Senovilla L Wang

Humeau J Perez-Lanzon

Martínez-Leal Jf

Mauseth B Wang

Wei Xie

Sabrina Forveille

Kristina Iribarren

Allan Sauvat

Laura Senovilla

Yan Wang

Juliette Humeau

Maria Perez-Lanzon

Heng Zhou

Juan F Martínez-Leal

Guido Kroemer
email: kroemer@orange.fr

Oliver Kepp

A L Orcid

Laura Mondragón

Brynjar Mauseth

Jonathan Pol

Sarah Lévesque

Takahiro Yamazaki

Johannes J Eksteen

Laurence Zitvogel

Baldur Sveinbjørnsson

Øystein Rekdal

Lurbinectedin synergizes with immune checkpoint blockade to generate anticancer immunity

Keywords: cancer, immunogenic cell death, immune checkpoint blockade, transcription, oncolytic peptide cancer, mort cellulaire immunogène, inhibiteur de point de contrôle immunitaire, transcription, peptide oncolytique Anticancer immunity, immunogenic cell death, checkpoint blockade, tumor clearance Oncolysis, anticancer therapy, immunogenic cell death, checkpoint blockade

Cancer is the second leading cause of death worldwide, despite the existence of standard treatment, innovative therapeutic strategies and drugs are still in urgent demand. The combination of immunogenic cell death (ICD) inducing drugs and immune checkpoint blockade (ICB) seems to be a promising modality.

In this thesis, we demonstrated Lurbinectedin, a transcription inhibitor newly approved for relapsed lung cancer treatment, triggers hallmarks of ICD in four different human and murine cell line in vitro. Vaccinated with Lurbinectedin-treated fibrosarcoma cell protects immunocompetent mice from rechallenge of syngeneic tumours. Lurbinectedin restrains transplanted fibrosarcoma growth in an immune dependent manner. Both transplanted MCA205 cancer and hormone/carcinogen induced breast cancer were sensitized by Lurbinectedin to PD-1 and CTLA-4 double ICBs. Of note, long-term immunological memory was generated in cured mice. Further, we evaluated the anticancer capacity of LTX-401, an oncolytic peptide designed for local immunotherapy. Sequential intratumoral injections of LTX-401 dramatically retards subcutaneous MCA205 and TC-1 tumour growth in immunocompetent host, yet shows limited therapeutic effect on abscopal syngeneic tumours. Single LTX-401 administration boosts the efficacy of anti-CTLA-4 or anti-PD-1/anti-CTLA-4 ICBs. Moreover, sequential LTX-401 treatment with double ICBs exhibits systemic antitumor immunity to both treated and abscopal tumour.

In conclusion, lurbinectedin and LTX-401 induce cancer cell immunogenic cell death and enhance the anticancer effects of immune checkpoint blockade. These results lay the experimental foundation of combination regiments and may facilitate the clinical trial designs.

as the 1 st leading cause of premature death (30 to 69 years old) in 55 countries, including USA, japan and the majority of European countries. In other 79 countries, cancer has been the second most important killer among premature populations. Specifically, more than 1.8 million Americans are estimated to be diagnosed as new invasive cancer cases in 2020, and nearly 600 000 deaths will occur in USA in the same year (Cancer statistics, 2020). In 2018, the top 3 common cancers in men are prostate cancer, lung cancer and colorectal cancer, which contributed almost 40% of total cancer incidence. Breast cancer (30%) is the most common cancer in women, followed by lung cancer (13%) and colorectal cancer (7%) (Cancer Facts & Figures 2018) (

Cancer immunology

The immune system has been defined as a host defence system which could recognise nonself-compartment and protect organism from a lot of diseases, especially for infectious disease.

The immune system can be divided into two parts: innate immunity and adaptive immunity. More and more researches demonstrated the pivotal function of innate and adaptive immunity in anticancer immune response [START_REF] Coca | The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma[END_REF][START_REF] Denardo | Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy[END_REF][START_REF] Donadon | Increased Infiltration of Natural Killer and T Cells in Colorectal Liver Metastases Improves Patient Overall Survival[END_REF][START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF][START_REF] Hildner | Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity[END_REF][START_REF] Ishigami | Prognostic value of intratumoral natural killer cells in gastric carcinoma[END_REF][START_REF] Keizman | The association of pre-treatment neutrophil to lymphocyte ratio with response rate, progression free survival and overall survival of patients treated with sunitinib for metastatic renal cell carcinoma[END_REF][START_REF] Pylayeva-Gupta | IL35-Producing B Cells Promote the Development of Pancreatic Neoplasia[END_REF].

From cancer immunosurveillance to cancer immunoediting

In the early 1900s, Paul Ehrlich firstly hypothesized that immune system could recognises cancer cells as extrinsic components and eliminates them effectively [START_REF] Burnet | The concept of immunological surveillance[END_REF][START_REF] Ehrlich | Ueber des jetzigen Stand der Karzinomforschung[END_REF].

After around half centenary, the theory of 'immunosurveillance' was formally proposed by Macfarlane Burnet and Lewis Thomas based on the availability of inbred strain of mice and accumulated evidence of 'tumour-specific antigen', they speculated that the non-stop emerging transformed cells could provoke immune response and be recognised and eliminated by lymphocytes before it becomes clinically detectable [START_REF] Burnet | Cancer: a biological approach. III. Viruses associated with neoplastic conditions. IV. Practical applications[END_REF][START_REF] Klein | Tumor antigens[END_REF][START_REF] Old | Immunology of Experimental Tumors[END_REF].

In the next dozens of years, the concept of immunosurveillance was in great debate. One of the representative studies which challenged this theory was conducted by Osias Stutman. Stutman observed the phenomenon that compared to genetically matched wild-type mice, carcinogen injection neither induced more tumours nor shorten tumour latency period in CBA/H strain nude mice [START_REF] Stutman | Tumor development after 3-methylcholanthrene in immunologically deficient athymic-nude mice[END_REF]. Moreover, similar results obtained by different groups conclusively destroyed the bases of immunosurveillance theory and led to the abandonment of this hypothesis [START_REF] Outzen | Spontaneous and induced tumor incidence in germfree "nude" mice[END_REF]Rygaard and Povlsen, 1974a, b;[START_REF] Stutman | Chemical carcinogenesis in nude mice: comparison between nude mice from homozygous matings and heterozygous matings and effect of age and carcinogen dose[END_REF]Thomas, 1982). Due to the solid data aforementioned, the concept of cancer immunosurveillance faded away since then.

The resuscitate of cancer immunosurveillance started from 1990s, due to the application of immunodeficiency mice on pure genetic backgrounds.

Between 1990 and 2000, the function of two fundamental immunological components, interferon γ (IFN-γ) and perforin, in controlling primary tumour formation were revealed. In the first finding, researchers observed that transplanted fibrosarcoma grows faster when host endogenous IFN-γ was neutralized by blocking antibodies [START_REF] Dighe | Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma receptors[END_REF]. Also, sarcoma cells which express dominant negative IFN-γ receptors (IFNGR1) showed increased tumorigenicity when implanted into syngeneic immunocompetent mice [START_REF] Dighe | Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma receptors[END_REF]. Both IFN-γ receptor deficient mice and STAT1 -/-mice in 129/SvEv background are much more sensitive to MCA carcinogen-induced fibrosarcoma [START_REF] Kaplan | Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice[END_REF]. Another study which employed different genetic background's IFN-γ -/-mice, reinforced the protective function of endogenous IFN-γ in restraining spontaneous tumour formation and transplanted tumour growth [START_REF] Street | Perforin and interferon-gamma activities independently control tumor initiation, growth, and metastasis[END_REF].

The next demonstrated key factor is perforin. Perforin is a glycoprotein which is mainly produced by natural killer (NK) cells and CD8 + T cells, upon interacting with target cell's membrane, channels and pores formed subsequently [START_REF] Osinska | Perforin: an important player in immune response[END_REF][START_REF] Russell | Lymphocyte-mediated cytotoxicity[END_REF]. Similar to IFN-γ -/-mice, perforin -/-mice developed more tumours and showed shorter tumour latency time post MCA incubation [START_REF] Smyth | Differential tumor surveillance by natural killer (NK) and NKT cells[END_REF][START_REF] Street | Perforin and interferon-gamma activities independently control tumor initiation, growth, and metastasis[END_REF][START_REF] Van Den Broek | Decreased tumor surveillance in perforin-deficient mice[END_REF]. In conclusion, these findings indicated the indispensable role of immunological components in subverting cancer development.

In 2001, a seminal study conducted by R.D. Schreiber's group in Washington University renovated the cancer immunosurveillance concept [START_REF] Shankaran | IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity[END_REF]. In this 5-pages short assay, firstly they showed inbred mice lacking either lymphocytes (RAG2 -/-) or IFN-γ response (IFNGR1 -/-/STAT1 -/-) were prone to grow MCA-induced tumour. Second, all of the RAG2 -/-or RAG2 -/-/STAT1 -/-mice developed spontaneous tumours in the age of 15-21 months, supported the theory of immunosurveillance. Next, they observed an interesting phenomenon that when transplanted MCA induced tumours into wild type naïve mice, all of the tumours (17/17) derived from immunocompetent mice grew normally. On the contrary, 40% of tumours (8/20) derived from RAG2 -/-immunodeficient mice were rejected post transplantation. The tumour rejection effect was abrogated when RAG2 -/-mice were used as recipients. This ingeniously designed experiment proved the qualitative differences between tumours from immunocompetent mice and immunodeficient mice, strongly supports the existence of tumour immunogenicity alteration during cancer development in immunocompetent hosts. In the last part, the authors proved that tumour rejection was T cell dependent. Consistent results were obtained by other group by using nude mice, SCID mice or TCR Jα281 -/-mice [START_REF] Engel | MCA sarcomas induced in scid mice are more immunogenic than MCA sarcomas induced in congenic, immunocompetent mice[END_REF][START_REF] Smyth | Differential tumor surveillance by natural killer (NK) and NKT cells[END_REF]Svane et al., 1996).

Given the fact that cancer occurs in the presence of immunosurveillance, and cancer cells with lower immunogenicity are more likely to survive compared to highly immunogenic ones, R.D.

Schreiber and his colleagues proposed a more comprehensive immune theory named Immunoediting. This term was further elucidated as three different phases 'elimination,'

'equilibrium,' and 'escape' [START_REF] Kim | Cancer immunoediting from immune surveillance to immune escape[END_REF][START_REF] Schreiber | Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion[END_REF] (Figure 2).

The elimination phase could be regarded as updated immunosurveillance, in this phase transformed cells undergo stress and release danger signals such as reactive oxygen species and inflammatory cytokines [START_REF] Kim | Cancer immunoediting from immune surveillance to immune escape[END_REF][START_REF] Matzinger | Tolerance, danger, and the extended family[END_REF]. These compartments could be sensed by different types of innate immune cells like dendritic cell, macrophage and NK cell, subsequently promote their activation. The activated innate immune cells could either directly kill transformed cells or trigger adaptive immune response to achieve clearance. Of note, both innate immune system and adaptive immune system are indispensable in eliminating transformed cell. If early tumours are eliminated, this phase is the end of the whole process.

However, if a small fraction of transformed cells survived from elimination phase, the subsequent equilibrium phase starts. In this phase, tumour cells are in a special state named 'functional dormancy' and under consistent immune pressure. In this circumstance, cancer cells keep evolving to gain resistance to immune recognition or induce an immunosuppressive tumour microenvironment, resulting in the generation of less immunogenic tumour clones. Importantly, cytokine IL-12 and IL-23 were reported to play opposing roles in this process (Teng et al., 2012).

This phase is a prolonged process, which may last from months to years, even to the end of host's life. Anti-tumour immunity and tumour promoting factors are in dynamic balance. As a result, tumour growth is strictly controlled by adaptive immune system and tumour immunogenicity is sculpted in the meantime. Particularly, innate immune response seems inessential in this period [START_REF] Kim | Cancer immunoediting from immune surveillance to immune escape[END_REF][START_REF] Mittal | New insights into cancer immunoediting and its three component phases--elimination, equilibrium and escape[END_REF].

In the escape phase, evolved tumour variants evade immunological control and grow progressively, leading to visible tumours. The escaping mechanisms are extensively discussed but not fully elucidated, the balance is towards to tumour progression perhaps due to poor immunogenicity or immunologic cytotoxicity resistance of cancer cells, or by the gradually established immunosuppressive tumour microenvironment [START_REF] Khong | Natural selection of tumor variants in the generation of "tumor escape" phenotypes[END_REF][START_REF] Radoja | Mice bearing late-stage tumors have normal functional systemic T cell responses in vitro and in vivo[END_REF]. The vast majority of clinically diagnosed cancers are in escape phase and immune system can no longer work alone to control tumour growth, therefore appropriate therapeutic strategies should be taken to achieve favourable outcomes. suppressor mechanisms have failed. In its most complex form, cancer immunoediting consists of three sequential phases: elimination, equilibrium, and escape [START_REF] Schreiber | Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion[END_REF].

Cancer immunoediting in humans

To investigate the existence of cancer immunoediting in humans, cancer incidence in immunodeficient or immunosuppressive populations are studied. Compare to general population, acquired immune deficiency syndrome (AIDS) patients are prone to several types of cancer, especially for virally induced tumour like Kaposi's sarcoma (KS) [START_REF] Mitsuyasu | AIDS-related malignancies[END_REF].

As a consequence of immune-suppressive treatment, patients who received transplant organs show higher risk of cancer. Birkeland analysed the cancer risk of 4178 patients who received renal transplantation between 1964 to 1986 in the Nordic countries and demonstrated an overall 3-to 5fold increase in the combined cohort. Specifically, the incidence of lip cancer, colon and rectum cancer, non-melanoma skin cancer, melanoma, lung cancer, thyroid cancer, non-Hodgkin's lymphoma elevated strikingly in both men and women [START_REF] Birkeland | Cancer risk after renal transplantation in the Nordic countries, 1964-1986[END_REF]. In another transplant recipients' study, the malignancy incidence of 905 patients who received heart or/and lung between 1989 and 2004 were evaluated. Among which 102 patients were diagnosed with cancer, more than 7 times higher than general population. The report revealed that lymphoproliferative disorders, head and neck cancer and lung cancer rank the top three common cancer types in the cohort [START_REF] Roithmaier | Incidence of malignancies in heart and/or lung transplant recipients: a single-institution experience[END_REF]. Of note, a review report from Cincinnati

Transplant Tumour Registry showed two-fold higher melanoma incidence in transplant patients as compared with general population [START_REF] Penn | Malignant melanoma in organ allograft recipients[END_REF]. Apart from that, other highly convincing results demonstrated the correlations between infiltrating immune cells and prognosis of patient, which strongly supported the principle of immunoediting theory [START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF].

Immunogenic cell death in cancer therapy

Depending on the capacity of activating an adaptive immune response, regulated cell death can be categorized as either immunogenic cell death (ICD) or non-immunogenic cell death [START_REF] Galluzzi | Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018[END_REF]. As a host defence approach, cells infected with virus or intracellular bacterial undergo ICD release antigens and alarmins, which stimulates immune response to eliminate pathogens. The very first demonstrated ICD inducing drug was doxorubicin, other chemotherapeutic agents like oxaliplatin, cyclophosphamide, or some physical approaches (radiotherapy, photodynamic therapy, high hydrostatic pressure) were also proved to be able to induce ICD (Apetoh et al., 2007;Casares et al., 2005;[START_REF] Fucikova | High hydrostatic pressure induces immunogenic cell death in human tumor cells[END_REF]Garg et al., 2012a;Obeid et al., 2007a;Tesniere et al., 2010;[START_REF] Vacchelli | Trial Watch: Chemotherapy with immunogenic cell death inducers[END_REF]. These agents could trigger the emission of damage-associated molecular patterns (DAMPs) from cancer cells, resulting in innate and adaptive immune cell activation and immunological memory generation [START_REF] Kepp | Consensus guidelines for the detection of immunogenic cell death[END_REF][START_REF] Lotze | Damage associated molecular pattern molecules[END_REF]. The major DAMPs and cytokines involved in ICD include calreticulin (CALR), adenosine triphosphate (ATP), high-mobility group box 1 (HMGB1), Type I interferon (IFN), CXCchemokine ligand 10 (CXCL10), annexin A1 (ANXA1) and so on. (Galluzzi et al., 2020b)(Table1). Of note, the same pathway in different forms of ICD may exhibits opposing effects [START_REF] Garg | ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death[END_REF].

In the context of ICD, CALR translocate from endoplasmic reticulum (ER) to cell surface, facilitating antigen uptake by dendritic cell (DC) (Gardai et al., 2005;Obeid et al., 2007b). ATP released by dying tumour cell could be sensed by DC through purinergic receptor P2Y2 (P2RY2)

and purinergic receptor P2X7 (P2RX7), which mediate DC recruitment and activation respectively [START_REF] Elliott | Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[END_REF]Ma et al., 2013;Michaud et al., 2011). HMGB1 emitted from cancer cell nuclear binds to Toll-like receptor 4 (TLR4) to promotes DC maturation [START_REF] Sims | HMGB1 and RAGE in inflammation and cancer[END_REF].

Tumour cell secreted Type I IFN interacts with self-interferon α/β-receptor subunit 1 (IFNAR1)

to trigger autocrine and paracrine circuitries, leading to the release of CXCL10. As a chemoattractant, CXCL10 recruits both αβ and γδ T cells to tumour site [START_REF] Harding | Mitotic progression following DNA damage enables pattern recognition within micronuclei[END_REF][START_REF] Mackenzie | cGAS surveillance of micronuclei links genome instability to innate immunity[END_REF]Sistigu et al., 2014;[START_REF] Vanpouille-Box | DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity[END_REF]. Extra extracellular ANXA1 from cancer cell engages with formyl peptide receptor 1 (FPR1) on DC, resulting in the formation of corpse/DC synapses [START_REF] Baracco | Contribution of annexin A1 to anticancer immunosurveillance[END_REF]Vacchelli et al., 2015). Together, these processes convert dying tumour cells into 'cancer vaccines', which activates DC and subsequently mediate T cell priming and memory T cell induction (Galluzzi et al., 2017)(Figure 3).

Table 1. Major immunostimulatory DAMPs and cytokines mechanistically linked to ICD in cancer. Adapted from (Galluzzi et al., 2020b). IFNAR1; interferon α/β-receptor subunit 1; IL, interleukin; LRP1, LDL receptor related protein 1; P2RX7, purinergic receptor P2X7; P2RY2, purinergic receptor P2Y2; TLR4, Toll-like receptor 4 (Galluzzi et al., 2017).

Major DAMPs of ICD Calreticulin exposure

As one of the most abundant proteins in ER, CALR plays fundamental role in calcium homeostasis and protein folding [START_REF] Michalak | Calreticulin: one protein, one gene, many functions[END_REF]. Upon ICD treatment, CALR translocate from ER to cell surface in cooperate with other chaperone proteins [START_REF] Asadzadeh | Current Approaches for Combination Therapy of Cancer: The Role of Immunogenic Cell Death[END_REF]Fucikova et al., 2011;Garg et al., 2012b;[START_REF] Panaretakis | The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death[END_REF](Figure 4). As an 'eat me' signal, cells exhibiting CALR on surface can be recognised by phagocytes through low density lipoprotein receptor-related protein 1 (LRP1; also known as CD91), resulting in antigen uptake and clearance of cellular components [START_REF] Bergmann | Endoscopic evaluation of the effect of ketoprofen, ibuprofen and aspirin on the gastroduodenal mucosa[END_REF]Garg et al., 2012b). On the contrary, this process is impaired when CD47 (a don't eat me signal) co-expressed on cancer cells [START_REF] Chao | Programmed cell removal: a new obstacle in the road to developing cancer[END_REF].

Also, CALR-CD91 conjunction on antigen-presenting cells (APCs) primes Th-17 cell response in an Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) dependent manner [START_REF] Pawaria | CD91-dependent programming of T-helper cell responses following heat shock protein immunization[END_REF]. Of note, CALR ablation by siRNA or neutralising antibody abolish the immune response towards dying tumour cell in vivo, while incubation with recombinant CALR protein restores the immunogenicity of the cells (Liu et al., 2019;Obeid et al., 2007b). Moreover, the immunogenicity deficiency mediated by PERK, caspase-8 or SNAREs depletion in anthracyclines induced cell death can also be restored by absorbing recombinant CRT to the cell surface [START_REF] Panaretakis | Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death[END_REF]. Altogether, these results definitely indicated that the immunogenicity of ICD is defined by cell surface CALR.

