs cibles très rapidement et ne nécessitent pas une préactivation par exposition préalable aux l'antigènes. Pour ces raisons, les cellules NKs sont fréquemment considérées comme une des premières lignes de défense contre le cancer. Les cellules NKs reconnaissent les cellules malignes en détectant une perte des molécules inhibitrices du complexe majeur d'histocompatibilité de classe I (CMH I) et/ou une surexpression de ligands activateurs induit par le stress, tels que les ligands du récepteur NKG2D comme ULBP1-6 et MICA/B. La balance entre les signaux inhibiteurs et activateurs déterminent en grande partie l'activation de la cytotoxicité des cellules NKs, même si des mécanismes complémentaires ont été décrits. Outre leur cytotoxicité directe et la production de cytokines pro-inflammatoires, les cellules NKs modulent l'activité d'autres cellules immunitaires et peut amorcer les cellules dendritiques pour activer les réponses anti-tumorales des lymphocytes T. Si les cellules NKs ont une activité cytotoxique forte contre les cellules transformées, leur nombre dans les tumeurs solides est fréquemment faible. Afin de résoudre ce problème, le transfert de cellules NKs autologues ou allogéniques suivi de doses élevées d'IL-2 a été cliniquement évalué. Les deux approches thérapeutiques se sont avérées partiellement efficaces, mais n'ont pas réussi à induire une rémission durable. Les principaux obstacles à une efficacité clinique demeurent, notamment l'absence d'expansion des cellules NKs in vivo ainsi que les mécanismes immunosuppresseurs induits par les tumeurs. Avec l'avènement d'immunothérapies basées sur des cellules T autologues et approuvées par la FDA, l'intérêt de l'applicabilité des cellules NKs en immunothérapie contre le cancer a été réactivé. Des modifications génétiques similaires à celles employées pour générer des cellules T couplées à un récepteur antigénique chimérique(''CAR T cells''), pourraient être appliquées aux cellules NKs pour augmenter leur efficacité contre les tumeurs. Cependant, les mécanismes d'évasion insuits par les cellules cancéreuses et les microenvironnements tumoraux immunosuppresseurs demeurent des obstacles majeurs. Après la reconnaissance des cellules tumorales, les cellules NKs vont suivre une série d'étapes spécifiques conduisant à la sécrétion orientée de granules lytiques préformés contenant des molécules cytotoxiques, tels que la perforine et la GzB. Un événement clé et précoce conduisant à la mort cellulaire des cellules tumorales est la formation d'une synapse immunologique entre les cellules immunitaires et leurs cibles, dont la fonction est de focaliser la sécrétion des granules lytiques vers les cellules cibles. La formation et l'activité de la synapse impliquent des réarrangements importants du cytosquelette d'actine dans les cellules NKs. Les premières étapes de la formation de la synapse sont associées à une accumulation importante de filaments d'actine, qui facilite la formation et la stabilisation de ''peripheral supramolecular activation clusters'' (pSMAC). Après réorganisation du cytosquelette, les granules lytiques convergent vers le centre organisateur des microtubules le long des microtubules. Par la suite, ces granules lytiques se polarisent vers la synapse. Les études d'imagerie à haute résolution ont démontré qu'un réseau dynamique d'actine est situé au centre de la synapse et que ce réseau contrôle la distribution de granules associés à la myosine IIA vers zones spécifiques de la membrane. Les deux principales classes de nucléateurs d'actine (le complexe ARP2/3 et formines) se sont avérées avoir des rôles critiques mais distincts au cours de la cytotoxicité médiée par les cellules NKs. Récemment, il a été montré que la coronine 1A a été impliquée dans la déconstruction du réseau d'actine corticale au niveau de la synapse, un processus aboutissant à la création d'un espace où la sécrétion de granules se produit préférentiellement. Par rapport aux les rôles bien établies du cytosquelette d'actine des cellules NKs pendant la reconnaissance et la destruction des cellules cibles, les configurations et fonctions de l'actine dans les cellules tumorales restent énigmatiques au cours de ces processus. Dans l'étude présente, nous démontrons de manière directe et pour la première fois que le cytosquelette d'actine des cellules tumorales joue un rôle clé dans la résistance à la lyse médiée par les cellules NKs.

