Marcin Jakub Domagala

Marcin Jakub

Doctorat De L'université De Toulouse

Mary Poupot

M Antonio Sica

Rapporteur M Alan Ramsay

Rapporteur Mme

Fatima Teixeira-Clerc

Rapporteure M Jean-Pierre

Examinateur M Delord

Examinateur Jean-Jacques Fournié

Mary Mme

Directrice Poupot

De

Dr Frédéric Lagarrigue

Vera Pancaldi

Nina Verstraete

Malvina Marku

Laetitia Ligat

Frédéric Pont

Juan-Pablo Cerapio Arroyo

Carine Valle

Emeline Sarot

Carine Froment

Chloé - Remi

Joli Radkowi

Ani I Piotrowi

Rafałowi

Ewie Halinie

Konie

Kocham Cię

Characterization of a novel antibody targeting tumor-associated macrophages

est destinée au dépôt

Pauline -I wanted to thank you for everything. You are truly a lovely soul with incredible goodwill, eagerness to help and unmatched ability to multi-task. Don-Marc -huge thanks for answering my never-ending questions, especially scientific ones, but also sharing many good stories. Your diligence and will when it comes to your job, will always remain an inspiration for me.

Olivia -I will never forget your warmness and smile, that always made me feel at home. Big thanks for help the experiments, especially microscopy and dreaded electroporation. I hope I will finally be able to apply at least a percent of your sense of organization and composure.

Sophie -I will always remain grateful for our collaboration on the project! You are a great person and I am happy you have found a good position in the north Sarah -thank you for many great memories, I hope you are satisfied with your new job! Cathy -even if we didn't have time to know each other well, I really appreciate your good energy.

Especially big thanks to dear Carla, Chloé and Nico -my dear companions, who also shared with me their own ups and downs of PhD journey. You are truly remained the best sports! Chloé -I remember I was a bit afraid that there might remain some reserve between each other in the beginning. Gladly I could not be more mistaken. You turned out to be a great person, who showed me a lot of support and heart.

Carla -sometimes I don't know, what I would have done without you! You allowed me to find some true connections in France. You are truly the best! I am sure you will do exceptional on your defense and find an interesting post-doc in Switzerland.

Nico -thank you for our long discussions, both scientific ones and also geekier oriented. I truly keep my fingers crossed for your soon to be finished PhD and unfold of your scientific carrier! Fabien -thank you for your kindness, steadiness and willingness to help! Benoit -big thanks to the best organizer of all the social event and thus a countless great memories! I am really happy, that despite only few months in CRCT, you have remained an integral part of "our group"

Résumé

Les macrophages associés aux tumeurs (TAM) sont une des principales populations cellulaires du microenvironnement tumoral. Ils favorisent la croissance et les métastases tumorales et confèrent aux cellules cancéreuses une résistance à l'apoptose, aux attaques du système immunitaire et à la thérapie. Le ciblage thérapeutique des TAM dans les cancers reste difficile, en raison de leur grande plasticité et de l'absence de marqueurs exclusifs. L'équipe 9 du CRCT a alors développé un anticorps monoclonal appelé 6-25, en utilisant des cellules de type Nurse-like (NLC) comme modèle de TAM. Les NLC sont le type de TAM rencontrés dans la leucémie lymphoïde chronique (LLC), sont cruciales pour la survie des cellules leucémiques et peuvent être facilement générées in vitro. L'anticorps 6-25 a été caractérisé pour se lier spécifiquement aux TAM de différentes tumeurs en reconnaissant la protéine sidéroflexine-3 (SFXN3) à leur surface. L'anticorps 6-25 et sa cible ont été brevetés (WO2018/020000 et WO2018/19990).

L'objectif global de cette thèse était de caractériser davantage la spécificité et le potentiel thérapeutique de l'anticorps 6-25 ainsi que le rôle biologique de sa cible, la SFXN3. La première partie de la thèse visait à évaluer la fonctionnalité de l'anticorps 6-25. La version IgG1 nue de l'anticorps 6-25 n'a montré aucune toxicité envers les PBMC provenant à la fois de donneurs sains et de patients atteints de LLC. Il n'a pas causé de déplétion des NLC et n'a montré qu'un effet mineur sur le phénotype de ces cellules et des macrophages dérivés des monocytes (MDM) in vitro. Cependant, l'anticorps 6 25 s'est avéré être efficacement internalisé par les NLC.

La deuxième partie de la thèse a été consacrée à la cible proposée reconnue par l'anticorps 6-25, la SFXN3. En raison du manque général d'informations concernant SFXN3, la caractérisation plus approfondie de son expression dans les macrophages a été étudiée. De nombreuses expériences sur MDM traitées avec divers agents polarisants M1 et M2, ont prouvé que la cible du 6 25 est spécifiquement exprimée à la surface des macrophages de type M2. Les résultats obtenus lors du criblage de la cible 6-25 sur MDM ont en outre été utilisés pour étudier son modèle d'expression dans la NLC. L'augmentation de l'expression de la cible du 6-25 à la surface des NLC a été corrélée à leurs fonctions protectrices, ce qui en fait un marqueur de TAM pro-tumoraux. De plus, des expériences sur la polarisation des NLC ont permis de mieux comprendre le rôle du TNF et de l'IL-10 sur la biologie de ces cellules et en particulier la fonction protectrice vis-à-vis des cellules cancéreuses LLC.

La réalisation des expériences complémentaires sur la cible reconnue par l'anticorps 6 25 a conduit à la remise en cause initiale et finalement à la disqualification de la SFXN3 en tant qu'épitope potentiel. La réidentification ultérieure de la cible a été effectuée à l'aide d'une immunoprécipitation optimisée/spectrométrie de masse quantitative (IP-MS) et d'une approche RNAseq, qui a fourni la liste des cibles potentielles. Dans le processus de criblage, le récepteur du folate β (FRβ) a été identifié comme la cible de l'anticorps 6-25. Les résultats présentés dans cette thèse montrent que l'anticorps 6-25 représente une voie prometteuse pour cibler les TAM. Le FRβ a été montré comme étant un marqueur M2 spécifique des TAM. En raison des propriétés fonctionnelles du 6 25, y compris une spécificité élevée et une internalisation lors de la liaison aux macrophages de type M2, une version conjuguée à une drogue (ADC) de celui-ci a été générée.

Comme les premières expériences avec le 6-25 ADC ont montré des résultats prometteurs de déplétion des macrophages exprimant le FRβ, elles seront poursuivies à l'avenir. Toujours en perspective, les expériences in vivo avec l'anticorps 6-25 sont prévues pour évaluer son efficacité dans la modulation du TME, afin d'améliorer les résultats de la thérapie anticancéreuse. antibody targets. In the process of screening, folate receptor β (FRβ) was identified as the target of 6-25 antibody. This result was successfully validated by: 1 -knock-out (KO) of FRβ gene (FOLR2) in primary macrophages, and 2 -overexpression of FOLR2 in HEK-T cells (wild type HEK-T cells do not express 6-25 target).

Results presented in this thesis show that the 6-25 antibody represents a promising way of targeting TAM.

They also fully support FRβ, as a specific M2 marker of TAM. Because of functional properties of 6-25, including high specificity and internalization upon binding to M2-like macrophages, antibody-drug conjugate (ADC) version of it have been generated. As initial experiments with 6-25 ADC showed promising results in depletion of macrophages expressing FRβ, they will be continued in the future. Also in perspective, the in vivo experiments with 6-25 antibody are planned to evaluate its efficacy in modulating tumor microenvironment (TME), in order to improve the outcomes of anticancer therapy.

1 Introduction

The Role of Tumor Microenvironment in Cancer

For a long time, cancer research and therapy have been centered on deciphering the complexity of oncogenesis from the perspective of cancer cells itself. It has led to better understanding the mechanisms leading to emergence of the cancer cells, but also their heterogeneity, ability to evade immunesurveillance and resist therapeutic targeting. Nonetheless, this knowledge made evident that cancer cell focused approach is not enough to efficiently control the disease. It has been known for a long time that oncogenesis, beside cancer cells, involves also non-tumoral cells and extracellular components, which all form a complex ecosystem 1 . Already in 1863 Rudolf Virchow discovered leukocytes infiltrating neoplastic tissue thereby finding connection between tumor and inflammation 2 . Later in 1899 Pagets developed the theory of "seed and soil", where he proposed that spread of cancer cells (seed) is controlled by cooperation with the host organ (soil) 3 . Interestingly, these concepts are still being explored leading to better understanding of biological mechanisms controlling the cancerogenesis. With an advent of more advanced methods it has been finally possible to uncover a multitude of cell types infiltrating tumors, their phenotypes and functions, spatial distribution and density 1 . Among cells found in tumors we can distinguish -immune cells: T and B lymphocytes, NK cells, macrophages, MDSC, neutrophils, basophils, eosinophils, mast cells; stromal cells: fibroblast, mesenchymal stromal cells, osteoblasts and chondrocytes and non-stromal cells: endothelial cells, pericytes, adipocytes 4 and neurons 5 . Additionally, stromal cells with extracellular matrix (ECM) are constituting tumor stroma (some consider stroma to be formed also by blood vessels and inflammatory cells). All of these components together are forming the so-called tumor microenvironment (TME) 4,6 . TME can be further divided into different niches, to signify stimuli influencing/controlling particular part of the tumor: hypoxic niche, acidic niche, immune microenvironment, metabolism microenvironment, innervated microenvironment and mechanical microenvironment. 5 It has been accepted that TME has a crucial influence on multiple hallmarks of cancer, actively supporting all stages of oncogenesis and also having central role in cancer resistance to the treatment. Additional layer of complexity comes from the observation that TME is constantly changing along with cancer cells, adapting to new situations and stages of the cancer. Thus TME of metastases differ from the environment of primary tumor 3,7 . Continuous focus on deciphering the interplay of TME with tumor cells and finding a way to efficiently target components of TME seems to be a key in better controlling the disease and improving curative efficiency of cancer therapy 4 .

Recently the concept of TME is being revisited/expanded, with the emergence of the tumor-organismal environment (TOE) containing a multilayer view on TME 8 .

Formation of tumor stroma

Tumor stroma is composed of extracellular matrix and stromal cells and is one of the essential components of TME serving as a scaffold for tumoral and non-tumoral cells. Amount of stroma between the tumors shows extreme variations depending on the nature of the malignancy. At one side of the spectrum there are solid tumors, where stroma can represent up to ~90% of the tumor mass, as in case of some of the breast, pancreas and stomach carcinomas, whereas in case of hematological malignancies its content is much smaller [START_REF] Dvorak | Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing[END_REF] and often restricted to lymphoid organs 10 . Dependency of the solid tumors from the stroma, comes from the nature of surrounding tissue, that restricts availability of the nutrients and oxygen.

It has been established that if the tumor is growing beyond 1-2 mm, it requires establishment of nutrients influx, removal of metabolism byproducts and gas exchange for its further growth [START_REF] Dvorak | Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing[END_REF] . As mentioned previously the observation of complexity of tumor stroma and inflammatory processes has been made already in the XIX century. It was however much later, in the 1970s that molecular mechanisms underlying the formation of TME were uncovered. Researchers showed that mechanisms of tumor stroma formation share striking similarities to regenerative processes found in wound healing and chronic inflammatory diseases like rheumatoid arthritis (RA) or psoriasis 4,[START_REF] Dvorak | Tumors: Wounds That Do Not Heal--A Historical Perspective with a Focus on the Fundamental Roles of Increased Vascular Permeability and Clotting[END_REF] . As a continuation of these discoveries, in 1986 Dvorak came up with a very picturesque concept, describing tumors as a "wounds that do not heal" [START_REF] Dvorak | Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing[END_REF][START_REF] Dvorak | Tumors: Wounds That Do Not Heal--A Historical Perspective with a Focus on the Fundamental Roles of Increased Vascular Permeability and Clotting[END_REF][START_REF] Dvorak | Tumors: Wounds that do not heal-redux[END_REF] .

He pointed out that tumor microvasculature is characterized by incredible hyperpermeability, which can be compared to injured blood vessel. The crucial factor responsible for this state is vascular permeability factor (VPF) [START_REF] Dvorak | Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing[END_REF] . VPF was later identified to be VEGF [START_REF] Dvorak | Tumors: Wounds that do not heal-redux[END_REF] . Action of VEGF leads to extravasation/leaking of vascular exudate rich in plasma proteins and clotting agents into the tumor proximity. Tumor cells itself are also able to release substantial amount of tissue factors and enzymes that after mixing with this vascular exudate lead to formation of fibrin gel serving as an early/provisional stroma and a perfect medium for subsequent infiltration of accessory cells/non-cancer cells [START_REF] Dvorak | Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing[END_REF] . Early stroma is subjected to constant remodeling, overgrowth of new blood vessels/angiogenesis/vasculo-and arterio-genesis. With degradation of fibrin, the stroma forms granulation tissue, rich in blood vessels. The last stage of this process is connected to "resorption of vasculature and synthesis of collagen resulting in formation of dense and fibrous connective tissue" that in case of cancer is called desmoplasia. Desmoplasia corresponds to scar tissue in wound healing process [START_REF] Dvorak | Tumors: Wounds That Do Not Heal--A Historical Perspective with a Focus on the Fundamental Roles of Increased Vascular Permeability and Clotting[END_REF][START_REF] Dvorak | Tumors: Wounds that do not heal-redux[END_REF] .

Recently Dvorak has refined the previous concept of "wound that never heals" taking into account the heterogeneity of the tumor. As distinctive parts of the tumor might be in the different stages of tumorigenesis/healing, there might be a presence of "stable" parts, where tumor cells are embedded in mature, poorly vascular stroma representing "healed" parts. On the other hand, the invading front of the tumor keeps initiating previously described healing cycle, with release of permeability factors and fibrin gel deposition [START_REF] Dvorak | Tumors: Wounds That Do Not Heal--A Historical Perspective with a Focus on the Fundamental Roles of Increased Vascular Permeability and Clotting[END_REF][START_REF] Dvorak | Tumors: Wounds that do not heal-redux[END_REF] . The similarity between tumor and regenerative processes is reflected not only on the phenotypic/protein level, but also on the gene expression level. Microarray analysis of murine models of renal cell carcinoma and renal regeneration and repair, showed 77% of similarity in overexpressed genes.

The common genes were implicated in regenerative processes, cell proliferation and immune responses 13 .

Profound observation of molecular mechanisms harnessed by cancer to sustain its growth and progression, shows striking analogies with one utilized by physiological cells, suggesting that cancer cells do not rely on cancer-specific tools. Instead, they hijack tools normally utilized in physiological processes.

It holds a very important implication that the knowledge of the physiological processes can be used to better understand and fight cancer [START_REF] Dvorak | Tumors: Wounds That Do Not Heal--A Historical Perspective with a Focus on the Fundamental Roles of Increased Vascular Permeability and Clotting[END_REF][START_REF] Dvorak | Tumors: Wounds that do not heal-redux[END_REF][START_REF] Riss | Cancers as wounds that do not heal: Differences and similarities between renal regeneration/repair and renal cell carcinoma[END_REF][START_REF] Suijkerbuijk | From good to bad : Intravital imaging of the hijack of physiological processes by cancer cells[END_REF] .

Cancer-related inflammation (CRI)

Physiological role of inflammation

Inflammation is a defensive response involving recruitment and activation of immune cells in order to reestablish tissue homeostasis [START_REF] Freire | Natural resolution of inflammation[END_REF][START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] . While this process is most often associated with fighting the infection, it is equally important in controlling the damage and regenerating the tissue after injury [START_REF] Freire | Natural resolution of inflammation[END_REF][START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF] . In physiological conditions (often referred as acute inflammation), the process leads to elimination of noxious material, resolution of inflammation followed by initiation of repair phase and restoration of homeostasis. On the mechanistic level the inflammation response starts with detection of pathogen-associated molecular patterns (PAMP) or damage-associated molecular pattern (DAMP), that activates macrophages and mast-cells, which in turn secrete: pro-inflammatory cytokines, vasoactive amines and eicosanoids [START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] . These pro-inflammatory mediators cause dilatation and increased permeability of blood vessels, followed by recruitment of leukocytes. First lane of defense is represented by polymorphonuclear neutrophils, which are specialized in phagocytosis and release of enzymes that destroy the pathogen. Subsequently the inflamed site is cleared from apoptotic neutrophils by monocytes and macrophages. Clearance of dying cells and additional release of lipid-derived resolvins and protectins, contribute to quenching of the inflammation. Resolving inflammation is a crucial phase, that allows the regenerative processes to further restore the local homeostasis [START_REF] Freire | Natural resolution of inflammation[END_REF] .

The physiological inflammation response proceeds in a highly-programmed manner. However, if any stage of the process is disturbed or the response is unable to neutralize the triggering agent it may convert into chronic inflammation. This pathological condition may eventually lead to fibrosis, formation of granulomas or development of auto-immunological conditions [START_REF] Freire | Natural resolution of inflammation[END_REF] .

Chronic inflammation in cancer

Inflammatory processes are nowadays recognized to play a crucial role in the cancerogenesis. While acute inflammation, especially at the early steps of tumor formation might play an anti-tumoral role, it changes into chronic state upon failure to remove the source of the response [START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] . Process of chronic inflammation often referred to as "smoldering inflammation" [START_REF] Balkwill | Smoldering and polarized inflammation in the initiation and promotion of malignant disease[END_REF] is recognized as one of the main driver and hallmark of oncogenesis, that allows tumors to acquire all the characteristics of cancer and escape immunesurveillance [START_REF] Freire | Natural resolution of inflammation[END_REF][START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] . Inflammation in cancer is characterized as non-resolving and involves constant presence of various immune cells like tumor-associated macrophages, MDSCs, Tregs, but also inflammatory mediators, including: cytokines, chemokines, vasoactive amines and prostaglandins [START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] .

It is important to mention that the tumor environment is especially non-hospitable, characterized by hypoxia, acidosis, scarce of nutrients and growth factors, but also surveillance from the immune system 16 . To counterbalance these adversities inflammation can promote cancer both directly and indirectly.

Direct promotional effect of inflammation is based on the supply of cytokines to cancer cells that can serve as growth factors, enabling cell proliferation and resistance to cell death. This is especially crucial at the early stage of survival of transformed clones. [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] . Additionally, inflammation can shape plasticity of cancer cells by inducing epithelial-mesenchymal transition and expression of adhesion molecules, thus promoting metastasis processes [START_REF] Mantovani | Cancer-related inflammation[END_REF] .

Indirectly, inflammation can promote cancer by constantly shaping the TME, which include controlling of cellular composition and maintaining immunosuppressive status of TME to protect malignant cells from immune attacks 1,[START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF][START_REF] Mantovani | Cancer-related inflammation[END_REF] , activation of accessory cells to promote angiogenesis and ECM remodeling, thus modifying availability of key metabolites and facilitating metastasis process [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] .

Importance of CRI in cancer promotion has been proven by the numerous experiments and clinical studies of both murine and human tumors. In general, modulation of immune responses by the transfer of immune cells or induction of cytokines promotes progression of the disease, while targeting of immune cells and mediators of inflammation can impair cancer metastasis. Also limiting inflammation by administration of non-steroid anti-inflammatory drugs (NSAIDs) proved to lower the incidence or mortality of some malignancies [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] .

Chronic inflammation in cancer initiation

There are two main pathways connecting inflammation with cancer initiation and establishment of immunosuppressive TME -extrinsic (microenvironment driven) and intrinsic one [START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF][START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] or tumor-elicited inflammation (TEI) [START_REF] Suijkerbuijk | From good to bad : Intravital imaging of the hijack of physiological processes by cancer cells[END_REF][START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] .

Extrinsic pathway involves chronic inflammatory processes induced long before tumor formation. They can be an effect of: bacterial/viral infection or autoimmune disease, environmental pollution, but also unhealthy lifestyle -including tobacco smoking or obesity [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF][START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] . These stimuli induce an inflammatory milieu involving: constant activation of master-regulatory transcription factor (TF) NF-κB, presence of proinflammatory cytokines including Il-1, IL-6, TNF and reactive-oxygen species (ROS) or reactive-nitrogen species (RNS). ROS and RNS can induce genetic mutations in the adjacent cells that may affect the function of oncogenes and tumor-suppressor genes. Long-term exposure to these conditions may lead to malignant transformation of the cells [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF][START_REF] Mantovani | Cancer-related inflammation[END_REF][START_REF] Allavena | Pathways connecting inflammation and cancer[END_REF] . It is recognized that chronic inflammation caused by infection or autoimmunity is responsible for induction of about 20% of the malignancies, usually at the same site of the body. One of the examples is inflammatory bowel disease (IBD) that may predispose to colorectal cancer (CRC) [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF][START_REF] Galdiero | Cancer inflammation and cytokines[END_REF] . On the other hand, a relatively common skin condition like psoriasis is not considered to increase the risk of skin cancer [START_REF] Sica | Cancer related inflammation: The macrophage connection[END_REF] , but is associated with increased risk of development and mortality of cancer at the other sides of the body [START_REF] Trafford | Association of Psoriasis with the Risk of Developing or Dying of Cancer: A Systematic Review and Meta-analysis[END_REF] . Inhalation of fine particles, asbestos or tobacco smoke is also responsible for site specific inflammation and may lead to development of malignancies related to respiratory system. On the other hand, conditions like obesity and hyperglycemia are associated with induction of systemic inflammation, thus increasing the risk of developing malignancies in the various sites of the body [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] .

While extrinsic pathway remain an important driver of tumorogenesis, a majority of the primary tumors and cancers form without pre-existent inflammation [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF][START_REF] Wang | Tumor-Elicited Inflammation and Colorectal Cancer[END_REF] . Instead the tumor itself can promote an inflammatory environment to further drive it's progression, which is referred to as tumor-elicited inflammation (TEI) [START_REF] Suijkerbuijk | From good to bad : Intravital imaging of the hijack of physiological processes by cancer cells[END_REF][START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] or intrinsic pathway [START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF][START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] .

In the intrinsic pathway, the tumor cells themselves initiate the inflammation by the action of mutated oncogenes. These oncogenes are not only leading to neoplastic transformation, but are also responsible for expression of pro-inflammatory mediators that attract immune cells, further prolonging their survival and retention. RET, RAS and MYC are the main oncogenes that drive formation of inflammatory responses/milieu. For example, activation of protein tyrosine kinases like RET, drives expression of among others: colony-stimulating factors (CSFs) that attracts leukocytes to the tumor site, subsequently increasing their survival and differentiation; chemokines CLL2 and CCL20 that attracts monocytes and dendritic cells; IL-1β -which is one of the main pro-inflammatory cytokines; COX2 responsible for synthesis of prostaglandins; IL8/CXCL8 -chemokine that drives angiogenesis and CXCR4 attracting lymphocytes [START_REF] Mantovani | Cancer-related inflammation[END_REF][START_REF] Allavena | Pathways connecting inflammation and cancer[END_REF] .

Tumor-elicited inflammation can be also induced as a side-effect of nascent tumor formation. As previously mentioned growing tumors tend to induce angiogenic responses, as the oxygen demands from the near vessels are insufficient. The lack of oxygen causes formation of hypoxic niche in the tumor and stabilization of hypoxia-inducible factors (HIFs). These transcription factors cause induction of a plethora of genes, implicated in processes like: angiogenesis, cell survival and proliferation. At the same time HIFs support reprogramming of metabolism of cancer cells to rely mainly on glycolysis. It is important to mention that even in normoxic regions of the tumor, cancer cells are undergoing glycolytic switch to generate energy. This process is referred to as aerobic glycolysis [START_REF] Brassart-Pasco | Tumor Microenvironment: Extracellular Matrix Alterations Influence Tumor Progression[END_REF] or Warburg's effect 6 . Even though glycolysis generates low net ATP in comparison to oxidative phosphorylation, it allow cancer cells to accumulate metabolic intermediates to build macromolecules for biosynthesis and to support quick cell proliferation 7 . One of the side effects of these phenomenon is release of a substantial amount of lactate by cancer cells, which lowers the pH of surrounding tissue and leads to formation of acidic niche [START_REF] Brassart-Pasco | Tumor Microenvironment: Extracellular Matrix Alterations Influence Tumor Progression[END_REF] . Both hypoxia and acidosis may cause cells death and release of DAMPs that further fuel inflammation and wound-healing processes 7 . Additionally, acidosis is important in repolarization of immune cells, promoting Th2 responses in TME to further attenuate anti-tumor processes. These cascades of events contribute to non-resolving inflammation state and finally formation of pro-tumoral TME [START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] .

Establishment and types of TME

Irrespective of the pathway leading to cancer initiation, CRI leads to recruitment of a rich population of tumor-promoting inflammatory cells. By the action of selective pressure of inflammatory molecules present in TME, the recruited cells will adopt pro-tumoral roles, helping to enforce the supportive character of TME [START_REF] Binnewies | Understanding the tumor immune microenvironment (TIME) for effective therapy[END_REF] .

Depending on the genetic background and phenotype of the tumor, the TME can have a different proportion and spatial distribution of specific subtypes of immune and stromal cells [START_REF] Binnewies | Understanding the tumor immune microenvironment (TIME) for effective therapy[END_REF] . We can differentiate immunologically "hot" tumors, strongly infiltrated with cytotoxic T lymphocytes (CTLs) and CD103+ DC, that in some cases can even lead to formation of tertiary lymphoid structures in the tumor.

Neoplastic cells in these tumors are characterized by high genetic instability that may lead to release of numerous neoantigens that further increase overall immunogenicity. In general these tumors are representing favorable prognosis, as anti-tumoral potential of CTLs can be reactivated therapeutically [START_REF] Binnewies | Understanding the tumor immune microenvironment (TIME) for effective therapy[END_REF] .