Importantly, CALR exposure is correlated with clinical outcomes. In human non-small cell lung cancers (NSCLC), high expression of CALR correlates with increased antitumor immune response and favourable prognosis (Fucikova et al., 2016a). CALR level cloud also be used as an independent prognostic factor in neuroblastoma [START_REF] Hsu | Calreticulin expression in neuroblastoma--a novel independent prognostic factor[END_REF]. Similarly, high level of CALR is associated with more infiltrated CD45RO + cells in colon cancer, which correlates with a higher 5-year survival rate of patients [START_REF] Peng | Expression of calreticulin is associated with infiltration of T-cells in stage IIIB colon cancer[END_REF]. In acute myeloid leukaemia (AML) patients, CALR exposure on the membrane of malignant cells positively correlated with leukaemia specific immune response, further associated with both improved relapse-free survival and superior overall survival (Fucikova et al., 2016b). Moreover, low CALR level was associated with immunogenic chemotherapeutic resistance and poor survival rate in endometrial cancer patients [START_REF] Xu | Significance of calreticulin as a prognostic factor in endometrial cancer[END_REF]. BCL2-associated X protein, Bak; Bcl-2 homologous antagonist/killer, SNARE; soluble N-ethylmaleimidesensitive fusion protein-attachment protein receptor [START_REF] Asadzadeh | Current Approaches for Combination Therapy of Cancer: The Role of Immunogenic Cell Death[END_REF].

ER stress

ER stress is considered to initiate CALR translocation in response to ICD induction. During homeostatic conditions, protein translation goes smoothly in ER, while the three unfolded protein response (UPR) mediators are bind with heat shock protein 70 (Hsp70)-type chaperone protein Bip and stay in inactive state. In response to exogenous or endogenous ER stress, Bip preferably binds with misfolded proteins thus dissociates with these UPR mediators, leading to the activation of Inositol-requiring enzyme 1α (IRE1α), PKR-like ER-resident kinase (PERK) and activating transcription factor 6α (ATF6α). Upon Bip dissociation, 1) IRE1α forms a homodimer and undergo autophosphorylation, activating its C-terminal endoribonuclease activity. IRE1α splices the XBP-1 mRNA and facilitates XBP-1 expression, which further promotes the transcription of protein chaperones, glycosylase, and ER-associated degradation (ERAD) system [START_REF] Lee | XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response[END_REF].

Additionally, IRE1α could directly modulate the abundance of ER-localized mRNAs through regulated IRE1α-dependent decay (RIDD) pathway; 2) As an ER-resident eukaryotic translation initiation factor 2 (eIF2α) protein kinase, PERK phosphorylates the translation initiation factor eIF2α under ER stress conditions, abolishing 5′-cap assembly-dependent translation [START_REF] Harding | Perk is essential for translational regulation and cell survival during the unfolded protein response[END_REF][START_REF] Harding | Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase[END_REF]. Meanwhile, the general translation inhibition enhances the activating transcription factor 4 (ATF4) production, driving the expression of downstream transcription factor CHOP [START_REF] Ma | Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response[END_REF][START_REF] Vattem | Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells[END_REF]; 3) Post dissociation with Bip, ATF6α translocates into Golgi and activated by proteolytic cleavage, promoting the accumulation of ERAD components and chaperones [START_REF] Bailey | Transmembrane bZIP transcription factors in ER stress signaling and the unfolded protein response[END_REF][START_REF] Haze | Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress[END_REF][START_REF] Shen | ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals[END_REF]. Although these three sensors could coordinate to combat ER stress, the individual UPR signalling pathways varies in different circumstances [START_REF] Bettigole | Endoplasmic reticulum stress in immunity[END_REF][START_REF] Shoulders | Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments[END_REF].

Interestingly, ICD inducers like anthracyclines and oxaliplatin specifically induce PERKdependent eIF2α phosphorylation, yet neither activating downstream ATF4 nor other two ER stress branches (Bezu et al., 2018a)(Figure 5). But the precise mechanism of ICD induced split ER stress response remains to be investigated. It's demonstrated that PERK mediated eIF2α phosphorylation is mandatory for CALR exposure in response to ICD induction in several mouse and human cell lines (Bezu et al., 2018a;[START_REF] Panaretakis | Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death[END_REF]. However, it is protein kinase R (PKR) and general control nonderepressible 2(GCN2), rather than PERK, trigger eIF2α phosphorylation and CALR exposure in mitoxantrone (MTX) or doxorubicin (DOXO) treated melanoma cells (Giglio et al., 2018). Moreover, eIF2α phosphorylation and caspase-8 activation are dispensable in Hyp-PDT mediated CALR exposure, indicating that different ICD inducers may functions distinctively (Garg et al., 2012b). Of note, tumoral p-eIF2α and CALR expression indicate positive prognostic value in NSCLC patients (Fucikova et al., 2016a). Altogether, p-eIF2α could be employed as a hallmark of ICD and promising biomarker in clinical prognosis prediction although exceptions exist. without BiP occupation by misfolded proteins and/or second, ICD-inducers inhibit UPR downstream signals such as ATF4 translation, ATF6 cleavage and the IRE1α-mediated XBP1 splicing [START_REF] Bezu | eIF2alpha phosphorylation: A hallmark of immunogenic cell death[END_REF].

ATP release

As a multi-functional molecular expelled by dying cells in ICD event, the extracellular ATP plays an important role in immune response. Upon secretion, ATP binds to P2X7 and P2Y2 on APCs, promoting their maturation and facilitates chemotactic effect respectively [START_REF] Elliott | Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[END_REF]Ghiringhelli et al., 2009;Michaud et al., 2011). In DCs, ATP triggers the aggregation of NLRP3-ASC-Casp-1 inflammasome and facilitates IL-1β release, favouring the prime of antigenspecific cytotoxic T cell and IL-17 producing-γδ T cell. Thus, deficiency of any of these genes (Nlrp, P2rx7, Casp1) attenuates the establishment of adaptive immune response in chemotherapeutic ICD (Ghiringhelli et al., 2009;[START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF]. Besides, ATP could modulate DC activity simultaneously by stimulating the expression of costimulatory molecules include CD40, CD80, and CD86 [START_REF] Idzko | Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells[END_REF].

The molecular mechanism of ATP secretion during ICD is often related to autophagy, a tightly regulated pathway mediates the recycle of cytosolic components in response to stress [START_REF] Martins | Molecular mechanisms of ATP secretion during immunogenic cell death[END_REF]Michaud et al., 2011). When treated with ICD inducer MTX or oxaliplatin, autophagy deficient cancer cells (by knock-down or knock-out of essential autophagy proteins or pharmacological inhibition) secrete much less ATP yet release similar amount of HMGB1 and expose equal level of CALR on cell surface . Immunization with MTX treated autophagy deficient CT26 or MCA205 cells failed to protect immunocompetent mice from re-challenging of the same cell type, while its immune stimulating capacity could be restored by co-injection of rIL-1β but not rCALR (Michaud et al., 2011). Moreover, autophagy-deficient tumour exhibits impaired tumour-infiltrating lymphocytes recruitment in response to immunogenic chemotherapy, but intratumoral administration of ecto-ATPase inhibitor regenerate therapeutic immune response in immunocompetent hosts [START_REF] Chao | Programmed cell removal: a new obstacle in the road to developing cancer[END_REF]. Accordingly, these results indicate that autophagy is required for ATP release during chemotherapy. Following research revealed a more complex machinery in regulating ATP secretion by dying cancer cell. Several relatively independent molecules include autophagy related proteins, caspases, Rho-associated, coiled-coil containing protein kinase 1 (ROCK1), PANX1 channels and LAMP1 are involved in ICD-associated ATP release. In other words, autophagy, lysosomal exocytosis, apoptosis, membrane blebbing and plasma membrane permeabilization are orchestrated in ATP secretion [START_REF] Martins | Molecular mechanisms of ATP secretion during immunogenic cell death[END_REF].

Apart from autophagy, the extracellular ATP level could also be regulated by CD39 and CD73

ectonucleotidase. CD39 and CD73 convert ATP to immunosuppressive adenosine, resulting in the establishment of a tumour promoting microenvironment [START_REF] Chalmin | Stat3 and Gfi-1 transcription factors control Th17 cell immunosuppressive activity via the regulation of ectonucleotidase expression[END_REF]Sun et al., 2010).

Mice lacking CD73 shows resistance to MCA-induced fibrosarcoma and increased anti-tumour immunity, CD73 antibody treatment restrains prostate tumour growth and metastasis [START_REF] Stagg | CD73-deficient mice are resistant to carcinogenesis[END_REF](Figure 6).

Breast cancer patients bearing the loss-of-function (Glu496Ala) P2RX7 allele are prone to cancer metastasis post anthracyclines treatment, comparing to P2RX7 normal counterpart (Ghiringhelli et al., 2009). In a cohort of colorectal cancer (CRC) patients, absence of Beclin-1 expression is associated with poor prognosis [START_REF] Choi | Absence of autophagy-related proteins expression is associated with poor prognosis in patients with colorectal adenocarcinoma[END_REF]. Similarly, another CRC study

shows that the LC3 level is positively correlated with long-term survival [START_REF] Wu | Expression and clinical significances of Beclin1, LC3 and mTOR in colorectal cancer[END_REF].

HMGB1 exodus

HMGB1 is the first identified HMGB family protein which locates in the nuclear as nonhistone chromatin-binding protein in steady state [START_REF] Palumbo | Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation[END_REF]. The function of HMGB1 mainly depends on its sublocation, namely nuclear, cytoplasm or extracellular milieu [START_REF] Garg | Immunogenic cell death, DAMPs and anticancer therapeutics: an emerging amalgamation[END_REF]. Within nuclear, HMGB1 binds to chromatin to stabilize the structure and modulates transcription [START_REF] Garg | Immunogenic cell death, DAMPs and anticancer therapeutics: an emerging amalgamation[END_REF][START_REF] Scaffidi | Release of chromatin protein HMGB1 by necrotic cells triggers inflammation[END_REF]. In cytoplasm, HMGB1 can interacts with

Beclin-1 to affect autophagy [START_REF] Huang | Targeting HMGB1-mediated autophagy as a novel therapeutic strategy for osteosarcoma[END_REF]Tang et al., 2010). It's reported that HMGB1

regulates the expression of various genes including p53 and nuclear factor-κB (NF-κB) [START_REF] Krysko | Immunogenic cell death and DAMPs in cancer therapy[END_REF]. Extracellular HMGB1 could directly interact with different surface receptors like RAGE (receptor for advanced glycation end-products), TLR2 and TLR4, to induce significant immune response in a cytokine-like manner [START_REF] Sims | HMGB1 and RAGE in inflammation and cancer[END_REF]. HMGB1 stimulates the production of a series of proinflammatory cytokines in human monocytes, but not lymphocytes. Accordingly, intraperitoneal administration of rHMGB1 increase TNF concentration in mice serum [START_REF] Zhang | HMGB1 induced inflammatory effect is blocked by CRISPLD2 via MiR155 in hepatic fibrogenesis[END_REF]. In the context of ICD, TLR4 seems to be the main HMGB1 receptor which contributes to tumour eradiation. HMGB1 inhibition and TLR4 deletion compromise the protective efficiency of dying tumour in vaccination experiment. Besides, the immune response to dying tumour cell is largely depends on TLR4 expression on DC. Moreover, both myeloid differentiation primary response 88 (MYD88) -/-mice and TLR4 -/-mice failed to response to chemotherapy or radiotherapy, demonstrating the contribution of HMGB1/TLR4/MyD88 axis in anticancer immunity (Apetoh et al., 2007). It should be noticed that the function of HMGB1 is determined by its redox modification, HMGB1 acts as chemoattractant DAMP, pro-inflammatory cytokineinducing DAMP or inactivated DAMP depending on actual reduced to oxidized forms [START_REF] Venereau | Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release[END_REF][START_REF] Yang | Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1)[END_REF])(Figure 7).

HMGB1 can be released in two different fashions, named active secretion or passive liberation.

Macrophages and monocytes secret HMGB1 through a cytokine secreting manner post IL-1β, TNF or lipopolysaccharides (LPS) stimulation [START_REF] Garg | Immunogenic cell death, DAMPs and anticancer therapeutics: an emerging amalgamation[END_REF][START_REF] Muller | Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function[END_REF]. Primary necrosis cells could passively release large amount of HMGB1 to extracellular space [START_REF] Scaffidi | Release of chromatin protein HMGB1 by necrotic cells triggers inflammation[END_REF].

Also, Cells undergo secondary necrosis discharge HMGB1. In particular, Z-VAD-fmk (a broad caspase inhibitor) treatment reduces the release of HMGB1 from ICD (Apetoh et al., 2007;[START_REF] Bell | The extracellular release of HMGB1 during apoptotic cell death[END_REF].

A study showed both TLR4 Asp299Gly and Thr399Ile polymorphisms were positively associated with cancer development and progression in CRC patients, and the expression of these mutations impact patients' survival [START_REF] Messaritakis | Evaluation of the detection of Toll-like receptors (TLRs) in cancer development and progression in patients with colorectal cancer[END_REF]. Also, Thr399Ile polymorphism was significantly associated with an elevated gastrointestinal cancer risk [START_REF] Jing | Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms in cancer: a meta-analysis[END_REF]. Breast cancer patients bearing mutated TLR4 alleles relapse faster post anthracycline-based chemotherapy and local irradiation (Apetoh et al., 2007). However, controversial results showed no correlations between CRC risk and Asp299Gly or Thr399Ile alleles [START_REF] Davoodi | Variant Toll-like receptor4 (Asp299Gly and Thr399Ile alleles) and Toll-like receptor2 (Arg753Gln and Arg677Trp alleles) in colorectal cancer[END_REF]. state, receptors and their interactive partners [START_REF] Chen | Emerging role of highmobility group box 1 (HMGB1) in liver diseases[END_REF].

Methods to detect ICD

As aforementioned, ICD inducers cause cancer cell death in parallel with the emission of DAMPs, which includes split ER stress induced CALR translocation, autophagy dependent ATP release, HMGB1 exodus and Type I IFN secretion. These DAMPs define its adjuvanticity and triggers adaptive immune response in company with antigenicity. One thing should be emphasized here is the precise mechanisms of ICD are not fully addressed, the immunogenicity of agents should be tested both in vitro and in vivo.

A drug discovery pipeline was constructed to identify ICD inducers from large libraries (Bezu et al., 2018a;Kepp et al., 2019)(Figure 8). This pipeline consists four procedures, 1) use artificial intelligence to pre-select drug candidates; 2) high content screening to find hits; 3) in vitro validation and 4) in vivo validation. In vivo vaccination assay is employed as the 'Gold Standard' to evaluate immunogenicity. In brief, tumour cells were treated with cytotoxic ICD inducer candidates in vitro, then immunocompetent naïve mice were injected with these dying tumour cells without adjuvant in the flank subcutaneously, after one to two weeks these vaccinated mice were rechallenged by the syngeneic living tumour cell in the contralateral flank, tumour emergence and growth were monitored. The 'real' ICD inducer treated cells could protect mice from growing tumours, or at least delay tumour growth and extends lifespan compare to 'PBS' group and 'non-ICD' group [START_REF] Humeau | Gold Standard Assessment of Immunogenic Cell Death in Oncological Mouse Models[END_REF](Figure 9). Given this in vivo vaccination experiment is only applicable for mouse cancer cell line, the generation of humanized mice would be particularly useful in predicting ICD in human cancers.

ICD inducers

Doxorubicin was the first identified ICD inducer in 2005, as tumour cells treated by DOXO could effectively elicit anticancer immunity in the absence of adjuvant (Casares et al., 2005). Since then, numerous ICD inducers were discovered by different research groups. ICD inducers could be categorized into four categories: chemotherapy, physical approach, immunotherapy and target therapy. As a well-studied ICD inducer cluster, chemotherapeutic drugs such as anthracyclines, platinum derivatives, alkylating agents and proteasome inhibitors are proved to trigger ICD potently (Casares et al., 2005;[START_REF] Panaretakis | Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death[END_REF][START_REF] Schiavoni | Cyclophosphamide synergizes with type I interferons through systemic dendritic cell reactivation and induction of immunogenic tumor apoptosis[END_REF][START_REF] Spisek | Bortezomib enhances dendritic cell (DC)-mediated induction of immunity to human myeloma via exposure of cell surface heat shock protein 90 on dying tumor cells: therapeutic implications[END_REF].

Radio therapy and Photodynamic therapy are also effective approaches to induce ICD [START_REF] Demaria | Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated[END_REF][START_REF] Formenti | Radiotherapy induces responses of lung cancer to CTLA-4 blockade[END_REF][START_REF] Korbelik | Photodynamic therapy-mediated immune response against subcutaneous mouse tumors[END_REF][START_REF] Korbelik | Involvement of damage-associated molecular patterns in tumor response to photodynamic therapy: surface expression of calreticulin and highmobility group box-1 release[END_REF][START_REF] Krosl | Induction of immune cell infiltration into murine SCCVII tumour by photofrin-based photodynamic therapy[END_REF][START_REF] Paz-Ares | Outcomes with durvalumab by tumour PD-L1 expression in unresectable, stage III non-small-cell lung cancer in the PACIFIC trial[END_REF]. Some oncolytic virus and oncolytic peptides induce traits of ICD [START_REF] Koks | Newcastle disease virotherapy induces longterm survival and tumor-specific immune memory in orthotopic glioma through the induction of immunogenic cell death[END_REF]Yamazaki et al., 2016;[START_REF] Zamarin | Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy[END_REF]Zhou et al., 2016a;Zhou et al., 2018). Only specific target therapy drugs like Crizotinib and Cetuximab are capable to generate ICD (Liu et al., 2019;[START_REF] Pozzi | The EGFR-specific antibody cetuximab combined with chemotherapy triggers immunogenic cell death[END_REF]. Confirmed ICD inducers are listed in

Induction of ICD sensitize cancers to immune checkpoint blockade

Immunotherapy, particularly immune checkpoint blockade (ICB), is now largely applied to treat various cancers. But due to the immunosuppressive nature of tumour microenvironment, the objective response rates (ORRs) of standard ICB treatment in solid tumours are relatively low [START_REF] Lim | Pembrolizumab for the treatment of non-small cell lung cancer[END_REF][START_REF] Rosenberg | Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial[END_REF]Topalian et al., 2012). ICD induction in tumour sites enables to recruit antigen presenting cells and CD8 T cells to the tumour bed, thus convert immunologically 'cold' tumours into 'hot' lesions. Given that ICB's therapeutic function dedicated to the presence of CD8 T cells, pre-treatment with ICD inducers sensitize tumours to ICB in several studies (Liu et al., 2019;Pfirschke et al., 2016;[START_REF] Yamazaki | PT-112 induces immunogenic cell death and synergizes with immune checkpoint blockers in mouse tumor models[END_REF].

In a cohort of NSCLC patients, chemoradiotherapy plus programmed death-ligand 1 (PD-L1) blockade dramatically prolonged the overall survival of combination treatment group against placebo group [START_REF] Antonia | Overall Survival with Durvalumab after Chemoradiotherapy in Stage III NSCLC[END_REF]. In another study involves metastatic triple-negative breast cancer patients, doxorubicin treatment upregulates the expression of key genes which related to programmed cell death protein 1 (PD-1)/PD-L1 and T cell cytotoxicity pathways, thus evaluated the objective response rate to PD-1 blockade [START_REF] Voorwerk | Immune induction strategies in metastatic triple-negative breast cancer to enhance the sensitivity to PD-1 blockade: the TONIC trial[END_REF]. In addition, bunch of clinical trials that involves the combination of immunogenic chemotherapy and ICBs are still ongoing (Galluzzi et al., 2020a)(Figure 10).

Altogether, both preclinical and clinical studies reveal that ICD-inducing chemotherapy may bring benefits to cancer patients in some immunotherapy included settings. But the optimal therapeutic combination regiments remain to be critically evaluated. FDA-approved immune checkpoint inhibitors in patients with cancer that were initiated after 1 January, 2019. CTLA4, cytotoxic T lymphocyte-associated protein (Galluzzi et al., 2020a).

Transcription inhibitor Lurbinectedin

Lurbinectedin (PM01183) is a novel synthetic marine-derived anticancer agent developed by PharmaMar Company. It was recently approved by Federal Drug Administration (FDA) for the salvage treatment of small-cell lung cancer (SCLC). As an alkaloid, lurbinectedin could binds to the DNA minor groove, induces double-strand DNA breaks, selectively blocks RNA polymerase II activity and promotes its degradation, disrupts mitosis and eventually induces cell death [START_REF] Leal | PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity[END_REF][START_REF] Santamaria Nunez | Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells[END_REF][START_REF] Vidal | Lurbinectedin (PM01183), a new DNA minor groove binder, inhibits growth of orthotopic primary graft of cisplatin-resistant epithelial ovarian cancer[END_REF]. As an analogue of trabectedin, the structure of lurbinectedin is similar to trabectedin except the modification in C subunit [START_REF] Leal | PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity[END_REF]. Thus, lurbinectedin gains different pharmacokinetic and pharmacodynamics properties from trabectedin [START_REF] Bueren-Calabuig | Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations[END_REF][START_REF] Leal | PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity[END_REF][START_REF] Soares | Trabectedin and its C subunit modified analogue PM01183 attenuate nucleotide excision repair and show activity toward platinum-resistant cells[END_REF].