  A c t -m E G F P . C e l l s w e r e s u b s e q u e n t l y s u b j e c t e d t o w e s t e r n b l o t a n a l y s i s t o e v a l u a t e L i f e A c t -m E G F P e x p r e s s i o n l e v e l . A c t i n w a s u s e d a s a l o a d i n g c o n t r o l . ( B ) E x p r e s s i o n o f t h e e p i t h e l i a l m a r k e r E -c a d h e r i n a n d t h e m e s e n c h y m a l m a r k e r v i m e n t i n i n M C F -7 , T 4 7 D , M D A -M B -2 3 1 a n d H s 5 7 8 T b r e a s t c a n c e r c e l l l i n e s a s e v a l u a t e d b y w e s t e r n b l o t . G A P D H w a s u s e d a s a l o a d i n g c o n t r o l . ( C ) R e p r e s e n t a t i v e i m a g e s s h o w i n g t h e m a s k s ( d a r k b l u e a n d d o t t e d l i n e s ) g e n e r a t e db y t h e I D E A S s o f t w a r e a n d a p p l i e d t o t h e t a r g e t ( T ) a n d e f f e c t o r ( E ) c e l l s t o c a p t u r e t h e s y n a p t i c r e g i o n a n d t h e a c t i n r e s p o n s e . T h e s y n a p t i c r e g i o n ( l i g h t b l u e m a s k o n t h e b r i g h t fi e l d c h a n n e l ) i s d e f i n e d a s t h eo v e r l a p p i n g a r e a b e t w e e n t h e t a r g e t c e l l m a s k ( L i f e A c t -m E G F P ) a n d t h e N K c e l l m a s k ( C D 5 6 -P E -C y 7 ) . S c a l e b a r , 7 µ m . ( D ) M e t h o d t o d e t e r m i n e t h e p e r c e n t a g e o f t a r g e t c e l l s w i t h ( + A R ) a n d w i t h o u t ( -A R ) a n a c t i n r e s p o n s e . A n e x a m p l e i s g i v e n f o r b o t h M C F -7 a n d M D A -M B -2 3 1 c e l l l i n e s .

  r e g i o n w e r e s c o r e d a s p o s i t i v e f o r t h e a c t i n r e s p o n s e . 7 D o u b l e t s -M C F 7 P e r f r o t i

  e n s i t y _ A c t i n i n t a r g e t I n t e n s i t y _ A c t i n a t t h e m a s k , I n t e n s i t y _ A c t i n i n t a r g e t e A c t -m E G F P i n t e n s i t y i n t a r g e t c e l l L i f e A c t -m E G F P i n t e n s i t y i n t a r g e t c e l l I n t e n s i t y o f L i f e A c t -m E G F P i n t h e s y n a p t i c r e g i o n I n t e n s i t y o f L i f e A c t -m E G F P i n t h e s y n a p t i c r e g i o n e l l -M C F -7 c e l l d o u b l e t s N K c e l l -M D A -M B -2 3 1 c e l l d o u b l e t s ls . C y t o c h a l a s i n D ( C D ) t r e a t m e n t ( c o n c e n t r a t i o n a n d d u r a t i o n ) w a s o p t i m i z e d t o e f f i c i e n t l y p r o m o t e F -a c t i n d i s r u p t i o n i n t a r g e t c e l l s w i t h o u t a f f e c t i n g c e l l v i a b i l i t y . O n l y M D A -M B -2 3 1 c e l l s a r e s h o w n h e r e . ( A -C ) T y p i c a l a c t i n c y t o s k e l e t o n o r g a n i z a t i o n i n c o n t r o l , D M S Ot r e a t e d , c e l l s ( A ) , C D -t r e a t e d c e l l s ( B ) , a n d C D -t r e a t e d c e l l s a f t e r 1 h o u r r e c o v e r y ( C ) . A r r o w s i n d i c a t e t h e r e a p p e a r a n c e o f F -a c t i n b a s e d s t r u c t u r e s . S c a l e b a r , 1 0 µ m .