On the other hand, immunologically "cold" tumors can be distinguished. They are characterized by exclusion of CTLs from the tumor core and prominent infiltration with tumor-associated macrophages (TAM), monocyte-derived suppressor cells (MDSC) and regulatory T cells (Tregs) [START_REF] Binnewies | Understanding the tumor immune microenvironment (TIME) for effective therapy[END_REF] . These cells are especially important for establishment of an immunosuppressive environment by a potent inhibition of anti-tumor responses of adaptive immunity [START_REF] Mantovani | Cancer-related inflammation[END_REF][START_REF] Binnewies | Understanding the tumor immune microenvironment (TIME) for effective therapy[END_REF] . They can block antigen presentation by DC, inhibit proliferation and activation by T and B lymphocytes [START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF] . Additionally, "cold" tumors are characterized to be more genetically stable -poor in neoantigens and generally harder to target by immune system or therapy [START_REF] Binnewies | Understanding the tumor immune microenvironment (TIME) for effective therapy[END_REF] .

Presented information signifies the importance of classification of not only an absolute number of immune cells in the tumor, but also their activation status and spatial distribution 1 .

CRI and TME in tumor progression and metastasis

With the tumor growth and progression the diseases can approach an invasive and finally metastasis state 7 . Establishment of cancer metastasis is a critical event in tumorogenesis, as metastatic disease is responsible for 90% of overall cancer-related mortality. Numerous clinical studies spot the role of inflammation and TME in driving cancer spreading in the body. Also long term administration of NSAID have shown to decrease colonization events and help remove micrometastases [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] .

Depending on the tumor progression that leads to metastasis begins with disruption of the basal membrane or ECM capsule around the tumor, that can be further breached by cancer cells. Remodeling of the ECM is mostly induced by the action of TAMs, which release among others matrix metaloproteinases MMP, facilitating local invasion or spread through blood or lymphatic vessels [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] . In general, the metastasis process is highly inefficient and among numerous cancer cells that leave primary tumor only few will succeed in colonizing a new site. It might be caused from both intrinsic characteristic of cancer cells, but also from the properties of the potential seeding site, that might not support the metastasis growth [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF][START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF] .

In order to increase the chances of a successful metastatic event, cancer cells need to be properly conditioned by either intrinsic genetic factors or influence of TME. It is important to mention that cancerstem cells (CSC) are much more likely to colonize distant tissues, as they are more plastic and resistant in comparison to remaining primary tumor cells. Inflammatory milieu of the tumor can stimulate NF-κB and STAT3 signalling that induce stemness of cancer cells and increase proportion of CSC in the tumor [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] . This process is partially overlapping with induction of epithelial-mesenchymal transition (EMT) of cancer cells that drastically increases their migratory capabilities [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] and stemness [START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF] . Upon colonizing a distant site, under the influence of "new" TME cancer cells often undergo reverse transition, back to epithelial phenotype in order to form a secondary tumor. This illustrates how TME can influence plasticity of malignant cells [START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF] .

Additionally, as metastasis happens mostly by lymphatic or blood vessels, cancer needs to be able to attach to vessel walls and further intravasate. For that they need to be able to express specific adhesion molecules, including integrins and chemokine receptors that confer tropism of cancer cells to specific tissue [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] . For example in case of melanoma cells, expression of CCR7 will be associated with lymph-node metastasis, while CCR9 with targeting of small intestine [START_REF] Mantovani | Cancer-related inflammation[END_REF] .

Another interesting mechanism that increases efficiency of metastasis is an ability of cancer cells to form complexes with other cancer cells, monocytes or neutrophils. These complexes are not only more likely to evade immune attacks, but also have a broader tissue tropism [START_REF] Greten | Inflammation and Cancer: Triggers, Mechanisms, and Consequences[END_REF] .

CRI is not restricted to the tumor site though, as the neoplastic disorders are associated with defective inflammatory responses at the other parts of the body 2 . The systemic inflammatory processes along the factor released by the tumor cells itself are essential for establishment of pre-metastatic niches at distant parts of the body. It has been shown that before arrival of neoplastic cells, potential metastatic sites can be already colonized by inflammatory cells, that will first prepare permissive environment [START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF] .

Role of macrophages in oncogenesis

Macrophages represent a major population of the immune cells within the TME and their presence is usually associated with a bad prognosis. Understanding both the origin and physiological roles of macrophages provide improved insight into their role in cancer. It also shows the importance of macrophages-targeting specificity when it comes to development of new anti-TAM therapies.

Origin and physiological roles of macrophages

There are two main sources of macrophages in the body: tissue resident ones and derived from circulating cells, including monocytes and bone-marrow progenitors. It was recently shown that most resident macrophages (MPs) in normal tissues are not only derived from bone marrow (BM) progenitors, as previously thought, but also from yolk sac or fetal liver, and are maintained by self-renewal [START_REF] Wynn | Macrophage biology in development, homeostasis and disease[END_REF][START_REF] Ginhoux | perspective New insights into the multidimensional concept of macrophage ontogeny , activation and function[END_REF] . However, during adult life, the rate of resident MPs can also be maintained by the infiltration of monocytes or early bone-marrow progenitors, with exception for/of microglia in the brain [START_REF] Ginhoux | The mysterious origins of microglia[END_REF][START_REF] Ajami | Local self-renewal can sustain CNS microglia maintenance and function throughout adult life[END_REF] . Monocytes from blood and bone marrow are therefore able to infiltrate tissues and to differentiate into specific tissue MPs but whether they can be considered as tissue-resident MPs is still a debate. In human blood, three main monocyte populations have been identified, a classical one, CD14 ++ CD16 -, a non-classical one, CD14 int CD16 + , and an intermediate one, CD14 + CD16 + 33 . These two latter populations are differentiated from a uniform population CD14 ++ CD16 -egressing from the bone marrow [START_REF] Patel | The fate and lifespan of human monocyte subsets in steady state and systemic inflammation[END_REF] .

Resident macrophages display key roles in growth, tissue development, homeostasis and remodelling, and have site-specific phenotypes and functions. It was proposed that the specialization of the resident MPs takes place inside the target tissue due to close contacts with tissue-specific cells as well as to soluble factors in the tissue environment [START_REF] Gautier | Understanding macrophage diversity at the ontogenic and transcriptomic levels[END_REF] . Microglia, in the brain and spinal cord, contribute to synaptic maturation during brain development and clearance of immature or defective neuronal synapses [START_REF] Amit | review The role of the local environment and epigenetics in shaping macrophage identity and their effect on tissue homeostasis[END_REF] . In the lung, alveolar MPs mediate approximately 30% of the surfactant lipid metabolism Langerhans cells with cutaneous MPs in the skin are specialized in the formation of the extracellular matrix and in the skin layer differentiation. Cardiac-resident MPs are required during heart development and take part in the regulation of the cardiac rhythm [START_REF] Hulsmans | Macrophages Facilitate Electrical Conduction in the Heart Article Macrophages Facilitate Electrical Conduction in the Heart[END_REF] . Kupffer cells in the liver are involved in the modulation of metabolism in hepatocytes, preventing the pathogenic accumulation of lipids [START_REF] Odegaard | Article Alternative M2 Activation of Kupffer Cells by PPAR d Ameliorates Obesity-Induced Insulin Resistance[END_REF] . Tissue-resident MPs located in the red-pulp region of the spleen have important functions in iron processing connected with the clearance of damaged red blood cells and the erythropoiesis [START_REF] Kohyama | Role for Spi-C in the development of red pulp macrophages and splenic iron homeostasis[END_REF][START_REF] Bruggen | Of macrophages and red blood cells ; a complex love story[END_REF] . White-pulp of the spleen also contains specialized MPs in both T cells zones and B-cell follicles. Role of the latter, called tangible MPs is especially important, as they remove apoptotic B-cells cumulating during germinal selection, thus preventing inflammation and autoimmunity [START_REF] Den Haan | Innate immune functions of macrophage subpopulations in the spleen[END_REF] .

Apart from physiological functions, monocytes/MPs also participate in infection/inflammation contexts, tissue repair and cancer.

Pathological functions of macrophages

MPs are very plastic cells which are able to respond to molecular or cellular signals from the tissue environment. The molecular signals can be endocrine or paracrine signals originating from phagocytosed cells or microorganisms and from the extracellular matrix/proteins. MPs can also directly interact with other tissue-resident cells such as immune cells recruited during injury. Indeed, monocytes and MPs are recruited from the bone marrow to the tissue injury site via the chemoattractant CCL2 secreted by resident MPs, endothelial cells, myocytes and fibroblasts [START_REF] Yoshimura | Non-myeloid cells are major contributors to innate immune responses via production of monocyte chemoattractant protein-1 / CCL2[END_REF] . In humans, classical monocytes (CD14 + CD16 -) display a high expression of CCR2 and are involved in responses to bacterial infection and inflammation, inflame masome signaling and in low density lipoprotein uptake. In contrast, non-classical monocytes (CD14 int CD16 + ) display a high expression of genes involved in cytoskeletal dynamics, tissue invasion during inflammation and genes suggesting terminal differentiation and cellular maturity [START_REF] Anbazhagan | Transcriptomic Network Support Distinct Roles of Classical and Non-Classical Monocytes in Human[END_REF] .

Monocytes-derived and tissue-resident MPs therefore co-localize to take part in healing and then to the resolution, thanks to the production of cytotoxic and pro-inflammatory mediators, clearance of invading microorganisms or removal of apoptotic and damaged cells [START_REF] Laskin | Macrophages and Tissue Injury : Agents of Defense or[END_REF] . On arrival at the injury site, blood-derived MPs can adopt a pro-inflammatory/M1/classical or anti-inflammatory/M2/alternative phenotype depending on the cytokines that are present in the microenvironment [START_REF] Martinez | Macrophage activation and polarization[END_REF][START_REF] Macrophages | M-1/M-2 Macrophages and the Th1/Th2 Paradigm 1[END_REF] . Regarding the plasticity of MPs, although the framework of M1/M2 polarization is a very useful system for in vitro studies, it is unclear how similar clear-cut phenotypes can be appended during in vivo injury and repair [START_REF] Xue | Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation[END_REF] . This M1/M2 paradigm is well pictured by the high expression of M2 markers by tissue-resident MPs compared to the mature phenotype of monocyte-derived MPs 48,[START_REF] Mould | Single cell RNA sequencing identifies unique inflammatory airspace macrophage subsets Single cell RNA sequencing identifies unique inflammatory airspace macrophage subsets[END_REF] . Early on after damage or injury, infiltrated Ly6C+ monocytes scavenge apoptotic debris or pathogens or infected cells thanks to the expression of pattern recognition receptors (PRR), Toll like receptors (TLR), scavenger receptors and Fc receptors that respectively recognize microbial antigens, danger signals, or immunoglobulins. These events lead to activation of transcription factors such as interferon (IFN) regulatory factors and nuclear factor kappa B (NFκB), inducing an M1 polarization and initiation of the inflammatory response [START_REF] Kawai | review The role of pattern-recognition receptors in innate immunity : update on Toll-like receptors[END_REF] . These blood-derived M1 MPs then release proinflammatory cytokines (e.g., IL-1β, IL-6, IL-12, IL-23, and TNF-α) and type-1 cell-attracting chemokines (e.g., CXCL9 and CXCL10), favoring the recruitment of more macrophages and leukocytes to help with injury resolution [START_REF] Mantovani | The chemokine system in diverse forms of macrophage activation and polarization[END_REF] . Once the acute injury has been resolved, MPs are in charge of suppressing inflammation and initiating wound repair. After clearing debris, MPs produce growth factors and mediators which abrogate the pro-inflammatory function of T cells and other immune cells [START_REF] Vannella | Mechanisms of Organ Injury and Repair by Macrophages[END_REF] . This is accompanied by a progressive repolarization of the blood-derived MPs towards a phenotype and functions increasingly similar to those of homeostatic tissue-resident MPs [START_REF] Olingy | Non-classical monocytes are biased progenitors of wound healing macrophages during soft tissue injury[END_REF] .

Pro-and anti-inflammatory pathways in MPs are induced by activation of specific plasma membrane receptors. If IFN-γ mediates the classical/M1 activation with upregulation of MHCII antigens, induction of nitric oxide synthase (iNOS) and production of pro-inflammatory molecules, interleukin-4 and -13 (IL-4 and IL-13) induce the alternative/M2 phenotype characterized by upregulation of CD206, transglutaminase 2, arginase and production of IL-10 and chemokines such as CCL17, CCL22 and CCL24 [START_REF] Mantovani | The chemokine system in diverse forms of macrophage activation and polarization[END_REF][START_REF] Martinez | Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages : similarities and differences[END_REF] . Like other immune cells, specific functions of MPs are therefore coupled to specific phenotypes even considering their plasticity, MPs can display intermediate phenotypes in certain inflammatory diseases and cancer [START_REF] Vogel | Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status[END_REF][START_REF] Pettersen | [END_REF] .

Macrophages in cancer

MPs in cancer are called tumor associated macrophages (TAM) and represent one of the major cellular component of the TME. They play a major role in tumor growth, metastatic dissemination, and therapy failure by inhibiting anti-tumor immune responses, promoting angiogenesis and extracellular matrix remodeling and providing nutrients for cancer cells. High TAM infiltration is generally correlated with poor outcomes in several types of cancer [START_REF] Mantovani | Tumour-associated macrophages as treatment targets in oncology[END_REF] .

Origin and phenotype of TAM

Until recently, TAM were considered to originate exclusively from blood-derived MPs undergoing differentiation upon tissue infiltration in response to chemokine and growth factors produced by stromal and tumor cells in the TME. Colony-stimulating factor 1 (CSF-1), vascular endothelial growth factor A (VEGF-A) and different CCL, especially CCL-2 [START_REF] Anbazhagan | Transcriptomic Network Support Distinct Roles of Classical and Non-Classical Monocytes in Human[END_REF][START_REF] Pettersen | [END_REF][START_REF] Schreiber | Cancer immunoediting: Integrating immunity's roles in cancer suppression and promotion[END_REF] were found to act as chemotactic molecules for monocytes in various cancers [START_REF] Linde | Vascular endothelial growth factor-induced skin carcinogenesis depends on recruitment and alternative activation of macrophages[END_REF][START_REF] Nandi | Stromal CCR6 drives tumor growth in a murine transplantable colon cancer through recruitment of tumor-promoting macrophages[END_REF][START_REF] Manuscript | Cellular and Molecular Origin of Tam[END_REF] . However, evidence shows that tissue-resident MPs can coexist in tumors with blood-derived MPs and their phenotype can rapidly evolve depending on the stage of the tumor and the characteristics of the molecular and cellular actors in the TME.

Depending on the tumor stage, state of microenvironment (oxygen ratio), TAMs phenotype is distributed somewhere in between M1 and M2 of the polarization spectrum, depending on the tumor stage and microenvironmental context. While generally considered to be protumoral, macrophages can exhibit some anti-tumoral activity, especially at the early stages of the tumor formation [START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF][START_REF] Mantovani | Tumour-associated macrophages as treatment targets in oncology[END_REF] . Also in vitro experiments demonstrated that macrophages activated with M1 promoting cytokines or bacterial products, including LPS can kill malignant cells [START_REF] Mantovani | Tumour-associated macrophages as treatment targets in oncology[END_REF][START_REF] Griffith | Necrosis Factor -related Cytokine[END_REF] . For example, IFN-γ can polarize resident MPs towards an M1 phenotype and promotes maturation of blood derived-M1 MPs. These MPs release pro-inflammatory factors that activate Th1 and Th17 immune responses and can also produce TNF-related apoptosis-inducing ligand (TRAIL) that result in TRAIL-induced cancer cell apoptosis [START_REF] Griffith | Necrosis Factor -related Cytokine[END_REF] . However, during most of the oncogenesis, especially in more advanced tumors, TAMs show strong protumoral influence and are characterized by M2 directed polarization. This polarization occurs thanks to anti-inflammatory mediators produced by the tumor cell and by stromal and immune cells in the TME, including MPs themselves. CSF-1, CCLs -2, -3, -14 and ILs -4, -10, -13 are common tumor-derived factors driving recruitment, proliferation and M2polarization of MPs [START_REF] De | CSF1 Overexpression Promotes High-Grade Glioma Formation without Impacting the Polarization Status of Glioma-Associated Microglia and Macrophages[END_REF][START_REF] Li | Tumor-associated macrophages secrete CC-chemokine ligand 2 and induce tamoxifen resistance by activating PI3K / Akt / mTOR in breast cancer[END_REF][START_REF] Zhao | Response gene to complement 32 ( RGC-32 ) expression on M2-polarized and tumorassociated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4[END_REF] . Other factors are more specific to the type of cancer such as prostate cancerderived cathelicidin-related antimicrobial peptide [START_REF] Zhao | Response gene to complement 32 ( RGC-32 ) expression on M2-polarized and tumorassociated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4[END_REF] or hypoxic cancer cell-derived cytokines Oncosatin-M and Eotaxin 66 . Besides, migration-stimulating factor (MSF) and CXCL12 can be secreted by MPs to promote self-polarization [START_REF] Solinas | Tumor-Conditioned Macrophages Secrete Migration-Stimulating Factor: A New Marker for M2-Polarization[END_REF][START_REF] Sica | Autocrine Production of IL-10 Mediates Defective IL-12 Production and NF-κ B Activation in Tumor-Associated Macrophages[END_REF][START_REF] Sa | The chemokine CXCL12 regulates monocytemacrophage differentiation and RUNX3 expression[END_REF] . Finally, low pH, hypoxia-inducible factors (HIF-1α and 2α), high-mobility group box 1 protein (HMGB1), extracellular ATP or tumor-derived ECM components are also potential factors that promote M2 polarization [START_REF] Sa | The chemokine CXCL12 regulates monocytemacrophage differentiation and RUNX3 expression[END_REF][START_REF] Doedens | Macrophage Expression of Hypoxia-Inducible Factor-1 α Suppresses T-Cell Function and Promotes Tumor Progression[END_REF] .

Pro-tumoral function of TAMs

There are several pro-tumoral functions displayed by TAMs at practically all stages of tumorogenesis.

These cells are essential in maintenance of immunosuppressive TME, especially by inhibiting adaptive anticancer responses [START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] . First of all TAM-derived TGF-β decreases antigen presentation and migration of DC, increasing their apoptosis at the same time [START_REF] Ito | Tumor-Derived TGF β -1 Induces Dendritic Cell Apoptosis in the Sentinel Lymph Node[END_REF] . IL10 and TGF-β released by macrophages can directly suppress recruitment and functions of cytotoxic CD8 T-cells and natural killer cells [START_REF] Kato | Lenvatinib plus anti-PD-1 antibody combination treatment activates CD8 + T cells through reduction of tumor-associated macrophage and activation of the interferon pathway[END_REF][START_REF] Takenaka | Control of tumor-associated macrophages and T cells in glioblastoma via AHR and CD39[END_REF] . Also IL-10, alone or in concert with IL-6, causes upregulation of macrophage B7-H4 expression, responsible for suppression of tumor-associated antigen-specific T cell immunity [START_REF] Chen | Induced expression of B7-H4 on the surface of lung cancer cell by the tumorassociated macrophages : A potential mechanism of immune escape[END_REF] . Moreover, IL-10 and PGE2 can induce expression of immune-checkpoint ligands (PD-L1) in myeloid cells which can inhibit cytolytic T lymphocytes responses 75,76 . Finally, IL-10 acts in an autocrine circuit in TAM to restrain their expression of IL-12 and also inhibits the release of IFN-γ [START_REF] Sica | Autocrine Production of IL-10 Mediates Defective IL-12 Production and NF-κ B Activation in Tumor-Associated Macrophages[END_REF] . Production of CCL17 and CCL22 by TAM promotes infiltration of Th2 and Treg populations in tumors, that are another important immunosuppressive cells [START_REF] Ito | Tumor-Derived TGF β -1 Induces Dendritic Cell Apoptosis in the Sentinel Lymph Node[END_REF] . TAM-derived prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO) and arginase 1 (ARG1) play important roles in further suppression of cytolytic T lymphocytes and in the induction of Treg function [START_REF] Ito | Tumor-Derived TGF β -1 Induces Dendritic Cell Apoptosis in the Sentinel Lymph Node[END_REF][START_REF] Lievense | Pleural Effusion of Patients with Malignant Mesothelioma Induces Macrophage-Mediated T Cell Suppression[END_REF][START_REF] Wang | Research trends in pharmacological modulation of tumor-associated macrophages[END_REF] . TAMs are also a potent producers of reactive oxygen species (ROS) and reactive nitrogen species (RNS), that are not only an important inducers DNA damage, but are also leading to modification of MHC on the surface of tumor cells. This reaction might lower binding of antigenic peptides to MHC and thus recognition of malignant cells by CTL [START_REF] Lu | Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice[END_REF] . All of these effect combined lead to situation where TAM form almost a "physical barrier", completely excluding CD8 T cells from the center of the tumor, leading to formation of previously described "cold" tumors [START_REF] Binnewies | Understanding the tumor immune microenvironment (TIME) for effective therapy[END_REF] .

TAM can secrete IL-17 and IL-23 that promote cancer cell proliferation. IL-23 signaling in tumor cells is important for the intra-tumoral production of downstream cytokines, which are either direct (IL-6, IL-22) or indirect (IL-17A) STAT3 activators [START_REF] Hung | products drive IL-23 / IL-17-mediated tumour growth[END_REF] . IL-6 produced by TAM also promotes tumor cells proliferation and invasive potential via STAT3 signalling [START_REF] Kong | Deletion of interleukin-6 in monocytes / macrophages suppresses the initiation of hepatocellular carcinoma in mice[END_REF] . TAMs also represent an important source of iron, which is essential for tumor cell division, growth and survival [START_REF] Jung | Iron as a Central Player and Promising Target in[END_REF] . TAM also take part in the promotion of the formation of blood vessels within the tumor providing nutrition for tumor growth [START_REF] Qian | Review Macrophage Diversity Enhances Tumor Progression and Metastasis[END_REF] . Several proangiogenic factors such as TGF-β, VEGF, PDGF are produced by TAM in the TME, especially in hypoxia conditions [START_REF] Wen | Increased metabolites of 5-lipoxygenase from hypoxic ovarian cancer cells promote tumor-associated macrophage in fi ltration[END_REF] . CCL18 produced by TAM promote synergically with VEGF the endothelial cell migration and angiogenesis [START_REF] Lin | CCL18 from tumor-associated macrophages promotes angio -genesis in breast cancer[END_REF] . Other chemokines such as CXCL1, 8, 12, 13 and CCL2, 5 produced by TAM help with the angiogenic switch in tumor tissues [START_REF] Mantovani | The chemokine system in diverse forms of macrophage activation and polarization[END_REF] .

TAM are also responsible for production of TNF which is a key player in NFκB upregulation, and induces migration and invasion potential of cancer cells, which is essential for subsequent metastasis [START_REF] Jung | Iron as a Central Player and Promising Target in[END_REF] . Various metalloproteinases (MMPs) and cathepsins produced by TAM are crucial for ECM remodeling and that further allows cancer cells dissemination [START_REF] Wang | Lung Cancer Tumor-associated macrophages provide a suitable microenvironment for non-small lung cancer invasion and progression[END_REF][START_REF] Hong | Pathology -Research and Practice Tumor-associated macrophages promote the metastasis of ovarian carcinoma cells by enhancing CXCL16 / CXCR6 expression[END_REF] . Some chemokines such as CCL18 produced by TAM, can induce clusterization of integrins in cancer cells, increasing their adherence to ECM and promoting migration and metastasis [START_REF] Chen | CCL18 from Tumor-Associated Macrophages Promotes Breast Cancer Metastasis via PITPNM3[END_REF] . A mouse study showed that mouse MPs-derived insulin growth factor 1 (IGF-1) induces migration of epithelial ovarian cell lines [START_REF] Liu | Upregulation of IGF1 by tumor-associated macrophages promotes the proliferation and migration of epithelial ovarian cancer cells[END_REF] . Finally, it has been shown that MPs-derived microRNA (miR-223) also regulates tumor invasion [START_REF] Yang | Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells[END_REF] . Additionally, TAM plays an active role in induction of ETM of tumor cells, by both direct interaction and release of specific molecules, like EGF that induces the transition by activating the EGFR/ERK1/2 signalling pathway [START_REF] Gao | Tumor associated macrophages induce epithelial to mesenchymal transition via the EGFR / ERK1 / 2 pathway in head and neck squamous cell carcinoma[END_REF] .

Pro-tumoral functions of TAMs lead also to cancer cell resistance to various therapies. TAM-derived exosomes have been shown as involved in mediating the resistance of gastric cancer cells to cisplatin [START_REF] Lu | Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice[END_REF] .

Endocrine resistance in breast cancer cells can be increased by TAM-derived CCL2 through activation of the PI3K/Akt/mTOR signalling pathway [START_REF] Li | Tumor-associated macrophages secrete CC-chemokine ligand 2 and induce tamoxifen resistance by activating PI3K / Akt / mTOR in breast cancer[END_REF] . Autophagy in hepatocellular carcinomas cells can be induced by TAM leading to oxaliplatin resistance [START_REF] Fu | Tumorassociated macrophages modulate resistance to oxaliplatin via inducing autophagy in hepatocellular carcinoma[END_REF] . It was recently shown that depletion of TAM by an anti-CSF1R enhanced the anti-tumor effect of docetaxel in a murine epithelial ovarian cancer [START_REF] Lu | Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice[END_REF] . High levels of TAM in the TME was also shown to increase the resistance of tumor cells to Padeliporfin, Vascular-targeted photodynamic (VTP) therapy in a prostate mouse model 93 . Anticancer immunotherapies may also be reduced by TAM when their suppression of TME correlates with an increase of DC-vaccination therapy in a malignant mesothelioma mouse model [START_REF] Of | Role of the microenvironment in chronic lymphocytic leukaemia[END_REF] . Finally TAMs are getting recognition for the low frequency of response to Immune Checkpoint Blockade (ICB) therapies, as they limit both CD8 T cell infiltration and activity [START_REF] Ceci | Targeting tumor-associated macrophages to increase the efficacy of immune checkpoint inhibitors: A glimpse into novel therapeutic approaches for metastatic melanoma[END_REF][START_REF] Peranzoni | Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti -PD-1 treatment[END_REF][START_REF] Panni | Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies[END_REF] .