It's reported that lurbinectedin has potent antiproliferative activity in numerous cancer cell lines in vitro, including cisplatin-sensitive and resistant cells [START_REF] Leal | PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity[END_REF][START_REF] Santamaria Nunez | Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells[END_REF][START_REF] Soares | Trabectedin and its C subunit modified analogue PM01183 attenuate nucleotide excision repair and show activity toward platinum-resistant cells[END_REF]. Single lurbinectedin treatment exhibits antitumor/antimetastatic activity in MNMCA1, M5076 murine reticulosarcoma, HOC8 and MNBPTX human ovarian carcinoma xenograft in vivo [START_REF] Romano | Comparison of in vitro and in vivo biological effects of trabectedin, lurbinectedin (PM01183) and Zalypsis(R) (PM00104)[END_REF]. In preclinical uterine cervical cancer models, lurbinectedin inhibits tumour growth by eliminating both cancer stem cell and non-cancer stem cell [START_REF] Yokoi | Lurbinectedin (PM01183), a selective inhibitor of active transcription, effectively eliminates both cancer cells and cancer stem cells in preclinical models of uterine cervical cancer[END_REF]. Lurbinectedin inactivates oncoprotein EWS-FLI1 and synergized with irinotecan to retard Ewing sarcoma xenograft growth [START_REF] Harlow | Lurbinectedin Inactivates the Ewing Sarcoma Oncoprotein EWS-FLI1 by Redistributing It within the Nucleus[END_REF]. Also, lurbinectedin therapy overcomes cisplatin resistance in in epithelial ovarian cancer [START_REF] Vidal | Lurbinectedin (PM01183), a new DNA minor groove binder, inhibits growth of orthotopic primary graft of cisplatin-resistant epithelial ovarian cancer[END_REF]. In particular, lurbinectedin depletes tumour-associated macrophages and modulates inflammatory microenvironment in both mouse fibrosarcoma tumour model and patient-derived (AVATAR)

PDA xenografts [START_REF] Belgiovine | Lurbinectedin reduces tumourassociated macrophages and the inflammatory tumour microenvironment in preclinical models[END_REF][START_REF] Cespedes | Lurbinectedin induces depletion of tumor-associated macrophages, an essential component of its in vivo synergism with gemcitabine, in pancreatic adenocarcinoma mouse models[END_REF][START_REF] Farago | ATLANTIS: a Phase III study of lurbinectedin/doxorubicin versus topotecan or cyclophosphamide/doxorubicin/vincristine in patients with small-cell lung cancer who have failed one prior platinum-containing line[END_REF](Figure 11). The approval of lurbinectedin for second line SCLC treatment was based on several clinical trials. In a Phase I study enrolled 27 relapsed SCLC patients, the combination regiment of lurbinectedin and doxorubicin showed remarkable anticancer activity. As second-line treatment, 11 out of 12 patients with platinum-sensitive disease and 3 out of 9 patients with platinum-resistant disease were responded to the combination treatment [START_REF] Calvo | Antitumor activity of lurbinectedin (PM01183) and doxorubicin in relapsed small-cell lung cancer: results from a phase I study[END_REF]. In a single-arm, openlabel phase II basket trial, 105 patients with relapsed SCLC were treated with lurbinectedin alone.

The median overall survival (OS) was 9.3 months, 11.9 months, 5.0 months in the whole cohort, patients with chemotherapy sensitive or resistant disease respectively. Among all patients the overall response rate (ORR) was 35.2%, whereas 45.0% in chemotherapy sensitive subgroup and 22.2% in chemotherapy resistant subgroup. Further, lurbinectedin was of good safety, no patient dead due to treatment related advise effects (Trigo et al., 2020). It's notable that the combination therapy of lurbinectedin and atezolizumab (anti-PD-L1 antibody) in patients with progressed SCLC is under evaluation (ClinicalTrials.gov, NCT04253145). Apart from SCLC, lurbinectedin is in therapeutic trials for multiple cancer types, including breast cancer, ovarian cancer, sarcoma, acute leukaemia and different advanced solid tumours (Markham, 2020)(Table 3).

Table 3. Key clinical trials of lurbinectedin. mCRC metastatic colorectal cancer, NSCLC non-small cell lung cancer, SCLC small cell lung cancer [START_REF] Markham | Lurbinectedin: First Approval[END_REF].

Oncolytic peptide LTX-401

Antimicrobial peptides (AMPs) are a series of short cationic amphipathic peptides that protect hosts from various microorganisms [START_REF] Fjell | Designing antimicrobial peptides: form follows function[END_REF][START_REF] Hancock | Antimicrobial and host-defense peptides as new antiinfective therapeutic strategies[END_REF]. More than 2000 kinds of natural AMPs were discovered currently, but no conserved motif or sequence has been identified yet [START_REF] Wang | APD3: the antimicrobial peptide database as a tool for research and education[END_REF]. Apart from anti-microorganism activity, some AMPs exert anticancer property by directly lysing cancer cells (Al-Benna et al., 2011;[START_REF] Gaspar | From antimicrobial to anticancer peptides. A review[END_REF].

Bovine lactoferricin (LfcinB) is the best studied natural AMP which is derived from milk protein.

It's reported that LfcinB could induce cancer cell necrosis and apoptosis by destabilise membranes [START_REF] Eliassen | The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo[END_REF][START_REF] Eliassen | Evidence for a direct antitumor mechanism of action of bovine lactoferricin[END_REF][START_REF] Mader | Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines[END_REF][START_REF] Yoo | Apoptosis in human leukemic cells induced by lactoferricin, a bovine milk protein-derived peptide: involvement of reactive oxygen species[END_REF]. Based on the structure of LfcinB, a large number of oncolytic peptides were designed and synthetized to treat cancer. One of these oncolytic peptides is LTX-401, an amphiphatic β (2,2)-amino acid derivate that is able to lysis different type of cells (Eike et al., 2016) (Figure 12). Previous data demonstrated that LTX-401 preferentially enriches in Golgi apparatus and destroys its structure, subsequently inducing cell death. The presence of brefeldin A (BFA) reduces the cytotoxicity of LTX-401 not only due to the prophylactic dissociation of Golgi, but also through decreasing mitochondrial permeabilization. BAX/BAK deficient or mitochondrial depleted cells show partial resistance to LTX-401 mediated killing, while pan-caspase inhibitor Z-VAD-fmk doesn't exhibit any protective effect [START_REF] Zhou | The oncolytic compound LTX-401 targets the Golgi apparatus[END_REF]. In addition, LTX-401 treatment impairs lysosomal integrity and induces the release of reactive oxygen species (ROS).

Massive DAMPs are released following LTX-401 treatment, including ATP, Type I IFN, HMGB1, cytochrome c and CALR exposure. Intra-tumour injection of LTX-401 causes focal necrosis and T lymphocytes infiltration (Eike et al., 2016;[START_REF] Mauseth | The Novel Oncolytic Compound LTX-401 Induces Antitumor Immune Responses in Experimental Hepatocellular Carcinoma[END_REF][START_REF] Zhou | The oncolytic compound LTX-401 targets the Golgi apparatus[END_REF].

The antitumor capacity of LTX-401 were verified in several cancer models. Local administration of LTX-401 in 3 consecutive days cured more than 80% of B16F1 melanoma (Eike et al., 2016). In subcutaneous JM1 tumour model, 7 out of 10 tumours were completed regressed post LTX-401 treatment. All cured animals were resistant to both subcutaneous and intrahepatic rechallenge of the same tumour cell. More convincing date was obtained from rat orthotopic JM1 liver cancer model, a preclinical model of human hepatocellular carcinoma (HCC). 5 out of 9 mice were cured from orthotopic JM1 liver tumour, by intrahepatic injection of LTX-401 on day 6 and day 8 post tumour incubation. Again, cured mice generated long-term antitumor immunity [START_REF] Mauseth | The Novel Oncolytic Compound LTX-401 Induces Antitumor Immune Responses in Experimental Hepatocellular Carcinoma[END_REF].

AIMS OF THE THESIS

Cancer cells undergo immunogenic cell death effectively provoke anticancer immune response in immunocompetent host. Therefore, ICD inducers could somehow convert dying tumour cells to therapeutic 'vaccines', attracting leukocytes to tumour beds and thus switching from the immunosuppressive tumour microenvironment to a proinflammatory context. In addition, both preclinical study and clinical trials support the hypothesis that pre-treatment with a panel of ICD inducers sensitize tumours to immune checkpoint blockade.

Based on this rationale, it's of great value to evaluate the capacity of drugs/compounds in inducing ICD and potential benefits in combination with ICBs.

Lurbinectedin is a RNA polymerase II inhibitor which was newly approved by FDA for cancer treatment. The majority of previous studies investigated its anticancer ability in immunodeficient host, thus the immunogenicity of lurbinectedin induced cell death is still unknown.

As an oncolytic peptide, LTX-401 is designed for local administration. Past research shows that LTX-401 could selectively concentrate in and destroy Golgi apparatus, inducing all the hallmarks of ICD in vitro. But the capacity of LTX-401 to stimulate anticancer immune responses remains to be elucidated.

The specific aims of this thesis are:

To evaluate the capacity of lurbinectedin in stimulating the hallmarks of ICD.

To investigate the anticancer immune responses of lurbinectedin and LTX-401 in different experimental cancer models.

To explore the combination therapeutic regiments in cancer treatment.

RESULTS

Lurbinectedin synergizes with immune checkpoint blockade to generate anticancer immunity

Accumulating results indicate that chemotherapeutic drugs which efficiently inhibit transcription and promote eIF2α phosphorylation are potent ICD inducers, indicating the potential of lurbinectedin in inducing ICD. Both preclinical data and clinical trials demonstrate that pretreatment with certain ICD inducers sensitizes to immune checkpoint blockade, it's worthwhile to investigate the role of lurbinectedin in the era of immunotherapy.

To evaluate the immunogenicity of lurbinectedin, different parameters of ICD hallmarks were measured by robotized epifluorescence microscopy followed by automated image analysis.

Lurbinectedin triggered CALR exposure, ATP release, HMGB1 exodus, Type I IFN release and cell death in a dose-and time-dependent manner in 4 human or mouse cancer cell lines. Also, lurbinectedin activated two ICD pathways named split ER stress and transcription inhibition, featured by elevated eIF2α phosphorylation level and decreased co-localization of nucleolin and fibrillarin. Thus, Lurbinectedin induced all traits of ICD in vitro. To further confirm its immunogenicity in vivo, we employed the vaccination experiment in immunocompetent mice.

Lurbinectedin-treated cells significantly reduced tumour growth and led to increased overall survival.

Next, we investigated the therapeutic efficacy of lurbinectedin in immunocompetent and immunodeficient mice. Lurbinectedin significantly retarded MCA205 tumour growth and extended survival span in immunocompetent host but failed to do so in immunodeficient counterpart. Implying that the anticancer efficiency of lurbinectedin in MCA205 cancer depends on immune system.

Moreover, lurbinectedin, αCTLA-4 and αPD-1 triple combination regiment dramatically inhibited tumour progression and induced complete regression in 3 out of 8 animals. Cured mice exhibited systemic antitumor immunity to the same type of cancer cell. In particular, T lymphocyte depletion abolished the effect of lurbinectedin with αCTLA-4/αPD-1 dual checkpoint blockade.

Finally, we tested the therapeutic effect of lurbinectedin in MPA/DMBA-induced spontaneous breast cancer model. Both lurbinectedin alone and in combination with double ICBs significantly reduced tumour growth and increased overall survival.

Introduction

Primary or transplantable tumors react to anthracycline-based chemotherapy with durable response in syngeneic immunocompetent mice yet fail to do so in immunodeficient hosts. 1-3 Consistently, retrospective clinical studies in patients with solid tumors subjected to chemotherapy showed that severe lymphopenia negatively affects prognosis, 4,5 which points to the fact that chemotherapy-elicited anticancer immunity plays a critical role for the outcome of anticancer therapy. 6,7 Based on these findings, 1-3 we introduced the hypothesis that some chemotherapeutic agents can induce immunogenic cell death (ICD) in tumors and convert them into a therapeutic vaccine, hence stimulating an immune response that can control residual cancer cells.

Selected chemotherapeutics such as anthracyclines and oxaliplatin are able to induce ICD 1-3 while many other antineoplastic agents including cisplatin and mitomycin C fail to do so. Cancer cells undergoing ICD can evoke anticancer immunity and protect against a subsequent challenge with living cells exhibiting the same antigenic profile in mice 1-3 or elicit anticancer immune responses during chemotherapy in patients. 8 Distinctive properties of immunogenic cell death include the exposure of calreticulin (CALR) at the cytoplasmic surface, 3,8,9 the autophagy-dependent liberation of ATP from stressed and dying cells, 10,11 the cell death-associated exodus of nuclear high mobility group box 1 (HMGB1) 12,13 and the stimulation of an autocrine or paracrine type-1 interferon response. 14 CALR serves as a de novo uptake signal and stimulates the engulfment of dying cancer cells by dendritic cells (DCs). 3 HMGB1 binds to toll-like receptor-4 (TLR4) entities on DC, eliciting MYD88-dependent signaling that facilitates tumor antigen processing. 3,15 ATP ligates purinergic receptors of the P2X type and thus activates the NLRP3 inflammasome to stimulate the production of interleukin-1β (IL-1β) by DC and eventually interferon-γ (IFNγ) by CD8 + cytotoxic T lymphocytes (CTL). 10,16 The sum of danger associated molecular patterns (DAMP) emitted during ICD is necessary to generate anticancer immunity, thus tumors growing in Tlr4 -/-, P2rx7 -/-, Myd88 -/-, Nlrp3 -/- , Il1r -/-, Ifnγ -/-, Ifnγr -/-, Fpr1 -/-, athymic or CD8 + Tcell-depleted mice fail to respond to immunogenic chemotherapeutic regimens. Loss-of-function mutations of FPR1, P2RX7 or TLR4 in breast cancer are negatively correlated with clinical response to adjuvant chemotherapy with anthracyclines. 3,10,13,14,17-19 These results imply the obligate contribution of anticancer immune responses to the success of ICD-inducing chemotherapies.

Lurbinectedin is a selective inhibitor of active transcription of protein-coding genes 20 that is currently undergoing clinical investigation and has recently gained orphan drug approval for the treatment of small cell lung cancer (SCLC). Here, we investigated the capacity of lurbinectedin to stimulate the emission of immunogenic DAMPs and tested anticancer immune responses in three experimental in vivo models. Our results support the contention that lurbinectedin causes immunogenic cell death in tumors and creates anticancer immunity.

Results and discussion

Emission of immunogenic signals by lurbinectedin

The known parameters determining ICD are the translocation of CALR to the surface of the plasma membrane, the autophagy-dependent liberation of ATP and the release of the nonhistone binding protein HMGB1, which occur before, during and after apoptosis, respectively. The production of type I interferons (IFNs) has been added to the list of ICD hallmarks as it controls autocrine or paracrine circuits that underlie cancer immunosurveillance.

In a systematic screening campaign, the capacity of lurbinectedin to induce immunogenic cell death in cancer cells was assessed in human osteosarcoma U2OS cells stably expressing fluorescent biosensors for the detection of CALR-relocation (as a surrogate marker for CALR surface exposure), HMGB1 release and Type I IFN responses together with U2OS WT cells stained with the ATP-sensitive dye quinacrine. ICDrelated parameters were measured at 4, 8, 16 and 32 hours post exposure to lurbinectedin from 1 nM to 1 µM by robotized epifluorescence microscopy followed by automated image analysis (Figure 1). The induction of cell death was evaluated based on changes in the nuclear morphology visualized by means of the DNA intercalating dye Hoechst 33342. Lurbinectedin caused a dose-and time-dependent drop in cellular viability comparable to mitoxantrone (MTX) that was used at 1 and 3 µM as a positive control throughout all experiments. The translocation of a CALR-GFP (green fluorescent protein) fusion protein from the perinuclear ER to the cellular periphery was measured by assessing cytoplasmic "granularity" (see Materials and Methods) as an indicator for the formation of CALR-containing vesicles and as a surrogate marker for CALR exposure. Lurbinectedin, similar to MTX, induced a time-and dose-dependent increase in CALR-granularity as compared to untreated controls. The reduction of intracellular ATP (as an indicator for ATP release) was assessed by measuring the decrease in the cytoplasmic granularity of ATP containing vesicles stained with the fluorescent probe quinacrine. As compared to untreated controls a significant decrease in ATP signal similar to MTX was detectable for lurbinectedin. The effect was dosedependent and decreased over time in line with the fragile nature of the metabolite. HMGB1 release was detected as a loss in the nuclear fluorescence of an HMGB1-GFP chimera. A significant decrease in nuclear GFP signal was detected for MTX and lurbinectedin at medium to late time points. Type I interferon (IFN) production was measured using U2OS biosensor cells stably expressing a GFP under the control of the MX1 (a Type I IFN response gene) promoter. To this aim the supernatant of U2OS cells following treatment and additional 48 hours incubation with fresh media was used to treat the biosensor cells. Following the type 1 IFN response was monitored by means an increase in GFP fluorescence intensity. A significant increase in de novo GFP signal intensity was detected for both lurbinectedin and MTX throughout all time points (Figure 1(a)). Similar results were obtained when the approach was repeated in human breast cancer HCC70 cells (Figure 1(b)), human colon carcinoma HT29 (Figure 1(c)) and mouse fibrosarcoma MCA205 cells (Figure 1(d)). Next, we investigated the capacity of lurbinectedin to activate two additional characteristics of common ICD inducers, the phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α) and the inhibition of general transcription. Indeed, lurbinectedin led to a dose-dependent phosphorylation of eIF2α monitored by fluorescence microscopy upon immunostaining with a phosphoneoepitope-specific antibody (Figure 2(a,b)). Lurbinectedin also inhibited mRNA transcription at a level comparable to a known transcription-inhibitor, as assessed by visualizing the dissociation of nucleolin and fibrillarin by microscopy (Figure 2(b,c)), an accepted proxy of suppressed transcription. 21 Lurbinectedin holds many of the described in vitro parameters of ICD, thus qualifying for further in vivo investigations in immunocompetent animals, which remains the gold standard assay for the determination of ICD-mediated anticancer immunity.

Anticancer immunity induced by lurbinectedin

In order to assess the capacity of lurbinectedin to stimulate anticancer immunity in a monotherapeutic approach and to convert tumor cells into a therapeutic vaccine we exposed murine fibrosarcoma cells to the drug in vitro (in conditions previously established to induce a sufficient amplitude of cell death) and then injected the dying cancer cells into syngeneic immunocompetent mice. One week later, the animals were rechallenged injecting live tumor cells of the same kind into the opposite flank, (Figure 3(a)). In this setting, a decrease of tumor growth can be interpreted as sign of a productive anticancer immune response. Indeed, lurbinectedin-treated cells significantly reduced tumor growth (p = .0094) (Figure 3(b)) and led to an increase in overall survival (Figure 3(c)). As compared to know ICD inducers 1-3 the vaccination effects observed here were rather limited yet statistically significant. Next we evaluated the effect of lurbinectedin on established cancers growing on immunocompetent or immunodeficient mice. MCA 205 tumors were implanted subcutaneously on immunocompetent C57BL/6 as well as in athymic nu/nu mice. When the tumors became palpable, the animals were treated with three consecutive intravenous injections of 0.18 mg/kg lurbinectedin on day 1, 7 and 14. (Figure 4(a)). The treatment with lurbinectedin had significant therapeutic benefit in immunocompetent animals. The tumor growth was significantly reduced as compared to control animals (p < .0001) (Figure 4(b)) and overall survival was increased (Figure 4(c)). This effect was exclusively observed when tumors grew on immunocompetent mice, yet was lost when the tumors proliferated on athymic (nu/nu)m i c e( Figure 4(d,e)). These results underscore the obligate contribution of the immune system to the chemotherapeutic activity of lurbinectedin.