L

  'échappement tumoral aux cellules immunitaires cytotoxiques est un obstacle majeur à de nombreuses immunothérapies. Dans cette étude, nous démontrons le rôle critique du cytosquelette d'actine dans la mise en place de la résistance des cellules du cancer du sein à la lyse induite par les cellules NKs. Grâce à l'utilisation de la cytométrie de flux en image à haut débit, nous avons trouvé que les lignées cellulaires tumorales communes contiennent deux sous-populations de cellules différant par leur capacité à répondre à l'attaque des cellules NKs via une accumulation forte et rapide d'actine filamenteuse dans la région de la synapse. De façon Remarquable, le taux de ''réponse actine'' dans une lignée cellulaire donnée est inversement corrélé avec la susceptibilité globale de cette lignée cellulaire à la lyse induite par les cellules NKs. L'imagerie en temps réel de cellules vivantes a démontré de manière directe que les cellules tumorales présentant une réponse actine survivent à l'attaque des cellules NKs et restent en vie après la séparation des conjugués, tandis que les cellules tumorales sans réponse actine sont efficacement lysées. Ces observations sont en accord avec nos analyses indiquant que le taux d'apoptose dans les cellules cibles conjuguées aux cellules NKs est considérablement réduit dans la souspopulation cellulaire compétente pour la réponse actine. De plus, l'inhibition de la réponse actine suffi à convertir les lignées cellulaires résistantes, telles que 1001 et MDA-MB-231, en un phénotype hautement susceptible. Au total, ces résultats établissent un lien de causalité entre la réponse d'actine et la résistance aux cellules NKs. La réponse d'actine est un processus remarquablement rapide et localisé qui a lieu presque immédiatement après le contact physique entre les cellules cibles et les cellules effectrices. Le suivi des conjugués cellulaires individuels au cours du temps a révélé que la réponse actine dure pendant toute la durée de l'interaction et s'arrête rapidement après le détachement de la cellule effectrice. Ceci suggère un modèle dans lequel un signal de la synapse est transmis vers le cortex des cellules tumorales où il induit une polymérisation active et continue d'actine. Bien que les composants en amont de la voie de signalisation restent à identifier, l'inhibition robuste de la réponse actine induite par le knockdown de N-WASP ou de Cdc42 suggère que le complexe ARP2/3 est un effecteur clé responsable de la polymérisation de l'actine suite à l'attaque des cellules NKs. Le rôle de N-WASP et de Cdc42 dans la polymérisation de l'actine dépendante du complexe ARP2/3 à la membrane a été largement documenté. Notamment, Cdc42 est un régulateur clé des projections membranaires (comme les filopodes) et de la polarité cellulaire. Il est intéressant de noter que la réponse actine est associée à des projections membranaires de type filopodes. Comme dans les filopodes, les nucléateurs d'actine de la famille des formines et des protéines de bundling, telles que la fascin, sont probablement nécessaires pour l'extension des projections membranaires associées à la réponse d'actine. D'un point de vue fonctionnel, la réponse actine est associée à une réduction significative des niveaux de GzB dans les cellules cibles. De plus, l'inhibition pharmacologique ou génétiques (ablation de Cdc42 ou N-WASP) de la réponse actine