Adapted and updated from our review on Cancer Cells Resistance Shaping by Tumor Infiltrating Myeloid Cells [START_REF] Domagala | Cancer cells resistance shaping by tumor infiltrating myeloid cells[END_REF]

Targeting of TAM

As mentioned previously, classical anticancer therapy has been mostly focused on chirurgical removal of the tumors, systemic treatment involving chemo-or radiotherapy and finally targeted treatment focusing on specific mechanisms driving tumor cells survival and proliferation. Unfortunately, these treatments are often associated with low efficiency, egress of multi-drug resistant clones of cancer cells that leads to relapse and further cancer progression [START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF] .

To overcome the shortcomings of current cancer treatment, the focus has been shifted from cancer cells to TME. As it has been proven numerous times that TME is crucial for tumor immune evasion and failure of anti-cancer therapy 1,4,[START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF] . Additionally, it has been shown that in the right circumstances, tumorinfiltrating cells have capabilities to normalize growth of cancer cells. Immune cells of TME also have a big anti-cancer potential, that after efficient re-activation might lead to a long lasting and efficient way of controlling the progress or even treating the disease 4,[START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF] . Beside, targeting of TME represents a good therapeutic target, as accessory cells of TME are genetically stable in comparison to tumor cells, thus lowering the chance of developing resistance 1 .

Reactivation of the immune system to fight cancer cells is extensively studied, yielding first successes in the form of immune checkpoint inhibitors (ICI) and specific CAR T cells. These relatively new therapies led to some spectacular results in melanoma, non-small cell lung cancer (NSCLC), and other cancers that were previously associated with poor prognosis [START_REF] Mun | The M-CSF receptor in osteoclasts and beyond[END_REF] . While undoubtedly, these therapies can be considered a breakthrough in the field of oncology, they show efficacy in a very limited number of patients 100,101 , and especially in case of CAR T cells are associated with significant expenses, further limiting their accessibility 101 .

Limited efficiency of ICB is sought in the presence of immunosuppressive cells of TME. It further stresses the importance of targeting the remaining populations of TME, especially innate immunity cells 101 . Among these cells, TAM are cited as one of the most numerous and influential groups [START_REF] Sica | Cancer related inflammation: The macrophage connection[END_REF][START_REF] Mantovani | Tumour-associated macrophages as treatment targets in oncology[END_REF] and their presence in TME is partially responsible for failure of many anticancer therapies 102,103 . Various therapeutic approaches targeting TAM are being developed right now. We can differentiate three main strategies, aiming to: deplete TAM population; re-educate TAM to reverse their pro-tumoral phenotype and actively attack tumor cells; inhibit recruitment of monocytes/TAM to the tumor site 104,105 . Illustration 1 summarizes main strategies to target TAM presented in chapters below. . Several antagonists and antibodies to CSF1R were developed for the therapeutic modulation of infiltrating TAMs and tested in preclinical models. In a preclinical breast cancer model, anti-mouse CSF1R antibodies were shown to deplete macrophages and to reduce tumor size 108 . Treatment with RG7155l, a humanized monoclonal antibody, was associated with a reduction in tumor-infiltrating TAMs and an increase in infiltration of effector T-cells in a fibrosarcoma mouse model, as well as in colorectal cancer and cancer patients with advanced tenosynovial giant cell tumors 107 . Alongside antibodies blocking CSF1R, small-molecule antagonists to CSF1R, capable of penetrating the blood-brain barrier, were developed. For example, tests with PLX3397 (pexidartinib) were associated with a reduction in the number of tumorassociated microglia and glioblastoma invasions in glioma mouse models 109 . Moreover, PLX3397 was able to potentiate the therapeutic effects of radiotherapy in a preclinical mouse xenograft model of intracranial human glioblastoma through the neutralization of CSF-1, which was upregulated by radiotherapy 110 . In an ovarian cancer model, GW2580 -selective CSF1R inhibitor, induced a reduction of ascite volume and a decrease in the number of infiltrating macrophages 111 . In a phase II clinical trial, patients with recurrent glioblastoma displayed a good tolerance to GW2580, but only 8% of 37 patients showed a six-month progression-free survival 112 . To enhance the potential of these inhibitors, combination strategies with conventional chemotherapy were proposed. The efficacy of paclitaxel in a mammary adenocarcinoma mouse model was enhanced by the blockade of CSF1R/CSF-1, with paclitaxel upregulating the production of CSF-1 113 . On the other hand, phase I study using a combination of a monoclonal anti-CSF1R with paclitaxel in patients with advanced and metastatic solid tumors revealed TAM depletion but no clinically relevant anti-tumor activity 114 . Combination of GW2580 with another chemotherapeutic -gemcitabine, showed a synergistic effect in mouse model of pancreatic adenocarcinoma. Action of GW2580 allowed to stop production of cytidine deaminase by TAM -an enzyme that was responsible for cancer cell resistance to gemcitabine 115 . More recently, combination of anti-CSF1R with docetaxel (microtubule-stabilizing agent) led to TAM depletion in a murine epithelial ovarian cancer model, which improved the anti-cancer effect of docetaxel 116 . Finally, it was shown that CSF1R targeting by the BLZ945 inhibitor (more potent and selective inhibitor of CSF1R) in a mouse model of glioma, led to significant decrease of tumor size.

Interestingly, while the CSF1R blockade led to depletion of macrophages in the peripheral tissues, it did not affect viability of the TAM in the tumor. It has been shown that the TAM has been rescued by factors produced by cancer cells, including: GM-CSF, IFN-γ and CXCL10, that however led to loss of M2 protective phenotype of macrophages 117 .

Another way to deplete TAMs is to use bisphosphonates (BPs), which induce apoptosis of myeloid cells by inhibiting the essential farnesyl diphosphate synthase, a key enzyme for cholesterol synthesis and protein prenylation. These compounds have a high affinity for bone hydroxyapatite, where they are internalized by bone osteoclasts leading to their apoptosis, and they are, thus, indicated as treatment in pathologies with abnormal bone resorption such as osteoporosis or multiple myeloma 118 . However, all tissue-resident macrophages, including TAMs, can be targeted by BPs such as clodronate using a proper formulation and administration strategy 119 . In case of cancer treatment clodronate is used as encapsulated in liposomal formulation, which not only prolong its half-life in circulation, but further target it toward mononuclear phagocytic system (APS) and thus macrophages 119 . Clodronate is approved to use in the clinics, showing to lower the incidence of bone and visceral metastasis in human mammary carcinomas treated with a combination of clodronate/chemo-or hormonal therapy 120 . Zoledronate, another type of BPs reduced skeletal-related events in patients with pancreatic cancer and improved overall survival in patients with bone metastases 121 . Effect of BPs is still studied in pre-clinical models, to check their usefulness in other types of cancers showing positive results correlated with TAM depletion 122 . In a murine xenograft model for cutaneous T-cell lymphoma, the reduction of tumor growth was associated with a decrease in pSTAT3 and macrophage number 123 ; the same association was observed in other preclinical mouse models of bone metastasis from lung cancer 124 , lung metastasis from breast cancer 125 , or melanoma xenograft models 126 .

Finally, trabectedin a tetrahydroisoquinoline alkaloid based chemotherapeutic, approved for the treatment of advanced-stage tissue sarcoma and relapsed ovarian cancer, was shown to also cause a partial depletion of circulating monocytes and TAMs 127 . Trabectedin is a DNA-binding agent isolated from a sea squid. It can bind to a minor DNA groove of DNA, inhibit DNA repair and transcription, and induce cell cycle arrest 128 . Trabectedin activates a TRAIL-dependent pathway of apoptosis, and myeloid cells, especially monocytes express high level of TRAIL receptor, which makes them sensitive to TRAIL-triggered apoptosis 129 .

Reprogramming

Even if TAMs generally display a pro-tumor role, they can potentially be converted to directly kill cancer cells or mobilize the immune system against the malignant cells. TAMs can be reprogrammed toward a tumoricidal phenotype by the targeting of surface markers, signaling molecules, or by modifications of their metabolism. Several markers at the surface of TAMs can be targeted to switch their phenotype such as the scavenger receptor MARCO, TLRs, MerTK, CD40, or CCR5 [START_REF] Wang | Research trends in pharmacological modulation of tumor-associated macrophages[END_REF][130][131][132][133] . The pattern recognition scavenger receptor MARCO has been implicated in various human cancers, and has been finally identified to be specifically expressed by a subset of TAM 130 . Use of an anti-MARCO monoclonal antibody with modified Fc receptor, showed anti-tumor activity in breast and colon carcinoma, and also in melanoma models. It turned out that by binding to inhibitory Fc-receptor (FcγRIIB), mAb led to reprogramming of TAMs populations to a pro-inflammatory phenotype, leading to tumor regression 130 . The pro-inflammatory phenotype can also be activated by specific ligands of TLRs or of CD40. Some TLR synthetic ligands were tested in cancer, such as the TLR3 agonist Poly(I:C) which activates the NF-κB pathway, leading to proinflammatory M1 polarization with production of type I IFN 134 . Intratumoral delivery of a TLR9 agonist, such as cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN), or a TLR7/8 agonist (R848) showed a tumoricidal activity in mouse models of melanoma and breast cancer 131,135 . Clinical trials with IMQ and a DNA-based TLR9 immunomodulator in metastatic cancer also showed histological tumor regression and an increase in lymphoid immune infiltration 136 . TLRs can also be activated by sensing bacterial ligands such as the attenuated ∆actA/∆inlB strain of Listeria monocytogenes, which, when introduced into the aggressive ID8-Defb29/Vegf-A murine ovarian carcinoma, is preferentially phagocytosed by TAMs inducing their anti-tumoral phenotype 137 . CD40 is also an important receptor at the TAM surface, as its ligation with the CD40 ligand at the T-cell surface stimulates T-cell-based antitumor responses. In preclinical studies, a rat anti-mouse CD40 antibody showed remarkable therapeutic activity in the treatment of CD40 + B-cell lymphomas 138 . The first class of modified anti-CD40 antibody, presenting five point mutations in the Fc domain (CP-870,893), showed efficient immunostimulation of effector T-cells in mouse models but little clinical effect in advanced cancer patients 139 . Recently, the same CD40 agonist was shown to increase pancreatic carcinoma sensitivity to chemotherapy in a PDAC mouse model associated with a switch of infiltrated macrophages due to an increase in CCL2 expression and IFN-γ in these mice 133 . This last study opened promising perspectives for combination therapies. CCR5 antagonists such as maraviroc provided an anti-tumor effect in a phase I trial in patients with liver metastasis of advanced refractory colorectal cancer correlated with a macrophage repolarization 132 . Furthermore, a majority of cancer cells express a high amount of CD47 at their surface, which interacts with signal regulatory protein alpha SIRPα on myeloid cells such as macrophages, to transmit the "do not eat me" signal. This inhibition of phagocytosis of autologous cells exists in homeostatic conditions by the prevention of myosin IIA accumulation at the phagocytic synapse. Anti-CD47 antibodies or engineered SIRPα-Fc fusion were shown to restore macrophage ability to phagocytose cancer cells and prime cytotoxic CD8 T-cells, leading to the tumor regression in several tumor models and in clinical trials. Two anti-CD47 antibodies (Hu5F9-G4 and CC-90002) and one soluble recombinant SIRPα-crystallizable fragment (Fc) fusion protein (TTI-621) were tested in phase I clinical trials in AML and other hematological malignancies, pediatric brain tumors, and some multiple solid tumors (trial numbers NCT02678338 and NCT03957096). To minimize the off-target toxicity (transient anemia), bispecific antibodies targeting both CD47 and tumor-associated antigens were recently developed, yielding promising results 140 . Specific pathways involving STAT3 or STAT6 characteristic of an anti-inflammatory response can be targeted to modify M2 characteristics of TAMs. For instance, through its ligation to the IL-10 receptor, IL-10 and IL-6 induce the activation of STAT3 necessary to drive an anti-inflammatory effect. Formerly, microtubule-stabilizing agents such as paclitaxel were shown to promote polarization of MDSC to macrophages with the M1 phenotype via suppression of STAT3 phosphorylation 141 . The STAT3 axis was then more specifically targeted with an intravenous injection of a STAT3-small interfering RNA (siRNA)-CpG conjugate which was shown to silence the immune suppressor gene STAT3, reprogramming the functions of TAMs correlated with an immune-mediated tumor rejection 142 . A recent in vitro study showed that corosolic acid, packaged within long-circulating liposomes and coupled to an anti-CD163 antibody, inhibited the activation of STAT3 in human M2 type macrophages and their IL-10-induced gene expression 143 . In the same way, STAT6, one of the major signal transducers activated by IL-13, involved in M2 polarization, can be inhibited by different synthetic molecules (AS1517499, TMC-264, A771726), leading to an inhibition of tumor growth in the 4T1 mammary tumor model and modification of genetic markers for TAM infiltration 144 . Furthermore, microRNA (miRNA)-155 is best characterized as a pro-inflammatory miRNA since it enhances M1-like macrophage activation by decreasing inhibitors of pro-inflammatory responses and targets the IL-13 receptor to reduce alternative macrophage activation 144 . Currently, several miRNAs are implicated in the regulation of macrophage activation and polarization in cancer 145 , and developments of pharmacological formulations that either suppress or enhance the activity of selected miRNAs to reprogram the TAM phenotype are emerging. For instance, targeting of miRNAprocessing enzyme DICER in macrophages with lysozyme-M-driven Cre prompts M1-like TAM reprogramming in lung carcinoma and MC38 tumors 146 . The switch of M2 TAMs toward an M1-like phenotype can also be driven by rapamycin, which is an mTORC1-specific inhibitor, inducing an anti-tumor effect in a hepatocarcinoma mouse model 147 . Moreover, inhibitors of PI3kγ, a molecule upstream of mTOR, are able to promote TAM-immunostimulatory responses in several cancer models 148 . Activation of MAPKs via production of ROS, thanks to a copper chelator, CuNG (N-(2-hydroxy acetophenone) glycinate), can convert suppressive TAMs toward a pro-inflammatory phenotype 149 . M1 genes such as iNOS and CXCL9 can also be upregulated concomitantly with the downregulated expression of M2 genes, arginase-1 and CD206, thanks to the inhibition of the CYP4X1 monooxygenase or the activation of the autophagyinduced RelB/p52 by flavonoids in glioma or hepatocellular carcinoma models 150,151 . Several synthetic molecules can also actively reverse the M2 phenotype of TAMs such as TMP195, an HDAC inhibitor, in breast cancer mouse models, or paclitaxel, an anti-microtubule agent, in patients with ovarian carcinoma 152,153 . Combination strategies were also reported to have a good effect on TAMs reprogramming. Anti-CD40 was combined with anti-CSF1R to create a pro-inflammatory environment eliciting T-cell responses before depletion of TAMs 154 or with chemotherapies such as imatinib (tyrosine kinase inhibitor) with strong tumor regression in a gastrointestinal tumor mouse model 154 . A TLR9 agonist (IMO-2055) combined with chemotherapies and anti-VEGF showed anti-tumor activity in advanced or metastatic lung cancer 155 . Moreover, TLR9 agonists were also able to enhance radiofrequency-induced CTL responses, potentiating the inhibition of primary tumor growth and lung metastasis 156 . Regulating macrophage mitochondrial function is another way to activate the reprogramming of TAMs. Indeed, downregulation of the gene encoding pyruvate dehydrogenase is avoided by dampening the nuclear respiratory factor 1 degradation under hypoxia, which minimizes the Warburg effect and promotes M1 polarization of TAMs 157 . Moreover, cancer cells secrete lots of lactate through glycolysis, which is recognized by Gpr132 at the surface of TAMs and promotes M2 polarization. PPARγ agonists, which are suppressive for the Gpr132 axis, or siRNA silencing of Gpr132, was successfully used to desensitize TAMs to lactate stimulation in a breast tumor model 158,159 . Finally, recent studies identified novel drug targets for further investigation in the future.

For instance, loss of Gadd45b, a gene involved in the regulation of growth and apoptosis in myeloid cells, restored activation of pro-inflammatory functions of TAMs and intra-tumor immune infiltration in a fibrosarcoma mouse model 160 . Interfering with angiopoietin family molecules might be an effective strategy for reprogramming TAM polarization in NSCLC 161 . CaMKK2 was defined as a new myeloidselective checkpoint and could be blocked to facilitate a favorable reprogramming of immune cells in the TME 162 . Several studies also proposed to reprogram the energetic metabolism in TAMs, in which fattyacid oxidation is preponderant, decreasing lipid and cholesterol intake and metabolism 163,164 .

Another interesting approach allowing to harness pro-tumoral potential of TAM is development of chimeric antigen receptor macrophages (CAR-Ms). Based on pre-clinical experiments, CAR-Ms were shown to efficiently phagocyte cancer cells in a directed manner. Additionally, they possess stable M1 phenotype and ability to convert other bystander M2 macrophages into M1 side of the spectrum, thus leading to disruption of pro-tumoral microenvironment 165 . Further development of CAR-Ms should soon yield the first results of its efficacy in the clinical trials.

Inhibition of recruitment

Modulation of the recruitment of monocytes and macrophages to the tumor site in order to disrupt TAM populations, represents a promising complementary therapeutic strategy. Inhibition of the CCR2/CCL2axis is a major focus in this field as it represents the main pathway responsible for monocyte recruitment.

Targeting of CCR2/CCL2 pathway showed positive outcomes in various experimental cancer models [166][167][168][169][170] .

In contrast to animal studies, clinical translation proved to be problematic. Clinical trials with agents depleting CCL2 (carlumab or CNTO888) or blocking CCR2 (MLN1202) showed poor therapeutic responses when used as monotherapies 171,172 . While administration of an anti-CCL2 antibody initially showed response, leading to decreased levels of CCL2, in a long term it led to significant elevation of this chemokine. Further precaution must be taken, as discontinuation of CCL2 therapy in murine models of breast cancer showed increased monocyte mobilization and tumor progression 172 . Phase I and II clinical trials with carlumab were carried out in patients with different types of tumors and indicated that CCL2 levels were only partially suppressed, leading to lack of therapeutic effect 173,174 . On the other hand, the blockage of CCR2 by small-molecule drug PF-04136309 in combination with folfirinox chemotherapy in a phase Ib clinical trial in patients with pancreatic cancer led to promising outcomes. While monotherapy turned out to be ineffective, 97% of patients with the combined treatment showed an objective tumor response and remaining patients showed a local tumor response 175 . To lower the chance of negative outcomes of direct inhibition of CCR2/CCL2 pathway, it has been proposed to target pathways responsible for upregulation of CCR2 on the cells. It was observed that, in a mouse model of glioblastoma, TAMs express aryl hydrocarbon receptor (AHR), which, upon activation by tumor released kynurenine (KYN), upregulates CCR2 expression. Blocking the AHR led to decreased TAM infiltration and tumor growth with concomitant increased survival of the animals [START_REF] Takenaka | Control of tumor-associated macrophages and T cells in glioblastoma via AHR and CD39[END_REF] .

Targeting of CSF1R also showed a decrease in macrophage/monocyte recruitment 176 . In spite of this, numerous clinical trials showed a limited therapeutic effect when CSF1R was targeted alone 177 , with the exception of benign tenosynovial giant cell tumors, where a decrease in TAM infiltration and disease stabilization was achieved 178 . Efficacy of CSF1R inhibitors is still being tested in anticancer therapies, also in the form of broad-spectrum kinase inhibitors such as chiauranib (CSF1R, VEGF1-3, PDGFRα, c-Kit, Aurora B inhibitor) 179 and pexidartinib (CSF1R, KIT, FTL3 inhibitor), with potential anticancer results 180 . Research on a mouse model of lung adenocarcinoma showed that FGF released by CAF can induce TAM recruitment.

Blocking of FGF receptor (FGF-R) with the Fgfr tyrosine kinase family inhibitor AZD4547 showed strong TAM abolishment and tumor regression, making this receptor a potential therapeutic target 181 . In addition, targeting other important molecules for monocyte/macrophage recruitment in cancer, i.e., catecholamines by 6-hydroxydopamine 182 and complement components such as C5a, may also bring positive results 183 .

Adapted and updated from our review on Latest Advances in Targeting the Tumor Microenvironment for Tumor Suppression 106

Conclusions

Several strategies have been proposed to target TAM, including: inhibition of recruitment, reprogramming into an antitumor phenotype, blocking of functions or depletion of these cells 104,105 . Targeting of CSF1R-CSF1 axis remains the leading approach, showing mixed responses in the clinic, with most promising effect in diffuse-type tenosynovial giant cell tumors 178,184 . Problem of this approach is associated with targeting apart from TAMs, also monocytes and tissue macrophages [START_REF] Mun | The M-CSF receptor in osteoclasts and beyond[END_REF] . Similar situation concern administration of Clodronate and other BPs, which potentially target the whole mononuclear phagocytic system (MPS) 119 . Blocking of CCL2 axis responsible for recruitment of monocytes to the tissue sites, has been associated with low efficiency and intensified monocytes mobilization upon the treatment termination 171,172 . Alternative to "systemic" targeting of macrophages, would be therapies aiming to address only discrete populations of pro-tumoral TAMs. Unfortunately, specific targeting of macrophages remains a major challenge, mostly because of the remarkable plasticity of these cells, and shortage of exclusive markers. Emergence of a relatively new macrophage-specific markers like: MARCO 130 , VISTA 185 , Tyro3, Axl and MerTK 186 may offer a more accurate TAM-targeting in the future, opening a possibility for more precise elimination or reprogramming of TAM. Also continuation of finding new specific markers of TAM should remain a priority.

Chronic lymphocytic leukemia

TME is often associated with solid tumors, but it plays an equally important role in case of blood cancers.

While the amount of tumor stroma and location of TME elements may differ drastically in comparison to localized tumors 9 , all the interactions between cancer cells and accessory cells are also present in hematological malignancies. One of such examples can be observed in case of chronic lymphocytic leukemia (CLL) 187 .

CLL belongs to the group of B cells malignancies and is characterized by the accumulation of small, CD5+, CD19+ mature-appearing neoplastic lymphocytes in the circulation and both primary and secondary lymphoid organs. Depending on the stage of the disease it leads to lymphocytosis, lymphadenopathy or splenomegaly. CLL is an extremely heterogenous disease. Freshly diagnosed patient might require only monitoring of the disease, otherwise remaining fully active for decades. On the other side, there are individuals, who will progress rapidly, which despite treatment might lead to death. In most cases however, patients land somewhere in between of these two extremes 187 . 5-year relative survival of CLL is around 87.2% 188 .

Epidemiology of CLL

CLL is stated as a most frequent adult leukemia of "western world", with frequency reaching around 0,06% Additionally, there are plethora of flow cytometry markers with prognostic value, regarding agresivness of the disease, likelihood of treatment necessity etc. These include: CD49d 190,191 ,CD38,ZAP70,CD69 192 . Also measurement of b2-microglobulin (β2-m) levels in the serum, represents an important predictive value, and since this protein is constitutively released by CLL B cells it can be used to evaluate overall burden of cancer cells and thus response to therapy 193 .

Illustration 2. U-CLL and M-CLL classification depends on maturation status of CLL cells. Unmutated or mutated status of CLL cells, depends on the maturation stage from which malignant clone originates. Naïve B cell before somatic hypermutation (SHM)

can give rise to U-CLL cell, analogically more mature B cell, after SHM and class switching can potentially lead to M-CLL cell expressing IgG. Adapted from 187 , permission granted via RightsLink.

Pathophysiology

CLL is associated with plethora of chromosomal alternations, somatic mutations, epigenetic modifications and miRNAs expression. Around 80% of patients have one of 4 chromosomal abnormalities: a deletion in chromosome 13q14. 3 (del(13q)), del(11q), del(17q) and trisomy 12. Del(13q) is present in >50% of patients and is associated with positive prognosis. Alternations in this region are connected to expression of BCL2 gene, that has big implication in resistance to apoptosis and cell cycle progression. Del(11q) is detected in around 18% of patients and is usually connected to mutation in ATM gene. Del(17q) is found in 7% of patients, leading to loss of tumor suppressor gene TP53 187 . Finally, trisomy 12 occur in 20% of patients, is frequently associated with NOTCH1 mutations 194 . Both Del(11q) and del(17q) are both associated with adverse prognosis 187,195 , while trisomy 12 with intermediate one 187,194 in the CLL pathophysiology will be discussed in detail in the next chapter). MSCs also supports cancer cell survival through release of CXCL12, WNT and directly by VCAM1 and CD49d/CD29 interaction. Activated cancer cells in turn release CLL2-4 to recruit more T-cells, and monocytes that will differentiate into NLC, thus further strengthening the TME 187,197 .

Looking globally, at any given moment only few percent of CLL B cells reside in the proliferation centers.