Combinatorial effects of lurbinectedin and αPD-1/αCTLA-4 double immune checkpoint blockade Given the capacity of lurbinectedin to induce immunedependent anticancer effects on established tumors, we investigated whether this agent could sensitize cancers to therapy with immune checkpoint blockers targeting CTLA-4 or PD-1. For this, established MCA205 fibrosarcomas were treated with Lurbinectedin as before and subjected to immunotherapy with antibodies specific for CTLA-4, PD-1 or a combination of both on day 6, 9 and 12, when the anticancer immune c) and murine methylcholantrene-induced fibrosarcoma MCA205 cells (d) were treated with lurbinectedin (Lurbi, 1 nM, 10 nM, 100 nM and 1 µM) for the indicated times. Subsequently, the cells were stained with 1 µM Hoechst 33342 and 1 µM propidium iodide and assessed for the loss of viability by automated image acquisition. Images were segmented, cellular debris was excluded and the number of cells with normal nuclear morphology was enumerated. Cells stably expressing CALR-GFP were treated as above. Following the cells were fixed with 3.7% of PFA, stained with 1 µM Hoechst 33342 and assessed by automated image acquisition. Images were segmented, cellular debris was excluded and CALR-GFP granularity (a surrogate marker of CALR exposure) was evaluated in the cytoplasmic region of cells with normal nuclear morphology. Wild type cells were treated as above and then assessed for cytoplasmic quinacrine granularity (after staining with the ATP-sensitive dye quinacrine together with Hoechst 33342) by automated image acquisition, segmentation and analysis. Cells stably expressing HMGB1-GFP were treated as above and then assessed for nuclear HMGB1-GFP fluorescence intensity. The cells were fixed and stained with Hoechst 33342 and images were acquired, segmented and analyzed. WT cells were treated as above and following the media was changed and the cells were incubated for 48 hours before the supernatant was used to treat MX1-GFP biosensor cells for additional 48 hours. The cells were fixed and stained with Hoechst 33342 before type 1 IFN responses were monitored by means of automated microscopy as an increase in GFP fluorescence intensity. Mitoxantrone (MTX, 1 and 3 µM) was used as a positive control. The means of quadruplicate assessments and p-values are depicted as heat maps. (*p < .01; **p < .005; ***p < .001, two-tailed Student's t test).

response in the tumor peaks (Figure 5(a)). Tumor monitoring led to the deduction that the most efficient therapeutic regimen was a combination of all three anticancer agents (lurbinectedin, αCTLA-4 and αPD-1) in contrast to single-ICB therapies that appeared to be relatively inefficient in this setting (Figure 5(b-e)). The combination of lurbinectedin with αCTLA-4/αPD-1 dual checkpoint blockade in tumorbearing animals significantly extended life expectancy and, moreover, led to tumor clearance in 3/8 mice in the time frame of the experiment (Figure 5(e)). The effect of lurbinectedin with αCTLA-4/αPD-1 dual checkpoint blockade was abrogated in conditions in which CD4 + and CD8 + cytotoxic T lymphocytes (CTLs) were depleted. Mice that had been rendered tumor-free for more than 50 days rejected tumors upon rechallenge with the same cancer cell type from which they had been cured (MCA205), yet developed cancers when rechallenged with TC1 tumor cells (Figure 5(f,g)). Thus, mice that had been cured by a combination of systemic lurbinectedin-based chemotherapy and immunotherapy had established a specific anticancer immune response that generated immunological memory.

Lurbinectedin retards the growth of carcinogen-induced and spontaneous breast cancer

To explore the potential lurbinectedin for the therapy of breast cancer, we took advantage of a hormone/carcinogen induced breast cancer model activated by the continuous stimulation of MCA205 cells treated for 20 h with 1 µM lurbinectedin were inoculated subcutaneously (s.c.) into immunocompetent C57BL/6 mice, which were rechallenged 7 days later s.c. with living cells of the same type. The tumor growth was measured until endpoints were reached and overall survival was evaluated regularly for the following 30 days (n = 6). (*p < .01, two-tailed Student's t test, compared to all other groups). Data were analyzed with TumGrowth.

progesterone receptors by medroxyprogesterone acetate (MPA) and the repeated exposure to the DNA-damaging agent dimethylbenzantracene (DMBA). This induced model of breast cancer is known to be modulated by the immune system. 22 We treated mice with palpable MPA/DMBA-induced tumors by systemic injection with lurbinectedin alone or in combination with double immune checkpoint blockade neutralizing CTLA-4 and PD-1 (Figure 6(a)). Both interventions significantly reduced tumor growth and increased overall survival. However, only the combination with αCTLA-4/αPD-1 yielded tumor clearance in the time frame of the experiment (Figure 6(b-d)).

Concluding remarks

The results of this study suggest that lurbinectedin efficiently induces cell death in a broad panel of solid tumors. This procedure likely does not only cause the cells to succumb to disintegration but rather triggers traits of immunogenic cell death, including the phosphorylation of eIF2a and the release of danger associated molecular patterns (DAMPs). Irrespective of the exact molecular mechanisms accounting for these effects, there are a number of evidences advocating for lurbinectedin-triggered cancer-specific immunogenicity. Thus, animals that had been cured by lurbinectedin from established cancers became resistance to rechallenge with the same cancer type. The therapeutic effect of lurbinectedin was neutralized in conditions in which either the host was immunocompromised or T-cell had been depleted. Furthermore, the recapitulation in a heterogeneous spontaneous tumor model of effects that were previously observed in homogenous transplanted tumors indicates that the results presented here hold a high translational value.

Altogether, these results convincingly demonstrate that lurbinectedin mediated immunochemotherapy may be advantageously combined with clinically established immune checkpoint blockade regimens.

Materials & methods

Cell culture and chemicals

All media and cell culture supplements were from Thermo Fisher Scientific (Carlsbad, CA, US). Lurbinectedin was provided by PharmaMar (Madrid, Spain). Cell culture plastics and consumables were purchased from Greiner Bio-One (Kremsmünster, Austria). Human osteosarcoma U2OS cells previously genetically altered as described earlier, 23 murine methylcholanthreneinduced fibrosarcoma MCA-205 cells and murine lung cancer TC-1 cells were cultured in Glutamax®-containing DMEM medium supplemented with 10% fetal bovine serum (FBS), and 10 mM HEPES. Cells were cultured in a temperature-controlled environment at 37°C with a humidified atmosphere containing 5% CO 2 .

Automated image acquisition and analysis

One day before the experiment 5 × 10 3 cells were seeded in 96well µClear imaging plates (Greiner BioOne) and let adhere under standard culture conditions. The following day cells were treated with lurbinectedin at 0.001, 0.01, 0.1 and 1 µM for 4, 8, 16 or 32 hours. Then cells were fixed with 3.7% formaldehyde supplemented with 1 μg/ml Hoechst 33342 for 30 min at RT. The fixative was changed to PBS and the plates were analyzed by automated microscopy. For the detection of ATP enriched vesicles, the cells were labeled after 4, 8, 16 or 32 hours of incubation with the fluorescent dye quinacrine (as described before 23 ). In short, cells were incubated with 5 µm quinacrine and 1 µg/ml Hoechst 33342 in Krebs-Ringer solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 0.7 mM KH 2 PO 4 ,2m mC a C l 2 ,6m M Live MCA205 cells were injected subcutaneously (s.c.) into immunocompetent C57BL/6 mice or immunodeficient nu/nu mice as depicted in the scheme in (a) When tumors became palpable, mice were intravenously (i.v.) injected with 0.14 mg/Kg lurbinectedin (on day 1,7 and 14). Tumor growth was assessed regularly for the following 30 days. Data is depicted as tumor growth curves (b,d) and overall survival plots (c,e). Data were analyzed with TumGrowth.

glucose and 25 mM Hepes, pH 7.4) for 30 minutes at 37°C. Thereafter, cells were rinsed with Krebs-Ringer and viable cells were microscopically examined. For automated fluorescence microscopy a robot-assisted Molecular Devices IXM XL BioImager (Molecular Devices, Sunnyvale, CA, USA) equipped with SpectraX light source (Lumencor, Beaverton, OR, USA), adequate excitation and emission filters (Semrock, Rochester, NY, USA) and a 16-bit monochromes sCMOS PCO.edge 5.5 camera (PCO Kelheim, Germany) and a 20 X PlanAPO objective (Nikon, Tokyo, Japan) was used to acquire a minimum of 9 view fields, followed by automated image processing with the custom module editor within the MetaXpress software (Molecular Devices). Depending on the utilized biosensor cell line the primary region of interest (ROI) was defined by a polygon mask around the nucleus allowing for the enumeration of cells, the detection of morphological alterations of the nucleus and nuclear fluorescence intensity. Cellular debris was excluded from the analysis and secondary cytoplasmic ROIs were used for the quantification of CALR-GFP or quinacrine containing vesicles. For the latter, the images were segmented and analyzed for GFP granularity by comparing the standard deviation of the mean fluorescence intensity of groups of adjacent pixels (coefficient of variation) within the cytoplasm of each cell to the mean fluorescence intensity in the same ROI using the MetaXpress software (Molecular Devices).

In vivo experimentation

Six-to eight-week-old female wild-type C57BL/6 and nu/nu mice were obtained from Envigo France (Huntingdon, UK) and were kept in the animal facility at the Gustave Roussy Campus Cancer . Sequential lurbinectedin treatment with double immune checkpoint blockade exhibits systemic antitumor immunity C57BL/6 mice were inoculated subcutaneously (s.c.) with murine fibrosarcoma MCA205. Palpable tumors were treated with sequential intravenous (i.v.) injections of 0.14 mg/Kg lurbinectedin (Lurbi) as indicated in (a). Single-or double-immune checkpoint blockade was mounted by sequential intraperitoneal (i.p.) injections of monoclonal antibodies targeting CTLA-4 or PD-1 at day 6, 9 and 12 post treatment and tumor growth (b,c) and overall survival (d,e) were assessed regularly for the following 30 days. (f,g) The generation of immunological memory was assessed in cured animals by rechallenged with MCA205 and TC-1. Naïve animals were used as controls. Individual tumor growth curves are depicted. Data were analyzed with TumGrowth.

in a specific pathogen-free and temperature-controlled environment with 12 h day, 12 h night cycles and received food and water ad libitum. Animal experiments were conducted in compliance with the EU Directive 63/2010 and protocols 2013_094A and were approved by the Ethical Committee of the Gustave Roussy Campus Cancer (CEEA IRCIV/IGR no. 26, registered at the French Ministry of Research). As described, 24,25 MCA205 tumors were established in C57BL/6 mice by subcutaneously (s. c.) injection of 5 × 10 5 cells. When tumors became palpable, 0.18 mg/Kg lurbinectedin was injected sequentially once a week intravenously into the tail vein and animal well-being and tumor growth were monitored. A total of 0.5 mg of anti-CD8 (clone 2.43 BioXCell BE0061) and anti-CD4 (clone GK1.5 BioXCell BE0003-1) intraperitoneal (i.p.) injections were repeated every 7 days to assure the complete depletion of both T cell populations during t h ew h o l ee x p e r i m e n t .M i c ew e r es a c r i f i c e dw h e nt u m o rs i z e reached end-point or signs of obvious discomfort associated to the treatment were observed following the EU Directive 63/2010 and our Ethical Committee advice. Tumor-free animals were kept for more than 30 days before testing the generation of immunological memory by s.c.rechallengewith5×10 5 TC-1 in one flank and 5 × 10 5 MCA205 cells injected in the contralateral flank. Animals were monitored and tumor growth documented regularly until end-points were reached. Statistical analysis was performed by applying 2-way ANOVA analysis followed by Bonferroni's test comparing to control conditions (* p <. 0 5 ,* * p < .01 and ***p < .001). Murine fibrosarcoma MCA205 cells were incubated with 1 µM lurbinectedin for 24 h, resulting in approximately 70% cell death. For vaccination experiments, 3 × 10 5 dying MCA205 cells were inoculated s.c. into the left flanks of six-weekold female C57BL/6 mice. Seven to ten days later, animals were rechallenged in the opposite flank with 3 × 10 5 living MCA205 cells, and tumor growth and incidence were monitored. Six-week-old female C57BL/6 mice (n = 12 per group) underwent surgical implantation of slow-release medroxyprogesterone acetate (MPA) pellets ( 50 Medroxyprogesterone acetate (MPA) pellets (50 mg, 90-day release) were implanted subcutaneously into the interscapular area of immunocompetent C57BL/6 mice. Then the animals received 1 mg dimethylbenzantracene (DMBA) administered by oral gavage 6 x during 7 weeks. When spontaneous tumors became palpable mice were randomly assigned to receive 0,14 mg/Kg lurbinectedin (Lurbi) alone or in combination with double immune checkpoint blockade with monoclonal antibodies targeting CTLA-4 and PD-1 at day 6, 9 and 12 post treatment (a). The tumor area and overall survival were measured regularly until ethical endpoints were reached (b,c,d). Data were analyzed with TumGrowth (https://github.com/kroemerlab).

ANOVA analysis followed by Bonferroni's test comparing to control conditions (* p < .05, ** p < .01 and ***p < .001).

Statistical procedures

Unless otherwise specified, experiments were performed in quadruplicate instances. Data were analyzed with the freely available software R (https://www.r-project.org). Significances were calculated using a student t-test with Welch correction. Thresholds for each assay were applied based on the Gaussian distribution of positive and negative controls. In vivo tumor growth was analyzed with the help of the TumGrowth software package 26 freely available at https://github.com/kroemerlab.

Tumor lysis with LTX-401 creates anticancer immunity

Previous results demonstrated that LTX-401 could induce ICD hallmarks in vitro, and intratumour injection of LTX-401 showed potent antitumor capacity in few cancer models (Eike et al., 2016;[START_REF] Mauseth | The Novel Oncolytic Compound LTX-401 Induces Antitumor Immune Responses in Experimental Hepatocellular Carcinoma[END_REF][START_REF] Zhou | The oncolytic compound LTX-401 targets the Golgi apparatus[END_REF]. The capacity of LTX-401 to stimulate anticancer immune response and combination effect with ICBs remains to be elucidated.

Since LTX-401 is designed for future clinical usage, we first tested its safety profile at therapeutically effective dose. LTX-401, similar to LTX-315 that is under clinical evaluation in patients with transdermal accessible tumours, did not depict any signs of adverse toxicity in vivo.

We then investigated the therapeutic effect of LTX-401 in subcutaneous MCA205 fibrosarcoma and TC-1 lung cancer. Intratumoral delivery of LTX-401 reduced tumour growth and increased survival rate in both models. 3 out of 12 and 8 out of 12 mice were cured in MCA205

and TC-1 settings, respectively. All these cured mice were resistant to the rechallenge of same cell type.

To study the abscopal effects of LTX-401-mediated tumour lysis, mice were implanted with MCA205/TC-1 subcutaneously on one flank and four days later on the contralateral flank. Only primary tumour was treated by LTX-401 and the growth of contralateral tumour was monitored.

The local treatment had a significant tumour growth reducing effect on primary tumours yet only limited effects on secondary contralateral tumours.

Given

Introduction

The word 'oncolysis' usually evokes oncolytic viruses, i.e. viruses that selectively infect and kill cancer cells. 1,2 However, oncolysis can also be achieved by applying physical, chemical or pharmacological agents to tumor in a way that the cancer is locally destroyed. For example, cationic ampholytic peptides or peptide mimetics can be used to target cellular membranes, causing their disruption by detergentlike effects. We have been characterizing the mode of action of several oncolytic reagents of this class such as LTX-315, LTX-401, DTT-205 and DTT-304. LTX-315 turned out to preferentially target mitochondrial membranes; 3,4 while LTX-401 destroys Golgi apparatus-associated membranes, 5-7 thus setting off a different cell-destroying cascade. 7 DTT-205 and DTT-304, instead, have a tropism for lysosomal membranes, thus activating yet another cell death subroutine. 8 Despite their diversity, these oncolytic peptide and peptide derivatives all appear to be able to stimulate anticancer immune responses, at least in specific circumstances. Thus, cancer cells treated with LTX-315 or LTX-401 in vitro can stimulate a protective antineoplastic immune response when injected into rodents. 3,7,9-13 Moreover, LTX-315 treatments sensitize to subsequent immune checkpoint blockade with αCTLA-4 antibodies, 11 while tumors that are successfully cured with DTT-205 or DTT-304 established a long-term immune response rendering them resistant against later rechallenge with the cancer cells that had been eliminated from them. 8 Here, we compared the safety profiles of different lytic peptides to discover that LTX-401 (similar to LTX-315) was far less toxic than DTT-205 and DTT-304. We also investigated the capacity of LTX-401 to stimulate anticancer immune responses in three different experimental setups. Our results support the hypothesis that oncolysis by LTX-401 is highly immunogenic.

Results and discussion

Differential toxicity of oncolytic agents

In a retrospective study, the safety profiles of oncolytic peptides and peptidomimetics at their therapeutically effective doses were compared. The onset of manifest discomfort or death that was defined as an endpoint for in vivo experimentation was evaluated for the sequential treatment of tumors established on C57Bl/6 mice with therapeutically effective doses of LTX-315, LTX-401, DTT-205 and DTT-304 (Figure 1A). Data from in vivo experimentation utilizing oncolytic compounds in C57BL/6 mice bearing palpable subcutaneous solid tumors were evaluated by enumerating global survival (Figure 1B). Whereas both experimental compounds DTT-205 and DTT-304 led to death in a certain number of animals, LTX-401, similar to LTX-315, that is under clinical evaluation in patients with transdermally accessible tumors, did not depict any signs of adverse toxicity in vivo.

Stimulation of anticancer immunity by LTX-401

To identify the capacity of LTX-401 to stimulate anticancer immune responses, we treated established MCA205 fibrosarcomas growing subcutaneously (s.c.) in C57BL/6 mice by daily intratumoral injection (up to 4 times) of the compound (or, as a control, its vehicle PBS) until macroscopically detectable tumors disappeared or persisted (Figure 2A). This procedure greatly reduced tumor growth (Figure 2B) and extended the longevity of the mice, yielding a survival rate of ~22% at 60 days postdiagnosis (Figure 2C). At this point, tumor-free mice were rechallenged by s.c. inoculation of the same tumor type (MCA205, injected into the opposite flank) or antigenically unrelated mammary carcinoma AT3 cells (injected into the same flank, though at a site distant from the primary MCA205 fibrosarcoma). Importantly, all mice that were cured by LTX-401 treatment from their primary MCA205 tumor became resistant to rechallenge with MCA205 (but not AT3) cancer cells (Figure 2D), supporting the idea that they had developed a long-term immunological memory. Very similar results were obtained when MCA205 fibrosarcomas were replaced by non-small cell lung adenocarcinoma TC-1 cells (that have been engineered to express the human papilloma virus antigen E6). Again, local therapy with L T X -4 0 1i n j e c t i o n sw a sa b l et og r e a t l yr e d u c et h eg r o w t ho f primary TC-1 carcinomas (Figure 2E), yielding a 60% survival rate at 60 days (Figure 2F). Again, mice that were cured became resistant to rechallenge with TC-1 (but not MCA205) cells (Figure 2G), confirming the contention that a tumor-specific memory immune response had been established.

Limited abscopal effects of LTX-401-mediated tumor lysis

In the next step, we interrogated the experimental system for the induction of abscopal effects, i.e. a presumably immune-mediated long-distance effect of the injected LTX-401 peptide that would reduce the growth of a non-injected tumor. For this, we inoculated mice with MCA205 fibrosarcomas into the right flank to create a primary tumor (that became palpable 8 days later) and shortly later (4 days after the first injection) a similar amount of MCA205 cells into the left flank to create a secondary tumor. Only the primary tumor was injected for up to 4 days until tumor disappearance with LTX-401 (or PBS as a vehicle control) and the growth of both the primary (treated) and secondary (untreated) tumors were monitored. The local treatment had a significant tumor growth reducing effect on primary tumors (Figure 3A,B,D) yet only limited effects on secondary contralateral tumors (Figure 3C,E). Hence, LTX-401-mediated oncolysis alone is not sufficient to induce a therapeutically relevant abscopal effect. Rechallenge of animals cured from MCA205 fibrosarcoma (pooled from several experiments) with MCA205 several weeks after the initial therapy on the contralateral flank and challenged with syngeneic mouse AT3 breast cancer cells on the ipsilateral side resulted in efficient rejection of MCA205 but aggressive tumor growth of AT3 (D). Thus, LTX-401 caused the generation of immunological memory that sufficed in rejecting isogenic tumors. Similar effects were obtained when mouse lung cancer TC-1 cells were inoculated subcutaneously in syngeneic C57BL/6 mice and tumors and were treated when palpable with repeated injections of 0.25 mg LTX-401 intratumorally as shown in (A). LTX-401 induced efficient oncolysis and tumor control in some animals reflected in reduced tumor growth and cure of some animals (E), and increased overall survival (F) (Chi 2 test, ***p < 0.001). Some of the animals cured from subcutaneous TC-1 lung cancers were rechallenged with TC-1 several weeks after the initial therapy on the contralateral and challenged with syngeneic mouse MCA205 fibrosarcoma cells on the ipsilateral side. This maneuver resulted in efficient rejection of TC-1 but aggressive tumor growth of MCA205 (G). Thus, also in this model system LTX-401 caused the generation of immunological memory in cured animals that sufficed in rejecting isogenic tumors.

subjected to immunotherapy with antibodies specific for CTLA-4, PD-1 or a combination of both (Figure 4A). Continuous tumor monitoring led to the conclusion that the most efficient therapeutic regimen consisted in a combination of all three anticancer agents (LTX-401, αCTLA-4 and αPD-1). In contrast, single-agent therapies appeared to be relatively inefficient in this setting (Figure 4B-F). When the survival of tumor-bearing animals was monitored, the combination of LTX-401 and dual checkpoint blockade (αCTLA-4 plus αPD-1) or the combination of LTX-401 and αCTLA-4 (but not αPD-1) alone were able to significantly extend life expectancy (Figure 4G-H). Again, mice that had been rendered tumor-free for more than 50 days resisted rechallenge with the same cancer cell type from which they had been cured (MCA205), yet readily developed TC-1 cancers (Figure 4I). Thus, as to be expected, mice that had been cured by a combination of local oncolysis and systemic immunotherapy had established a specific anticancer immune response. Sequential treatments of LTX-401 in combination with dual checkpoint blockade increased this effect and was able to achieve 100% clearance of treated tumors (Figure 5A,B,D). Of note in six out of nine animals the combination of LTX-401 with dual checkpoint blockade not only cleared the treated tumor but also exhibited abscopal neoplastic effects on distant tumors (Figure 5C,E).