restaure des niveaux élevés de GzB dans les cellules cibles. En revanche, nous n'avons trouvé aucune association entre la réponse d'actine et une polarisation anormale du MTOC ou le processus de dé-granulation au niveau des cellules effectrices. Ceci sugère un mécanisme où la réponse actine obstrue la pénétration du GzB dans les cellules cibles et/ou facilite la dégradation du GzB à l'intérieur des cellules cibles plutôt qu'un mécanisme où la réponse actine inhibe l'activation des cellules NKs. À cet égard, nous avons récemment rapporté que l'autophagie favorise la dégradation du GzB dérivée des cellules NKs dans les cellules MCF-7 hypoxiques, réduisant ainsi la sensibilité des cellules cibles à la lyse induite par les cellules NKs. Dans le même sens, l'inhibition de l'autophagie dans les cellules cibles améliore l'élimination des tumeurs par les cellules NKs dans des modèles murins in vivo de BC et de mélanome. Considérant les rôles multiples et critiques du cytosquelette d'actine dans les différentes étapes de l'autophagie, les études futures devront élucider le lien entre la réponse d'actine et la dégradation de GzB par l'autophagie dans les cellules tumorales. Le rôle de l'EMT dans l'évasion des cellules tumorales aux cellules immunitaires cytotoxiques, telles que les cellules T et NKs est de plus en plus reconnu. Nos résultats indiquent que les lignées cellulaires de type mésenchymal ont une capacité largement supérieure aux lignées de type épithelial à générer une réponse actin. En outre, une EMT expérimentale augmente à la fois la compétence des cellules tumorales à la réponse actine et la résistance à la lyse induite par les cellules NKs. Au cours de l'EMT, de vastes modifications du cytosquelette d'actine sont nécessaires pour conduire les adaptations morphologiques et fonctionnelles, telles que l'acquisition de propriétés migratrices et invasives. Ainsi, les changements de propriétés du cytosquelette d'actine (organisation, dynamique) qui accompagne le processus de l'EMT confèrent probablement aux cellules tumorales une capacité accrue à mobiliser rapidement leur actine en réponse à une attaque immunitaire. Une observation intrigante de notre étude est que les lignées cellulaires du cancer du sein contiennent deux sous-populations de cellules différant par leur capacité à mobiliser le cytosquelette d'actine en réponse à l'attaque des cellules NKs et de survivre à cette attaque. Une implication directe et importante de cette nouvelle facette de l'hétérogénéité intra-lignée tumorale est que la susceptibilité apparemment élevée d'une lignée donnée, telle que MCF-7, peut en réalité masquer l'existence d'une sous-population mineure de cellules immuno-résistantes (et compétentes pour la réponse d'actine). Cette information devrait être prise en compte dans des études basées sur des analyses de cytotoxicité in vitro, et les efforts de recherche devraient se concentrés sur la sous-population cellulaire compètent pour la réponse d'actine. La caractérisation des voies de signalisation qui contrôle la réponse actine et l'identification de cibles moléculaires pour altérer ce processus sont des axes de recherche prometteurs. Les résultats pourraient contribuer au développement de nouvelles stratégies visant à accroitre la susceptibilité des cellules tumorales aux cellulaires immunitaires et à améliorer l'efficacité des immunothérapies.