After activation and proliferation in LN, cancer cells return to circulation where they acquire resting status or be driven into anergy. Returning to circulation will cause CLL cells to upregulate expression of CXCR4 and surface IgM. After reaching certain level, the cells can again follow the CXCL12 gradient that will lead them back to LN, thus repeating the cycle. The "activation cycle" of CLL B cells can be followed by the CXCR4/CD5 expression pattern 198 . Engagement of CXCR4 with CXCL12 leads to its downregulation, thus CLL B cell right after leaving proliferation centers are CXCR4 dim and CD5 bright and resting cells are CXCR4 int/bright and CD5 int/dim respectively 187,198 (Illustration 3). The role of CD5 in this process is not exactly clear, however it has been suggested that CD5 might prevent negative BCR signaling and also induce expression of CCR7, activation of STAT3 and expression of IL-10 197 . CCR7 is crucial for homing to LN 198 and IL-10 has a very important role in CLL, as it not only increase survival of cancer cells themselves 187,199 , but also induce immunosuppressive environment and polarization of NLC.

Illustration 3 Representation of hypothetical model of CLL cell lifecycle, signifying migration of cancer cells between proliferation center (solid tissue) and blood stream. 1) CLL cell enter proliferation center, and get in contact with accessory cells, including NLC, stromal cells and T cells. There cancer cells get activated, proliferate and upregulate expression of CD5 and downregulate CXCR4.

CD5 high CXCR4 dim cancer cells reach the blood stream. 2) in the bloodstream CLL cells gradually lose expression of CD5 and upregulate CXCR4, leading to CD5 int CXCR4 int and finally CD5 dim CXCR4 high populations. 3) CD5 dim CXCR4 high cells will most likely detect CXCL12 gradient and re-enter the proliferation center/solid tissue to repeat the cycle (otherwise they die from exhaustion). Adapted from 198

Nurse-like cells (NLC)

Nurse-like cells represent one of the main component of CLL-specific TME found in lymphoid organs, especially LN where they participate in formation of "proliferation centers", as introduced previously. NLC differentiate from CD14+ monocytes, attracted to the tissue site, mostly by cancer cells producing chemokines like CCR2 200,201 . The exact factors involved in NLC differentiation and maturation remains unknown, none the less CSF-1 202,203 , extracellular nicotinamide phosphoribosyltransferase (NAMPT) produced by CLL cells 204 , HMGB1 205 released by dying CLL cells and direct contact with CLL B cells are all necessary 206 . NLC can be obtained in vitro during long-term culture of PBMC isolated from blood of CLL patient. PBMC culture leads to outgrowth of large, adherent NLC with numerous cancer cells attached to their surface. Sometimes also pseudo-emperipolesis process can be observed, where CLL B cells are crawling under the macrophages without being internalized (Illustration 4). This phenomenon also granted NLC their name, based on nurse cells present in the thymus, which display emperipolesis and have a crucial role in thymocytes maturation 201 . Furthermore, transcriptomic analyses of monocytes, M1, M2 macrophages, TAM and NLC revealed that NLC show very close resemblance to M2-like TAM. Thus NLC can be classified as specialized form of tumor-associated macrophages in CLL 207 . Recently also term leukemia-associated macrophages (LAM) is quite often utilized in context of blood malignancies 208 . NLC can be characterized by expression of CD14 + , CD68 + , CD163 ++ and CD206 + markers 201,209 . Ibrutinib exhibit also immunomodulatory functions, leading to normalization of T cells populations and inhibition of BTK expressed by NLCs. Interestingly treatment with ibrutinib potentiates M2 phenotype of NLCs by: blocking STAT1 phosphorylation with concomitant activation of STAT6; increased expression of CD163 and CD206; stimulation of IL-10 expression. It also decreases the phagocytic potential of NLCs by downregulation of Macrophage-1 antigen (MAC1)/CD11b/CD18 expression 216 . However, it was not shown if ibrutinib can also affect the efferocytosis process which is an especially relevant form of phagocytosis in tumorogenesis 217 . In vitro studies on monocyte to macrophage differentiation upon treatment with ibrutinib, showed: promotion of spindle shape morphology of mature cells; decrease expression of CD11b; increased oxidative metabolism. On the other hand, it did not change the expression of important M2 markers, like CD163 and CD206 218 . In the end, better characterization of the effect of ibrutinib on NLCs function is required, especially as these cells are suggested to be an important factor in the resistance to this treatment. So far the results imply that ibrutinib, actually promote protective phenotype of NLCs and alternative targeting of these cells would help improve the outcome of the therapy in case of resistant patients.

Illustration 4. Visualization of interaction between CLL cells and NLCs

All the drugs presented below are not yet approved in the clinic for treatment of CLL and have been mentioned upon general TAM targeting. The first one -Lenalidomide is an immunomodulatory agent (IMID) with non-defined molecular mechanism of action, involving increased actin polymerization and cell adhesion. This agent turned out to affect several cell types forming TME of CLL, including monocytes and All of the drugs presented above affect NLCs by modulating their phenotype and potentially disrupting their nursing properties toward CLL cells. However, they are working on the broad spectrum of cell types, displaying low specificity. More direct approach in targeting of NLCs could involve targeting of CSF1R signaling or treatment with Clodronate. For example, treatment of primary CLL samples in vitro with agents blocking CSF1R signaling, led to depletion of NLCs, increased mortality of cancer cells and also sensitization of cells to idelalisib and ibrutinib in case of resistant cells 202,203 . Also in vivo treatment of leukemic Eμ-TCL1 mouse model with Clodronate resulted in: depletion of both monocytic and M2 macrophage populations; stimulation of proinflammatory cytokines production including GM-CSF; increased number of DCs -all of which led to decreased survival of CLL B cells 224 .

Finally, an interesting strategy in disruption of NLCs properties, could be achieved by repolarization of TME with use of cytokines, like IFN-γ. One study on in vitro effect of IFN-γ, showed that NLCs could be partially repolarized into M1-side of the spectrum, leading to decreased viability of CLL cells and increased antibody-dependent cell phagocytosis (ADCP) potential of NLCs 225 .

All things considered, targeting of NLCs display similar problems, as in case of TAM in other cancer -overall lack of specificity. It leads to not only weaker response to therapy, but also might increase toxicity as seen with drugs like Lenalidomide. If we consider that in most cases targeting of TME, including TAM should be used in combination with agents targeting cancer cells, it is necessary limit side-effects of the complementary treatment. For that, it remains crucial to find specific markers for TAM in different types of cancers.

Aim of the thesis

Tumor-associated macrophages (TAMs) belong to the major cell population of tumor microenvironment where they support tumor growth and metastasis, and also confer cancer cells resistance to apoptosis, immune system attacks and therapy. Therapeutic targeting of TAM in cancer remains challenging, owing to their high plasticity and lack of exclusive markers. To address these problems, Team 9 of CRCT used a Hybridoma technology, immunizing mice with Nurse-like cells (NLCs) as a model of TAM. NLCs are the type of TAM encountered in chronic lymphocytic leukemia (CLL), are crucial for the survival of cancer B-CLL cells and can be easily generated in vitro. This approach resulted in generation of monoclonal antibody, called 6-25, that was further characterized to specifically bind to TAM of different human malignancies by recognizing sideroflexin-3 (SFXN3) protein on their surface. Both 6-25 antibody and its target have been patented (WO2018/020000 and WO2018/19990). SFXN3 represented a very interesting and unique target as it is a poorly defined, ubiquitous inner-membrane mitochondrial protein, that for an unknown reason was detected on the subpopulation of TAM.

The overall aim of this thesis was to further characterize 1) the specificity and therapeutic potential of 6-25 antibody toward TAM in vitro and 2) study the biology of 6-25 target (SFXN3), a potential new marker of TAM. These results will further support project aiming to use 6-25 antibody to target TAM in human malignancies, in order to improve the outcome of anticancer therapies.

2 Material and methods

Cell culture in vitro

Both primary cells after isolation and cell lines cultured in the complete medium containing: RPMI 1640 with GlutaMAX (Gibco, France) supplemented with 10% of FBS (Life Technologies, France) and 100 µg/ml of penicillin/streptomycin (Sigma-Aldrich, France). Only in the case of HEL92, THP-1, HEP-G2 and HEK-T cells, DMEM with GlutaMAX (Gibco, France) was used instead. All cultures were performed in Tissue Culture Treated Plates (Corning, USA). Cells were kept in 37°C, at the atmosphere of 5% CO2. Purity and viability of the cells after isolation were evaluated by flow cytometry, following staining with anti-CD5/CD19 antibodies and Annexin-V/7-AAD respectively. For each experiment both values exceeded 95% (data not shown).

Normal human PBMC

Normal human PBMC were isolated from healthy donor buffy coats, using density-gradient centrifugation on Ficoll-PaqueTM PLUS (GE Healthcare, Sweden). Whole PBMC were used for further in vitro culture or for subsequent isolation of CD14+ monocytes.

PBMC for toxicity assay with the 6-25 mAb, were used freshly after isolation. Cells were cultured at 1 × 10 6 c/mL, in 0.5 mL of complete medium, per well of 24-well plate.

Isolation of CD14 + monocytes

Isolation of CD14 + monocytes from normal human PBMC was performed using CD14 MicroBeads positive selection kit and MS/LS columns (Miltenyi Biotec, France), according to the adjusted manufacturer's protocol. In the process of optimization, the amount of positive selection beads was reduced 6-times leading to: retention of satisfactory cell yield, reduced monocyte clumping and improved cell purity.

Following the isolation, cell purity was measured by flow cytometry following staining with α-CD14 antibody. Isolated monocytes were used right away for subsequent generation of monocyte-derived macrophages (MDM) or heterologous NLC.

For experiments with RNP (ribonucleoprotein) CRISPR Cas9 gene knock-out, monocytes were also isolated with RosetteSep negative selection kit (STEM cell, France) according to the manufacturer's protocol.

Monocyte-derived macrophages (MDM) differentiation

In order to study expression pattern of the 6-25 target, multiple protocols of generation of MDM were tested. As a result of optimization a following protocol was chosen for further experiments -monocytes after isolation were seeded in a 12-well plate, at 0.5 × 10 6 c/mL in 1.5mL of complete medium..

To obtain M1 or M2-like macrophages, monocytes were activated with 50 ng/mL of GM-CSF (research grade) or 25ng/mL CSF-1 (premium grade) respectively. In both cases cytokines were administrated at day 0 and again on day 3. On day 6, half of the culture medium was replaced and cells were supplemented with 10 ng/mL of IFN-γ to generate M1 macrophages or 25 ng/mL of IL-4 and IL-13 to generate M2 macrophages (Illustration 5). In case of M1 MDM, 18h later, cells were treated with 10ng/mL of LPS.

In chapter 3.3.1 and 3.3.2, IL-34 was used at 50 ng/mL with the same schedule as CSF-1 and GM-CSF. In case of TNF treatment, the cytokine was added at 10ng/mL at day 0, 3 or 6 (according to descriptions in specific chapters). All cytokines were bought at Miltenyi, and after resuspension, aliquoted at 0.1 mg/mL and stored in -20°C.

NLC and MDM detachment protocol

Non-enzymatic detachment was performed using Versene solution (0.48 mM EDTA in PBS, Gibco, France).

In case of NLCs, non-adherent cells were first removed by gentle pippeting. Then remaining cells were washed twice with cold PBS, covered with Versene solution and incubated at 4°C for 20-40 min (depending on the degree of cell rounding). Subsequently FBS was added as a protectant, and cells were gently scrapped with 2-position, 16 cm cell scraper (Sarstedt, USA).

Enzymatic detachment was performed using Accutase® Cell Detachment Solution (Ozyme, Biolegend).

Briefly, cells were washed twice with PBS at RT, covered with pre-warmed Accutase solution and placed on orbital rocker, in cell culture incubator (37°C, 5% CO2) for 15-20 min. Afterwards FBS was added, and if needed cells were gently scrapped (analogically as in non-enzymatic procedure).

For both procedures cell rounding and detachment were controlled with bright-field microscopy (BFM).

Following cell detachment, cells were centrifuged (200g, 5min, RT), counted and re-suspended in appropriate buffer for subsequent assay.

Note: non-enzymatic procedure was used in chapter 3.1, 4.1, 6.2 and 6.3. Enzymatic procedure was used for remaining experiments as it was characterized with better cell recovery. As both procedure can affect cell phenotype, only one technique was used in given set of experiments.

Cell lines

Various cell lines used for screening of the 6-25 target expression, including: MV4-11, THP-1, HEP-G2, HMC-1, HEL92 were purchased previously from the ATCC collection. MOLM-14, OCI-AML-3, U-373 and U-937 cells were a kind gift of Dr. Thomas Farge (Team METAML, CRCT, France). HEK-T cells used for overexpression of the FOLR2 gene, were a kind gift of Pauline Herviou (Team RNAreg, CRCT, France). All cell lines were split every 2-3 days, until reaching passage 20-25, before returning for fresh frozen vial. In case of adherent cultures, cells were treated with trypsin for 5-10 min in 37°C, followed by inactivation of the enzyme with FBS.

Cryopreservation of cells

For cryopreservation procedures: cell lines, CLL PBMC for isolation of CLL cells and purified CLL cells were re-suspended in cold, freshly prepared FBS containing 10% of DMSO. CLL PBMC for whole PBMC culture and isolated human CD14+ cells were suspended in cold Cryostor CM10 medium (STEMCELL Technologies, France). 1-1.5mL aliquots were placed in Cryotubes, transferred to a cryofreezing container (Mr. Frosty, Nalgene) and placed in a -80 °C freezer (short term) or in liquid nitrogen (long term). Note: only Cryostor CM10 medium allowed to obtain viable monocytes after thawing procedure. Nevertheless, NLC from cryopreserved CLL PBMC showed reduced yield and modified phenotype, hence their use was limited to the necessary minimum.

Flow cytometry

List of antibodies used for phenotyping of normal human PBMC, CLL and myeloid cells.

Basic cell staining

Collected macrophages were re-suspended in flow cytometry staining buffer (2% FBS, 1mM EDTA in PBS) containing 2.5 µg/mL of Human BD Fc Block™ (BD Pharminge, France) and incubated for 15 min at 4°C.

Subsequently cells were stained with antibodies at saturating concentrations, for 20 min at 4°C. After washing, cells were re-suspended in PBS, containing 0.8 µg/mL 7-AAD (Sony, USA) and immediately analyzed by flow cytometry.

For CLL cell viability analysis, flow cytometry staining buffer contained no EDTA. After Fc Block incubation, cells were stained with 1 µg/ml of CD5 and CD19 antibodies for 20 min at 4°C. Next cells were washed in Annexin-V buffer and stained with Annexin-V-FITC according to manufacturer protocol. 5 minutes before the analysis cells were re-suspended in Annexin-V buffer containing 0.8 µg/mL 7-AAD.

6-25 staining

Indirect

Data acquisition and analysis

All presented results were acquired on an LSRII flow cytometer (BD Biosciences, France). Obtained data was analyzed by Flow Logic 700.2A (Inivai Technologies, Australia), further calculations were performed in Graph Pad Prism 9.1.2 or Microsoft Excel 2016.

Statistical analysis

All statistical analyses were performed using Graph Pad Prism 9.1.2.

Western blot analysis

Cell for preparation of lysates, were washed ×2 in PBS and lysed with RIPA buffer supplemented with protease inhibitors (Roche), for 15 minutes at 4°C. Cell lysates were centrifuged at 10000g for 15 min at 4°C. Supernatants were collected and protein concentration was evaluated with BCA assay. Then appropriate amount of protein was mixed with Laemmli Sample Buffer (Bio-Rad #161-0747: 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 1% LDS, 0.005% Bromophenol Blue) and heated at 60°C for 15 min. Samples were then resolved by SDS-PAGE, during ~2h at 120V, using in-house prepared 4/12% gel. After separation, proteins were transferred to Nitrocellulose membrane using wet transfer procedure (400 mA, 2h, 4°C).

Afterwards, membrane was incubated in blocking buffer (5% milk, in TBS-T) for 1h at RT. Then membrane was incubated with primary antibody, O/N at 4°C, followed by ×4 washing with TBS-T. Next, membrane was incubated with appropriate secondary antibody conjugated with HRP, for 1-2h at RT. After ×4 washing in TBS-T, followed by ×1 wash in TBS, membrane was covered in ECL™ Western Blotting Detection Reagents (Amersham TM , GE Healthcare, Sweden) reagent for 5 minutes, and chemiluminescence was detected by ChemiDoc Imaging System (Biorad, Germany).

Ribonucleoprotein-mediated CRISPR-Cas9 gRNA delivery to primary myeloid cells

In order to obtain gene KO in primary monocytes or MDM we used Ribonucleoprotein (RNP)-mediated CRISPR-Cas9 gRNA delivery. To target specific gene of interest (SFXN3, FOLR2) we used predesigned crRNA sequences https://eu.idtdna.com/site/order/designtool/index/CRISPR_PREDESIGN, provided by Integrated DNA Technologies (IDT). For each gene we used three different crRNA targeting different coding region.

Before each experiment a freshly isolated human monocytes were activated with 25 ng/mL of CSF-1 and cultured in complete medium without antibiotic. In case of experiment on monocytes, cells were used 16h after isolation. For mature MDM, cells were allowed to grow for 5-6 days in medium with CSF-1. At the day of electroporation, RNP complexes were prepared, according to protocol provided by IDT and Thermo Fisher. Briefly, crRNA and tracrRNA were reconstituted in IDTE buffer (IDT, USA). Then crRNA:tracrRNA duplexes were mixed in equimolar concentrations, followed by heating at 95°C for 5 min. Generated duplexes (gRNA) were used freshly, or were stored at -20°C for up to 6 months. Then each gRNA was mixed with Cas9 protein at a 1:1.2 molar ratio and incubated at 37°C for 15-20 min. Afterward obtained RNP complex was moved to RT. In the meantime, cells for electroporation were detached, washed with PBS, counted by haemocytometer and resuspended in buffer T (monocytes) or buffer R (mature MDM) at desired quantity. Usually from 1-5 × 10

RNAseq analysis

RNAseq analysis was performed to detect differentially expressed genes in CSF-1 activated MDM, upon a treatment with TNF (that was previously shown to abrogate 6-25 target expression, as measured by FC).

RNA isolation and quality control

Total RNA from CSF-1 activated MDM (5 control and 5 TNF treated groups) at day 8 of culture were isolated using Direct-zol TM RNA MiniPrep (Zymo Research, France), according to manufacturer instruction and stored in -80°C until further processing. Before sequencing, quality control of RNA was performed with

Fragment analyzer (Agilent) and concentration of the RNA was evaluated using Ribogreen.

RNA sequencing

Total RNA after quality control was subjected to: purification of mRNA, temperature fragmentation, generation of cDNA by reverse transcription, adapter ligation and indexing and finally amplification. Next RNA library was sequenced using NextSeq 550 Illumina system, with 2 × 75bp paired-end reads and 25M reads per sample.

Differential gene expression (DGE) analysis

Raw data after sequencing was subjected to normalization and batch corrections with ComBat algorithm (sva R package). Generated datasets were then collapsed using Fred's Softwares algorithm. A pathwaycentric approach using SES was then applied based on four curated databases from Fred's Softwares: KEGG, Reactome (reactome_2018803), Gene OntologyCellular Component (C5_CC), and MSigDB

Hallmarks (H. Fred's Software source codes and databases are available at: https://sites. google.com/site/fredsoftwares/products. A gene-centric approach was applied to identify DEGs using limma R package with a p-value of 0.05.

IP-MS validation of 6-25 target

To re-identify protein recognized by 6-25 mAb, IP-MS approach was applied.

Immunoprecipitation

For IP, CSF-1 activated MDM at day 8 of the culture were taken. ~ 15 × 10 6 cells per sample were used.

Expression of 6-25 target was confirmed each time, using FC. At day 8 of culture, cells were washed with PBS and subsequently lysed with RIPA buffer supplemented with Benzonase (2mM) and protease inhibitors (Roche), for 15 minutes at 4°C. Cell lysates were centrifuged at 10000g for 15 min at 4°C.

Supernatants were collected and protein concentration was evaluated with BCA assay. 1,5mg of supernatant with concentration of 1mg/mL was mixed with 10μg of 6-25 antibody (6-25 condition) or isotype control antibody (IC condition) and incubated O/N at 4°C on the rocker. Next day samples were mixed with 0.5 mg of Pierce Protein A Magnetic Beads and incubated further for 4 h at 4°C on the rocker unit. Subsequently magnetic beads were washed 5 times with RIPA buffer (with 0,25x detergent content), re-suspended in 30μl of ×4 Laemmli Sample Buffer (Bio-Rad #161-0747: 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 1% LDS, 0.005% Bromophenol Blue), incubated for 30 min in 37°C and subsequently frozen in -20°C before further processing.

Protein digestion and MS analysis

For mass spectrometry analysis, samples immunoprecipitated with 6-25 antibody (6-25 condition) or isotype control antibody (IC condition), prepared in quadruple biological replicates for each condition, in 30µl of ×4 Laemmli buffer, were reduced with 1M DTT (to final conc. of 25 mM) during 30 min at 56°C under agitation, cooled to room temperature and alkylated with 1M iodoacetamide (to final conc. 60 mM) during 30 min in the dark. Each reduced/alkylated protein sample was then digested using the S-Trap™ Mini spin column protocol 225 . Briefly, equivalent volume of 10% SDS were added to each sample in order to reach 5% SDS final concentration. Undissolved matter was removed by centrifugation for 8 min at 13,000g. 12% aqueous phosphoric acid was added at 1:10 to the protein sample for a final concentration of ~1.2% phosphoric acid followed by seven volumes of S-Trap binding buffer (90% methanol, 100 mM TEAB, pH 7.1). After gentle mixing, the protein solution was loaded onto an S-Trap filter in several times, each separated by a 4,000 g centrifugation step until all the SDS lysate/S-Trap buffer has passed through the column. Afterwards, the captured proteins were washed six times with 400 µL S-Trap binding buffer.

Digestion was performed for 1h at 47°C and then over-night at 37°C by addition of 125 µL of trypsin (Sequencing Grade Modified Trypsin, Promega) at 8ng/µL in 50 mM ammonium bicarbonate. The digested peptides were eluted by addition of 80 µL of 50 mM ammonium bicarbonate and 1 min centrifugation at 4,000 g followed by 80 µL of 0.2% formic acid and 4,000 g centrifugation (1 min) and finally 80 µL of 50% aqueous acetonitrile containing 0.2% formic acid and a last 1 min centrifugation step at 4,000 g. The pooled eluates were dried down, resuspended with 17µl of 0.05% trifluoroacetic acid (TFA) in 2% acetonitrile (ACN) and sonicated for 10 min before analyzing by online nanoLC using an UltiMate® 3000 RSLCnano LC system (ThermoScientific, Dionex) coupled to an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Scientific, Bremen, Germany) operating in positive mode. Five microliters of each sample were loaded onto a 300 μm ID × 5mm PepMap C18 pre-column (Thermo Scientific, Dionex) at 20 µL/min in 2% ACN, 0.05% TFA. After 3 min of desalting, peptides were on-line separated on a 75 μm ID × 50 cm C18 column (inhouse packed with Reprosil C18-AQ Pur 3 μm resin, Dr. Maisch; Proxeon Biosystems, Odense, Denmark) equilibrated in 90% of buffer A (0.2% formic acid (FA)), with a gradient of 10 to 30% of buffer B (80% ACN, 0.2% FA) for 100 min then 30% to 45% for 20 min at a flow rate of 300 nl/min. The instrument was operated in data-dependent acquisition (DDA) mode using a top-speed approach (cycle time of 3 s). Survey scans MS were acquired in the Orbitrap over 350-1400 m/z with a resolution of 120,000 (at 200 m/z), an automatic gain control (AGC) target value of 4e5, and a maximum injection time of 60 ms. Most intense multiply charged ions (2+ to 6+) per survey scan were selected at 1.7 m/z with quadrupole and fragmented by Higher Energy Collisional Dissociation (HCD). The monoisotopic precursor selection was turned on, the intensity threshold for fragmentation was set to 25,000 and the normalized collision energy was set to 28%. The resulting fragments were analysed in the Orbitrap with a resolution of 30,000 (at 200 m/z), an automatic gain control (AGC) target value of 5e4, and a maximum injection time of 54 ms. Dynamic exclusion was used within 60 s with a 10 ppm tolerance, to prevent repetitive selection of the same peptide. For internal calibration the 445.120025 ion was used as lock mass.

MS-based protein identification and label-free quantification

Acquired MS and MS/MS data as raw MS files were converted to the mzDB format 226 using the pwiz-mzdb converter (version 0.9.10, https://github.com/mzdb/pwiz-mzdb) executed with its default parameters.

Generated mzDB files were processed with the mzdb-access library (version 0. For label-free relative quantification across samples, raw MS signal extraction of identified peptides was performed using Proline. The cross-assignment of MS/MS information between runs was enabled (it allows to assign peptide sequences to detected but non-identified features). Each protein intensity was based on the sum of unique peptide intensities and was normalized across all samples by the median intensity. To eliminate false-positive hits from quantitation of low intensity signals, following filters are applied on proteins: the minimum number of values identified by MS/MS (in whole experiment) was set to 2 and the minimum percentage of defined values (in one condition) was set to 50%. After independently replacing missing values for each run with the Gaussian method (random drawing around the median of the intensity values for each protein in a same condition), data were log2-transformed. For pairwise comparison, an unpaired two-tailed Student's t-test was performed and proteins were considered significantly enriched when their absolute log2-transformed fold change was higher than 1 and their pvalue lower than 0.05. Volcano plots were drawn to visualize significant protein abundance variations between the two compared conditions. They represent -log10 (p-value) according to the log2 ratio.