Concluding remarks

The results of this study suggest that LTX-401 can provide oncolysis with a relatively favorable safety profile when the agent is injected into established tumors. Indeed, LTX-401 could be injected daily on 4 subsequent days with zero fatalities. Oncolysis by LTX-401 yielded a rapid macroscopic response and allowed most of the tumors to become undetectable following

Tumor clearance n=8

Figure 3. LTX-401 treatment of subcutaneous MCA205 fibrosarcoma induces limited abscopal effects. C57BL/6 mice were inoculated with murine fibrosarcoma MCA205 subcutaneously on one side and four days later on the contralateral flank. Palpable primary tumors arising from the first injection were treated with sequential intratumoral injections of 0.25 mg LTX-401 as indicated in (A). LTX-401 induced efficient oncolysis in primary tumors which is reflected in reduced tumor growth and tumor clearance in some animals (B). In parallel tumors arising from untreated secondary lesions were monitored and abscopal effects were documented as tumor growth in separate curves (C). Lung cancer TC-1 cells were injected subcutaneously into C57BL/6 mice on one flank and four days later on the contralateral side. Palpable primary tumors arising from the first injection were treated with sequential injections of 0.25 mg LTX-401 intratumorally as indicated in (A). LTX-401 induced efficient oncolysis in primary tumors which is reflected in reduced tumor growth and tumor clearance in some animals (D). In parallel tumors arising from untreated secondary lesions were monitored and abscopal effects were documented as tumor growth curves (E). several local injections. This procedure likely does not simply cause necrotic (and passive) lysis of neoplastic and stromal cells. Rather, it triggered some immunogenic events, be it the induction of immunogenic cell death or by the liberation of tumor-associated antigens from their tumor towards the draining lymph nodes. 14 Irrespective of the exact molecular mechanisms . Combination of single LTX-401 treatment with immune checkpoint blockade increases its efficacy. C57BL/6 mice subcutaneously implanted with MCA205 fibrosarcoma were injected intratumorally when tumors became palpable with a single injection of 0.25 mg LTX-401 as indicated in (A). Immune checkpoint blockade was mounted by sequential injections of monoclonal antibodies targeting CTLA-4 or PD-1 alone or in combination at day 6, 9 and 12 post treatment. LTX-401 induced efficient oncolysis which is reflected in reduced tumor growth and tumor clearance in some animals. This effect was increased in combination with CTLA-4 and PD-1cotreatmentas depicted in separate tumor growth curves (B-E) and overall survival plots (Chi 2 test, **p < 0.01) (F-H). Rechallenge of animals cured from MCA205 fibrosarcoma with MCA205 several weeks after the initial therapy on the contralateral and challenged with syngeneic mouse TC-1 lung cancer cells on the ipsilateral side resulted in efficient rejection of MCA205 but aggressive tumor growth of TC-1 (I). Thus, LTX-401 caused the generation of immunological memory that led to the rejection of isogenic tumors. accounting for these effects, there are at least three lines of evidence pleading in favor of LTX-401-triggered cancer-specific immunogenicity. First, animal st h a th a db e e nc u r e df r o mt h e i r established cancers by LTX-401 became resistance to rechallenge with the same cancer type. Second, one single intratumoral injection of LTX-401 (which turned out to be inefficient on its own in reducing tumor volume) sensitized the cancers to subsequent immunotherapy with antibodies blocking CTLA-4 or, more so, a combination of antibodies targeting CTLA-4 and PD-1. Third, in mice bearing two neoplastic lesions, one which was treated and another one that was left untreated, the combination of LTX-401 with double immune checkpoint blockade did not only reduce the growth of the locally injected tumor but also mediated long-distance (abscopal) effects resulting in the control of most of the non-injected cancers.

Altogether, these results convincingly demonstrate that LTX-401 mediated oncolysis may advantageously combine with established immunotherapeutic regimens.

Materials & methods

Chemicals, cell culture media and supplements

All supplements and cell culture media were purchased from Thermo Fisher Scientific (Carlsbad, CA, US). LTX-401 was provided by Lytix Biopharma (Oslo, Norway). Cell culture support and consumables were obtained from Greiner Bio-One (Monroe, CA, US). Mouse fibrosarcoma cells MCA-205 and murine lung cancer TC-1 cells were cultured in Glutamax®-containing DMEM medium supplemented with 10% fetal bovine serum (FBS), and 10 mM HEPES. Cells were maintained in a humidified incubator at 37°C with an atmosphere containing 5% CO 2 .

In vivo experimentation

Female wild-type C57BL/6 mice at the age of 6-8 weeks were obtained from Harlan France (Gannat, France) and kept under controlled conditions in the animal facility at the Gustave Roussy Campus Cancer in specific pathogen-free and temperature-controlled environment with 12 h day, 12 h night cycles and received food and water ad libitum. Animal experiments were conducted in compliance with the EU Directive 63/2010, and protocols 2013_094A and were approved by the Ethical Committee of the Gustave Roussy Campus Cancer (CEEA IRCIV/IGR no. 26, registered at the French Ministry of Research). MCA205 or TC-1 tumors were established in C57BL/6 hosts by subcutaneously inoculating 5 × 10 5 cells. When tumors became palpable, either 0.25 mg (for each injection of sequential treatment) or 0.4 mg (for single injection) of LTX-401 was injected intratumorally. Remaining tumor tissue was treated on subsequent days accordingly and animal well-being and tumor growth were monitored.

Mice were sacrificed when tumor size reached end-point or signs of obvious discomfort associated with the treatment were observed always following the EU Directive 63/2010 and our Ethical Committee advice. Surviving and tumor-free animals were analyzed and kept for more than 30 days before subcutaneous rechallenge with 5 × 10 5 live TC-1 in one flank and 5 × 10 5 live MCA205 cells injected in the contralateral flank. In the case of previously injected TC-1 tumor-free animals, the location of the injected cells was inverted. Animals were monitored and tumor growth documented regularly until end-points were reached or signs of obvious discomfort were observed. Statistical analysis was performed employing two-way ANOVA analysis followed by Bonferroni's test comparing to control conditions (* p < 0.05, ** p < 0.01 and ***p < 0.001). Abscopal effects were analyzed by inoculating C57BL/6 hosts subcutaneously with 5 ×1 0 5 MCA205 or TC-1 cells. Four days later the same number of cells was inoculated on the contralateral side to establish a second syngeneic tumor. Following exclusively primary tumors were treated with the oncolytic LTX-401 as described above and tumor sizes of both primary and secondary tumor were monitored and documented regularly until end-points were reached or signs of obvious discomfort were observed.

Retrospective study

The adverse toxicity of sequential intratumoral injections of oncolytic peptides and peptidomimetics at their respective therapeutically effective dose such as LTX-315 (0.3 mg/injection), LTX-401 (0.25 mg/injection), DTT-205 and DTT-304 (both at 1.5 mg/injection) was evaluated. Data from in vivo experimentation utilizing oncolytic compounds in C57BL/6 mice bearing palpable subcutaneous solid tumors were collected from these two studies, 11,8 and unpublished data. Animals that depicted adverse toxic effects were enumerated and the percentage of mice that depicted an onset of discomfort upon treatment was plotted.

Immune checkpoint blockade

Immune checkpoint blockade was mounted by sequential intraperitoneal injections of 200 µg monoclonal antibodies targeting PD-1 (Clone 29F.1A12, BioXcell, West Lebanon, NH, USA) or CTLA-4 (Clone 9D9, BioXcell) alone or in combination at day 6, 9 and 12 post treatment. Animals were monitored and tumor growth documented regularly until end-points were reached or signs of obvious discomfort were observed. Statistical analysis was performed employing two-way ANOVA analysis followed by Bonferroni's test comparing to control conditions (* p < 0.05, ** p < 0.01 and ***p < 0.001).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

II. Does all of transcription inhibitors induce ICD? If negative, what determines the immunogenicity of transcription inhibitors?

A newly published paper shows that transcription inhibition may serve as an initial event for ICD induction (Humeau et al., 2020), but whether this stress pathway in sufficient to trigger ICD is not defined yet. To answer this question, the immunogenicity of transcription inhibitors needs to be systemically evaluated. By taking advantage of the ICD predication system (in silicon), fluorescent biosensor-based platform (in vitro) (Kepp et al., 2019), high-throughput screening could be launched to identify ICD candidates in this cluster. Further validated by vaccination assay (in vivo). Conclusions can be made only by this means.

III. Why lurbinectedin sensitize MCA205 cancer to ICBs but the synergistic effect is not significant in spontaneous breast cancer?

This could be further addressed by tumour infiltrated lymphocytes analysis. Maybe the immunostimulating effect of lurbinectedin induced cancer cell death was unable to neutralise the immunosuppressive microenvironment in this circumstance.

IV.

Perspective on clinical application of lurbinectedin-immunotherapy combination in cancer therapy.

As a newly approved drug in relapsed SCLC treatment, the potential benefits of lurbinectedinimmunotherapy combination regiments are under evaluation in 3 different clinical trials.

Lurbinectedin (PM01183) will combine with Atezolizumab (anti-PD-L1), or Pembrolizumab (anti-PD-1) or Nivolumab (anti-PD-1) plus Ipilimumab (anti-CTLA-4) to treat SCLC in NCT04253145, NCT04358237 and NCT04610658 respectively. Except SCLC, the application of lurbinectedin in breast cancer, sarcoma, and other advanced solid tumours treatment are still in clinical trials. In our ongoing study, we demonstrated that lurbinectedin not only facilitates cytotoxic T cell infiltration, but also relives the immunosuppressive nature of tumour microenvironment by depleting macrophage. As CD8 T cell is regarded as a positive prognostic factor while macrophage exhibits opposite function in multiple solid cancers, lurbenectedin shows great therapeutic potential in these cancer types. Besides, an induction of PD-1 and CTLA-4 expression was observed in tumour infiltrate CD8 T cell, which theoretically supported the hypothesis that immune checkpoint blockade synergize with lurbinectedin in different types of cancer (unpublished data).

In the second topic, we demonstrated that oncolysis by LTX-401 is highly immunogenic and elicits systemically anticancer response in combination with ICBs. Also, some questions need be answered to extend its future application.

I. Why LTX-401 preferably synergize αCTLA-4 rather than αPD-1 in this tumour model?

It's reported that within oncolytic peptide LTX-315 treated tumour, CTLA-4 molecule was unregulated while PD-1 expression was decreased on CD8 + TILs. This may explain the different outcomes in these two different combinatorial regimens. Maybe LTX-401 shares the same property with LTX-315 in modulating tumour microenvironment, TIL analysis may give some clues in this question.

II. How to achieve suitable and sustainable concentration of LTX-401 intratumorally?

This is a major obstacle for clinical application of oncolytic compounds, given its unspecific oncolysis property, local administration seems to be the only delivery technique. The best solution for this problem is to identify new oncolytic drugs or coating biomaterials which minimize the injection times but reach the same therapeutic effects. This improvement may benefit beyond superficial cancer patients.

12.

Autophagy induction by thiostrepton improves the efficacy of immunogenic chemotherapy

Yan Wang, 1,2,3,4,5,6,7,8,9 Wei Xie , 1,2,3,4,5,6,7 Juliette Humeau, 1,2,3,4,5,6,7 Guo Chen, 1,2,3,4,5,6,7,10 Peng Liu, 1,2,3,4,5,6,7 Jonathan Pol, 1,2,3,4,5,6,7 Zhen Zhang, 8,9 Oliver Kepp , 1,2,3,4,5,6,7 Guido Kroemer 1,2,3,4,5,6,7,11,12,13 To cite:

IntroduCtIon

Although it was thought for more than 50 years that anticancer cytotoxicants would kill tumor cells, thus yielding a direct antineoplastic effect, 1 it has become clear over the past 15 years that some particularly successful chemotherapeutics do not act in such a direct fashion but rather stress and kill a fraction of the tumor cells in a way that they elicit an immune response against tumor-associated antigens. 2 Thus, immunogenic cell death (ICD) 3 causes tumor growth control via the induction of antitumor immunity requiring the implication of dendritic cells and interferon-γ producing cytotoxic T lymphocytes (CTLs). This implies that some chemotherapeutics can be advantageously combined with immune checkpoint blockers targeting the programmed cell death death 1/ programmed death ligand 1 (PD-1/PD-L1) interaction. 4-7 Only a fraction of cytotoxicants are able to stimulate ICD, which requires the premortem induction of specific stress pathways, in particular, autophagy and partial endoplasmic reticulum (ER) stress response with the phosphorylation of eukaryotic initiation factor 2α (eIF2α). 8-10 Autophagy is required for the lysosomal secretion of ATP, a chemotactic factor that is released from stressed/ dying cancer cells and attracts dendritic cell precursors into the tumor bed via the action on purinergic receptors and may contribute to local inflammasome activation as well. 11 12 ER stress facilitates the surface exposure of the most abundant ER luminal protein, calreticulin (CALR). 9 10 13 14 Once present at the surface of the plasma membrane of stressed/ dying cancer cells, CALR serves as a potent 'eat-me' signal to facilitate the uptake of tumor antigens by immature dendritic cells. 15 High-mobility group box 1 (HMGB1), which is the most abundant non-histone chromatinbinding protein, is released from dying/dead cells to reach toll-like receptor 4 (TLR4) on dendritic cells, stimulating their maturation for the cross-presentation of tumor antigens to CTL. 16 17 Hence, this cascade (ATP, CALR, HMGB1) 2 18 contributes to the stepwise initiation of the anticancer immune response within the tumor bed and composes the triad of ICD.

MAterIAls And Methods

Cell culture and chemicals Culture media and supplements for cell culture were obtained from Life Technologies (Carlsbad, California, USA) and plasticware came from Greiner BioOne (Kremsmünster, Austria). PC12 cells stably expressing doxycycline-inducible Q74-GFP were cultured in Roswell Park Memorial Institute (RPMI)-1640 containing 5% fetal bovine serum and Open access 10% horse serum. 19 Human osteosarcoma U2OS TFE -/- cells carrying a knockout for TFE3 and TFEB, 20 MCA205 murine fibrosarcoma stably expressing shRNAs interfering with the expression of TFE3 and TFEB, 20 and all the other cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and both containing 100 units/mL penicillin G sodium and 100 mg/mL streptomycin sulfate. All cells were cultured at 37°C under 5% CO 2 . The Prestwick Chemical Library was obtained from Prestwick Chemical (Illkirch, France), flavonoids were purchased from Extrasynthese (Genay, France), fatty acids came from Larodan (Malmö, Sweden); known calorie restriction mimetics, cisplatin, dactinomycin, cycloheximide, staurosporine, thapsigargin, tunicamycin, bafilomycin A1, and cycloheximide were from Sigma-Aldrich (St. Louis, Missouri, USA).

high-content microscopy

Human osteosarcoma U2OS cells wild type or stably expressing GFP-LC3; GFP-TFEB; mRFP-GFP-LC3; mCherry-GFP-p62, HMGB1-GFP and CALR-RFP, or human glioma H4 cells stably expressing GFP-LC3 or PC12 cells expressing GFP-Q74 were seeded in 384-well black microplates. After treatment, cells were fixed with 4% paraformaldehyde (PFA, w/v in phosphate buffered saline (PBS)) overnight at 4°C and stained with 10 µg/mL Hoechst 33 342 in PBS. Images were acquired using an ImageXpress micro XL automated microscope (Molecular Devices, San Jose, California, USA) with a ×20 PlanApo objective (Nikon, Tokyo, Japan). A minimum of four view fields was acquired per well, and experiments involved at least triplicate assessments. Quantitation was done on 1000-2400 cells per condition. Following images were analyzed using the Custom Module Editor of the MetaXpress software (Molecular Devices). Briefly, cells were segmented and divided into nuclear and cytoplasmic regions based on the nuclear Hoechst staining and cytoplasmic GFP or RFP signal. GFP-LC3 dots were measured in the cytoplasmic compartment. For quinacrine assays, cells were incubated with Krebs-Ringer solution containing 5 µM quinacrine and 20 µg/mL Hoechst 33 342 at 37°C for 30 min, then cells were washed and imaged alive. Quinacrine dots were measured in the cytoplasmic compartment. GFP-TFEB or HMGB1 fluorescence intensities were measured in the nuclear compartment. CALR-RFP aggregation was measured perimembranous while excluding perinuclear signal. Data processing and statistical analyses were performed using the R software (http://www. r-project. org/).

In vivo experimentation

All mice were kept in an environmental-controlled and pathogen-free facility with 12 hours light/dark cycles and food and water ad libitum. Female wild-type C57BL/6 mice and athymic female nude mice (nu/nu) at the age of 6-8 weeks were obtained from Envigo, France (Envigo, Huntingdon, UK). For immunoblotting assessment of LC3, wild-type C57BL/6 mice were treated with thiostrepton, liver were thereafter collected and processed. For the GFP-LC3 assessment, 8-week-old GFP-LC3 transgenic (Tg-GFP-LC3) mice, containing a rat LC3-EGFP fusion protein under the control of the chicken b-actin, were treated with thiostrepton with or without 40 mg/kg of the autophagic flux inhibitor leupeptin 2 hours before the organs were collected and processed. For tumor growth experiments, MCA205 or TC-1 tumors were established in C57BL/6 hosts by subcutaneously inoculating 3×10 5 cells. Thiostrepton was dissolved in 3.34% dimethylsulfoxide (DMSO), 10.02 % PG400 (Sigma-Aldrich), 10.02% TWEEN80 (Sigma-Aldrich) and 76.6% corn oil (Sigma-Aldrich).

When tumors became palpable, mice were treated with thiostrepton in 200 mg/kg 1 day before, on, and 1 day after the chemotherapy (10 mg/kg oxaliplatin, Sigma-Aldrich) by intraperitoneal injection. On the following days, thiostrepton were given 40 mg/kg alone three times per week, and mice well-being and tumor growth were monitored and documented. Animals were sacrificed when tumor size reached endpoint or signs of obvious discomfort were observed following the EU Directive 63/2010 and our Ethical Committee advice.

detection of protein deacetylation U2OS-GFP-LC3 stable cells were seeded in 384-well microplates for 24 hours. After experimental treatments, cells were fixed with 4% PFA for 20 min at room temperature. Thereafter, cells were incubated with an antibody specific for acetyl-alpha-tubulin (#5335, 1:500, Cell Signaling Technology) in 5% bovine serum albumin (BSA, w/v in PBS) for 1 hour to block non-specific binding sites and acetylated tubulins, followed by overnight incubation at 4°C with specific antibody to detect acetylated proteins at lysines (#623402, 1:400, BioLegend, San Diego, California, USA). After washing several times with PBS, cells were incubated with Alexa Fluor conjugates (Life Technologies) against the primary antibody for 2 hours at room temperature. Nuclei were stained by incubating the cells with 10 µg/mL Hoechst 33 342 in PBS. Fluorescent images were acquired and analyzed as described before.

determination of extracellular hMGb1 concentration Cell culture supernatants were collected and centrifuge to remove debris. The concentration of HMGB1 was detected by ELISA (HMGB1 ELISA kit II; Shino Test Corporation, Tokyo, Japan) according to the manufacturer's instructions. The positive control was from the same kit. Absorbance was analyzed by means of an Paradigm I3 multimode plate reader (Molecular Devices).

Immunoblotting

After treatment, cells were collected and lysed in cold RIPA lysis and extraction buffer (Thermo Fisher Scientific, Carlsbad, California, USA) containing Pierce Protease and Phosphatase Inhibitor Mini Tablet (Thermo Fisher Scientific) on ice for 40 min. After centrifugation at 12 000g for 15 min, supernatants were heated in sample buffer (Thermo Fisher Scientific) at 100°C for 10 min. Protein samples were separated on precast 4%-12% polyacrylamide NuPAGE Bis-Tris gels (Thermo Fisher Scientific) and electrotransferred to Polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Burlington, Massachusetts, USA). Membranes were probed overnight at 4°C with primary antibodies specific to LC3 (#2775, 1:1000, Cell Signaling Technology), p62 (#ab56416, 1:10 000, Abcam, Cambridge, UK), Atg5 (#A2859, 1:1000, Sigma-Aldrich, St. Louis, Missouri, USA), GAPDH (#ab8245, 1:10 000, Abcam), TFEB (#4240, 1:1000, Cell Signaling Technology, Danvers, Massachusetts, USA), TFE3 (ab93808, 1:2000, Abcam), and β-actin (#ab49900, 1:10 000, Abcam) followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (Southern Biotech, 1:5000, Birmingham, Alabama, USA). Immunoreactive bands were visualized with ECL prime western blotting detection reagent (Sigma-Aldrich). Equal loading was controlled for all blots and one representative loading control is depicted, as indicated in the figure legends.

Immunofluorescence

Cells were incubated with 3.7% PFA for 20 min at room temperature. For TFE3 staining, fixed cells were then permeabilized with 0.1% Triton X100 on ice, and blocked with 5% bovine serum albumin (BSA, w/v in PBS) for 1 hour. For phospho-eIF2 alpha (Ser51) staining, fixed cells were directly blocked with 5% BSA for 1 hour. Then cells were incubated with antibodies specific to TFE3 (#PA5-54909, 1:500, Invitrogen) or phospho-eIF2 alpha (Ser51) (#ab32157, 1:500, Abcam) overnight (ON) incubation at 4°C. Thereafter, Alexa Fluor 647 nm conjugates (Thermo Fisher Scientific) against the primary antibody were applied for 2 hours at RT. Cells were then washed and imaged by high-content microscopy as described above. The nuclear intensity of TFE3 and cytoplasmic intensity of phospho-eIF2 alpha (Ser51) were measured and normalized on Ctrl.