Figure 22 |

 22 Figure 22 | Beclin-1 knockdown reverts breast cancer cell resistant to NK cellmediated cell lysis MCF-7, 1001 and MDA-MB-231 cell lines were transfected with control siRNA (siCtrl) or two different siRNAs targeting Beclin-1 (siBeclin-1 #1 and #2). Western blots (left panels) are representatives of three independent experiments and show the significant knockdown of Beclin-1 48 hours after cell line transfection. Actin is shown as a loading control. Cytotoxicity assays (right panels) were performed in triplicates with NK92MI cell as effectors at an E:T ratio 5:1. The two-tailed unpaired Student's t-test was applied to determine the statistical significance. * denotes p  0.05; ** p  0.01; ns: non-significant.

  similar function in normoxic resistant breast cancer cells. To test such a hypothesis, we quantified GzB levels in control and Beclin-1-depleted MCF-7, 1001 and MDA-MB-231 cells. Target cells were incubated for 30 min with NK-92MI cells prior fixation, permeabilization and staining. Imaging flow cytometry was used to quantify GzB levels in NK cell-conjugated target cells exclusively. Our results show that Beclin-1 depletion significantly increases (by up to two times) GzB levels in both 1001 and MDA-MB-231 cell lines (Figure 23 b and c). In contrast, Beclin-1 knockdown had no effect on GzB levels in MCF-7 cells (Figure 23 a). Together these results are highly consistent with the effects of Beclin-1 depletion on tumor cell susceptibility to NK cell-mediated lysis

Figure 23 |

 23 Figure 23 | LC3 staining in breast cancer cells and the Peak mask Representative panel of the Peak mask generated in the IDEAS software for LC3 puncta detection. The light blue dots represent LC3 puncta in an MCF-7 cell. Scale bar, 7 μm.

Figure 24 |

 24 Figure 24 | The actin response is associated with autophagosome polarization towards the synaptic region in target cells a) Representative panels obtained by imaging flow cytometry showing autophagosome distribution in MDA-MB-231 cells without (no AR, upper panels) and with (with AR, lower panels). The cyan area in the right field image depicts the mask applied to capture the synaptic region in the target cell. Scale bar, 7 μm. b) and c) relative autophagosome concentration at the synaptic region of MCF-7, 1001 and MDA-MB-231 cells with (+AR) or without (-AR; set to 1) actin response as evaluated by the BDI (b) and spot count (c) approaches. d) Percentage of total cancer cell autophagosomes in the synaptic region of MCF-7, 1001 and MDA-MB-231 cells with (+AR) or without AR (-AR), as calculated by the BDI approach. Data are graphed as mean ± SE from three independent experiments including a total of 300 conjugates. The two-tailed unpaired Student's t-test was applied to determine the statistical significance. * denotes p < 0.05.

Figure 25 |

 25 Figure 25 | Relative autophagosome concentration at the synaptic region of 1001 cells challenged by primary NK cellsPrimary NK cells were isolated from five healthy donors, activated with IL-2 (100u/ml) and incubated with 1001 cells for 30 minutes. The BDI (a) and spot count (b) approaches were used to quantify LC3 at the synaptic region of at least 200 conjugates without (-AR, light grey) or with (+AR, black) actin response. Data are expressed relative to synaptic LC3 levels in cancer cells without actin response (set to 1).

Figure 26 |

 26 Figure 26 | Effects of ICAM-1 and MICA/B blocking on the actin response MDA-MB-231 cells were treated with anti-ICAM-1 antibodies or an IgG1 control for 1-hour prior their presentation to NK92MI cells. (a) Percentage of target cells conjugated by NK cells. (b and d) Cytotoxicity assays performed with NK92MI cells and MDA-MB-231 treated with either anti-ICAM-1 antibodies (b), anti-MICA/B antibodies (d) or IgG1 controls at 1:1 and 5:1 effector:target ratios. (c-e) Percentage of NK cell-conjugated MDA-MB-231 cells with (black, +AR) and without (light grey, -AR) actin response after treatment with anti-ICAM-1, anti-MICA/B or IgG control antibodies. Data originate from three independent experiments. The two-tailed unpaired Student's t-test was used to measure the statistical significance. * denote p  0.05; ** p  0.01; ns: non-significant.

  unpaired Student's t-test in Microsoft Excel 2016 was used to determine the statistical significance of the results obtained. For fold-change results, a Student's t-test used on the logarithmic values to determine the statistical significance between samples. * p ≤ 0.05 and ** p ≤ 0.01. ns: non-significant. Based on our findings we propose the following 4-step-model for actin-responsemediated breast cancer resistance to NK cells (Figure. 31). The section below presents and discusses the model in detail and highlights important perspectives to my PhD work.

Fig. S2 .

 S2 Fig. S2. CSRP2 is not required for MDA-MB-231 cell migration under normoxia or hypoxia. Transwell migration assay with normoxic or hypoxic MDA-MB-231 cells transfected with control (siCtr) or two different CSRP2-targeting siRNAs (siCSRP2 #1and 2). Cells were plated on collagen-coated transwell for 18h and quantified via MTT staining. Results were expressed relatively to the migration of siCtr-transfected normoxic cells (set to 1). The data originate from 3 independent experiments. *p < 0.05.