3 Study I) specificity of the 6-25 antibody

The 6-25 antibody was developed with the purpose to selectively target pro-tumoral TAM. While the initial experiments confirmed the specificity of antibody toward TAM, not much was known about potential expression of the 6-25 target -SFXN3, on the other cells types. Because SFXN3 is a ubiquitous inner, membrane mitochondrial protein, that for an unknown reason was detected on the surface of subtype of macrophages, we had to use unsupervised approach in further study of the 6-25 mAb specificity. We decided to confirm the previous results on specificity of the antibody and further complement them with analysis of other cell populations present in the blood, including neutrophils or activated version of T-cells.

Additionally, we optimized protocol for monocyte-derived macrophage (MDM) culture, to further study expression pattern of the 6-25 target. Finally, we continued the screening of the various cell line with the 6-25 mAb. Apart from giving us more information about the specificity of the 6-25 mAb, the cell line with high expression of the 6-25 target, could serve as an important tool to study the role of the 6-25 target, especially as at that time we were unable to obtain SFXN3 KO in primary NLC or MDM.

Specificity of the 6-25 antibody toward primary blood cells

Initial studies on the 6-25 antibody, showed that it specifically binds to NLC generated in vitro, without targeting CLL B-cells (Fig. 1A) and normal human PBMC (Fig. 1B), besides slight affinity to monocytes.

Additionally, staining with the 6-25 antibody was tested on various cell lines (Fig. 1C), showing no specificity, with exception of weak staining on the MV4-11 cell line. Finally, staining of sections from human tumors with 6-25 antibody, suggested that the antibody can also recognize TAM from different human malignancies (Fig. 1D).

Figure 1. Initial evaluation of 6-25 mAb specificity. A) flow cytometry analysis of NLCs (CD163 + , CD206 + ) and CLL cell (CD5 + , CD19 + ). B) flow cytometry analysis of normal human PBMC: T (CD4 + and CD8 + ) and B (CD19 + ) lymphocytes, NK cells (CD56 + ) and monocytes (CD14 + ); C) and various human cell lines. For histogram plot red represents 6-25 and blue isotype signal respectively. D) Immunohistochemistry of tumour biopsies. Hematoxylin -blue staining; 6-25 mAb -brown staining.

In this part of the project we wanted to further study the specificity of the antibody toward both primary cells and established in vitro cell lines.

First, we have tested staining with the 6-25 mAb on monocytes from CLL patients (Fig. 2A and2B).

Depending on the patient (Fig. 2B) we detected a lack of or dim signal with the antibody, similarly to staining on normal human monocytes (Fig. 1B). Additionally, we performed the staining on normal human neutrophils (Fig. 2C), detecting no signal from the antibody. To be sure that the 6-25 target is not expressed following cell activation (as in the case of monocytes, that upregulate expression of the 6-25 target while differentiating into TAM), we tested staining with 6-25 antibody on primary  T cell lines. These cells were obtained by activation with phosphoantigen and IL-2 of naïve  T lymphocytes, isolated from normal human PBMC. No staining was detected with the 6-25 mAb on these activated  T cells (Fig. 2D).

Figure 2 The 6-25 mAb FC analysis of primary human cells. A) FC analysis of monocytes from CLL PBMC, gating strategy based on CD14 expression (red color on FSC/SSC dotplot depict monocytes after backgating on CD14 + ). Exemplary dotplots representing CD163 and 6-25 staining on CD14+ monocytes. B) FC analysis of 6-25 mAb signal of monocytes from PBMC of three different patients (P1-P3), histogram plots represents 6-25 signal on CD14 + gated cells. C) representative FC plots of normal human

neutrophils gated based on SSC/FSC difference (D) primary  T cell line.

3.1.1 Expression of the target on autologous NLC, NLC from patient after relapse and heterologous NLC.

NLC represented the main model to study 6-25 mAb in vitro. During our study, we routinely tested the 6-25 staining on NLC derived from various patient samples. By that, we wanted to better characterize consistency of the 6-25 target expression on these cells, also in relation to other myeloid cell markers. In order to do that, we cultured the CLL PBMC for 14 days and subsequently analyzed the phenotype of the NLCs by flow cytometry (Fig. 3).

For the most part, NLCs gated based on SSC-FSC showed high and uniform expression of 6-25 target (Fig. 3A). It was consistent with upregulation of typical M2 markers of NLC, including: CD163, CD206 and CD14. However, in some cases more heterogeneous, dim or even lack of expression of the 6-25 target of NLC was observed (Fig. 3B). In some cases, gating of NLC based on CD163 and CD206 markers, allowed to distinguish 3 main subpopulations of cells with varying expression of 6-25 target, that was positively correlated with CD163 and CD206 markers (Fig. 3C). Heterogeneity of the 6-25 target expression was further confirmed by confocal microscopy analysis with 6-25 and CD163 co-staining (Fig. 3D). In most cases co-expression of CD163 and 6-25 target on NLCs was observed (Fig. 3D, left picture), but sporadically presence of CD163 + and 6-25 -was detected (Fig. 3D, right picture). These results suggested that the 6-25 target can be specific to only subpopulation of NLCs and is preferentially co-expressed on more M2-like cells.

CLL PBMC samples for the project were generally taken from untreated patients. Hence, we wanted to confirm that NLC generated from PBMC from patients after relapse can also be recognized by 6-25 mAb.

This would be especially important in case of evaluation of therapeutic potential of 6-25 mAb. NLC generated from patient, after relapse from classical fludarabine, cyclophosphamide and rituximab (FCR) therapy, were prepared in the same fashion as before and analyzed by FC (Fig. 3E). Flow cytometry confirmed expression of the 6-25 target on NLCs from this patient.

NLCs can be also generated by co-culture of HD monocytes with isolated CLL cells (heterologous culture).

Previous results of the group, confirmed that heterologous NLCs are characterized by similar expression of the surface markers and ability to rescue CLL cells from spontaneous apoptosis in vitro as NLCs from autologous culture. So far they were not analyzed for expression of the 6-25 target though. Exemplary flow cytometry analysis of heterologous NLC (Fig. 3F), revealed that these cells also show expression of the 6-25 target.

Figure 3. Expression of 6-25 target on NLCs. A) FC analysis: NLCs were gated based on SSC-A and FSC-A parameters and further classified based on expression of CD14, CD163, CD206. B) FC analysis of 6-25 mAb signal on NLCs generated from PBMC of four different CLL patients (P1-P4), histogram plots represents cells gated on CD163 and CD206. C) Detailed analysis of the 6-25 signal in different subpopulation of NLCs. D) Confocal microscopy analysis of NLC: green -CD163, red -6-25, blue -DAPI. FC analysis of 6-25 staining on NLC generated from PBMC of patient after relapse and F) NLCs from heterologous co-culture of normal human monocytes with CLL cells.

In conclusion, autologous and heterologous NLCs generated in vitro, showed high expression of the 6-25 target, which was consistent with the presence of typical M2 markers, like CD163 and CD206. The difference in the expression of the 6-25 target and its potential connection with the functionality on NLC is described in the "chapter 4, study II" of this thesis.

Screening of AML cell line with the 6-25 antibody

In our work we were interested in finding the cell lines that would express the 6-25 target on their surface, which we could further use as a model to study this molecule in vitro. As previous results showed a dim staining with the 6-25 mAb on MV4-11 cell line (Fig. 1C), we continued to analyze other myeloid cell lines, in particular of acute myeloid leukemia (AML) origin. We included: U-937, THP-1, OCI-AML-13, MOLM-14, U-373 cell lines in the screening, and used MV4-11 cells as a positive control. Results presented on the fig. 4, show no targeting of OCI-AML-3 and U-373 cell lines with the 6-25 antibody. On the other hand, in case of U-937, MOLM-14 and THP-1 cell lines we detected a dim staining with the antibody, that in case of U-937 was comparable with MV4-11. where target cell express low level of antigen, including anti-CD20 targeting in CLL 227,228 . Moreover, AML represents a disease with poor overall prognosis and requires finding of a new markers that could improve therapeutic outcome in the future 229 . To pursue this path, it would be also important to evaluate 6-25 mAb staining of primary samples from AML patient.

Modulation of the 6-25 target in human monocyte-derived macrophages (MDM)

After confirming specificity of 6-25 antibody toward TAM, we wanted to study how to induce and modulate expression of 6-25 target in vitro. However, CLL PBMC model with its relative complexity was not suitable for the task. Moreover, working with the primary cells from CLL patients holds some disadvantages, including high variability of 6-25 target expression, overall low yield of NLCs and time-consuming experiments. Since we did not find a cell line with 6-25 expression comparable to NLCs that could also mimic functionality of these cells, we decided to develop and optimize primary human monocyte-derived macrophage (MDM) model. To obtain maximum information we tested various protocols of MDM differentiation and polarization. We evaluated polarization of macrophages, analyzing various surface markers by flow cytometry and comparison of morphology of the cells by bright-field microscopy.

Expression of the 6-25 target by MDM -effect of CSF-1, IL-34 and GM-CSF

As a starting point of the MDM protocol optimization, we decided to evaluate the effect of CSF-1 and GM-CSF, two of the most important cytokines inducing monocyte differentiation to macrophages 230 . As in the literature there is a lot of confusion involving the role of CSF-1 and GM-CSF itself on the polarization of macrophages [231][232][233] , we decided to optimize protocol, that would allow us to obtain M1 and M2 MDM.

First we checked the influence of CSF-1 and GM-CSF alone, on the morphology and phenotype of macrophages. In order to do that we isolated monocytes from HD PBMC and activated them with CSF-1 or GM-CSF. After 8 days of culture we analyzed the cell by flow cytometry (Fig. 5A).

Analysis of MDM obtained from CSF-1 activated monocytes showed high expression of M2 markers -CD163, CD206, but also CD14, CD16, CD64 and HLA-DR. In comparison, GM-CSF stimulated cells showed no expression of CD163, decreased level of CD206, CD14, CD16, but increased expression of CD86 (typical M1 marker). Interestingly, CSF-1, but not GM-CSF led to upregulation of the 6-25 target. Since CSF-1 action stimulated expression of the 6-25 target, we wanted to see, if IL-34 -second ligand of CSF1R (CD115)

would have similar effect. We activated monocytes with IL-34 in analogical way to CSF-1 and GM-CSF and analyzed the cells by flow cytometry. Flow cytometry analysis revealed that, IL-34-activated cells were characterized with high expression of CD163, CD206, HLA-DR, and the 6-25 target, thus resembling CSF-1 activated MDM, but with higher expression of CD115, CD86 and decreased expression of CD16.

Figure 5. Influence of GM-CSF, CSF-1 and IL-34 on polarization state of MDM and expression of 6-25 target. A) FC analysis representing histogram overlays between isotype control (blue) and GM-CSF (red), CSF-1 (green), IL-34 (yellow) activated MDM. B) Bright-field microscopy picture of MDM at day 8 of the culture, magnification = ×400.

Further distinction between GM-CSF, CSF-1 and IL-34 on activation of the monocytes could be also observed in morphology of the cells (Fig. 5B), as GM-CSF activated cells showed more amoeboid shape, while CSF-1 activated cells were characterized by more elongated, spindeloid shape indicative of M2 macrophages 234,235 . IL-34 activation, also led to appearance of spindle-shape like cells, but with higher number of roundish cells that could suggest less mature phenotype 235 . Notion of less differentiated IL-34 activated cells is also supported by the flow cytometry, as these cells were characterized with smaller size and decreased granularity compared to cells stimulated with GM-CSF or CSF-1 (data not shown).

By comparing the effect of CSF-1, IL-34 and GM-CSF on the maturation of MDM we observed that indeed, GM-CSF activation resulted in macrophages with more M1-like phenotype, lacking expression of CD163, decreased expression of CD206 and increase in the level of CD86. On the other hand, CSF-1 and IL-34 activated cells showed more M2-like phenotype, with high expression of CD163, CD206 and CD14.

Additionally, activation with CSF-1 and IL-34 (as oppose to GM-CSF) led to comparable and high expression of the 6-25 target. These results suggest that CSF1R stimulation is necessary for upregulation of the target of the 6-25 antibody.

3.3.2 The 6-25 target is a specific marker of M2-like macrophages and is upregulated by

CSF1R activation

Since the 6-25 target seemed to be preferentially expressed on M2-like macrophages, we wanted to further explore this hypothesis. In order to do that, we performed similar experiment to described in In conclusion, GM-CSF activated cells seemed to acquire a stable M1-like polarization state, with limited response to secondary treatment. On the other hand, both CSF-1 and IL-34 have similar M2-promoting effect on macrophages activation (at least regarding the expression of surface myeloid markers). At the same time, they maintained cell plasticity, as seen by the changes in MDM phenotype upon administration of M1 and M2 agents.

Regarding the effect of various treatments on 6-25 target expression, we confirmed that activation of CSF1R by either CSF-1 or IL-34 is necessary to induce expression of this molecule. While M2-polarizing agent, IL-4 and IL-13 (but also IL-10 and Dexamethasone -data not shown) can lead to further upregulation of 6-25 target in case of CSF-1 activated cells, they fail to do so in case of IL-34 and GM-CSF activated macrophages. Additionally, we observed overall downregulation of 6-25 target expression upon the treatment of MDM with M1-polarizing agents -IFN-γ and LPS. All these results strongly suggest that the 6-25 target could be categorized as a M2 marker of macrophages.

The 6-25 target expression is abrogated by TNF

The classical protocols usually suggest IFN-γ and LPS to generate M1 macrophages. While IFN-γ role is also indicated in cancerogenesis 236 , the effect of LPS stimulation is more relevant in case of bacterial infection.

As we were more interested in macrophage polarization in the context of cancer, we decided to evaluate the effect of TNF (another known M1-polarizing agent) on macrophage polarization. TNF has a very prevalent and ambiguous role in oncogenesis [START_REF] Zhao | Inflammation and tumor progression: signaling pathways and targeted intervention[END_REF]237,238 , but it was also shown as an important factor that "counterbalances emergence of M2 Tumor Macrophages" 232 . To study the effect of TNF on expression of 6-25 target, but also overall polarization state of macrophages we first performed a test, where we again treated monocytes with CSF-1 secondary M1 or M2-polarizing agents, but additionally challenged the cells with TNF from day 0.

Flow cytometry analysis of the macrophages treated with TNF, revealed changes in the maturation of the cells and subsequent response to classical M1 and M2 polarizing agents (Fig. 7). TNF led to an overall decrease in the expression of CD16, CD163 and CD206. In case of IL-4 polarized cells, we observed more pronounced response to TNF, compared to naïve macrophages (MDM activated only with CSF-1), with strong downregulation of CD16, CD163, CD206, CD14, CD64 but also partial upregulation of CD86. For IFN-γ stimulated cells, additional treatment with TNF led to further decrease of CD16 and CD206. Most strikingly, though, TNF caused also a complete abrogation of the 6-25 target expression in all of the tested conditions.

Figure 7. Effect of TNF on maturation and polarization of CSF-1 activated MDM, and expression of 6-25 target. FC results represented as overlay histograms for each measured marker. A) effect of TNF on M1-polarization of MDM, B) effect of TNF on M2-polarization of MDM. Legends for panels A and B are depicted on the figure.

Next we wanted to closer evaluate the effect of TNF on expression of the 6-25 target, also in cells initially activated with IL-34 or GM-CSF. Beside treatment at day 0, we also tested the effect of TNF when added from day 3. Combined results of 2 independent experiments performed with MDM from 4 different donors (Fig. 8), revealed that TNF abrogated the 6-25 target expression in both CSF-1 and IL-34 activated cells when added at day 0 and day 3. It also strongly downregulated expression of most analyzed markers, especially M2-specific: CD163, CD206 but also CD16. On the other hand, TNF treatment showed benign effect on the GM-CSF activated cells, with slight upregulation of HLA-DR, CD86 and downregulation of CD206 and CD64, suggesting further enforcement of M1 polarization.

To further systematize the effect of M1-polarizing agents on the 6-25 target expression we decided to compare the effect of IFN-γ, LPS and TNF on polarization of CSF-1 activated cells. In the previous experiment, IFN-γ and LPS were added late in the process of cell maturation -at day 6, when the 6-25 target was already highly present on the surface of the cells. So this time, we evaluated the effect of the Effect of LPS and TNF was less pronounced compared to IFN-γ, leading to downregulation of CD204, CD206

(beside LPS at day 0), HLA-DR (if added from day 3), CD86, CD16 (if added from day 0) and CD169 (if added from day 0). Additionally, LPS led to slight upregulation of CD206 (if added from day 0) and strong upregulation of CD169 (if added from day 3). While LPS, TNF and IFN-γ had various effects on the overall expression on both M2 and M1 macrophages markers, they all led to homogenous downregulation of the 6-25 target expression. This effect was stronger, the earlier the administration of one of the M1 agents (Fig. 9C).

In the end, we observed that LPS, TNF and IFN-γ lead to distinct M1 phenotypes of MDM, as in accordance with official guidelines of macrophages polarization 230 . We confirmed also that all three pro-inflammatory agents have similar effect on downregulation of 6-25 target.

Conclusions

Experiments performed in this chapter showed that the 6-25 target is driven by CSF1-R signaling and can be induced either by CSF-1 or IL-34. Expression of the 6-25 target can be further increased by M2-polarizing agents like IL-4, IL-13, IL-10 or Dexamethasone, but these agents alone do not induce its expression. Next, we have shown that the 6-25 target is a specific M2 marker of macrophages and its level is downregulated by M1-polarizing agents like LPS, TNF or IFN-γ.

We are aware that these analyses are not a definitive study of polarization state of human MDM, which would require additional analyses of: level of intracellular markers, including Arg1, iNOS, IDO; activity of transcriptional factors, like STAT1, STAT3 and STAT6; expression of specific cytokines 230 ; phagocytosis index 239 or ability of macrophages to suppress T cell responses 240 . Nevertheless, they allowed us to obtain a reliable model of the 6-25 target expression, and showed overall effect of most common polarization agents on the level of 6-25 target expression, also in relation to other myeloid markers.

4 Study II) The 6-25 target -a marker of protective NLC After we confirmed the specificity of the 6-25 antibody and explored expression pattern of the 6-25 target in MDM, we decided to turn back, to CLL PBMC model. As this model contains another layer of complexity -notably cancer cells, that can mimic interaction of malignant cells with NLC, we wanted to find a potential connection between the 6-25 target expression and functionality of NLCs. However, first we decided to get a closer look on the kinetic of the 6-25 target expression during maturation process of NLCs.

Kinetic of the 6-25 target expression in NLC

Since expression of the 6-25 target is highly upregulated during the differentiation of monocytes into NLCs, we wanted to evaluate the kinetic of this process. To do that, we analyzed the phenotype of monocytes by flow cytometry from the moment of CLL PBMC isolation until a late stage of the NLCs culture (day 14-20). During early stage of the culture we analyzed the phenotype daily, then every 2-3 days. Beside analysis of the 6-25 target expression, we measured expression of classical myeloid markers, including CD163 and CD206 (M2 phenotype) and CD11c, CD14 and CD16 (known to be highly expressed also on monocytes) to better follow the maturation of the cells. We further controlled the morphology of the culture by BF microscopy, to observe outgrowth of NLC.

The results of an exemplary analysis presented on the fig. 10 revealed that, during the first 24-48 h of the culture, there is no or a very dim expression of the 6-25 target on differentiating monocytes. Past the initial point, the level of the 6-25 target signal is gradually increasing, reaching maximum between day 9-15 of the culture (Fig. 10 B andC). The increase in the level of the 6-25 target in positively correlated with outgrowth of NLC as observed by BF microscopy (Fig. 10D) and negatively correlated with the MFI ratio of CD14, CD16 and CD11c. It was more difficult to correlate 6-25 target expression with CD163 and CD206.

In case of CD163, high fluctuation of the signal during NLCs maturation can be observed (this phenomenon was observed for all 8 experiments), which partially could be explained by potential shedding of the protein, which was reported as a regulatory mechanism for this receptor 214,241 . For CD206 marker, induction was observed after day 2 of the culture, reaching peak at day 3 and then from day 5 keeping stable level on the surface of NLCs. As NLC are known to support viability of CLL cells, we were also measuring changes in the viability and concentration of cancer cells during the culture, to further connect it with changes in the maturation of NLC. Analysis of CLL cell viability by flow cytometry (Annexin V, 7-AAD staining) (Fig. 11) showed, that during the first 3 days of the culture there is a decrease in the viability of the cells, followed by stabilization of this parameter until day 5. Then from day 5-8 viability of cells start to rise, oscillating around 90-95%

until the end of the culture. Measurement of the cell concentration by haemocytometer, revealed a continuous drop of the cell number until day 7-8 of the cultures, followed by stabilization of this parameter. Combining the results of NLC phenotype (Fig. 10) and CLL cells concentration and viability (Fig. 11), we noticed, that stabilization of CLL cell viability and concentration is tightly connected with maturation of NLC. We assumed that from around day 3-8 maturating macrophages are phagocyting dying cancer cells.

The intensified phagocytosis is most probably responsible for initial stabilization of cancer cells viability at day 3, while further increase of survival of cancer cells from day 5 is a resultant of both phagocytosis processes, but also anti-apoptotic action of NLC, that start to appear at that time. Based on both analyses we also concluded that the end of maturation of the NLC falls around day 8-10 of the culture, which is also associated with initial stabilization of the 6-25 target level. Results in this chapter suggest that 6-25 target is a marker of NLCs maturation, and its level might indicate their protective functions.

Connection of 6-25 target expression on NLC with viability of cancer B-CLL cells

As we continued to culture and analyze the CLL PBMC from different patients, we mostly observed NLC expressing a high level of the 6-25 target. In some rare instances however, we noticed that even though maturating monocytes were initially showing an increase in the 6-25 target expression, the level of this molecule started to drop again around day 4-7 of the culture (Fig. 12A). Interestingly, when viability of the CLL cells was measured at day 10 of the culture, a decrease in survival of the cancer cells was noticed in case, where NLC expressed low level of the 6-25 target (Fig. 12B). Additionally, there was no connection between percentage of monocytes at day 0, phenotype of NLC and viability of CLL cells (Fig. 12C). Previously we implied that phagocytosis is partially responsible for stabilization of CLL cell viability in vitro.

To support this hypothesis, we decided to take a closer look at the viability and cell concentration in the PBMC culture where NLC expressed low level of 6-25 target (Fig. 13). These results showed that the viability of the CLL cells decreases until day 3-5, followed by stabilization phase that last until the end of the culture. At the same time, cell concentration in this culture showed only minor decrease. Comparison of these results to analogical analysis from Fig. 11, show evidence on difference in functionality of NLC in cultures with high and low viability.

These results further support our hypothesis that expression of the 6-25 target might be connected with the protective functionality of NLCs. To confirm the consistency of this finding, we decided to further analyze phenotype of NLC and viability of CLL cells from different samples, focusing on later stages of the cultures (day 10-14). At the end, we gathered the results of PBMC cultures from 34 different CLL patients.

Next we divided the samples into two groups, based on viability of the CLL cells at day 10 of the culture:

"High CLL cell viability" -with CLL cells viability > 80% and "Low CLL cell viability" -with CLL cell viability <80% (Fig. 14A). Then we apposed the results of the 6-25 target MFI ratio on NLC from high vs low viability CLL PBMC cultures (Fig. 14B). Based on these analyses we observed a significant decrease in viability of CLL cells in PBMC cultures with NLC expressing low level of the 6-25 target. On the contrary, in the cultures with NLC expressing high expression of the 6-25 target, the viability of the CLL cells remained consistently high. These results further suggested a potential link between the expression of the 6-25 target, protective capabilities of NLC, and thus the viability of cancer cells. Beside analysis of the 6-25 target expression, we also characterized the level of other myeloid markers on the surface of these NLC. Afterward, we divided NLC into two groups: protective NLC (cells expressing high level of the 6-25 target) and non-protective NLC (cells expressing low level of the 6-25 target) (Fig. 15). The combined MFI from 8 and 6 independent experiment for protective vs non-protective NLC respectively, showed an upregulation of CD163, CD14, CD209, CD169, CD206 (to lesser extent) and downregulation of CD64, CD86 and CD71. Based on these results, protective NLCs land on the M2-, while non-protective on the M1-side of the polarization spectrum. Combining previous results, we concluded that 6-25 target is a marker of protective NLC, and that the M2polarization state of NLC is tightly connected with survival of cancer cells in these cultures.

4.3 Publication on Interplay between TNF and IL-10 on phenotype of CLL cells.

Taking into consideration previous results on the influence of cytokines on the polarization state of MDM, we decided to get insight into the effect of M1 and M2 cytokines on the polarization and functionality of NLCs. As we were still not ready to publish data on the 6-25 target, we decided to classify the NLCs based on the combined level of M2 and M1 markers.

Conclusions

Results presented in this paper shed light on the interplay between IL-10 and TNF on the functionality of NLC. Additionally, based on functionality assay of NLCs presented in the paper (cell grouping assay and phagocytosis assay), we confirmed a direct link between the protective role of NLCs and their M2 polarization state, thus confirming that indeed the 6-25 target is a specific marker of pro-tumoral NLC. At the same time, we still do not know if 6-25 have a direct role in pro-tumoral function of NLC, and what that role would be.

5 Study III) Determination of the 6-25 target 6-25 antibody was developed by hybridoma technology, using whole NLC for immunization procedure.