Autophagy measurement on tissue sections

Liver tissues were fixed with 10% neutral buffered formalin at overnight at 4°C and then transferred into 30% sucrose diluted in PBS for 24 hours at 4°C. Thereafter, the organs were embedded in optimal cutting temperature (OCT) solution, and consecutive tissue sections were cut by cryostat. Samples were stained with 4′,6-diamidino-2-phenylindole (DAPI) to detect nuclei, and images were acquired by confocal microscopy. GFP-LC3 dot area was measured by ImageJ. extracellular AtP level Cellular supernatants were collected and processed with the ENLITEN ATP Assay System Bioluminescence Detection Kit (Promega, Madison, Michigan, USA; #FF2000) following the manufacturer's methods. Fluorescence was detected by means of a Paradigm I3 multimode plate reader (Molecular Devices).

Open access

Flow cytometry analysis of CAlr surface exposure Cells were detached and collected and sequentially incubated with Zombie UV (#423114; Biolegend), anti-CALR rabbit monoclonal antibody (#ab2907; Abcam, Cambridge, UK) for 30 min at 4°C, followed by Alexa Fluor 488, 546 or 647 goat anti-rabbit IgGs (#A11034, Thermo Fisher Scientific) for 30 min at 4°C, then fixed with 4% PFA in PBS for flow cytometric assessment.

Flow cytometric analysis of annexin V and dAPI Cells were detached and collected and incubated with annexin V (Biolegende #640943) and DAPI according to the manufacturer's advice. Following cells were assessed by flow cytometry.

reactive oxygen species detection Cells were treated as indicated and menadione was used for 1 hour at 100 µM as a positive control. CellROX deep red (#C10422, Thermo Fisher Scientific) was added for 30 min at a final concentration of 5 µM. Then cells were fixed and stained with 4% PFA containing Hoechst for 30 min at room temperature and images were acquired and analyzed by automated microscopy.

ex vivo-phenotyping of the tumor immune infiltrate Tumors were harvested, weighed and transferred on ice into gentleMACS C tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) containing 1 mL of RPMI medium. Tumors were dissociated first mechanically with scissors, then enzymatically using Miltenyi Biotec mouse tumor dissociation kit and a GentleMACS Octo Dissociator following the manufacturer's instructions. Tumor homogenates were filtered through 70 µm MACS Smart-Strainer (Miltenyi Biotec) and washed twice with PBS. Finally, bulk tumor cells were homogenized in PBS at a concentration corresponding to 250 mg of the initial tumor weight per millilitre. Bulk tumor cell homogenates, each corresponding to 50 mg of the initial tumor sample, were stained with LIVE/DEAD Fixable Yellow dye (Thermo Fisher Scientific). Fc receptors were blocked with anti-mouse CD16/CD32 (clone 2.4G2, Mouse BD Fc Block, BD Pharmingen). Surface staining of murine immune cell populations infiltrating the tumor was performed with the following fluorochromeconjugated antibodies: ( 1 panel: anti-CD3 APC (clone 145-2 C11, BioLegend), anti-CD8 PE (clone 53-6.7, BD Pharmingen), anti-CD4 PerCP-Cy5.5 (clone RM4-5, Thermo Fisher Scientific), anti-CD25 PE-Cy7 (clone PC61.5, Thermo Fisher Scientific). Then, cells were fixed and permeabilized in eBioscience Foxp3/Transcription Factor Staining Buffer (Thermo Fisher Scientific). To complete the 'Tcell activation/exhaustion' panel, an intranuclear staining was performed with anti-FoxP3 FITC (clone FJK-16s, Thermo Fisher Scientific). Finally, stained samples were run through a BD LSR II flow cytometer. Data were acquired using BD FACSDiva software (BD Biosciences) and analyzed using FlowJo software (TreeStar). Absolute counts of leukocytes and tumor cells were normalized considering the following parameters: weight of the harvested tumor and total volume of the dissociated tumor cell suspension (cell concentration typically set to 250 mg/mL in PBS), proportion of the whole cell suspension and proportion of the cell suspension used for cytometry. (Representative gating strategies are depicted in online supplementary figure S5 and S6.) statistical analysis Data are reported as means±SD of triplicate determinations and experiments were repeated at least twice, yielding similar results if not otherwise reported. Statistical significance was assessed by Student's t-test. TumGrowth and GraphPad were used to analyze in vivo data arising from murine models. 21 results And dIsCussIon A pharmacologic screen identifies thiostrepton as an autophagy inducer To identify bona fide autophagy inducers, we screened a total of 1560 compounds contained in the Prestwick library, as well as home-made libraries containing flavonoids 18 20 and a range of saturated and unsaturated fatty acids 22 for their capacity to induce the formation of autophagic puncta in human H4 neuroblastoma cells that express a green fluorescent protein (GFP) fused to microtubule-associated proteins 1A/1B light chain 3B (best known as LC3) (figure 1A,B; online supplementary table S1). The 175 compounds that were most efficient at one of the chosen concentrations were re-tested on another cell line, namely, human U2OS osteosarcoma cells stably expressing the monomeric red fluorescent protein (mRFP)-GFP-LC3 tandem reporter 23 for their capacity to induce autophagic flux (meaning that they increase the ratio of RFP-positive over GFP-positive puncta) (figure 1C,D; online supplementary table S2). The 34 most effective candidate compounds (online supplementary table 1) were finally investigated for their capacity to reduce intracellular ATP concentrations measured with the fluorochrome quinacrine in combination with the prototypic ICD Open access inducers mitoxantrone, using 3,4-dimethoxychalcone 20 as a positive control (figure 1E,F; online supplementary table S3). This approach led to the identification of thiostrepton, an antibiotic produced by Streptomyces azureus and Streptomyces laurentii, 24-26 as a potential ICD enhancer.

Open access

Validation of autophagy induction by thiostrepton

When used at micromolar concentrations for 6 hours, thiostrepton had little or no cytotoxic activity on cultured cells and reduced cytoplasmic protein acetylation, as measured by immunofluorescence staining, as it induced cytoplasmic GFP-LC3 puncta in human and murine biosensor cells (figure 2A-D, online supplementary figure S1A,B). In the presence of bafilomycin A1, thiostrepton continued to increase the number of cytoplasmic GFP-LC3 puncta per cell (figure 2E,F, online supplementary figure S1A,B). Moreover, the autophagy-associated lipidation of LC3 was enhanced by thiostrepton, more so in the presence of bafilomycin A1 (figure 2G,H, online supplementary figure S1C,D), indicating that thiostrepton truly induced autophagy. Accordingly, thiostrepton acted on another biosensor that measures autophagic flux, namely, a mCherry-GFP-sequestosome-1 (SQSTM1, best known as p62) to preferentially reduce the GFP-dependent fluorescence, indicating an increase in autophagy (figure 2I,J). 27 Moreover, thiostrepton stimulated the degradation of a Q74-huntingtin-GFP fusion protein indicative of an increase of autophagic flux 28 (figure 2K,L). When systemically injected into mice, thiostrepton also caused increased LC3 lipidation in the liver, suggesting that it is active in vivo (figure 2M-P). Of note, thiostrepton lost its capacity to induce GFP-LC3 puncta in U2OS cells that lack the essential autophagy gene ATG5 or the two proautophagic transcription factors TFEB and TFE3 (figure 3A-D). Indeed, thiostrepton induced the translocation of TFEB and TFE3 from the cytoplasm to the nucleus, as did the positive control torin (figure 3E,F). Moreover, thiostrepton induced the phosphorylation of eIF2α, an event that is often associated with autophagy. 29 However, thiostrepton did inhibit transcription and translation in human cells cultured in vitro (see online supplementary figure S2A-D), indicating that it can interfere with protein synthesis in eukaryotes as it does in bacteria. 30 Thiostrepton did not lead to an overproduction of reactive oxygen species (see online supplementary figure S2E,F), yet had (limited) cytotoxic effects when cells were exposed to the drug for an extended amount of time (24 hours) (see online supplementary figure S2G-J). This effect was further increased when thiostrepton was combined with low-dose oxaliplatin. Altogether, these results confirm that thiostrepton is a potent autophagyinducing and cell stress-inducing agent.

thiostrepton amplifies ICd surrogate markers Mitoxantrone (MTX) and oxaliplatin (OXA) are potent ICD inducers. 31 As such, they elicit the ICD hallmarks in vitro, in cultured cancer cells, including the autophagydependent ATP release into the supernatant, calreticulin (CALR) exposure on the cell surface, as well as nuclear exodus of HMGB1. 32-34 We determined the capacity of thiostrepton to enhance these stigmata of ICD in a series of in vitro assays. The importance of thiostreptoninduced ATP release needs to be further validated in vivo. Nevertheless, it appears clear that thiostrepton reduced the abundance of quinacrine-detectable ATP in the cytoplasm of cultured cells, more so if combined with low concentrations of MTX or OXA, suggesting additive interactions (figure 4A,B). Accordingly, ATP concentrations in the supernatant increased with the thiostrepton/ MTX or thiostrepton/OXA concentration, and this ATP release was inhibited in cells in which ATG5 or both TFEB and TFE3 had been knocked down (figure 4C-I) even after extended incubation for 24 hours (see online supplementary figure S3A,B). Thiostrepton was able to cause the relocation of an RFP-CALR fusion protein to the periphery of the cells either alone or in the presence of MTX or OXA (figure 5A,B), and this effect did not require TFEB plus TFE3 (see online supplementary figure S4A). The capacity of thiostrepton to cause CALR exposure was validated by surface immunofluorescence staining (figure 5C,D). Finally, protracted incubation Open access (48-72 hours) with thiostrepton could cause HMGB1 release, more so when it was combined with low-dose MTX or OXA (figure 5E-G), which was reduced when TFEB plus TFE3 was knocked down (see online supplementary figure S4B). Hence, thiostrepton can amplify ICD surrogate markers in the context of low-dose MTX or OXA.

thiostrepton enhances the efficacy of immunogenic chemotherapy Although thiostrepton alone had no tumor growth reducing effect against TC1 non-small cell lung cancers or MCA205 fibrosarcomas established in immunocompetent C57/BL6 mice, it did increase the anticancer activity of the ICD inducer OXA (but not cis-diamminedichloridoplatinum(II), abbreviated CDDP, best known as cisplatin, which is unable to trigger ICD see online supplementary figure S5), 7 further diminishing tumor size and extending the survival of the animals (figure 6A,B). This thiostrepton/OXA combination effect depended on T lymphocytes because tumor developing in athymic nu/nu mice failed to reduce their growth in response to the treatment (figure 6C). Moreover, tumors formed by cancer cells lacking TFEB and TFE3 expression that were implanted in immunocompetent C57/BL6 mice, failed to exhibit the combination effect as well (although they responded to OXA) (figure 6D), supporting the idea that TFEB/TFE3dependent autophagy induction is critical for the anticancer action of thiostrepton. Analysis of the leukocyte infiltrate by immunophenotyping and cytofluorimetry (see online supplementary figure S6) revealed that thiostrepton had no significant effect on the abundance of CD8 + CTLs (figure 7A) but reduced the number of CD4 + CD25 + Foxp3 regulatory T cells (Tregs) if combined with OXA (figure 7B), thus improving the CTL/Treg ratio (figure 7C). Moreover, the combination treatment was particularly efficient in increasing the number of tumor infiltrating neutrophils (see online supplementary figure S7 andS8). In conclusion, thiostrepton can synergize with OXA to improve tumor control in an immune-dependent fashion, correlating with reprogramming of the tumor microenvironment.

Concluding remarks

Thiostrepton is a fully synthetic oligothiopeptide antibiotic that has been approved for veterinary but not human use, for the treatment of superficial infections including dermatitis and mastitis. 35 Preclinical experiment suggest that it may inhibit human breast cancer growth and overcome cisplatin resistance. 36 Here, we identified thiostrepton in an unbiased screen designed to identify autophagy inducers, and subsequently discovered that this agent can enhance the stigmata of ICD in cultured cells, thus enhancing ATP release, CALR exposure and HMGB1 exodus. Although thiostrepton had some activities on these ICD hallmarks when used alone, it was particularly active when combined with low doses of established ICD inducers such as MTX and OXA. Moreover, thiostrepton enhanced the anticancer activity of OXA in vivo. This adjuvant effect required the expression of proautophagic transcription factors by the cancer cells and was fully lost in the absence of T lymphocytes. Indeed, thiostrepton combined with OXA stimulated a major change in the tumor immune infiltrate, enhancing the density of CTL, but reducing that of Tregs and G-MDSC. Thiostrepton is an antibiotic, 24 and the intestinal microbiota has been shown to contribute to anticancer immune responses elicited by some immunogenic chemotherapies 37 38 or immunotherapy, meaning that broad-spectrum antibiotics causing sterilization of the intestinal tract reduce the efficacy of anticancer treatments. 39 40 For this reason, it appears unlikely that the immune-dependent anticancer effects of thiostrepton would be related to its antibiotic activity. Thus, thiostrepton could be repurposed as an enhancer of immunogenic chemotherapies, independent of its antibiotic activity.

Introduction

Peptides can be synthetically generated and potentially provide pharmacological leads or final agents for multiple purposes. In the field of cancer research, so-called oncolytic peptides have been conceived with the objective of selectively killing tumor cells. This may be achieved by fusing targeting sequences (that interact with proteins specifically expressed on the surface of malignant cells or tumor vasculature) with effector sequences (that cause the lysis of the targeted cell type) 1-3 , or alternatively by local administration of the oncolytic peptide into the neoplastic lesion, notably by direct injection 4-8 . Lytic peptides usually combine two physicochemical features, namely lipophilicity and cationic charge, meaning that they contain hydrophobic and positively charged amino acids (mostly arginine and lysine residues) that may be interspersed in a way to create an amphipathic structure 9 .Itis thought that this design facilitates the enrichment of the peptides within the cell and, in particular, the mitochondrial matrix as a result of their electrophoretic distribution following the Nernst equation 10 , hence allowing them to mediate local membrane-permeabilizing effects that compromise organellar and cellular integrity 11 .

Although the overall molecular design of oncolytic peptides follows the rules exposed above, there may be major, hitherto unexplained differences in the subcellular distribution of such agents. For example, the oncolytic peptide LTX-315 follows a classical pattern of mitochondrial distribution causing early permeabilization of this organelle with the dissipation of the mitochondrial transmembrane potential and the release of intermembrane proteins including cytochrome c and DIABLO through the outer membrane 11,12 . In sharp contrast, oncolytic, LTX-401, an amphipathic β(2,2)-amino acid derivative, tends to enrich in the Golgi apparatus and dismantles the organelle before mitochondrial integrity is compromised 4,8,13 . This mitochondrial step of the cell death cascade appeared to be important for cell death induction by both LTX-315 and LTX-401, because knockout of the proapoptotic multidomain BCL2 family proteins BAX and BAK attenuated cell killing by both LTX-315 and LTX-401 8,11 . These differences and similarities illustrate the complexity of pro-death signaling mediated by agents that apparently share comparable physicochemical properties.

Over the past few years, it has become increasingly clear that anticancer drugs should not only be optimized with respect to their capacity to kill a significant (and ideally close-to-total) fraction of malignant cells and hence to "debulk" the primary tumor and its metastases. Rather, antineoplastics should also be able to stimulate anticancer immune responses, an effect that can be achieved by a variety of mechanisms, namely, (i) killing of cancer cells in a way that leads to the release or exposure of danger-associated molecular patterns (DAMPs) that will alert the innate and later the acquired immune system to recognize tumor-associated antigens, (ii) direct stimulation of immune effectors or subversion of immunosuppressive mechanisms 14-16 .I n d e e d ,i ti s difficult to conceive that any kind of cancer treatment would be able to eliminate the very last malignant cell (among 10 10 to 10 12 neoplastic cells) and hence to achieve definitive cure, a scenario that would apply if anticancer immunosurveillance was not involved in long-term treatment outcomes.

One mechanism that appears to be particularly important for triggering antineoplastic immune responses is the induction of immunogenic cell death (ICD), tied to the cell surface exposure of the DAMP calreticulin, an "eatme" signal for dendritic cell (DC) precursors, as well as the release of several DAMPs including ATP and HMGB1 that attract DCs into the proximity of cancer cells and activate them for optimal tumor antigen crosspresentation to cytotoxic T lymphocytes 17-21 . Of note, both LTX-315 and LTX-401 are able to stimulate ICD, meaning that cancer cells killed by these agents in vitro can elicit protective antitumor immune responses upon their inoculation in vivo and that local administration of either LTX-315 or LTX-401 triggers signs of an immune response in established tumors 4-8,13,22,23 . For this indication, LTX-315 is currently undergoing clinical trials (NCT01986426; clinicaltrials.gov).

Based on the aforementioned premises, the development of oncolytic peptides is currently expanding. Recently, a new class of oncolytic peptides (DTT peptides) with antilymphoma activity were reported 24 . Several new analogues were designed and tested against hepatocellular and colorectal carcinoma cell lines (Table 1). In this report, we analyzed the cellular and immunological mode of action of two novel oncolytic DTT-analogues, DTT-205 and DTT-304. Here, we report that both agents selectively target lysosomes and share some but not all of their cytotoxic mechanisms of action. Importantly, both DTT-205 and DTT-304 can cause the total eradication of established mouse cancers as they induce a potent, therapeutically relevant antitumor immune response.

Results and discussion

Morphological effects of When added to human cancer cells, DTT-205 or DTT-304 stimulated a necrosis-like disruption of cellular morphology. In cells in which the plasma membrane was still intact and hence retaining the cytoplasm, lipid droplets in the cytoplasm were a prominent morphological feature (Fig. 1a) that was induced in a dose-dependent fashion (Fig. 1b,c), as determined by transmission electron microscopy. Staining with the red fluorescent lipophilic dye Nile red confirmed the formation of lipid droplets in the cytoplasm of cells treated with DTT-205 or DTT-304 that occurred in a time and dose-dependent fashion (Fig. 1d-f). Of note, these effects were only obtained when cellular metabolism and membrane trafficking were active at 37 °C, yet not at lower temperatures such as 14 or 22 °C (Fig. 1g,h). As for other oncolytic peptides such as LTX-315 25 and LTX-401 8 , DTT-205 and DTT-304 lost their cytotoxic potential in the presence of serum (Supplementary Figure S1). However, DTT-205 and DTT-304 differed from LTX-315 and LTX-401 in the sense that only the DTT peptides, but not the LTX compounds, induced lipid droplets (Supplementary Figure S2).

Lysosomal tropism of DTT-205 and DTT-304

Intrigued by the peculiar morphology of DTT-treated cells, we investigated the subcellular distribution of these agents, taking advantage of DTT-205 and DTT-304 that had been modified to attach a blue-fluorescent moiety to the molecules. Cells that express fluorescent biosensors in the nucleus (histone H2B fused to red fluorescent protein, RFP), endoplasmic reticulum (calreticulin, CALR fused to green fluorescent protein, GFP), Golgi apparatus (GALT1 fused to GFP), mitochondria (DIABLO fused to GFP), or lysosomes (LAMP1 fused to GFP) were incubated with the fluorescent derivatives of DTT-205 and DTT-304. Confocal fluorescence microscopy revealed that both DTT-205 and DTT-304 co-localized with the lysosomal marker LAMP1-GFP but not with any other organellar probe (Fig. 2a-d). Of note, this colocalization was abrogated upon preincubation of the cells with the vacuolar ATPase inhibitor bafilomycin A1 (BAFA1), which is known to abolish lysosomal acidification (Fig. 2b-d). As other lysosomotropic agents 26 , DTT-205 and (less so) DTT-304-affected lysosomal stability and let to a reduction in measurable organellar surface area as detected by LysoTracker TM (Fig. 2e-g). Next, we determined whether the lysosomotropism of DTT-205 and DTT-304 might explain their cytotoxic activity.

For this, we used BAFA1 to avoid their lysosomal accumulation (Fig. 2b), finding that this maneuver partially reduced cell killing by DTT-205 or DTT-304 (Fig. 2h,i).