Fig. S5 .

 S5 Fig. S5. CSRP2 and HIF-1α protein levels are highly correlated in MCF-7 and MDA-MB-231 tumour xenografts. (A) Example of scattered plots showing pixel intensity for CSRP2 versus HIF-1α signals in an MCF-7 tumour section. (Band C) Tables showing the correlation coefficients for CSRP2 and HIF-1α protein levels (extracted from scattered plots) in 22 MCF-7 (B) and 21 MDA-MB-231 (C) tumour xenograft sections. The sections originating from the same tumour were taken from distant sites. An average correlation coefficient value and a corresponding p value are given below each table.

H

  t . P a t i e n t s w e r e s t r a t i f i e d a c c o r d i n g t o t h e u p p e r ( A ) o r m e d i a n ( B ) C S R P 2 e x p r e s s i o n v a l u e s . T h e p a t i e n t s a m p l e s , h a z a r d r a t i o w i t h 9 5 % c o n f id e n c e i n t e r v a l , a n d p v a l u e ( L o g r a n k t e s t ) a r e d is p l a y e d o n e a c h c h a r t . u m i n a R N A s e q e x p r e s s i o n [ l o g 2 ( n o r m a l . c o u n t s + 1 ) ] C S R P 2 e x p r e s s i o n i n h u m a n b r e a s t c a n c e r s B D i s t a n t m e t a s t a s i s -f r e e s u r v i v a l C S R P 2 g e n e e x p r e s s i o n d a t a f o r t h e l a r g e T C G A i n v a s i v e , h u m a n b r e a s t c a n c e r d a t a s e t ( n = 1 , 2 1 5 ) w a s c o m p a r e d a c r o s s t h e d i f f e r e n t b r e a s t c a n c e r s u b t y p e s a n d f o u n d t o b e a n o r d e r o f m a g n i t u d e h i g h e r i n t h e b a s a l s u b t y p e . ( B ) K a p l a n -M e i e r s u r v i v a l a n a l y s i s w a s p e r f o r m e d ; p a t i e n t s w e r e s t r a t i f i e d a c c o r d i n g t o t h e m e d i a n C S R P 2 e x p r e s s i o n v a l u e a n d d i s t a n t m e t a s t a s i s -f r e e s u r v i v a l w a s u s e d a s a n e n d p o i n t . T h e p a t i e n t s a m p l e s , h a z a r d r a t i o w i t h 9 5 % c o n f i d e n c e i n t e r v a l , a n d p v a l u e ( l o g -r a n k t e s t ) a r e d i s p l a y e d o n t h e c h a r t . * * * d e n o t e s a p -v a l u e l e s s t h a n 0 . 0 0 1 . Citation: Mgrditchian T, Arakelian T, Paggetti J, Viry E, Al-Absi A, Medves S, et al. Hypoxia: A Key Feature of the Tumor Microenvironment Triggers Several Mechanisms of Evasion from Natural Killer and Cytotoxic T Lymphocytes Surveillance. J Immun Res. 2014;1(2): 8. J Immun Res -Volume 1 Issue 2 -2014 Submit your Manuscript | www.austinpublishinggroup.com Janji et al. © All rights are reserved

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  Research in tumor immunology has validated the concept of Review Article Hypoxia: A Key Feature of the Tumor Microenvironment Triggers Several Mechanisms of Evasion from Natural Killer and Cytotoxic T Lymphocytes Surveillance Immun Res 1(2): id1010 (2014) -Page -02 Bassam Janji Austin Publishing Group Submit your Manuscript | www.austinpublishinggroup.com

Figure 1 :