This protocol allowed to obtain an antibody, against conformational epitope 242 of potentially unique

antigen, but at the same time it created necessity for de novo identification of the target recognized by a given antibody. The initial identification of the 6-25 target was performed by combination of immunoprecipitation (IP) process combined with mass spectrometry (MS) analysis 155 . Briefly, the membrane fraction of NLC (that shown expression of the 6-25 target) were isolated and used for IP against agarose-protein A beads complexed with the 6-25 antibody or isotype control. Subsequently, the eluat from the IP was resolved by SDS-PAGE, protein bands were excised and analyzed by qualitative MS. List of proteins generated with the MS analysis was compared between the 6-25 and Isotype control group. In the process of selection, SFXN3 was chosen as a protein specifically detected in the 6-25 group. Results

were further validated by western blot (WB) with whole cell lysates from NLC and human recombinant SFXN3 protein fragment. Immunodetection was performed with the 6-25 antibody and commercial anti-SFXN3 antibody, showing in both cases the signal corresponding to MW of SFXN3 (~35 kDa). It must be mentioned however, that the 6-25 mAb was giving a much weaker signal compared to commercial anti-SFXN3 antibody, even though it was used in higher concentration of (10 µg/mL vs 1µg/mL). Also for the staining with recombinant SFXN3, it was possible to detect the signal only after loading at least 100 ng of pure protein.

Invalidation of SFXN3 as the target of the 6-25 antibody

During the time of this thesis, we intended to further validate SFXN3 as the target of the 6-25 antibody.

First, we performed another round of WB experiments, to confirm the previous findings, and test the 6-25

against the mouse version of the protein. It was important for us to know if the 6-25 antibody is specific toward the mouse variant of SFXN3, as in perspective we planned to test therapeutic potential of 6-25 mAb in vivo. Comparison of the protein sequence between mouse and human variants of SFXN3 showed 93.8% identity, so there was a good chance that antibody will recognize both versions. We performed immunodetection of previously tested fragment of human recombinant SFXN3 and full length recombinant mouse SFXN3, with the 6-25 mAb and commercial anti-SFXN3 antibody as a positive control (Fig. 16). WB analysis revealed that, as expected, commercial antibodies recognized both human and mouse SFXN3. On the other hand, with the 6-25 antibody a dim band was detected only in case of human fragment of SFXN3. Additionally, with the 6-25 antibody signal was observed only using an extremely high amount of recombinant protein -100 ng, and concentration of the antibody -10 µg/mL. Next we performed the WB analyses on the lysates from M1-and M2-MDM, but failed to detect any signal with the 6-25 antibody (results not shown).

Based on the WB analyses, we concluded that the 6-25 antibody is specific to the antigen in native conformation, which is lost during downstream processing of the samples. In the end, we had to consider following limitations while further validating SFXN3 as the target of the antibody: 1) at the time we were unable to obtain SFXN3 KO in NLC or primary MDM; 2) the 6-25 works reliably in techniques not involving denaturation, like extracellular staining by flow cytometry or immunofluorescence; 3) commercial anti-SFXN3 antibodies are suitable for WB or immunohistochemistry, involving antigen denaturation (most probably they recognize linear antigen) and they do not work in flow cytometry; 4) SFXN3 gene is expressed ubiquitously and its expression do not correlate with the 6-25 staining on the surface of the cells; 5) SFXN3 is a membrane protein, and its recombinant versions is likely lacking the native conformation required to study the interaction with the 6-25 by techniques like, surface plasmon resonance (e.g. Biacore).

To overcome these incompatibilities, we decided to try methods that could bypass limitations of both the 6-25 antibody and commercial antibodies against SFXN3, including: M2-MDM were cultured on glass chamber slides. 8 days after plating cells were stained in the native condition (for plasma membrane staining) or following fixation and permeabilization (intracellular staining), and analyzed using confocal microscope (Fig. 17). Detection of plasma membrane signal with the 6-25 mAb was confirmed with CD163 co-staining (Fig. 17A).

Interestingly, results showed only partial co-localization (indicated with orange patches). No signal from the 6-25 mAb detected after intracellular staining (Fig. 17B). Analysis in native conditions showed specific targeting of plasma membrane with the 6-25 mAb, as proved with α-CD163 co-staining. Results on Fig. 17A show M2-MDM co-stained with the 6-25 antibody and plasma membrane control -CD163. As expected, we detected the 6-25 staining localized to the surface of the cells as confirmed by CD163 signal. On the Fig. 17B we attempt to stain mitochondria with 6-25, however we failed to detect specific intracellular signal there. As intracellular staining of mitochondria requires the sample fixation and permeabilization, we concluded that it might have hindered potential detection with the 6-25 antibody.

In the next set of experiments, 6-25 mAb was replaced with commercial anti-SFXN3 antibody.

Results on the Fig. 18A andC, show intracellular signal with commercial antibody, that corresponds to mitochondrial staining. CD163 control signal, proved that there is no plasma membrane staining with anti-SFXN3 antibody (Fig. 18C). We also failed to detect any PM signal with anti-SFXN3 antibody when we stained the cells in native conditions (Fig. 18B). We knew that the best method to resolve these doubts, would be generation of gene KO in these cells. In order to do that, we incorporated we incorporated CRISPR-Cas9 ribonucleoprotein (RNP) electroporation technique, that finally allowed us to generate gene KO in primary myeloid cells.

To generate KO of SFXN3 in primary monocytes, three sgRNA targeting the gene of interest were combined for subsequent generation of RNP with Cas9. To increase the chances of the experiment, monocytes after negative and positive selection as well as CSF-1 activated MDM (at day 6 after plating) were used for electroporation. 5 days after the procedure, efficacy of the KO was evaluated by flow cytometry analysis of 6-25 staining, and by WB analysis of SFXN3 protein expression with commercial antibody (Fig. 19). The disqualification of SFXN3 as a target of 6-25 antibody required development of an alternative process aiming to identify a new target of the antibody.

Identification of a new target recognized by the 6-25 antibody

To evade possibility of another misidentification of 6-25 target, we decided to thoroughly retrace the initial analyses.

To re-evaluate the 6-25 target we have incorporated approaches:

1. indirect -RNA-seq based identification of differentially expressed genes (DEG) between control M2-MDM and cells treated with TNF (which abrogates expression of the 6-25 target), 2. direct -involving immunoprecipitation of M2-MDM lysates with 6-25 antibody combined with quantitative mass spectrometry.

Then we planned to combine results of both analyses and screen the list of potential target recognized by 6-25 antibody in CRISPR-Cas9 RNP-mediated KO of primary MDM.

Differential gene expression (DGE) analysis of MDM after treatment with TNF

According to optimization of MDM protocol (Study I), we knew that TNF abrogates expression of 6-25 target in CSF-1 activated cells. We decided to use this interaction and perform DEG analysis by RNAseq.

Briefly, monocytes isolated from HD were activated with CSF-1, or combination of CSF-1 and TNF. After 8 days of culture, expression of 6-25 target was tested by FC (Fig. 20A). After confirming downregulation of 6-25 target expression on TNF treated cells, RNA from the cells was extracted for subsequent RNAseq analysis. In order to perform quantitative analysis, RNA was extracted from MDM generated from 4 independent donors. Subsequently samples were analyzed by RNAseq, and data after processing ("MDM dataset") was used to perform DGE analysis. As a results of DEG analysis, list of significantly (p < 0.001) downregulated genes in TNF condition was extracted (list called D(MDM)). D(MDM) list, was then narrowed down to genes coding plasma membrane proteins (PM (DMD)). To increase the sensitivity of the analysis, analogous DGE analysis was performed on public data set (GSE95588) containing RNAseq results of monocytes activated for 24h with CSF-1 and another 24h with TNF. In the process additional two list of significantly downregulated genes in TNF conditions were generated -"D(Mo)" and "PM(Mo)". Finally, relation between all 4 gene lists were visualized with Venn diagram (Fig. 20B).

Comparison of 4 DGE lists, showed 199 genes coding plasma membrane proteins, that were commonly downregulated by the action of TNF in both datasets. One of this 199 gene was most probably responsible for expression of 6-25 target. We further defined the list based on the literature and experimental data on expression of various plasma membrane proteins.

Immunoprecipitation of M2 MDM membrane proteins with 6-25 antibody combined with quantitative mass spectrometry

After invalidation of the target, we come back to the results of initial IP-MS analysis that typed SFXN3 as protein recognized by the 6-25 antibody. We re-analyzed the list of remaining proteins and confronted them with the list of genes from RNA seq analysis (chapter 5.2.1) and data available in the literature. We did not find any potential candidates though. In this situation we decided perform a new IP-MS analysis for direct identification of the 6-25 target. First however, we wanted to confirm that 6-25 mAb is in fact recognizing protein antigen 243 . In order to do that, we generated the lysates from M2-MDM (after CSF-1 activation and IL-4 polarization) and subjected them to time-course of protease treatment. After inactivation of the proteases, we performed the dot blot analysis with the 6-25 detection (Fig. 21A). Dot blot analysis revealed that a signal of 6-25 mAb in untreated samples, that is partially diminished with Trypsin and completely with Proteinase K. Analogical results were observed with anti-CD206 antibody, used as a positive control in the assay. These results confirmed protein nature of the 6-25 target. We also performed second analysis to further support this results, but this time we subjected intact M2-MDM to protease treatment schedule and analyzed the cells by flow cytometry (Fig. 21B). From that analysis we observed that Trypsin had a little effect on staining with 6-25 antibody, on the other hand Proteinase K led to strong decrease of the staining. We believe that weaker effect of proteases in flow cytometry analysis, may be caused be decreased availability of the proteins in intact plasma membrane compared to the lysate. Nevertheless, both analyses confirmed that the 6-25 is recognizing protein antigen. Additionally, dot blot analyses showed that optimized lysis protocol, allowed to solubilize the target protein and preserve the binding with the 6-25 antibody, which was a crucial information for subsequent IP procedure.

Next, we decided to optimize the protocol of immunoprecipitation and downstream processing of the samples for MS. We used mouse 6-25 and matching isotype control for the procedure. For IP the same lysis protocol was used, as in case of dot blot analyses. After incubation of 6-25 or IC with the lysates antibodies were captured by magnetic Protein A beads. After extensive washing, samples were eluted with Laemmli buffer without reducing agent. For the downstream processing of the samples for MS, S-Trap™ spin column digestion protocol was used, instead of digestion of excised bands from SDS-PAGE analysis (which would more likely lead to loss of target protein in the process). To perform quantitative MS, 4

biological replicates for both 6-25 and IC control groups were used. As the result of the MS analysis, the list of significantly enriched proteins from both groups was obtained and results were plotted in the Volcano plot (Fig. 22). By comparing the results of the three staining, we observed that α-FRβ antibody is following the same pattern as the 6-25 antibody, irrespectively of the treatment. On the other hand, α-CD36 antibody showed increased staining in GM-CSF-only activated MDM and no changes after TNF treatment. Hence, we chose FOLR2 to perform the KO experiments first.

KO of FORL2 in primary monocytes and macrophages and ultimate target disqualification

In the next step of validation of FOLR2, we performed RNP CRISPR Cas9 KO of the gene coding FOLR2 in primary monocytes, following analogical procedure as in case of SFXN3 gene. We isolated monocytes form HD, activated them with CSF-1 and the next day we performed RNP electroporation. This time we chose sgRNA against SFXN3 gene as a negative control of the test. 5 days after electroporation, we stained the cells with the 6-25 mAb or α-FRβ commercial antibody and analyzed the samples by flow cytometry (Fig. 24). Based on the flow cytometry analysis we observed abrogation of the staining with both 6-25 and commercial anti-FRβ antibody in FOLR2 KO cells, compared to SFXN3 KO control group (Fig. 24A).

Additionally, downregulation of 6-25 and FRβ signal between UT and control electroporation (sgSFXN3) conditions was observed. As electroporation (ELP) is an aggressive procedure, the morphology (Fig. 24B) and phenotype (Fig. 24C) changes between UT and ELP cells were compared. It was noticed that ELP (even with control sgRNA) cause increase in size and granularity of the cells, and leads to comparable changes between FOLR2 and SFXN3 targeting sgRNA in expression of CD206, CD163 and CD14 markers. The specific decrease in the staining of anti-FRβ antibody and the 6-25 mAb in FOLR2 KO cells strongly suggested that

FRβ is the protein recognized by the 6-25 mAb.

Overexpression of FOLR2 gene in HEK-T cells

To ultimately validate the FRβ as a target of the 6-25 antibody, FOLR2 was overexpressed in cell line with no expression of this gene. For this purpose, HEK-T cell line was chosen as these cells: do not express FOLR2 Similarly, no effect on cell concentration was observed between treatment with 6-25 mAbs and corresponding isotype controls (Fig. 26 C). On the other hand, it was problematic to precisely evaluate the effect of the 6-25 mAb toward survival of NLC, as these cells usually represent ~0.5-2% of the whole culture, and their complete detachment requires scrapping (even after incubation with Trypsin) that inseparably damages the cells. Hence, the evaluation was limited to confirmation of the presence of NLC by the bright-field microscopy (BFM), considering distinct morphology of these cells, including increased size and adherence to plastic (Fig. 26 A). In the end no difference in NLC number was detected by BFM evaluation.

In the following series of experiments, we tested the effect of the 6-25 mAb on the viability of normal human PBMC. For that, PBMC were isolated from buffy-coats and plated at 1 × 10 6 c/mL. Then 10 µg/mL of either mouse or chimeric version of the 6-25 mAb and matching isotype controls were added to the cultures. Subsequently, the viability and cell number were measured at day 3, 6 and 9 of the culture. To evaluate effect of 6-25 antibody toward each of the main population of PBMC, the cells were first stained with anti-CD3, CD4, CD8, CD14 and CD56 antibodies, before incubation with Annexin V/7-AAD (Fig. 27). Flow cytometry analysis of healthy donor PBMC (HD PBMC), showed no significant difference in viability of the indicated cell subpopulations, when cultivated in the presence of 6-25 mAb, isotype control or in untreated control. In case of monocytes we analyzed their viability only at day 3, as later they were attached strongly enough to hinder more precise flow cytometry analysis. BFM observations showed no clear difference in the population of these cells though (data not shown).

To account for incomplete results on 6-25 toxicity toward NLCs and monocytes in previous experiments, we performed 6-25 toxicity assay on M1 and M2 MDM, using Resazurin assay. M1 and M2 macrophages were prepared by activation of monocytes with GM-CSF and CSF-1 respectively and subsequent polarization with IFN-γ and LPS for M1 MDM, and IL-4, IL-13 for M2 MDM. At day 8 mature MDM were treated with either chimeric 6-25, human IC at 10 µg/mL or left untreated. 6 days later potential toxicity was evaluated by addition of Resazurin, followed by reading of the changes in the fluorescence for 24 h (Fig. 28). Analysis of phenotype of NLCs treated with 6-25 from day 0 (Fig. 29B), also showed consistent staining with CD206, CD11c and HLA-DR (Fig. 29B). Level of CD163, CD64 and CD14 was decreased in case of cells treated with the 6-25 mAb, however the effect was not significant (and not consistent between the donors). As evaluation of the NLCs phenotype was challenging in this setup, owing to low number of the cells (further decreased by low recovery of the cells during detachment), heterogeneity of the samples and potential effect of other cells from CLL PBMC (that represented > 97% of the overall population) we decided to further study effect of 6-25 antibody in MDM model, as it represents more basic and isolated environment, which also solves the problem of macrophages shortage.

We decided to evaluate the effect of the chimeric version of 6-25 mAb, as it is more relevant in case of human cells. We generated M1-MDM by GM-CSF activation for 6 days, followed by IFN-γ and LPS (LPS added 18h after IFN-γ) polarization for 2 days, and M2-MDM by CSF-1 activation for 6 days, followed by IL-4, IL-13 polarization for 2 days. Subsequently we treated the cells with 10 µg/mL of the 6-25 mAb for 3 days and analyzed the phenotype of the cells by flow cytometry. As we observed no changes in the phenotype of M1-MDM upon the treatment (data not shown), we decided to further focus only on M2-MDM. We gathered the results from 9 independent experiments. To account for the heterogeneity between the phenotype of MDM from different donors, the MFI ratio of each tested marker was normalized to UT conditions for any given donor, and plotted together (Fig. 30) Results presented on Fig. 30, show significant decrease of signal of CD16 and CD169 molecules upon the treatment with 6-25 mAb, compared to the UT and IC conditions. Level of CD14 and CD163 markers was also significantly decreased between UT and 6-25, but not 6-25 and IC conditions, which suggest that the effect is more specific to FC fragment of the antibodies (effect of 6-25 on CD14 decrease is much stronger, compared to IC though). Decrease of CD163 expression upon IgG1 administration was already reported 229 .

Additionally, for some donors an increase in the expression CD86 was observed, upon administration of the 6-25 mAb, but the effect was not significant. As the CD14, CD16 and CD169 are overall associated with M2 macrophages, the IgG1 version of chimeric 6-25 mAb showed to slightly depolarize M2-MDM to M1 side of the spectrum. experiments, confirmed significant decrease of 6-25 signal, already after 30 min of incubation at 37°C (Fig. 31B). To further complement FC results, we performed similar analysis using confocal microscopy. Slides with NLCs were stained with 6-25 conjugated with fluorochrome and incubated or not at 37°C. After samples were analyzed with confocal microscope. Pictures taken after 1 min of incubation, show fluorescence signal from the 6-25 antibody, at the periphery of the cells. On the contrary, after 120 min of incubation, fluorescence can be detected in intracellular vesicles, further confirming internalization process (Fig. 31C).

Internalization of 6-25 antibody

Both flow cytometry and confocal microscope analyses confirmed that 6-25 antibody can be efficiently internalized in the NLCs.

Conclusions

Results presented in this chapter concern functionality of 6-25 target in the context of its effect on survival of primary human cells, polarization state of macrophages and internalization of antibody in the target cells. When tested on CLL and normal human PBMC, the 6-25 antibody showed no direct toxicity, leading to no changes in viability and cell concentration. While the antibody did not affect viability of the macrophages, it showed to modulate phenotype of M2

MDMs, by decrease of CD14, CD16 and CD169 markers. Finally, the 6-25 antibody turned out to be efficiently internalized in NLC. All of these characteristics indicated, that the 6-25 mAb might be a proper candidate for an antibody-drug conjugate (ADC) approach. And indeed, we generated the ADC versions of 6-25 antibody that was tested in the lab. Preliminary experiments indicated that 6-25-DC can selectively deplete M2 MDMs and NLCs in the in vitro cultures, without exerting toxic effect on healthy PBMC or M1 MDMs. These results are beyond the scope of this thesis however, and require additional development.

Discussion

TAMs belong to the major cell populations of TME [START_REF] Sica | Cancer related inflammation: The macrophage connection[END_REF][START_REF] Mantovani | Tumour-associated macrophages as treatment targets in oncology[END_REF] , that not only support cancer cells in every step of tumor development [START_REF] Mantovani | Tumour-associated macrophages as treatment targets in oncology[END_REF] , but are also regarded as one of the reasons for resistance of malignant cells to the treatment [START_REF] Wang | Research trends in pharmacological modulation of tumor-associated macrophages[END_REF][START_REF] Ceci | Targeting tumor-associated macrophages to increase the efficacy of immune checkpoint inhibitors: A glimpse into novel therapeutic approaches for metastatic melanoma[END_REF]102,103 . Multiple TAM-targeting strategies are currently in development or evaluation in I/II phases of clinical trials. The most advanced branch of these therapies revolves around targeting of CSF1R [START_REF] Wang | Research trends in pharmacological modulation of tumor-associated macrophages[END_REF][START_REF] Ceci | Targeting tumor-associated macrophages to increase the efficacy of immune checkpoint inhibitors: A glimpse into novel therapeutic approaches for metastatic melanoma[END_REF] , one of the major receptors controlling biology of monocytes and macrophages [START_REF] Mun | The M-CSF receptor in osteoclasts and beyond[END_REF] . Anti-CSF1R therapies were shown to deplete or reprogram TAMs in TME, leading to mixed clinical response [START_REF] Ceci | Targeting tumor-associated macrophages to increase the efficacy of immune checkpoint inhibitors: A glimpse into novel therapeutic approaches for metastatic melanoma[END_REF]178,248 .

At the same time, this receptor is also highly expressed by: monocytes, tissue-resident macrophages, and microglia, thus its targeting can lead to serious complications associated with decreased immunity, deregulation of the bone renewal 249 or neurotoxicity associated with microglia disruption 178 . This creates necessity to look for more specific markers of TAM and reduce toxicity of the potential therapeutic strategy (especially as TAM-targeting will most probably be part of combined therapy). With more specific targeting it will be also possible to deliver more potent drugs, that normally show unacceptable toxicity upon systemic administration.

The core results presented in this thesis revolves around the study of TAM-specific monoclonal antibody, called 6-25. 6-25 was previously generated by Team 09 of CRCT, using hybridoma technology with wholecell immunization protocol, using NLCs as a model of TAM. This approach not only increased a chance to generate an antibody against an accessible epitope with native conformation, but also had a potential to find a novel target on NLCs 242 . Initial target identification and validation, typed SFXN3 as a molecule recognized by 6-25 mAb. SFXN3 is a poorly identified, inner-membrane mitochondrial protein, ubiquitously present in mammalian cell 250,251 . 6-25 mediated detection of this protein in plasma membrane of NLCs and other TAMs, represented not only a novel way of targeting these cells, but also showed a rare example of translocation of inner-membrane mitochondrial protein to plasma membrane of the cells. There are already examples of such phenomenon, notably as in the case of mitochondrial F1Fo-ATP synthase 252,253 .

SFXN3 was also recognized to have a potential role in cancer. Recent findings identified SFXN3 to be an actor in one-carbon metabolism, as a transporter of serine 251 , making it a potential receptor involved in supporting cancer cells in TME 254 . Moreover, a presence of αSFXN3 autoantibodies was reported in patients with squamous cell carcinoma 255 . As much as all these characteristics typed SFXN3 to be a very attractive target for TAM targeting, in the course of this thesis growing body of evidence led us to initially doubt and ultimately invalidate SFXN3 as a potential target recognized by 6-25 antibody. Target disqualification was achieved thanks to CRISPR-Cas9 RNP mediated gene KO in primary MDM.

It is important to mention this technique, as primary macrophages represent problematic cells for gene manipulation, owing to their intrinsic ability to sense and destroy exogenous nucleic acid, initiate anti-viral response upon transduction procedure and general lack of proliferation 256,257 . RNP gene delivery is unique in that regard, as it ignores most of the mentioned obstacles 257 , and is characterized by high efficiency with minimization of the negative effect on phenotype of the cells 256 . Adopting this technique, allowed us to easily generate gene KO in both primary monocytes and MDM, with acceptable changes to phenotype of the cells, at least in regard to surface marker expression, after CSF-1 activation (we will have to evaluate effect of this procedure on functionality of macrophages though). We used RNP technology not only to disqualify SFXN3 as a target of 6-25 mAb, but also to validate a new target -FRβ. We plan to further utilize RNP technology in studying FRβ in primary human macrophages, including NLCs.

Identification of FRβ has a huge implication for the future of 6-25 project, as it validates a lot of our previous findings. Additionally, FRβ represents a relatively recent discovery in the field of TAM targeting, giving us an opportunity for further studies (especially in the field of CLL).

Expression of FRβ

The gene family coding folate receptor consists of 4 members, FOLR1-4, that code FRα, FRβ, FRγ and FRδ respectively. All of these proteins show affinity to folic acid (high in case of FRα, FRβ) 258 . Both FRα and FRβ, are glycosylated proteins, attached to the plasma membrane of the cell through glycosylphosphatidylinositol (GPI) anchors 259 . FRα and FRβ are also the most relevant in the context of cancer. FRα is overexpressed on many human tumors, while FRβ shows expression mostly on activated macrophages 260 and myeloid leukemia cells 258,261 . Regarding macrophages, FRβ was reported to be highly expressed on TAM, found in: melanoma 262 , breast cancer 262,263 , colon cancer 263 , hepatocarcinoma 264 , lung squamous cell carcinoma, adenocarcinoma 259 and various mouse models of cancer 258,264,265 . Level of FRβ was shown to be correlated with higher stage of cancer and presence of LN metastases 260 . Moreover, expression of FRβ on TAM in a mouse model of ovarian cancer was reported to increase with cancer progression 258 . During the course of this thesis we also confirmed that NLCs show high expression of FRβ and that level of this protein is correlated with increased survival of CLL cells in vitro (chapter 4.2, Fig. 14).

In the context of autoimmune diseases, FRβ showed to be highly expressed on macrophages of patient with various autoimmune diseases, including: Crohn's disease, Ulcerative Colitis, Psoriasis, systemic lupus erythematosus 266 , osteoarthritis (OA) and rheumatoid arthritis (RA) 267 . Besides, radiolabeled molecules specific to FRβ, showed that macrophages expressing this receptor are also enriched in atherosclerotic plaques 268 . All these findings signify connection of FRβ + macrophages with pathological inflammation.

Nevertheless, FRβ + tissue-resident macrophages were also reported in normal tissues, like tonsils, colon and muscle 263 . Moreover, FOLR2 was shown to be highly expressed on the Hofbauer cells and to lesser extent in the Kupffer and Langerhans cells 244 . Expression of FRβ on healthy macrophages, especially on the Kupffer cells, needs to be carefully considered in case of potential targeted therapy.

Expression of FRβ + in macrophages was reported to be driven by CSF-1 signaling and maintained by IL-4

and IL-10. On the other hand, GM-CSF, IFN-γ and LPS were shown to downregulate this protein 262 .

Additionally, PU.1 transcription factor was recently reported as an important element controlling expression of FOLR2 gene 263 . Another factor involved in control of folate transporters (including FRβ) is

Activin A. This cytokine can be upregulated in GM-CSF activated MDMs, leading to downregulation of FRβ and overall promotion of M1 polarization in macrophages 269 .