Apoptosis-related signaling induced by DTT-205 and DTT-304

Caspase-3 activation is one of the biochemical hallmarks of apoptosis 27 .Whileasignificant fraction of cells treated with the positive control, the pan-tyrosine kinase inhibitor staurosporine stained positively with an antibody recognizing the proteolyticallymaturefragmentof caspase-3, only a minor fraction of cells exhibited caspase activation after treatment with DTT-205 or DTT-304 (Fig. 3a-d). Both DTT-205 and DTT-304 caused nuclear shrinkage, though without the formation of apoptotic bodies, and a decrease in the number of analyzable cells (Fig. e,f; Supplementary Fig. 3). The pan-caspase inhibitor z-VAD-fmk failed to prevent cell killing by h), as did the ferroptosis inhibitor ferrostatin-1. In contrast, two antioxidants, namely N-acetylcysteine and glutathione strongly reduced the cytotoxic activity of Supplementary Figure 4). Apoptotic signaling also involves the mitochondrial membrane permeabilization that often depends on the expression of proapoptotic multidomain members of the BCL2 family such as BAX and BAK. Indeed knockout of BAX, alone or together with BAK, reduced killing by DTT-205 or DTT-304 to a variable extent (Fig. 3k,l). In synthesis,

Necroptotic signaling induced by DTT-205 and DTT-304

To investigate the potential role of necroptotic signaling in cell death induction by DTT compounds, we took advantage of a biosensor cell line that lacks MLKL (and hence cannot undergo necroptosis) yet expresses a RIP3-GFP fusion protein that can be monitored for its aggregation in the cytoplasm within so-called "necroptosomes" 28,29 . As a positive control, a combination of tumor necrosis factor-α (TNFα), DIABLO mimetic, and z-VADfmk (collectively abbreviated as 'TSZ') induced full necroptosome activation. DTT-205 was unable to induce this phenomenon, while DTT-304 turned out to cause necroptosome activation in a time-dependent and concentration-dependent fashion (Fig. 4a-d). Accordingly, cells treated with DTT-304 (but not DTT-205) manifested the phosphorylation of the RIP3 substrate MLKL (Fig. 4e,f). Cells rendered deficient for RIP3 or MLKL 30 became partially resistant against cell killing by DTT-304 (but not DTT-205) (Fig. 4g,h), supporting the notion that DTT-304 (but not DTT-205) can activate a cell death signaling pathway that involves the necroptotic cascade.

Immunogenic cell death triggered by DTT-205 and DTT-304

ICD is characterized by the cellular release/exposure of DAMPs (such as CALR, HMGB1 and type-1 interferons) that make dead-cell antigens recognizable to the immune system. Both DTT-205 and DTT-304 triggered CALR exposure on the cell surface (Fig. 5a,b), nuclear HMGB1 exodus (Fig. 5c,d), and the transcription of genes coding for type-1 interferons (Fig. 5e,f). Nevertheless, the treatment with oncolytic peptides did not lead to an increase in the expression of major histocompatibility complex class I molecules (MHC-I) (Supplementary Figure 5) and depicted a rather unspecific cytotoxicity on both cancer and immune cells (Supplementary Figure 6). Based on these in vitro characteristics of DTT compound-induced cell death, we next investigated whether these agents might mediate anticancer effects through the stimulation of an anticancer immune response. For this, established MCA205 fibrosarcomas growing on immunocompetent haploidentical C57BL/6 mice were injected with either DTT-205 or DTT-304, which both caused complete oncolysis in most surviving mice. Of note, prior depletion of T cells by injection of specific antibodies blocking CD4 and CD8 abolished these anticancer effects. These immune-dependent anticancer effects of DTT-205 and DTT-304 were evident both when tumor growth kinetics and overall survival of mice were monitored (Fig. 6a-g). Rechallenge of mice that had been cured from MCA205 cancers with the same tumor type (MCA205) was incompatible with the development of cancers, although antigenically distinct TC-1 lung adenocarcinoma grew in most (eight out of nine) cases (Fig. 6h-k). Very similar results were obtained when TC-1 carcinomas instead of MCA205 sarcomas were treated with DTT-205 or DTT-304. All animals that could be monitored were cured from their cancers (Supplementary Figure 7A-E), and none of the cured animals that were rechallenged with TC-1 developed cancers, although MCA205 tumors readily developed (Supplementary Figure 7F-H). Animals bearing TC-1 tumors on both flanks that were treated with the oncolytic peptides only on one side, but not on the other depicted abscopal effects that led to the reduction of tumor growth in the distant lesion (Supplementary Figure S8). These results indicate that immune-dependent cancer cure mediated by DTT-205 or DTT-304 is coupled to the establishment of systemic immunity that mediates long-term immune memory specific for the eradicated cancer type.

Concluding remarks

The results of this study suggest that the oncolytic peptides DTT-205 and DTT-304 both target lysosomes as indicated by their selective accumulation in this organelle. To this respect, DTT-205 and DTT-304 are rather distinct from other oncolytic peptides that often target the mitochondria. Despite their common lysosomotropic nature, DTT-205 and DTT-304 are to a certain extent different in their mode of action. While cell death induced by both agents strongly depends on the production of reactive oxygen species exclusively DTT-304 shows pronecroptotic features, such as the formation of the necroptosome and the phosphorylation of MLKL.

(see figure on previous page) Fig. 3 Cell death induced by DTT-205 and DTT-304. Human osteosarcoma U2OS cells were treated with 0.65 to 10 µM DTT peptides for the indicated time and following assessed for the activation of caspase-3 (CASP3). The pan-kinase inhibitor staurosporine (STS) was used as positive control. Representative images (a, b) and quantifications of CASP3 activation (c, d) are depicted while pyknosis was assessed by measuring the decrease in Hoechst 33342-stained nuclear area (e, f) by conventional microscopy (size bar equals 10 µm; mean ± SD of triplicate assessments, Student's t test, *p < 0.5, **p < 0.01, ***p < 0.001). In order to assess the cell death modality, U2OS cells were pretreated with the ferroptosis inhibitor ferrostatin-1 (FER-1), the pan-caspase inhibitor zVAD-fmk (zVAD) or the antioxidants N-actylcystein (NAC) and reduced glutathione (GSH) before the addition of 2.5 µM of DTT peptides (g, h). Exclusively the inhibition of reactive oxygen species (ROS) generation decrease the cytotoxic effects of both DTT-205 and DTT-304 in a dose-dependent fashion (i, j, mean ± SD of triplicate assessments, Student's t test, *p < 0.5, **p < 0.01, ***p < 0.001). Mouse embryonic fibroblasts (MEFs) that were either wild type, single-or double knockout for the proapoptotic proteins Bax and/or Bak were treated with 0.65 to 10 µM of DTT-205 or DTT-304 and viability was assessed by means of an exclusion dye in epifluorescence microscopy (k, l; mean ± SD of triplicate assessments. Student's t test, *p < 0.5, **p < 0.01, ***p < 0.001). Single as well as double knockouts were partially resistant to DTT-peptideinduced cell death Nevertheless, analogue to similar oncolytic peptides such as LTX-315 and LTX-401 4-8,12,23 both DTT-205 and DTT-304 triggered the emission of pro-immunogenic DAMPs in vitro and activated anticancer immune responses in vivo. Complete tumor lysis by DTT peptides required repeated intratumoral injections, which might become a challenging aspect during clinical translation but might facilitate to render the tumor into its own vaccine 31 . Thus, in this study animals that were cured from primary tumors generated long-term immunological memory that mediated the rejection of isogenic tumors, which underlines the immunotherapeutic potential of oncolytic peptides in general and DTT-205 and DTT-304 in particular.

Materials and methods

Chemicals and cell cultures

Cell culture media and supplements were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA) and chemicals came from Sigma-Aldrich (St. Louis, MO, USA) with the exception of LTX-315, LTX-401, DTT-205, and DTT-304 that were provided by Lytix Biopharma (Oslo, and FCS and HEPES as described above. Erythrolysis was conducted using ACK lysis buffer (Thermo Fisher Scientific).

HMGB1 release assays

Cells were seeded in 24-well plate and let adhere and adapt overnight, before they were treated with the DTT peptides. Supernatants were collected and centrifuged at 500 × g for 5 min in order to remove cellular debris. Released HMGB1 protein content was quantified in supernatants by ELISA (#ST51011; IBL, Hamburg, Germany) according to the manufacturer's recommendations, and samples were measured with a Paradigm E3 multilabel reader (Molecular Devices).

Western blot

Half a million cells were harvested from six-well plates resuspended in lysis buffer containing 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktails (Complete protease inhibitor cocktail, Roche, Basel, Switzerland) and incubated on ice for 30 min. To obtain supernatant, cell lysate was centrifuged at 12000×g for 20 min at 4 °C to remove insoluble materials. The lysate was mixed with 4 × NuPAGE® LDS sample buffer and 10 × sample reducing agent, and proteins were denaturated at 100℃ for 10 min. NuPAGE® Novex® 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific) were used for protein electrophoresis under a 100 V constant voltage mode. Separated proteins were transferred from gel to PVDF membrane (Merck-Millipore, Darmstadt, Germany). After blocking with 5% BSA in 1 × TBS containing 0.1% Tween®-20 (1 × TBST) for 1 h at room temperature, the membranes were probed with corresponding primary antibodies at 4 °C overnight: anti-mouse MLKL polyclonal antibody (AP14272b; Abgent, San Diego, CA, USA). To visualize phosphorylated MLKL (pMLKL), anti-mouse MLKL (phospho S345) monoclonal antibody (ab196436, Abcam) was used. The membranes were then washed and incubated with HRPconjugated secondary antibodies (SouthernBiotech, Birmingham, AL, USA) at room temperature for 1 h. The peroxidase activity was detected with ECL Western Blotting Detection Reagent (GE healthcare, Chicago, IL, USA) and images were acquired by ImageQuant LAS 4000 (GE healthcare).

RNA extraction, reverse transcription, and qRT-PCR Cells were collected for total RNA extraction using the RNeasy Mini kit (Quiagen, Hilden, Germany). Three micrograms of RNA were reverse-transcribed to cDNA using SuperScript® III First-Strand System (Thermo Fisher Scientific). Type I IFN-related gene expression was quantified with TaqMan® Gene Expression Assays using Universal Master Mix II (with UNG) on a StepOnePlus™ Real-Time PCR system (all from Thermo Fisher Scientific). GAPDH was used as house-keeping gene for normalization. Relative gene expression was quantified using the comparative Ct method and was calculated as fold change. All experiments were conducted in triplicate assessment.

High-throughput assessment of cell death

In total, 5 x 10 3 U2OS cells were seeded into black 96well µclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter the cells were treated with the DTT compounds and respective controls and incubated for additional 6 or 24 h before either 1 µM of DAPI or a mixture of 1 µM Hoechst, and 1 µM propidium iodide were added immediately before monitoring the uptake of the exclusion dye in a minimum of four view fields per well by means of an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20 × /0.75 NA objective (Nikon, Tokyo, Japan).

Immunostaining

In total, 5 x 10 3 U2OS cells were seeded into black 96well µclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter, the cells were treated with DTT compounds and respective controls and incubated for additional 6 or 24 h before fixation in 3.7% (w/v) paraformaldehyde (PFA) in PBS supplemented with 1 µM Hoechst 33342 for 20 min. Upon fixation, cells were permeabilized with 0.1% Triton in PBS for 10 min at RT. Unspecific binding was blocked with 2% BSA in PBS for 10 min at RT followed by primary antibody diluted in BSA 2% following the manufactures recommendations overnight on an orbital shaker at 4 °C. The cells were rinsed twice and stained with AlexaFluor-coupled secondary antibodies for 1 h at RT, rinsed twice and subjected to imaging using an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20 × /0.75 NA objective (Nikon).

High-content screening microscopy U2OS cells stably expressing GALT1-GFP, CALR-KDEL-GFP, H2B-RFP, LAMP1-GFP, and DIABLO-GFP or HT-29 cells stably expressing RIP3-GFP were seeded in 96-well black microplates for 24 h. After treatment, cells were fixed with 3.7% PFA for 20 min at room temperature and stained with 10 µg/ml Hoechst 33342 in PBS. Image acquisition was performed using an ImageXpress Micro XL automated microscope (Molecular Devices). A minimum of four view fields were captured per well. Upon acquisition, images were analyzed using the Custom Module Editor of the MetaXpress software (Molecular Devices). Briefly, cells were segmented and divided into nuclear and cytoplasmic regions based on the nuclear Hoechst staining and GFP or RFP cytoplasmic signals. GFP-LC3 and RIP3-GFP dots were detected using automated thresholding, and their number and surface were measured in the cytoplasmic compartment. Cooccurrence of GALT1-GFP, CALR-KDEL-GFP, H2B-RFP, LAMP1-GFP, and DIABLO-GFP fluorescence signals with Pacific Blue-labeled DTT compounds were systematically analyzed to assess subcellular targeting. Data processing and statistical analyses were performed using the R software (http://www.r-project.org/).

Transmission electron microscopy

For ultrastructural studies, human osteosarcoma U2OS cells were fixed in 1.6% glutaraldehyde (v/v in 0.1 M phosphate buffer) for 1 h, collected by scraping, centrifuged, and the pellet was postfixed 1% osmium tetroxide (w/v in 0.1 M phosphate buffer). Following dehydration through a graded ethanol series, cells were embedded in Epon™ 812 and ultrathin sections were stained with standard uranyl acetate and lead citrate. Images were taken using a Tecnai 12 electron microscope (FEI, Eindhoven, the Netherlands).

Data processing and statistical analyses

Unless otherwise specified, experiments were performed in triplicate parallel instances and repeated at least once, and data were analyzed with the R software (http://www. r-project.org/). Microscopy images were segmented and analyzed by means of the MetaXpress (Molecular Devices) software and numerical data was further processed with R. Unless otherwise specified, data are presented as means ± SD. Thresholds for the minimum number of events in each analysis necessary to apply further statistics were calculated based on a medium effect size (according to Cohen's conventional criteria) using the pwr package for R with a targeted value of 0.95. Samples that did not match the requirements were marked ND and were excluded from the analysis.

Determination of surface-exposed CALR or MHC-I by immunofluorescence

Cells were collected and rinsed twice with cold PBS. Following the cells were incubated with an anti-CALR antibody (ab2907; Abcam, Cambridge, UK) or anti-MHC-I antibody (12-5958-82, eBioscience, San Diego, CA, USA) diluted in cold blocking buffer (1% BSA in PBS) for 60 min on ice, followed by washing and incubation with AlexaFluor 488-conjugates (Invitrogen) in blocking buffer (for 30 min). Thereafter cells were washed in cold PBS, the vital dye propidium iodide (PI) or Zombie UV was added to a final concentration of 1 μg/mL, and samples were analyzed by means of a CyAn ADP (Beckman Coulter, Brea, CA, USA) coupled to a HyperCyt autosampler (IntelliCyt; Albuquerque, NM, USA). The analysis was limited to living (PI -) cells. Data were statistically evaluated using R (https://www.r-project.org).

Mouse experiments

Female wild-type C57BL/6 mice at the age of 6-8 weeks were obtained from Harlan France (Gannat, France) and maintained in the animal facility at Gustave Roussy Campus Cancer in specific pathogen-free conditions in a temperature-controlled environment with 12 h light, 12 h dark cycles, and received food and water ad libitum. Animal experiments were in compliance with the EU Directive 63/2010 and protocols 2013_094A and were approved by the Ethical Committee of the Gustave Roussy Campus Cancer (CEEA IRCIV/IGR no. 26, registered at the French Ministry of Research). MCA205 tumors were established in C57BL/6 hosts by subcutaneously inoculating 500.000 cells. When tumors became palpable, 1.5 mg of DTT peptides were injected intratumorally. Four days later, remaining tumor tissue was treated accordingly and mice well-being and tumor growth were monitored. Anti-CD8 and anti-CD4 i.p. injections were repeated every 7 days to assure the complete depletion of both T cell populations during the whole experiment. Animals were sacrificed when tumor size reached endpoint or signs of obvious discomfort associated to the treatment were observed following the EU Directive 63/ 2010 and our Ethical Committee advice. Surviving and tumor-free animals were analyzed and kept for more than 30 days before rechallenge with 5 × 10 5 live TC-1 and MCA205 cells injected contralateral in case of MCA205 previously-injected tumor-free animals. In case of previously-injected TC-1 tumor-free animals the location of the injected cells was inverted. Animals were monitored and tumor growth documented regularly until endpoints were reached or signs of obvious discomfort were observed. Statistical analysis was performed employing two-way ANOVA analysis followed by Bonferroni's test comparing to Ctr conditions (*p < 0.05, **p < 0.01, and ***p < 0.001).

Peptide synthesis

All peptides were synthesized on solid-phase with a Prelude instrument (Protein Technologies Inc. Tucson, AZ, USA) using standard Fmoc protocols and amino acid derivatives. All synthesized peptides were prepared as Cterminal amides by using a Rink amide resin (Novabiochem, Merck-Millipore, Billerica, MA, USA) as solid support. The Fmoc-amino acids used were standard derivatives from Novabiochem. Double couplings (2 × 30 min, 5 eq to the resin) were performed. The incoming Fmoc-amino acids were activated with 5 eq (2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate) (HCTU) and 10 eq diisopropylethylamine (DIPEA) with dimethylformamide (DMF) as solvent. Coupling reactions were concluded with a washing (DMF, 3 × 30 s) and Fmoc-removal step (20% piperidine in DMF, 5 + 10 min). For microscopy studies, the peptides were fluorescently labeled at their C-termini with Pacific Blue™ (Ex max = 410 nm, Em max = 455 nm). This was achieved by using a Universal NovaTag resin (Novabiochem, Merck-Millipore, Billerica, MA, USA) as solid support. Before removing the final Fmoc-group, the resin was treated with 2% trifluoroacetic acid (TFA) in dichloromethane (3 × 1 min) and washed with DMF (3 × 30 s) and 1 M DIPEA in DMF (1 × 30 s). The resin was then treated with Pacific Blue succinimidyl ester (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. The resin was washed with DMF (3 × 30 s) and the final Fmoc-group removed. Completed peptides were cleaved from the resin using a cocktail containing 95% TFA, 2.5% water, and 2.5% triisopropylsilane for 3 h. TFA was removed using a rotavapor (Hei-VAP Advantage rotavapor, Heidolph Instruments, Schwabach, Germany) and the fully deprotected peptides precipitated with diethyl ether as Cterminal amides. The ether was decanted and the precipitated crude peptide allowed to air dry before analysis and purification.

Peptide purification and characterization

The solvents used in the analytical and preparative systems were MilliQ water (Solvent A) and acetonitrile (Solvent B), both modified with 0.1% TFA. The crude and purified peptides were analyzed on an ACQUITY UPLC H-class system with a photodiode array (PDA) detector (Waters, Milford, MA, USA) equipped with an ACQUITY CEH C18 UPLC column (Waters, 2.1 × 50 mm, 1.7 µm). A gradient of 0-50% Solvent B over 30 min with a flow rate of 1 mL/min was used, and detection was set at 200-500 nm. The crude peptides were purified to >95% on a XSelect CSH C18 OBD prep column (19 × 250 mm,5 µm;Waters,Milford,MA,USA) installed in an AutoPurificayion System (Waters). A default gradient of 10-40% Solvent B over 30 min with a flow rate of 20 mL/min was used, but it was adjusted as required. The detection was set at 200-500 nm. The molecular weight of the peptides was confirmed on the Xevo G2 Q-TOF with ACQUITY UPLC I-Class system (Waters). The purified peptides were then freeze-dried (Labconco FreeZone 4.5 Plus, Kansas City, MO, USA) as TFA-salts and stored at -20 °C until further use.

In vitro cytotoxicity

The MTT assay was adopted to determine cell viability in a panel of cancerous and non-transformed cells after 4 h incubation with DTT peptides. Pre-cultured cells were seeded in 96-well plates at a density of 1 × 10 4 -1.5 × 10 4 cells/well and applied for experiment as previously described. In short, cells were washed once with serumfree RPMI 1640 and incubated with increasing concentrations of DTT peptides before adding 10 μl MTT solution to each well. Lastly, acidified isopropanol was added to facilitate formazan crystal solubilization. Absorbance was measured at 570 nm on a spectrophotometric microtiter plate reader (Thermomax Molecular Devices, NJ, USA). Cell survival was calculated as the A 570 nm of DTT-treated cells relative to the negative control (100% viable cells) using the mean of two or three independent experiments and expressed as a 50% inhibitory concentration (IC 50 ).

Hemolytic activity

The cytotoxic activity of two selected DTT compounds ( D T T -2 0 5a n dD T T -3 0 4 )a g a i n s th u m a nr e db l o o dc e l l s (hRBCs) was determined by a hemolytic assay using freshly isolated blood from healthy individuals who gave their signed informed consent. RBCs were resuspended to a 10% hematocrit solution before being incubated for 1 h at 37 °C with DTT compounds dissolved in PBS at concentrations ranging from 438-928 μM (1500-3000 μg/ml). RBCs with PBS and 1% Triton solution alone served as a negative and positive control, respectively. After centrifuging the samples at 4000 rpm for 5 min, the absorbance of the supernatant was measured at 405 nm on a spectrophotometric microliter plate reader (Thermomax, Molecular Devices). The protocol used for blood sampling and handling has been reviewed and approved by the Norwegian Regional Ethic Committee (REK)-approved protocol (2016/376).

Figure 1

 1

Figure 1 .

 1 Figure 1. Leading Sites of New Cancer Cases and Deaths -2018 Estimated (Cancer Facts & Figures 2018)

Figure 2 .

 2 Figure 2. The cancer immunoediting concept. Cancer immunoediting is an extrinsic tumor suppressor mechanism that engages only after cellular transformation has occurred and intrinsic tumor

Figure 3 .