 1 Figure 1: Hypoxia induces tumor cell resistance mechanisms leading to escape from CTL-or NK-mediated lysis. Under normoxic condition, the oxygen-sensitive prolyl hydroxylase domain protein 2 (PHD2)hydroxylatesHIF-1α subunit which is bound to Von Hippel-Lindau protein (VHL), ubiquitinated and subsequently degraded by the ubiquitin-proteasome system. Under hypoxia, PHD2 protein is inactivated, and HIF-1α is therefore stabilized. Stabilized HIF-1α translocated to the nucleus and formed with HIF-1β, a heterodimeric complex responsible for transcriptional activation of HIF-target genes including those responsible for the activation of autophagy. In addition, HIF induced the phosphorylation of the signal transducer and activator of transcription (STAT) 3 by a mechanism which is not fully described. The activation of autophagy leads to the degradation of the cargo protein p62, responsible for the elimination of phospho-STAT3 by the ubiquitin proteasome system. The degradation of phospho-STAT3 inhibits CTL-mediated lysis of tumor cells. Furthermore, excessive autophagy in hypoxic target cells leads to the selective degradation of granzyme B (GZMB), a serine protease contained into the cytotoxic granules released by natural killer (NK) cells, thereby inhibiting NK-mediated lysis.

Figure 2 :

 2 Figure 2: Hypoxia promotes tumor immune escape through actin cytoskeleton remodeling. Hypoxia can alter the expression of actin-binding proteins and/or Rho GTPase-mediated actin and adhesion signaling pathways either directly (in a HIF-dependent manner) or indirectly through extra cellular matrix (ECM) stiffening. The subsequent cytoskeletal changes modify cell adhesion, lateral diffusion of integrin ligands and promote epithelial-mesenchymal transition (EMT). This in turn drives tumor immune escape by compromising the formation of functional immunological synapses. In addition, the up regulation of inhibitory ligands at the surface of tumor cells compromise Rho GTPase activation signaling in immune cells, resulting in impaired cell migration and capacity to establish stable and functional immunological synapse.

  Zimmer for accepting to evaluate my thesis work. Thank you for your time and your effort. I am grateful to Dr. Hélène Heagel and Dr. Andreas Girod for being members of my mid-thesis committee. This work was completed at the Laboratory of Experimental Cancer Research, at the Luxembourg Institute of Health (LIH). It was a pleasure for me to work with all the great people at the LIH. I am very grateful to Dr. Guy Berchem for offering me the chance to proceed with my PhD thesis in his laboratory. I am also thankful to "Fond National de la Recherche" for funding my PhD project. Ernesto and each member of TSI and TM teams, it was really a pleasure to work with you. Thank you for all the sweet and funny moments we shared together. A special thank you for Maman Mano, Nassera and Victoria for your kindness and care. Malina, we met not long time ago but I am sure that we will laugh together again. Alexandra, Rita, Massimo, Sandrine, Angelina, Vanessa and Ning, each one of you took a different professional way but your presence around me during my PhD made my days. Thank you. Joanna, thank you a lot for all the support you gave me. I think we can make a comedy movie about our first 3 months in the lab! Hehe My gratitude also goes to my colleagues in NorLux lab. I really enjoyed working with you. Sabrina, Anna, Katrin, and my dear Monika thank you for everything. Mohammad, you are the coolest colleague I ever had. Keep it up. Coralie, I cannot thank you enough for all the support you have given to me. You let me discover new aspects that made my PhD a successful project. Lea, Maira and Anna, thank you a lot. Takouhie, thank you for every single funny moment we spent together. I smiled each time we talked and this smile will never go away. Thank you, Kris, for listening to me and helping me each time. Audrey, each Monday repeat the same sentence I used to say to you. Elodie, I will miss every single little thing we did together. Thank you for being a true friend. Even through distance, our hearts have remained together. My dad Farid and my mom Rouyida, you are my real heroes. I wouldn't have achieved nothing in my life without your love and support. I know you sacrificed a lot for me and my brothers to receive the best education. While I was doing my PhD, you were under fire in Syria. Every time I called you, you would still find the strength to support me. I am lucky I have parents like

Clément, thank you for being a great

(and cool)

supervisor. You integrated me in your laboratory and you were always my first supporter. We worked hard together to make a successful project. I wasn't always perfect, I had my ups and downs but I hope I made you proud of me. I am sure we will cross paths again, and until we do, I wish you and your family all the best. Thank you, Bassam, for your support during my thesis. I remember you telling me I will achieve great work when we first met, I hope that I was at your expectation.