Mentioned results on the role of CSF-1 and other cytokines on expression of FRβ are in perfect agreement with our findings presented upon study of 6-25 target/FRβ + expression in MDM model (chapter 5.3). In the future, we consider further research on a signaling pathway driving expression FRβ. Our DEG analysis of CSF-1 activated MDMs treated with TNF (chapter 5.2.1), showed that PI3K-ATK-mTOR pathway to be most likely responsible for controlling expression of FOLR2. We also performed some preliminary results to validate this hypothesis. We confirmed that wide spectrum inhibitor -Ly294002 and specific mTOR inhibitor -Rapamycin, lead to dose-dependent downregulation of FRβ in primary monocytes and MDM. However, further tests with compounds specific to different isoforms of class I PI3K did not give us much clarification, and would require further experiments (data not shown). Especially given the complexity of PI3K-ATK-mTOR pathway, which represents one of the master regulators of many crucial cellular processes 260 . Considering that expression of FRβ is abrogated by the action of LPS, TNF and IFN-γ (chapter 3.3.3, fig. 9), finding genes commonly downregulated in these three conditions. It might provide a more precise answer, compared to our previous DGE analysis (chapter 4.2.1). Alternatively, single cell study, for example in a form of spatial transcriptomic could also help in typing the genes controlling FOLR2 expression.

Finally, FRβ pool in the cell is distributed between extra-and intracellular compartments 258,270 , this is an aspect that we never considered during study on polarization of macrophages. Therefore, it is not clear if M1 agents like LPS, TNF and IFN-γ cause only gene downregulation, or also internalization of the receptor.

It would be important to also establish how stable is an intracellular pool of FRβ, after treatment with M1 agents. That would also stress the importance of distinction between extra-and intra-cellular staining, especially in techniques less precise in distinguishing between both compartments, like IHC. In autoimmune diseases like osteoarthritis (OA) and rheumatoid arthritis (RA), researchers confirmed increased infiltration of FRβ high , CD163 + and FRβ dim , CD163 -macrophages. Both of these populations were characterized with high phagocytosis capacity (based on a dextran uptake). Additionally, authors showed that both FRβ + and CD163 + macrophages can acquire mixed M1/M2 phenotype, based on the expression of TNF, iNOS (M1 signature) and IL-10, TGF-β (M2 signature), concluding that FRβ and CD163 markers, are not enough to classify polarization state of macrophages 267 . However, these results were performed only by 3 color IHC, with analysis of either CD163 or FRβ with M1 (TNF, iNOS) or M2 (IL-10, TGF-β) markers. It is well established, that polarization of macrophages is a continuum between M1-M2 side of the spectrum, and co-expression of classical M1 and M2 markers, such as TNF, iNOS and IL-10,TGF-β can be observed in some M2 macrophages 230,271 . In our studies, we observed that NLCs subjected to high doses of TNF, were able to maintain their M2-like/pro-tumoral phenotype, as long as they were co-treated with IL-10 (chapter 4.3), signifying an importance of interplay between various cytokines.

Perspectives for study of therapeutic potential of 6-25 mAb

As mentioned previously, all the results presented in this manuscript were designed and performed without knowledge of real specificity of 6-25 mAb. Hence we would like to discuss some of them from the perspective of FRβ, and propose possible improvements.

During our research we confirmed specificity of 6-25 mAb toward NLCs (Chapter 3.1.1), AML cell lines (3.2) and CSF-1 activated macrophages (3.3). Available data on FRβ expression further validated our finding.

Especially encouraging is confirmation of FRβ expression on TAMs from various human cancers 273 . As we have an access to neoplastic tissues from various human hematological cancers (thanks to cooperation with Universitaire du Cancer de Toulouse-Oncopole (IUCT-O)), we could try to study FRβ + TAMs in these malignancies, especially as the most of a data in this topic is focused on TAM in solid tumors sources.

Furthermore, we would like to continue deciphering the role of FRβ in CLL. Since we confirmed that FRβ is a marker of protective NLCs, we want to investigate potential involvement of this receptor in their protumoral activity. First we plan to evaluate an effect of FOLR2 KO on functionality of NLCs, especially ability to attract cancer cells and perform efferocytosis. Next, we would like to study an influence of folate deficiency on the CLL PBMC culture dynamics. It would be interesting to establish if there is an effect of folate on polarization of NLCs, as it was previously reported that this vitamin can blunt proinflammatory responses in macrophages 269,272 . Regarding CLL cells, folate was recently revisited as an important metabolite in various leukemia. Confirmation of CLL cell lines sensitivity to antifolate therapy (with methotrexate) further supports this hypothesis 278 . We suggest that NLCs could potentially serve as a reservoir of folate for cancer cells. We plan to evaluate this hypothesis using fluorescent conjugates of folate. First, we would like to confirm that NLCs can actually accumulate folate, and if so establish if they can share it with cancer cells. Trogocytosis was reported before as a process involved in interaction between NLCs and CLL cells 206 . Perhaps it is also involved in the potential delivery of folate. Moreover, folate was shown to be especially important for dividing cells 269 . Since these cells do not proliferate in normal CLL PBMC setup, we could stimulate their division with CpG oligodeoxynucleotide and IL-15 279 .

With folate studies in the CLL PBMC model, we could also re-evaluate our experiments on functionality of 6-25 mAb (chapter 6.2). Admittedly, our previous results showed an unclear effect of 6-25 on phenotype of NLCs, and no effect on viability of CLL cells. This time, we could study if 6-25 mAb can block binding of folate to FRβ on NLCs or disrupt transfer of this metabolite to cancer cells. Also using confocal microscopy, we could study an effect of the antibody on the contact between NLCs and CLL cells.

Apart from standard IgG1 version of 6-25 antibody, we have also ADCC enhanced, FC silent and two antibody-drug conjugate (ADC) versions at our disposal. As mentioned previously, we already performed some in vitro experiments with DC-6-25 which showed high selectivity only toward cells expressing FRβ.

In

Illustration 1 .

 1 Main therapeutic strategies aiming to reprogram, eliminate or inhibit recruitment of TAM. Main targets for each approach are listed with distinction to extra-or intracellular localization. Adapted from 106 . 1.3.1 Differentiation and Depletion Strategies based around TAMs depletion, represent the main body of focus regarding clinical trials. They are centered on targeting of CSF1R (CD115). CSF-R1 is expressed on monocytes, macrophages and some DC. It is a tyrosine kinase receptor, having two native ligands CSF-1 and IL-34, and upon activation it leads to signaling cascade promoting survival, proliferation and differentiation of monocytes and macrophages 107

  of population in Europe and United States to around 0,01% in people of Asian and Pacific Islands descent in US 187 . The median age of CLL diagnosis ranges from 70-72 years and risk of the disease development is 2-times higher in men, than women 187 . CLL is almost always preceded by an indolent stage called monoclonal B-cell lymphocytosis of CLL phenotype (CLL-like MBL). People with more than 500 of CLL-like B cells clones/ul of blood might develop therapy-requiring CLL, with frequency of 1-2%/year. Interestingly few CLL-like MBL/ul of blood can be already detected in 7-14% of Western adults > 40 years old. In comparison, for Taiwan population, this level reaches only 1.3% 189 . Additionally, first degree relatives of people with CLL have up to 8.5 higher risk of developing the disease 187 . These results signify importance of genetic background in CLL initiation. 1.4.2 Classification of CLL CLL B cells are characterized by expression of CD19+, CD5+, CD23+ and CD200 (which helps differentiating them from mantle lymphoma cancer cells), but low expression of CD20 and lack of CD10 187 . CLL can be divided into 2 distinct groups based on the presence of unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy-chain variable region gene (IGHV), thus reflecting typical maturation stage of B cells. CLL B cells with unmutated IGVH come from the cells which did not undergo the somatic hypermutation upon differentiation in germinal center zone. Hence, they are less mature leading to more aggressive disease with worse prognosis. On the other hand, CLL B cells with mutated IGVH represent the cells from post-germinal centers, expressing immunoglobulins after somatic hypermutation or in some cases also class switching (Illustration 2).

  .

  1.4.4 B cell signaling and life cycle of CLL cellsB cell receptor (BCR) signaling is required for survival of mature B cells and is also active in CLL B cells. It is composed of two parts: a membrane bound immunoglobulin (IgD, IgM, IgA, IgG or IgE) that serves as antigen-binding subunit and heterodimeric Ig-γ (CD79A)/Ig-β(CD79B), that is responsible for a signal transduction through SRC-family kinase-dependent phosphorylation (predominantly LYN) and spleen tyrosine kinase (SYK) 187 . CD19 serves as a co-receptor for BCR leading to Pi3K-AKT-mTOR, NFκβ and ERK activation187 . In CLL, malignant B cells can maintain continuous activation of BCR through engagement of autoantigen. Interestingly, BCR signaling in CLL can lead to completely opposite outcomes, from B cell activation that increases survival and proliferation to cell anergy. Outcomes of BCR engagement depends on which downstream pathway will be activated afterwards. In general, BCR signaling leads to cell activation in U-CLL and cell anergy in M-CLL. While the reason for cell anergy is not clearly known, it might be an outcome of chronic BCR activation with lack of proper T-cell engagement. Also, the difference in genetic and epigenetic background might be involved. Nevertheless, the cell anergy, at least partially explains the usual indolent character of M-CLL 100 . Another difference between M and U-CLL B cells is the repertoire and level of response to autoantigen recognized by BCR. In M-CLL, BCR signalling pathway show constant phosphorylation, and binding of antigens like β-(1,6)-glucans or rheumatoid factors (RF) lead to only mild response. On the other hand BCRs in U-CLL show high specificity toward much wider repertoire of targets, including: MYHIIA, vimentin, but also single and double-stranded DNA, oxidized low-density lipoprotein and bacterial antigens, like lipo-polysaccaride (LPS). Also, binding of the antigens in case of U-CLL b cells is leading to strong activation, especially in the presence of CD38 and ZAP70 196 . Beside direct activation of CLL B-cells, BCR control also activity of various surface receptors on cancer cells, including integrins. One of integrin modulated by BCR is α4β1 integrin composed of CD49d (alpha 4) and CD29 (beta 1), resulting in binding to vascular cell adhesion protein 1 (VCAM1) and fibronectin 100 , thus facilitating cell-cell and cell-ECM interaction respectively 190 . 1.4.5 Importance of TME and proliferation centers CLL represents quite a unique setting to observe dependency of cancer cells from TME. Because of their mature phenotype, CLL cells are unable to survive and proliferate on their own, instead they need to migrate from peripheral blood (PB) to lymphoid organs in order to obtain appropriate stimuli. These spots are mostly located in the lymph nodes (LN), are called proliferation centers (PC) and represents one of the hallmarks of CLL 187 . CLL cells migration to PC is driven by chemokine gradient created by non-malignant cells of TME. Mechanistically the main receptor responsible for CLL cells migration is CXC-chemokine receptor 4 (CXCR4), which binds CXC-chemokine ligand 12 (CXCL12), and CC-chemokine receptor 7 (CCR7), which binds CC-chemokine ligand 19 and 21 (CCL19/CCL21) 187 . Following the chemokine gradient, CLL cells enter lymphoid organs. In there, CLL B cells encounters various accessory cells, including: T-cells, Nurse-like cells and mesenchymal-derived stromal cells (MSCs). T-cells provide cancer cells with multiple proliferation stimuli, including engagement of CD40 by CD40L and release of IL-2, IL-4 and IL-10. It has been shown that IL-4 can upregulate expression of surface IgM on cancer cells, thus improving interaction with autoantigens. In turn, presence of autoantigens on both stromal cells and NLC 196 and stimulation from T cells, lead to proliferation of cancer cells (the role of NLC

  . A) Picture of NLCs (green) and CLL cells (red), 120 min after initiation of co-culture. Live-cell imaging with Operetta CLS High Content, x20 objective. B, C) Bright-field imaging of pseudo-emperipolesis event, with small round CLL cells gathered below big, adherent NLC. Most of the floating cells were removed by pipetting, right before taking the picture. B) ×20 objective, C) ×40 objective with focus on the indicated area on picture B. Similarly, to TAM, NLCs perform various processes to support cancer cells. Perhaps the most prominent function of NLCs is protecting CLL B cells from spontaneous apoptosis. NLCs are the main producers of SDF-1 (CXCL12), that beside stimulating survival of malignant cells, is also attracting cancer cells to the proliferation centers 187,201 . Beside NLC are protecting CLL B cells by expressing B cell-activating factor (BAFF), a proliferation-inducing ligand (APRIL) 210 , B cell-attracting chemokine (BCA-1 or CXCL13), WNT5A 187,201 and BDNF 211 . Even though APRIL continue to appear as an important molecule for CLL survival, some studies question its role in the process 212 . Apart from soluble factors, the NLC protective function is also mediated by direct contact mediated through CD31-CD38 196 and CD2-CD58 (lymphocyte functionassociated antigen 3/LFA3) interaction between macrophages and cancer cells respectively 206 . Beside, NLCs together with stromal cells are believed to be a source of autoantigen, necessary for stimulation of BCR signaling on cancer cells 213 . Similarly to other TAM, NLCs are believed to also participate in formation of immunosuppressive TME, as they are shown to produce: IL-10, CCL18, IL-6, IL-8 and also IDO 204 . Beside participation in pathophysiological function of CLL B cells, NLCs can also protect cancer cells from therapeutic insult. In vitro studies showed, that CLL cells co-cultured with NLCs were more likely to survive treatment with idelalisib 202 , ibrutinib, chlorambucil or dexamethasone 214 . In general, NLCs infiltration is associated with worse prognosis in CLL. Absolute monocyte count (AMC), which can indirectly reflect the number of NLCs in the LNs is associated with shorter time to first therapy (TTT) and overall survival (OS) 215 . In accordance, abundance of soluble CD163 (sCD163) in the blood of the patients is associated with disease progression and bad prognosis. Soluble CD163 can be used as a more direct indicator of NLCs infiltration level in the proliferation centers 214 . 1.4.7 Targeting of nurse-like cells in CLL NLCs represent an important therapeutic target, given their significance in the pathophysiology of CLL and resistance to treatment. So far no therapeutics directly targeting NLC in CLL has been approved. Nonetheless, multiple candidates indirectly affecting NLC are available. The first example of a drug indirectly modulating NLC is Ibrutinib. Ibrutinib belongs to the new generation of drugs for the treatment of CLL. It is an irreversible inhibitor of Bruton's tyrosin kinase (BTK), that shows exceptional efficacy in the clinics. Ibrutinib action causes impairment of BCR and NF-κβ signaling, which in turn modify mobility and adherence of the cancer cells, limiting access to prosurvival TME and finally leading to cell apoptosis 187 .

  NLCs. It is believed to indirectly decrease CLL B cell viability by: inhibiting migration of monocytes toward LN and repolarizing NLCs toward M1-like, anti-neoplastic phenotype, with increased ability to activate Tcells. On the other hand, results presented in the paper showed also increased proliferation and number of NLC upon treatment with Lenalidomide followed by stronger adhesion of cancer B cells to NLCs -which is one of the hallmarks of protective function of these cells. Also decrease of CD163 marker presented by the authors is negligible 219 . Additionally, use of Lenalidomide in clinical trials showed a high incidence of severe adverse effects 220 . In one of the phase III clinical trials of elderly patients with CLL, Lenalidomide showed therapeutic activity in the subset of patients. However, there was no improvement of PFS compared to Chlorambucil, and increased mortality of the patients in Lenalidomide led to early termination of the trial 221 .Trabectedin, described in a previous chapter, has been also proposed as an immunomodulatory drug that could be used in CLL. It showed activity to selectively kill CLL B cells and Mo-MDSC. Additionally, this drug, turned out to modulate function of immune cells of CLL TME, activating anti-tumoral activity of both T cells are macrophages. Trabectedin can also promote M1-like polarization of macrophages, inducing expression of many proinflammatory genes and impairing protective function of NLCs, serving as another potential drug to indirectly affect NLCs and TME of CLL 128 . While first clinical trials with Trabectedin in combination with Venetoclax for the treatment of CLL patients with resistance to BTK inhibition was terminated due to lack of participants, it will potentially lead to future attempts 222 .Another compound, that could be used in the treatment of CLL is Lurbinectedin (PM01183) -and analoque of Trabectedin. This compound is a selective inhibitor of protein-coding genes transcription, that leads to cell apoptosis. Both cancer B-cells and non-malignant immune cells dispaly increased sensitivity to the drug. It was shown, that Lubrinectedin can also change the profile of cytokine produced by monocytes and NLC, with increased production of IL-1β -which is overall a marker of good prognosis in CLL 223 .

2. 1 . 1

 11 Primary cell culture Chronic lymphocytic leukemia (CLL) cells Peripheral blood samples from CLL Patients were obtained from the Hematology Department with informed consent and referenced in the INSERM cell bank. According to French law, the INSERM cell bank has been registered with the Ministry of Higher Education and Research (DC-2013-1903) after being approved by the ethic committee (Comité de Protection des Personnes Sud-Ouest et Outremer II). Clinical and Biological annotations of the samples have been reported to the Comité National Informatique et Liberté (the Data Processing and Liberties National Committee). Blood samples were collected from previously untreated patients, if not stated otherwise. PBMC from CLL patient's blood were isolated by density-gradient centrifugation on Ficoll-PaqueTM PLUS (GE Healthcare, Sweden). If not stated otherwise, CLL PBMC were plated freshly after isolation at 10 × 10 6 c/mL and maintained for 12-14 days to allow maturation of NLCs. Culture of cells at such high concentration, allows to generate sufficient number of NLCs for downstream processing, and limits spontaneous apoptosis of CLL cells during early days of the culture. Experiments for flow cytometry analysis of NLCs were performed in 6-well plates in 4 mL of complete medium. To evaluate phenotype and viability of CLL cells only, cultures were downscaled to 48-or 96-well plates. 2.1.1.1.1 CLL B cells isolation CLL cells were isolated from fresh or frozen PBMC, using EasySep™ Human B Cell Enrichment Kit II Without CD43 Depletion (STEMcell, France) for negative cell selection, according to manufacturer instructions.

Illustration 5 .

 5 Scheme of generation classical M0, M1 or M2-MDM. To obtain M1 and M2 macrophages, monocytes were activated with GM-CSF and CSF-1 respectively.

3 .

 3 staining with 6-25 antibodyDuring early experiments an unconjugated version of 6-25 mAb was used. Briefly, after Fc block incubation, cells were incubated with 10ug/mL of 6-25 mAb for 30 min at 4°C. After washing cells were incubated with goat anti-mouse II-antibody for 20 min at 4°C. Next, cells were incubated with a specific antibody mix for 20 min at 4°C. After washing, cells were re-suspended in PBS, containing 0.8 µg/mL 7-AAD (Sony, USA) and immediately analyzed by flow cytometry. Protocol used in chapter 3, 4.1 and 6.Direct staining with 6-25 antibodyIn order to perform direct staining, 6-25 antibody was coupled with CF633 fluorophore, using Mix-n-Stain™ CF®633 Antibody Labeling Kit manufacturer instruction. During 6-25 conjugation, also matching isotype control antibody was prepared. Every new batch of antibody was tested by flow cytometry on M2-MDM or NLCs, to ensure consistency of the staining. 6-25 after labelling was used in the same fashion as other commercial antibodies conjugated with fluorophore.

Figure 4 .

 4 Figure 4. Flow cytometry analysis of the staining of AML cell lines with 6-25 mAb. Representative FC staining of MV4-11, U-937, THP-1, OCI-AML-13, MOLM-14 and U-373 cell lines. For histogram plots green represents 6-25 and blue IC signal respectively. As THP-1 cell line is often used as a model of macrophage differentiation, we attempted to upregulate expression of 6-25 target on these cells, by stimulation with PMA and further M2 polarization with IL-4. In the end however, differentiation of the cells failed to lead to any changes in the expression of the 6-25 target (data not shown). Additional screening of the AML cell lines, revealed that U-937, MOLM-14 and THP-1 cells are capable of expressing the 6-25 target, with U-937 cells showing comparable staining to MV4-11 cells. These results open a possibility to target AML cells with the 6-25 mAb. While the staining of these cell lines with the 6-25 mAb is nowhere near as high as in case of NLC, there are examples of successful biological therapies

  chapter 1.3.1, but additionally on day 6 we polarized cells with classical M1 agents (IFN-γ alone or in combination with LPS, added 18 h later) or M2 agents (IL-4 and IL-13) for 2 days. At the end of the maturation process, we collected the cells and analyzed their phenotype by flow cytometry. Results presented on fig. 6, show normalized expression of various myeloid surface markers in comparison to basal conditions ("naïve" macrophages, activated only with CSF-1). Starting with the results of CSF-1 activated cells, IL-4/IL-13 treated group show moderate upregulation of CD163, 6-25 target, CD11b, CD11c, HLA-DR and strong upregulation of CD206 compared to basal conditions. On the other hand, IFN-γ led to: strong upregulation of CD64, CD86, HLA-DR, mild upregulation of CD11c; strong downregulation of CD163, CD16; moderate downregulation of CD14, CD11b, CD115 and only mild downregulation of CD206 and 6-25 target. Addition of LPS (IFN-γ + LPS condition) resulted in further downregulation of previously mentioned markers, with concomitant decrease of CD64 and CD11c.In case of Il-34 activation, the effect of secondary polarization is weaker, as compared to CSF-1 activation.There are some interesting changes though, as IFN-γ causes abrogation of CD163 expression in CSF-1 activated cells, but in case of IL-34 cells cause slight upregulation of this marker. Also IL-4/IL-13 polarized, M(IL-34) cells show distinct phenotype, with downregulation of CD14, CD16, CD163, CD64, CD11b and CD11c and slight decrease to 6-25 target. Overall IL-34 activated cells show much higher expression of CD115 (CSF1R) compared with CSF-1 activated cells.

Figure 6 .

 6 Figure 6. Influence of secondary M1 (IFN-γ or LPS) or M2 (IL-4, IL-13) polarization on expression of 6-25 target in CSF-1, IL-34 or GM-CSF activated MDM. Heatmap plot with results of 2 independent experiments and MDM from 4 different donors in total. Data from FC analysis was processed as follow: median fluorescence intensity (MFI) ratio between signal from specific antibody and matching IC were calculated; MFI ratio values were normalized for each marker, within cells from each donor, and represented as percentage of basal condition's signal (MDM activated only with CSF-1); geometrical means of combined results were plotted on the heatmap. Green color represent upregulation (non-linear scale) and red color represents downregulation (each tic = 0.1 of basal signal) of each listed marker compared to basal condition. White color depicts signal of basal conditions. Representative histogram for CSF-1 showing basal expression of each tested marker (grey and green represents signal from isotype control and specific antibody respectively.Moving to GM-CSF activated macrophages, we observed the least changes upon secondary treatment with classical M1 and M2 agents. Irrespective of secondary stimulation, these cells did not express CD163 and 6-25 and showed very low expression of CD14 and CD16. Treatment with LPS led to slight decrease of the CD206 and CD64 expression, while IL-4 and IL-13 caused downregulation of CD64, and at the same time failed to upregulate CD206.

Figure 8 .

 8 Figure 8. TNF blocks M2-polarization of macrophages induced by CSF-1 and IL-34, and abrogates expression of 6-25 target on MDM. Heatmap plot with result of 2 independent experiments and MDM from 4 different donors in total. Data from FC analysis was processed as follow: median fluorescence intensity (MFI) ratio between signal from specific antibody and matching IC were calculated; MFI ratio values were normalized for each marker, within cells from each donor, and represented as percentage of basal condition's signal (MDM activated only with CSF-1); geometrical means of combined results were plotted on the heatmap.Green color represents upregulation (non-linear scale) and red color represents downregulation (each tic = 0.1 of basal signal) of each listed marker compared to basal condition. White color depicts signal of basal conditions. Representative histograms for CSF-1 column (grey -isotype control, green -specific antibody signal) show expression of each tested marker in basal conditions.

Figure 9 .

 9 Figure 9. Distinct effect of M1 polarizing agents (LPS, TNF and IFN-γ) on phenotype of CSF-1 activated MDM. A) Effect of LPS, TNF and IFN-γ administrated at day 0, 3 or 6 on morphology of MDM. IL-4 treated MDM at day 6 as a control of M2 phenotype. Analysis by BF microscopy at day 6 (grey) or 8 (yellow). Representation of M1-M2 spectrum of polarization, adapted from 230 . B) Comparison of MFI fold change of 6-25 target and various myeloid markers, in relation to the type and moment of treatment. Results are mean for 2 independent experiments and MDM from 4 different donors in total. C) Representative histogram plots, showing effect of LPS, TNF or IFN-γ on 6-25 target expression, depending on treatment time.

Figure 10 .

 10 Figure 10. Kinetics of the 6-25 target appearance on the surface monocytes during the maturation into NLC in relation to other myeloid markers. A) Gating strategy. B) Representative plot with MFI ratio calculated between specific marker and matching isotype control. Measurement point indicated with squares. C) histogram plots representing 6-25 signal (green) overlaid with isotype control (blue), each plot represents 1 measurement point from B). D) BF microscopy pictures of CLL PBMC over the first 9days of the culture, magnification x400.

Figure 11

 11 Figure 11 Changes in the viability and CLL cell concentration during typical CLL PBMC culture. PBMC isolated from the blood of CLL patients, were cultured at 10 × 10 6 c/mL. Viability and concentration of the cells were measured at indicated time points, by flow cytometry (Annexin V, 7-AAD staining) and haemocytometer respectively.

Figure 12 .

 12 Figure 12. Correlation of the 6-25 target expression on NLC and viability of CLL cells. A) Kinetics of 6-25 target expression on the surface of maturating NLC, depicted as MFI ratio (between signal from 6-25 and IC antibodies). B) viability of CLL cells at day 10 from the indicated cultures (Annexin V/7-AAD staining, analyzed by FC). C) Initial percentage of monocytes in the CLL PBMC, evaluated by FC (CD14 + , CD16 + staining, data not shown).

Figure 13 .

 13 Figure13. Changes in the viability and CLL cell concentration in CLL PBMC culture from patient 5 (Fig.12A). PBMC isolated from the blood of CLL patients, were cultured at 10 × 10 6 c/mL. Viability and concentration of the cells were measured at indicated time points, by flow cytometry (Annexin V, 7-AAD staining) and haemocytometer respectively.

Figure 14 .

 14 Figure 14. Correlation of 6-25 target expression on NLCs and viability of CLL cells. Comparison of A) MFI ratio of the 6-25 target on NLC from PBMC cultures with high and low CLL viability. B) relation between expression of 6-25 target on NLC and viability of CLL cells: 6-25 high/low indicates PBMC culture with NLCs expressing high/low level of6-25 target.

Figure 15 .

 15 Figure 15. Comparison of phenotype NLCs from high and low viability CLL PBMC cultures. Phenotype of the cells wasanalyzed by FC and level of each marker was represented as geometrical mean of MFI from 8 biological replicates for "Protective NLC" group and 6 biological replicates for "Non-protective NLC group".

Figure 16 .

 16 Figure 16. Western blot analysis of 1) fragment of human SFXN3 and 2) mouse SFXN3 recombinant protein. A) 20 ng of recombinant protein, detection with 1ug/mL rabbit pAb; B) 20 ng of recombinant protein, detection with 1ug/mL of mouse mAb;C) 100 ng of recombinant protein, detection with 6-25 mAb.

1 .

 1 immunoprecipitation (IP) of the M2-MDM lysates with the 6-25 antibody and subsequent detection of SFXN3 in the eluat by commercial antibody, 2. isolation of plasma membrane fraction from M2-MDM and detection of SFXN3 with commercial antibody, 3. immunofluorescence detection of intracellular SFXN3 with 6-25 and plasma membrane variant with commercial anti-SFXN3 antibodies.Note: we were unable to immunoprecipitate SFXN3 with the 6-25(1) and detect SFXN3 in plasma membrane fraction of M2-MDM (2) with commercial antibody (data now shown).5.1.1 Immunofluorescence with the 6-25 mAb and commercial anti-SFXN3 antibodiesAs both IP and plasma membrane isolation approaches failed in validating SFXN3 as a target of 6-25 antibody, we decided to perform immunofluorescence to attempt detection of mitochondrial SFXN3 with 6-25 antibody and plasma membrane staining with commercial anti-SFXN3 antibody in M2-MDM.

Figure 17 .

 17 Figure 17. Extracellular and intracellular immunofluorescence labelling of M2-MDM with 6-25 Ab. A) Plasma membrane staining in native conditions. B) Intracellular staining after fixation and permeabilization. Red signal -6-25 mAb, green signal -CD163 mAb (control staining of plasma membrane), blue signal -DAPI. Images were acquired with Zeiss 880 confocal microscope, using x63 objective.

Figure 18 .

 18 Figure 18. Intracellular and extracellular immunofluorescence labelling of M2-MDM with α-SFXN3 pAb. A) Intracellular staining of mitochondria with anti-SFXN3 pAb. B) Plasma membrane staining with anti-SFXN3 antibody. C) co-staining of anti-SFXN3 antibody with plasma membrane marker CD163. Images were acquired with Zeiss 880 confocal microscope, using x63 objective.

Figure 19 .

 19 Figure 19. CRISPR-Cas9 KO of SFXN3 in primary monocytes and macrophages. Monocytes (A, B) or M2-MDM (C) were electroporated with RNP targeting SFXN3 gene, and 5 days later analyzed by FC, after staining with 6-25 mAb (1) and by WB with anti-SFXN3 mAb (2). 1) US -unstained cells, IC -isotype control, SgCTL -sgRNA control, sgSFXN3 -sgRNA targeting SFXN3. 2) NaK-ATPase and α-Tubulin were used as a loading controls. Arrow indicates specific protein band. Based on flow cytometry analysis, no decrease in 6-25 staining in SFXN3 KO group was observed in comparison to cell treated with control sgRNA. On the other hand, WB results showed decrease of signal of SFXN3 protein in KO samples, confirming efficacy of the RNP-mediated KO. As downregulation of the SFXN3 gene did not affected staining of the 6-25 antibody, SFXN3 was ultimately invalidated as a target of 6-25 antibody.

Figure 20 .

 20 Figure 20. DEG analysis of plasma membrane-coding genes in CSF-1 activated-MDM treated with TNF. A) Control of 6-25 target expression in MDM used for RNAseq analysis after treatment with TNF. Mean results for MDM from 4 different donors, used for further RNA extraction (MDM dataset). B) Venn diagram comparing 4 DEG lists: 2 from "MDM dataset" and 2 from GSE95588 data set ["Mo dataset"]. D(Mo) and PM (Mo) -list of all downregulated genes, and genes coding plasma membrane proteins respectively from Mo dataset; D(MDM), PM(MDM) -list of all downregulated genes, and genes coding plasma membrane proteins respectively from MDM dataset.

Figure 21 .

 21 Figure 21. Confirmation of protein origin of the 6-25 target. A) Dot blot analysis of the MDM lysates treated with Proteinase K or Trypsin. C) Flow cytometry analysis of the CSF-1 activated MDM treated with Trypsin of Proteinase K.

Figure 22 .

 22 Figure 22. Volcano plot of proteins enriched in IP with 6-25 mAb. Lysates from M2-MDM were used in IP procedure with 6-25 and IC antibodies. Eluates from IP, were analyzed by MS and identified proteins were used for differential quantitative analysis. Statistical analysis performed with T-test, p <0.05 Overall 711 proteins were quantified during analysis, from which 6-25 group contained 59 significantly enriched protein compared to IC group. Moreover, most of the hits identified in the 6-25 group contained

Figure 23 .

 23 Figure 23. Comparison of staining of 6-25, αFRβ and αCD36 antibodies on MDM. Human monocytes were activated with CSF-1 or GM-CSF, and polarized with M1 or M2 agents, to induce various subpopulations of M1 and M2 MDM. At day 8 cells were collected, stained with indicated antibodies and analyzed by FC. US -unstained cells, IC -isotype control.

Figure 24 .

 24 Figure 24. Validation of new 6-25 target by RNP mediated KO of FOLR2 gene in MDM. Monocytes from HD were activated with CSF-1 for 24h and subsequently electroporated with RNP targeting FOLR2 gene. 5 days later cells were analyzed by FC, with 6-25 and α-FRβ (A). To account for electroporation stress, morphology (B) and phenotype (C) of UT and electroporated (ELP) cells were compared.

  244 and are susceptible to transfection. We chose to test three different transcripts variants of FOLR2 gene to additionally catch potential changes in staining between 6-25 and commercial anti-FRβ antibody, thus giving us potential information on the epitope recognized by 6-25 antibody. For transfection experiment, cells were plated 24h-prior the procedure in medium without antibiotics. Plasmid DNA transfection using Lipofectamine 2000 was performed according to manufacturer's protocol. 48h later cells were analyzed by FC, following staining with the 6-25 and α-FRβ antibodies (Fig.25).

Figure 25 .

 25 Figure 25. Overexpression of FOLR2 gene in HEK-T cell line. HEK-T cells were transfected with plasmids coding different versions of FOLR2 gene, and analyzed by FC 48h later. Results presented as histogram plots of 6-25 (A) and α-FRβ (B) stained cells. Results of the flow cytometry analysis showed a strong signal with the 6-25 and anti-FRβ antibodies in cells transfected with each variant of the FOLR2 gene, in comparison to untreated cells (UT). These results were an ultimate confirmation that indeed, the 6-25 antibody is recognizing FRβ protein. Additionally, three different transcripts resulted in the same staining pattern for both 6-25 and anti-FRβ antibodies, indicating that the epitope recognized by both antibodies is localized in the common region of the three protein variants.

Figure 26 .

 26 Figure 26. No toxicity of the 6-25 mAb toward CLL cells and NLCs. PBMC isolated from CLL patients were cultured in vitro in the presence of mouse (left panel) or chimeric (right panel) version of 6-25 mAb and matching isotype controls, at the concentration of 10µg/mL. On day 8 and 12, cell concentration and viability were monitored by haemocytometer and flow cytometry (Annexin V/7-AAD staining) respectively. A) Gating strategy for evaluation of CLL cell viability with representative dotplot for: UT (untreated), Ms/HuIgG1 (mouse/human isotype control) and 6-25 conditions. Above dotplots, BF microscopy imaging of the culture at day 12 to confirmed presence of NLCs. B) statistical analysis of CLL cells viability based on 6 independent experiments. C) combined results of CLL PBMC cell concentration at day 8 and 12. Statistical analysis with one-way ANOVA showed no difference between tested groups (for p=0.05).

Figure 27 .

 27 Figure 27. No toxicity of the 6-25 mAb toward healthy donor PBMC. PBMC were cultured in vitro in the presence of chimeric 6-25 mAb and matching isotype control at the concentration of 10µg/mL, or left untreated. Cell viability was monitored by flow cytometry (Annexin V/7-AAD staining) on day 3, 6 and 9. A) Gating strategy for detection of 5 main cell subpopulation in PBMC. B) Analysis of combined data from FC assessment of cell viability by Annexin V/7-AAD staining. Statistical analysis with one-way ANOVA showed no difference between tested groups (for p=0.05). Legend: UT -untreated; HuIgG1 -human IgG1 isotype control antibody.

Figure 29 .

 29 Figure 29. Effect of mouse 6-25 antibody on polarization (A) and maturation (B) of NLCs in vitro. CLL PBMC were cultured at 10 × 10 6 c/mL with 10µg/mL of 6-25 mAb administrated from day 0 (A) or day 6 (B). Phenotype of NLCs was analyzed by FC. NLC were gated based on SSC/FSC parameters, and level of each marker was depicted as representative histogram.

Figure 30 .

 30 Figure 30. Chimeric 6-25 mAb cause changes in phenotype of mature M2 MDM. CD14 + cells isolated from healthy donor PBMC were activated for 6 days with 50ng/mL of CSF-1 and polarized for 2 days with 25 ng/mL of IL-4 and IL-13 into M2 MDM. Subsequently mature macrophages were treated or not (UT) for 3 days with 10ug/mL of 6-25 or corresponding isotype control (IC) and phenotype of the cells was analyzed by flow cytometry. Subsequently MFI of each tested sample was calculated and results of antibody treated groups were normalized to MFI of untreated cells from each independent experiment. Statistical analysis was performed with one-way ANOVA (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001)

Figure 31 .

 31 Figure 31. The 6-25 mAb is internalized by NLCs. NLCs were incubated with6-25 mAb, and incubated at 37⁰C for: 5', 15', 30', 1h and 2h. Subsequently cells were stained with II-antibody conjugated with fluorochrome and analyzed by FC; 4°C condition, used as a no-internalization control. A) Histograms with % of NLCs positive for6-25, depending on incubation time at 37⁰C. B) Combined data from 5 independent experiments, represented by MFI ratio normalized to signal from 4°C control. Statistical significance calculated by one-way Anova. Confocal microscopy analysis (C) of 6-25 stained NLCs before (1) and after (2) two hours of incubation at 37°C. Another important functionality of the 6-25 we wanted to explore was internalization of the antibody in the NLC. To evaluate this phenomenon, we used in-house developed flow cytometry assay. Briefly, mature NLCs were collected, treated with FC block and incubated with unconjugated 6-25 mAb at 4°C. After extensive washing, cells were placed at 37°C to facilitate potential internalization for up to 2h. Subsequently cells were placed at 4°C and incubated with II-antibody conjugated with fluorochrome, to capture remaining 6-25 on the surface of the cells, and analyzed by FC. The results presented on Fig 31. A) show decrease of the signal from 6-25 antibody, that depends on the incubation time at 37°C. Statistical analysis of normalized data from 5 independent

7. 2

 2 Phenotype of FRβ + macrophagesMost studies classify FRβ + macrophages as M2-like, anti-inflammatory cells with high expression of CD163259,262,263 . In the context of cancer, FRβ + TAM were shown as immunosuppressive subpopulation, with increased secretion of IL-10 and defective production of proinflammatory cytokines (including: GM-CSF, IL-1α, IL-1β, IL-12p70, or IL-23) upon stimulation with LPS. Consistently with cytokine expression profile, these macrophages were shown to inhibit antigen-specific T cell functions 258 . This is in agreement with another study that connected FOLR2 expression with MS4A6A, LILRB5, CD209, CD163, C1QC, a geneset characteristic for immunosuppressive macrophages 263 . We also confirmed that FRβ + NLCs are characterized with an overall M2-like phenotype (chapter 4.2, Fig.15), ability to attract CLL cells (chapter 4.3, Fig.1E) and perform efferocytosis (chapter 4.3, Fig.2D).

FIGURE 1 .FIGURE 2 FIGURE 3 .FIGURE 4 FLOW

 1234 FIGURE 1. INITIAL EVALUATION OF 6-25 MAB SPECIFICITY. .............................................................................................................................. FIGURE 2 THE 6-25 MAB FC ANALYSIS OF PRIMARY HUMAN CELLS. ..................................................................................................................FIGURE 3. EXPRESSION OF 6-25 TARGET ON NLCS. ....................................................................................................................................... FIGURE 4 FLOW CYTOMETRY ANALYSIS OF THE STAINING OF AML CELL LINES WITH 6-25 MAB. ............................................................................... FIGURE 5 INFLUENCE OF GM-CSF, CSF-1 AND IL-34 ON POLARIZATION STATE OF MDM AND EXPRESSION OF 6-25 TARGET........................................

FIGURE 6 .

 6 FIGURE 6. INFLUENCE OF SECONDARY M1 (IFN-Γ OR LPS) OR M2 (IL-4, IL-13) POLARIZATION ON EXPRESSION OF 6-25 TARGET IN CSF-1, IL-34 OR GM-

FIGURE 14 CORRELATION

 14 FIGURE 14 CORRELATION OF 6-25 TARGET EXPRESSION ON NLCS AND VIABILITY OF CLL CELLS. ...............................................................................FIGURE 15. COMPARISON OF PHENOTYPE NLCS FROM HIGH AND LOW VIABILITY CLL PBMC CULTURES. ................................................................... FIGURE 16. WESTERN BLOT ANALYSIS OF 1) FRAGMENT OF HUMAN SFXN3 AND 2) MOUSE SFXN3 RECOMBINANT PROTEIN ......................................FIGURE 17. EXTRACELLULAR AND INTRACELLULAR IMMUNOFLUORESCENCE LABELLING OF M2-MDM WITH 6-25 AB. ............................................... FIGURE 18. INTRACELLULAR AND EXTRACELLULAR IMMUNOFLUORESCENCE LABELLING OF M2-MDM WITH Α-SFXN3 PAB. ........................................ FIGURE 19. CRISPR-CAS9 KO OF SFXN3 IN PRIMARY MONOCYTES AND MACROPHAGES. ...................................................................................FIGURE 20. DEG ANALYSIS OF PLASMA MEMBRANE-CODING GENES IN CSF-1 ACTIVATED-MDM TREATED WITH TNF. .............................................. FIGURE 21 CONFIRMATION OF PROTEIN ORIGIN OF THE 6-25 TARGET. ............................................................................................................. FIGURE 22. VOLCANO PLOT OF PROTEINS ENRICHED IN IP WITH 6-25 MAB.......................................................................................................FIGURE 23. COMPARISON OF STAINING OF 6-25, ΑFRΒ AND ΑCD36 ANTIBODIES ON MDM. .............................................................................. FIGURE 24. VALIDATION OF NEW 6-25 TARGET BY RNP MEDIATED KO OF FOLR2 GENE IN MDM. ......................................................................FIGURE 25. OVEREXPRESSION OF FOLR2 GENE IN HEK-T CELL LINE. .............................................................................................................. FIGURE 26. NO TOXICITY OF THE 6-25 MAB TOWARD CLL CELLS AND NLCS. ....................................................................................................FIGURE 27 NO TOXICITY OF THE 6-25 MAB TOWARD HEALTHY DONOR PBMC. .................................................................................................

FIGURE 28 .FIGURE 30 .

 2830 FIGURE 28. NO TOXICITY OF THE 6-25 MAB TOWARD M1 AND M2-MDM. ....................................................................................................FIGURE 29 EFFECT OF MOUSE 6-25 ANTIBODY ON POLARIZATION (A) AND MATURATION (B) OF NLCS IN VITRO.. .....................................................FIGURE 30. CHIMERIC 6-25 MAB CAUSE CHANGES IN PHENOTYPE OF MATURE M2 MDM. .................................................................................FIGURE 31 THE 6-25 MAB IS INTERNALIZED BY NLCS. .................................................................................................................................

  × 10 6 c/mL in 1mL of medium and placed in cell culture incubator. 90 min later, old medium was replaced to remove traces of electroporation buffer. Afterwards, cells were cultured for 4-5 days, before analyzing effectiveness of gene KO.

	well plate at 1
	Target	Species	Clonality	Cat. number	Dilution factor	Company
			Primary
	SFXN3	Rabbit	polyclonal	HPA008028	1000	Ozyme
	SFXN3	Rabbit	monoclonal	ab181163	1000	Abcam
	SFXN3	Rabbit	polyclonal	A12298	2000	Abclonal
	chimeric 6-25 Chimeric (human Fc fragment) monoclonal	-	10µg/mL	-
			Secondary
	anti-rabbit	Goat		#7074	5000	Cell signalling
	anti-mouse	Horse		#7076	5000	Cell signalling
	anti-human	Goat			5000	Sigma
			6 cells were used per 100µl Neon electroporation tip. Afterwards,
	RNP complex and cells were mixed together, aspirated by Neon Tip and electroporated by Neon
	Transfection System, using 1900V, 20ms, single pulse. Following electroporation cells were plated in 12-

  rank=1 and at 1 % FDR both at PSM level (on Adjusted e-Value criterion) and protein sets level (on Modified Mudpit score criterion) using the in-house-developed Proline software version 2.1.2 226 .

	7,
	https://github.com/mzdb/mzdb-access) to generate peaklists. Peak lists were searched against Swiss-Prot
	protein database with homo sapiens taxonomy (20,385 sequences) in Mascot search engine (version 2.7.0,
	Matrix Science, London, UK). Cysteine carbamidomethylation was set as a fixed modification and
	methionine oxidation as variable modification. Up to two missed trypsin/P cleavages were allowed. Mass

tolerances in MS and MS/MS were set to 10 ppm and 20 mmu, respectively. Search results were validated with a peptide

  TARGET IN CSF-1, IL-34 OR GM-CSF ACTIVATED MDM. ................................................................................................................................................................ FIGURE 7. EFFECT OF TNF ON MATURATION AND POLARIZATION OF CSF-1 ACTIVATED MDM, AND EXPRESSION OF 6-25 TARGET. ................................. FIGURE 8 TNF BLOCKS M2-POLARIZATION OF MACROPHAGES INDUCED BY CSF-1 AND IL-34, AND ABROGATES EXPRESSION OF 6-25 TARGET ON MDM .... FIGURE 9 DISTINCT EFFECT OF M1 POLARIZING AGENTS (LPS, TNF AND IFN-Γ) ON PHENOTYPE OF CSF-1 ACTIVATED MDM. ...................................... FIGURE 10. KINETICS OF THE 6-25 TARGET APPEARANCE ON THE SURFACE MONOCYTES DURING THE MATURATION INTO NLC IN RELATION TO OTHER MYELOID MARKERS. ................................................................................................................................................................................... FIGURE 11 CHANGES IN THE VIABILITY AND CLL CELL CONCENTRATION DURING TYPICAL CLL PBMC CULTURE.. .......................................................... FIGURE 12. CORRELATION OF THE 6-25 TARGET EXPRESSION ON NLC AND VIABILITY OF CLL CELLS. .........................................................................FIGURE 13. CHANGES IN THE VIABILITY AND CLL CELL CONCENTRATION IN CLL PBMC CULTURE FROM PATIENT 5 (FIG. 12A)........................................

Acknowledgements

Both mouse and chimeric variants of 6-25 mAb showed no significant in vitro toxicity toward CLL and normal human PBMC and also M1 and M2-MDM.

Effect of naked 6-25 antibody on the maturation and polarization of macrophages

The next step in characterization of 6-25 functionality, was evaluation of its effect on maturation and polarization of NLCs. For that, we cultured CLL PBMC at 10 × 10 6 c/mL, and treated the cells with 10 µg/mL of the mouse 6-25 mAb. We tested two different approaches: 1) with cells treated at day 6, when NLC are already advanced in the maturation process, in order to evaluate potential effect of the antibody on the polarization of NLC; 2) at day 0, right after the beginning of the culture to evaluate the effect of the antibody on the maturation of NLCs. At day 13, phenotype of NLCs was analyzed by FC (Fig. 29). Analysis of NLC from CLL cultures treated at day 6 (Fig. 29A), showed consistent staining with CD206, CD14, CD11c and HLA-DR between the cells cultured with the 6-25 antibody or isotype control. In case of CD163, DCSign (CD209), we observed very slight decrease in the signal and for CD16 increase in the signal in NLC cultured with the 6-25 mAb. In the end however, these changes were not significant.

Role of folate and FRβ in macrophages

Folate (folic acid, vitamin B9) is an essential nutrient for humans, and plays a role in one-carbon metabolism, DNA synthesis and cell proliferation 269 . Folate was shown to be a donor of methyl groups and contribute to inflammatory responses of monocytes and macrophages, by downregulating expression of pro-inflammatory genes, including interleukin 1β and TNF 272 . Prolonged treatment with folic acid (synthetic form of folate) showed decreased production of pro-inflammatory cytokines in humans 272 .

Another study indicated that folic acid diminished expression of TNF, IL-6, CSF-1, and CCL2 in GM-CSF activated M1 MDM upon LPS stimulation 269 .

Role of FRβ in macrophages is still not entirely clear. The receptor is obviously responsible for binding of folate, and its high expression in M2-like macrophages suggest involvement in preservation of this phenotype. Especially as FRβ high cells were shown to accumulate folate more efficiently, compared to M1-like macrophages, and folate was shown to blunt inflammatory responses 263 . This hypothesis is further supported by finding that Activin A, cytokine released by among others, GM-CSF activate macrophages that leads to downregulation of FOLR2 269 . Besides, FRβ might have a role in modulation of macrophage adhesion, by functional interaction with CD11b/CD18 that in turn led to decreased attachment to collagen 273 . Another potential role of FRβ could be associated with bacterial microbiota. Many bacteria in the gastro-intestinal tract are responsible for synthesis of folate, and there FRβ could help in sensing the uncontrolled overgrowth of microbes 263 .

Current state of FRβ-centered therapies

There are various characteristics that make FRβ a promising therapeutic target. This protein is exclusively expressed on myeloid cells, in particular on M2-like, immunosuppressive macrophages, connected with pathological inflammation 262,263 . In the context of cancer, these cells are shown to display protumoral functionality and their presence is correlated with bad prognosis and disease progression 258,260,262,263 .

Additionally, FRβ shows high affinity to folate and undergo quick internalization upon binding of this vitamin, making it a great tool to deliver therapeutic agents into the target cells 258 . All these yield multiple therapeutic strategies, targeting FRβ in cancer and auto-immune diseases.

We can differentiate different therapeutic approaches in targeting FRβ in cancer. One of them resolves around an α-FRβ monoclonal antibody, called "m909" that showed ADCC-mediated efficacy in elimination of primary human TAM and AML cells in dose-dependent manner in vitro. Additionally, authors demonstrated that m909 mAb treatment has a considerable impact on tumor growth in the humanized mouse model of AML, even without additional transfer of human NK cells 261 . In another study, authors used chimeric antigen receptor (CAR)-T cells, to selectively eliminate FRβ + TAM in the syngeneic mouse model of ovarian cancer. Targeting of FRβ + TAM, led in turn to increased infiltration with CD8 + cytotoxic Tcells and tumor regression, further showing importance of TAM in immunosuppression. Also, authors stressed importance of targeting of FRβ + as preparative adjuncts, before administration of classical immunotherapies, to observe synergistic effect of both treatments 258 . Another approach, utilized FRβ + antibody fragment (Fv), coupled with Pseudomonas exotoxin A, to form immunotoxin specific toward cells expressing FRβ + . This approach led to not only elimination of TAM, but also decreased tumor growth in the xenograft glioma mouse model 274 . Another study involved folate-modified lipoplex delivering BIM-s plasmid to FR expressing cells in a model of lung cancer. BIM stands for BCL-2-interacting mediator of cell death, which is a potent inducer of apoptosis. Administration of lipoplex with BIM-s plasmid turned out to effectively eliminate TAM and reduce tumor growth 259 .

Compared to cancer treatment, targeting of FRβ in autoimmune diseases has an even longer history. It utilizes analogous strategies, including monoclonal antibodies 272 , immunotoxins 275 and folic acid-drug conjugates 270,276 . Targeting of FRβ is these diseases demonstrated normalization of pathological inflammation, departure of pro-inflammatory cells, and symptom improvement in disease like rheumatoid arthritis (RA) 272,275,276 .

Finally, different folate-conjugates are utilized to visualize either FRβ + macrophages or FRα + cancer cells.

Compounds like [ 99 mTc]EC20 266 and [1 8 F]fluoro-PEG-folate 277 have been successfully used to monitor active disease in joints affected with RA. Folate-conjugates with near-infrared fluorophores, are also tested for fluorescence-guided cancer-resection surgeries, showing positive results in case of superficial imaging 270 .

All these promising results support the FRβ targeting in cancer, both as a mono-and combination therapy.

Moreover, imaging techniques utilizing folate-conjugates confirm selectivity of potential treatment to pathological tissues. Hopefully soon we will be able to see the first clinical trials involving FRβ-targeting.

List of abbreviations