My colleagues in Cytoskeleton and

Cancer Progression team, I cannot thank you enough. MERCI. Flora, with your positive energy, you were always eager to help me and I would be lost in the lab without you. I learned a lot from you and I will be always grateful! Céline, you transmit to me your passion for the microscope. Thank you for always being there to enhance my work. Hannah, thank you for all your help last year. I am sure you will do a great PhD. Good luck in all your endeavors. Thank you XianQing for not only being a colleague but a friend too. Josh, thank you for being a source of motivation. Carla, Ludo and Florian, I really enjoyed working with you all. I am grateful for your help. Etienne, Jerome, Sandrine, Delphine, Marina, Anne, Thank you Malou for supporting me and helping all the PhD students from the bottom of your heart. Tsolère (TsoTso), we began the PhD together, finish it together and we will stay always together. You are not just a colleague or a friend, you are my sister. My close friends, thank you from the bottom of my heart for supporting me in every single step. I am lucky to have each one of you in my life. Yasmine, Mohammad and Kamel, thank you for giving me hope, support and fun. My undying gratitude is to my big family for their support and encouragement. Even though war separated us from each other, I always felt your support. Nayla, my lovely aunt, I would have never achieved anything without your love and care. Thank you for being always by my side and most caring person. One personal "Thank you" for a very special person in my life, because you were beside me since the first day we met. Francesco, my love, my friend and my partner, thank you for supporting me each day more. We are strong together. I love you. you. My brothers, Dany and Naji, thank you for always encouraging me. You are the real reason for me to smile. My lovely little family, this PhD is dedicated to you. I hope I made you proud of me.

3.1. A general overview of breast cancer

  Breast cancer (BC) is the second most common form of cancer behind lung cancer. It is the first common cancer and accounts for 30% of all new diagnoses in women. Each year, 1.3 million cases are reported worldwide225 . Although advanced treatment strategies are applied, BC is still the second leading cause of death related to cancer in women today. Several risk factors are implicated in BC such as inheritance, age, hormonal status, and origins 226 .

Abbreviations

Results

Published OnlineFirst August 13, 2018

Supplementary materials and methods

Most of the materials and methods related to my studies was included in the article in Cancer Research (Result section 1.2) The materials and methods related to the supplementary result sections are given below.

Beclin-1 knockdown

Beclin-1 was knocked-down transfecting MCF-

Blocking antibodies

The blocking antibodies used in this study were purchased from Biolegend and

Contributions to articles

Résumé en anglais

Tumor immune evasion plays a central role in cancer progression and is a major hurdle to effective immunotherapy. In this Thesis, we examine the role of the actin cytoskeleton in breast cancer cell resistance to natural killer (NK) cell-mediated cell lysis. We found that resistant breast cancer cells escape from NKcell attack through a rapid and prominent accumulation of actin near the immunological synapse, a process we termed the "actin response". Our mechanistic investigations suggest that the actin response drives autophagosome polarization toward the immunological synapse and thereby facilitates the autophagymediated degradation of NK cell-derived cytotoxic molecules such as granzyme B. In addition, the actin response was associated with inhibitory ligand clustering at the immunological synapse, suggesting that it is a common driver of different immune evasion mechanisms. Taken together, our data lays the groundwork for therapeutic approaches aimed at interfering with the actin response and restoring an effective anti-tumor immune response.

Key words: Actin cytoskeleton, breast cancer, cytotoxicity, granzyme B, immune escape, natural killer.