 3 Figure 3. Mechanisms of chemotherapy-driven ICD. In response to inducers of immunogenic cell death (ICD), such as doxorubicin or oxaliplatin, malignant cells expose calreticulin (CALR) and other endoplasmic reticulum chaperones on their surface, secrete ATP, initiate a cell-intrinsic type I interferon (IFN) response culminating in the production of CXC-chemokine ligand 10 (CXCL10), and release highmobility group box 1 (HMGB1) and annexin A1 (ANXA1). Upon binding to cognate receptors on the surface of myeloid or lymphoid cells, these damage-associated molecular patterns favour the uptake of cell corpses and debris thereof by antigen-presenting cells, including dendritic cells (DCs) in the context of robust immunostimulatory signals, eventually leading to the priming of an adaptive immune response involving both αβ and γδ T cells. In addition to being associated with the establishment of immunological memory, such a response has the potential to eradicate malignant cells that survive chemotherapy via an IFNγ-dependent mechanism. CXCR3, CXC-chemokine receptor 3; FPR1, formyl peptide receptor 1;

Figure 4 .

 4 Figure 4. Mechanism of CRT exposure. Several steps are involved in the exposure of CRT in response to immunogenic cell death inducers including activation of PERK, the phosphorylation of eIF2α, caspase-8-dependent cleavage of BAP31, and the activation of Bax and Bak. Finally, a pool of CRT that has transited the Golgi apparatus is secreted by SNARE-dependent exocytosis. Abbreviations: CRT; calreticulin, PERK; protein kinase R-like ER kinase, BAP31; B cell receptor-associated protein 31, Bax;

Figure 5 .

 5 Figure 5. eIF2α phosphorylation: A hallmark of immunogenic cell death. (A) The unfolded protein response of the endoplasmic reticulum in normal circumstances. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) provokes the release of heat shock protein family A (Hsp70) member 5 (HSPA5, best known as binding immunoglobulin protein, BiP) and activates simultaneously the three arms of the unfolded protein response (UPR) ((i) EIF2AK3, (best known as PERK)-mediated phosphorylation of eukaryotic initiation factor α (eIF2α), which in turn halts general protein translation but favors the expression of activating transcription factor 4 (ATF4), (ii) the translocation of the ER transmembrane protein activating transcription factor 6 (ATF6) to the Golgi, proteolytic cleavage and the nuclear translocation of its cytosolic fraction (iii) the endoplasmic reticulum to nucleus signaling 1 (ERN, best known as IRE1α)-mediated splicing of X-box binding protein 1 (XBP1) to the XBP1s isoform. The signal transduction issued by the activation of the transcription factors ATF4, ATF6 and XBP1s aim at reestablishing ER homeostasis and cell survival (or the induction of cell death in conditions of enduring ER stress). (B) The unfolded protein response in the context of immunogenic cell death (ICD). First, the noncanonical activation of PERK and possibly other eIF2α kinases triggers the phosphorylation of eIF2α

Figure 6 .

 6 Figure 6. Modulation of immune cell responses by extracellular ATP in the tumour microenvironment (Di Virgilio et al., 2018).

Figure 7 .

 7 Figure 7. HMGB1 release and extracellular function. HMGB1 can be actively secreted by immune inflammatory cells or passively released by dead, dying or injured cells into the extracellular milieu by several different mechanisms as indicated. Extracellular HMGB1 acts as a DAMP molecule and plays a vital role in several pathophysiological processes. The activity of extracellular HMGB1 depends on redox

Figure 8 .

 8 Figure 8. Discovery pipeline.(Kepp et al., 2019)

Figure 9 .

 9 Figure 9. Assessing the ability of a cytotoxicant to stimulate cancer ICD in vivo.

(

  [START_REF] Humeau | Gold Standard Assessment of Immunogenic Cell Death in Oncological Mouse Models[END_REF]

Figure 10 .

 10 Figure 10. Overview of ongoing clinical trials involving one or more immunogenic cell death inducers in combination with immune checkpoint inhibitors in patients with cancer. As of 1 March, 2020, ClinicalTrials.gov listed 55 ongoing (defined as those that are not completed, withdrawn, suspended, terminated or have unknown status) clinical trials in which patients are receiving chemotherapy plus agents with known immunogenic cell death (ICD)-inducing potential in combination with one or more

Figure11.

  Figure11. Chemical structure and schematized actions of lurbinectedin. (A) Chemical structure of lurbinectedin. (B) Schematic representation of lurbinectedin mechanism of action for binding DNA, inhibiting active transcription and inducing DNA breaks. (C) Schematic representation of lurbinectedin interactions with the tumor microenvironment. Lurb: Lurbinectedin (Farago et al., 2019).

Figure 12 .

 12 Figure 12. Chemical structure of the small amphipathic β(2,2)-amino acid-derived antitumor molecule LTX-401 (MWnet = 367.53) (Eike et al., 2016).

Figure 1 .

 1 Figure1. Immunogenic cell death assessment in solid tumors. (a) Human osteosarcoma U2OS (a), human breast cancer HCC70 (b) human colon cancer HT29 cells (c) and murine methylcholantrene-induced fibrosarcoma MCA205 cells (d) were treated with lurbinectedin (Lurbi, 1 nM, 10 nM, 100 nM and 1 µM) for the indicated times. Subsequently, the cells were stained with 1 µM Hoechst 33342 and 1 µM propidium iodide and assessed for the loss of viability by automated image acquisition. Images were segmented, cellular debris was excluded and the number of cells with normal nuclear morphology was enumerated. Cells stably expressing CALR-GFP were treated as above. Following the cells were fixed with 3.7% of PFA, stained with 1 µM Hoechst 33342 and assessed by automated image acquisition. Images were segmented, cellular debris was excluded and CALR-GFP granularity (a surrogate marker of CALR exposure) was evaluated in the cytoplasmic region of cells with normal nuclear morphology. Wild type cells were treated as above and then assessed for cytoplasmic quinacrine granularity (after staining with the ATP-sensitive dye quinacrine together with Hoechst 33342) by automated image acquisition, segmentation and analysis. Cells stably expressing HMGB1-GFP were treated as above and then assessed for nuclear HMGB1-GFP fluorescence intensity. The cells were fixed and stained with Hoechst 33342 and images were acquired, segmented and analyzed. WT cells were treated as above and following the media was changed and the cells were incubated for 48 hours before the supernatant was used to treat MX1-GFP biosensor cells for additional 48 hours. The cells were fixed and stained with Hoechst 33342 before type 1 IFN responses were monitored by means of automated microscopy as an increase in GFP fluorescence intensity. Mitoxantrone (MTX, 1 and 3 µM) was used as a positive control. The means of quadruplicate assessments and p-values are depicted as heat maps. (*p < .01; **p < .005; ***p < .001, two-tailed Student's t test).

Figure 2 .Figure 3 .

 23 Figure 2. Traits of immunogenic cell death. Human osteosarcoma U2OS cells were treated with 10, 50 or 100 nM lurbinectedin (Lurbi) for 6 hours. Thapsigargin (Thaps, 3 µM) was used as a positive control. The cells were fixed with 3.7% PFA and DNA was stained with 1 µM Hoechst 33342. Following the phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2a) was assessed with phosphoneoepitope-specific antibody and was monitored by means of automated microscopy as an increase in cytoplasmic fluorescence intensity. (a,b) The level of transcription was measured in U2OS cell treated as above with Lurbi. The transcription inhibitor actinomycin D (ActD) was used as a control. The cells were fixed as above and following the colocalization of nucleolin and fibrillarin was assessed as an indicator for transcriptional activity (c,d) Scale bar equals 10 µm and bar charts depict depict mean values ± SD of quadruplicate assessments (*p < .01; ***p < .001, two-tailed Student's t test).

Figure 4 .

 4 Figure 4. Therapeutic efficacy of lurbinectedin in immunocompetent and immunodeficient hosts. Live MCA205 cells were injected subcutaneously (s.c.) into immunocompetent C57BL/6 mice or immunodeficient nu/nu mice as depicted in the scheme in (a) When tumors became palpable, mice were intravenously (i.v.) injected with 0.14 mg/Kg lurbinectedin (on day1,7 and 14). Tumor growth was assessed regularly for the following 30 days. Data is depicted as tumor growth curves (b,d) and overall survival plots (c,e). Data were analyzed with TumGrowth.

  Figure 5. Sequential lurbinectedin treatment with double immune checkpoint blockade exhibits systemic antitumor immunity C57BL/6 mice were inoculated subcutaneously (s.c.) with murine fibrosarcoma MCA205. Palpable tumors were treated with sequential intravenous (i.v.) injections of 0.14 mg/Kg lurbinectedin (Lurbi) as indicated in (a). Single-or double-immune checkpoint blockade was mounted by sequential intraperitoneal (i.p.) injections of monoclonal antibodies targeting CTLA-4 or PD-1 at day 6, 9 and 12 post treatment and tumor growth (b,c) and overall survival (d,e) were assessed regularly for the following 30 days. (f,g) The generation of immunological memory was assessed in cured animals by rechallenged with MCA205 and TC-1. Naïve animals were used as controls. Individual tumor growth curves are depicted. Data were analyzed with TumGrowth.

Figure 6 .

 6 Figure 6. Lurbinectedin retards the growth of spontaneous tumors. Medroxyprogesterone acetate (MPA) pellets (50 mg, 90-day release) were implanted subcutaneously into the interscapular area of immunocompetent C57BL/6 mice. Then the animals received 1 mg dimethylbenzantracene (DMBA) administered by oral gavage 6 x during 7 weeks. When spontaneous tumors became palpable mice were randomly assigned to receive 0,14 mg/Kg lurbinectedin (Lurbi) alone or in combination with double immune checkpoint blockade with monoclonal antibodies targeting CTLA-4 and PD-1 at day 6, 9 and 12 post treatment (a). The tumor area and overall survival were measured regularly until ethical endpoints were reached (b,c,d). Data were analyzed with TumGrowth (https://github.com/kroemerlab).

  that LTX-401 could induce anticancer immune response and facilitate T cell infiltration in tumour bed, we combined LTX-401 and ICBs to explore their potential synergic effects. Pretreatment with LTX-401 greatly improved the therapeutic efficiency of αCTLA-4, αPD-1 or αCTLA-4 plus αPD-1. The combination of LTX-401 and dual checkpoint blockade (αCTLA-4 plus αPD-1) or the combination of LTX-401 and αCTLA-4 (but not αPD-1) alone were able to significantly extend life expectancy. Again, cured tumour-free mice generated tumour specific immunological memory. Particularly, sequential LTX-401 treatment with double ICBs exhibits superior systemic antitumor immunity. 100% of treated tumours and 6 out of 9 contralateral tumours were completely regressed.

Figure 1 .Figure 2 .

 12 Figure1. Absence of adverse toxicity in LTX-401-treated mice. In a retrospective study, the adverse toxicity of sequential intratumoral injections of oncolytic peptides and peptidomimetics at they are respective therapeutically effective dose such as LTX-315 (0.3 mg/injection), LTX-401 (0.25 mg/injection), DTT-205 and DTT-304 (both at 1.5 mg/injection) was evaluated. Note that these doses have been optimized to obtain an optimal antitumor effect. Data from in vivo experimentation utilizing oncolytic compounds in C57BL/6 mice bearing palpable subcutaneous solid tumors were evaluated and global survival was enumerated (A). The percentage of animals that survived without severe discomfort requiring their euthanasia is indicated (B).

Figure 4

 4 Figure 4. Combination of single LTX-401 treatment with immune checkpoint blockade increases its efficacy. C57BL/6 mice subcutaneously implanted with MCA205 fibrosarcoma were injected intratumorally when tumors became palpable with a single injection of 0.25 mg LTX-401 as indicated in (A). Immune checkpoint blockade was mounted by sequential injections of monoclonal antibodies targeting CTLA-4 or PD-1 alone or in combination at day 6, 9 and 12 post treatment. LTX-401 induced efficient oncolysis which is reflected in reduced tumor growth and tumor clearance in some animals. This effect was increased in combination with CTLA-4 and PD-1cotreatmentas depicted in separate tumor growth curves (B-E) and overall survival plots (Chi 2 test, **p < 0.01) (F-H). Rechallenge of animals cured from MCA205 fibrosarcoma with MCA205 several weeks after the initial therapy on the contralateral and challenged with syngeneic mouse TC-1 lung cancer cells on the ipsilateral side resulted in efficient rejection of MCA205 but aggressive tumor growth of TC-1 (I). Thus, LTX-401 caused the generation of immunological memory that led to the rejection of isogenic tumors.

Figure 5 .

 5 Figure 5. Sequential LTX-401 treatment with double immune checkpoint blockade exhibits systemic antitumor immunity C57BL/6 mice were inoculated with murine fibrosarcoma MCA205 subcutaneously on one side and four days later on the contralateral flank. Palpable primary tumors arising from the first injection were treated with sequential injections of 0.25 mg LTX-401 as indicated in (A). Immune checkpoint blockade was mounted by sequential injections of double immune checkpoint blockade with monoclonal antibodies targeting CTLA-4 or PD-1 at day 6, 9 and 12 post treatment. Immune checkpoint inhibition alone did not control tumor growth of LTX-401-treated primary (B) and distant (C) tumors. In contrast, combination of sequential LTX-401 treatment with double immune checkpoint blockade cleared 100% of LTX-401-treated primary tumors (D) and depicted abscopal effects on distant tumors (E).

background

  Immunogenic cell death (ICD) is a peculiar modality of cellular demise that elicits adaptive immune responses and triggers T cell-dependent immunity. Methods Fluorescent biosensors were employed for an unbiased drug screen approach aiming at the identification of ICD enhancers. results Here, we discovered thiostrepton as an enhancer of ICD able to boost chemotherapy-induced ATP release, calreticulin exposure and high-mobility group box 1 exodus. Moreover, thiostrepton enhanced anticancer immune responses of oxaliplatin (OXA) in vivo in immunocompetent mice, yet failed to do so in immunodeficient animals. Consistently, thiostrepton combined with OXA altered the ratio of cytotoxic T lymphocytes to regulatory T cells, thus overcoming immunosuppression and reinstating anticancer immunosurveillance. Conclusion Altogether, these results indicate that thiostrepton can be advantageously combined with chemotherapy to enhance anticancer immunogenicity.

Figure 1 Figure 2 Figure 3

 123 Figure 1 Identification of thiostrepton as autophagy inducer and immunogenic cell death enhancer. (A,B) Human neuroglioma H4 stably expressing GFP-LC3 were treated for 6 hours at two to five concentrations with 1428 chemical compounds originating from a selection of the Prestwick library (at 0.5, 5 and 50 µM), a flavonoids library (at 10, 30, and 90 µM) and a fatty acid library (at 31.25, 62.5, 125, 250 and 500 µM), together with their respective control vehicles (complete medium, DMSO or ethanol). This resulted in 4147 tested conditions in total. Torin 1 at 300 nM was used as positive control for autophagy induction. The number of GFP-LC3 positive puncta was measured as a proxy for autophagy and the number of cells that confer a regular nuclear phenotype was assessed as an indicator for viability. Data were normalized to controls and are depicted as ranked percentage of the normalized GFP-LC3 dots. A total of 175 compounds with increased GFP-LC3 dots count (and number of healthy cells >0.8) were selected for further experiments. Normalized data are shown as a dot plot in (A) and representative images are depicted in (B). (C, D) Human osteosarcoma U2OS cells stably expressing a tandem mRFP-GFP-LC3 biosensor were treated with the selected compounds to assess autophagic flux. Bafilomycin A1 (BAFA1) at 100 nM was used as a prototype flux inhibitor. The number of GFP positive and RFP positive dots was assessed and normalized to untreated controls. Cellular viability was assessed and normalized as described above. Data are depicted as ranked percentage of the ratio of RFP/GFP dots. A total of 34 autophagic flux inducing compounds (with numbers of healthy cells >0.8) were selected for further experiments. Normalized data are shown as a dot plot in (C) and representative images are depicted in (D). (E,F) U2OS wild-type cells were tested with the selected hit compounds at 50 µM in the presence of low-dose oxaliplatin (OXA at 60 µM) for 6 hours. High-dose OXA (400 µM) was used as positive control for ATP release. Quinacrine positive dots were assessed by microscopy and then values were normalized to controls. Normalized data are shown as a dot plot in (E) and representative images are depicted in (F). Scale bar equals 10 nm.

Figure 4

 4 Figure 4 Validation of thiostrepton facilitated ATP release with mitoxantrone (MTX) and oxaliplatin (OXA). (A,B) Thiostrepton (THIOS; 0.5, 1, 2, 3, 4, 6 µM) decreased intracellular ATP content in human osteosarcoma U2OS wild-type cells within 6 hours in the presence of low doses of the ICD inducers mitoxantrone (MTX, 1 µM) and oxaliplatin (OXA; 200 µM) detected by quinacrine. High-dose MTX (5 µM) and OXA (400 µM) were used as positive controls. Representative images are shown in (A). Scale bar equals 10 µm. Data are means±SD of four replicates (compared with vehicle control (CTRL), *p<0.05, **p<0.01, ***p<0.001 compared with CTRL; ##p<0.01, ###p<0.001 compared with cells treated with low-dose MTX; &p<0.05, &&&p<0.01 compared with cells treated with low-dose OXA; unpaired Student's t-test). (C-E) Thiostrepton increased extracellular ATP levels in murine fibrosarcoma MCA205 wild-type cells in the presence of low doses of the ICD inducers MTX (0.5 µM) and OXA (150 µM), but much less efficient in MCA205-TFE knockdown or ATG5 knockdown cells. Cells were treated with THIOS (1.56, 3.12, 6.25, 12.5 µM) alone or in combination with low-dose MTX or OXA for 6 hours as described above. Data are means±SD of at least three replicates (*p<0.05, **p<0.01, ***p<0.001 compared with thiostrepton single treatment; #p<0.05 compared with cells treated with low-dose MTX alone, &&&p<0.01 compared with cells treated with low-dose OXA alone; unpaired Student's t-test).

Figure 5

 5 Figure 5 Thiostrepton facilitates calreticulin (CALR) exposure and high-mobility group box 1 (HMGB1) release in the presence of immunogenic cell death (ICD) inducer. (A,B) Thiostrepton increased CALR exposure in human osteosarcoma U2OS cells stably expressing CALR-RFP. Thiostrepton (THIOS: 0.5, 1, 2, 3, 4, 6 µM) was employed in the presence of lowdose mitoxantrone (MTX, 1 µM) and oxaliplatin (OXA, 200 µM). High-dose MTX (5 µM) and OXA (400 µM) were used as positive controls for 18 hours. Representative images are shown in (A). Scale bar equals 10 µm. Data are means±SD of four replicates (*p<0.05, **p<0.01, ***p<0.001; Student's t-test; compared with vehicle control (CTRL)). (C,D) Thiostrepton increased CALR exposure in MCA205 wild-type cells detected by flow cytometry. Cells were treated with THIOS (12.5 µM) alone or in combination with OXA (150 µM) cells for 6 hours. Representative histograms are shown in (L). Data are means±SEM of at least three replicates (***p<0.001, compared with CTRL; ###p<0.001, compared with OXA: Student's unpaired t-test). (E) THIOS alone or in combination (COMB) at the same concentration as in (A-B) increased extracellular HMGB1 after 48 hours in MCA205 wild-type cells monitored by ELISA. Data are means±SEM of at least three replicates ((*p<0.05, **p<0.01, ***p<0.001, compared with CTRL; ##p<0.01, compared with OXA: unpaired Student's t-test). (F-G) Thiostrepton increased extracellular HMGB1 in U2OS cells and decreased HMGB1 content in U2OS HMGB1-GFP biosensor cells. Cells were treated as in (A,B) for 72 hours. Representative images are shown in (F). Scale bar equals 10 µm. Data are means±SD of four replicates (*p<0.05, **p<0.01, ***p<0.001, compared with CTRL; Student's t-test).

Figure 6

 6 Figure 6 Thiostrepton improves the efficacy of anticancer chemotherapy in vivo. (A,B) Effects of thiostrepton (THIOS) on tumor growth of TC-1 murine lung cancers (A) and MCA205 mouse fibrosarcomas cancers (B) in syngeneic immunocompetent C57Bl/6 mice. From left to right: (i) growth curves (mean±SEM); (ii) tumor size distributions at day 21 for TC1 tumor or day 25 for murine MCA205 fibrosarcoma of the data shown in (i); (iii) individual tumor growth curves of mice treated with oxaliplatin (OXA) alone or in combination with THIOS of the data shown in (i); (iv) Kaplan-Meier curves with tumor size exceeding 250 mm 2 as an endpoint. (B) n=8 for mice treated with OXA and n=6 without OXA), (C) n=10 for single treatments or untreated animals, n=13 for the group receiving combination treatment). (C,D) Effects of THIOS on the growth of MCA205 fibrosarcomas in immunodeficient nu/nu mice (C) and MCA205-TFE knockdown cells in immunocompetent C57Bl/6 mice (D). From left to right: (i) growth curves (mean±SEM); (ii) tumor size distributions at day 19 on nude mice or day 37 on C57Bl/6 mice of data shown in (i); (iii) individual tumor growth curves of mice treated with OXA alone or in combination with THIOS of data shown in (i); (iv) Kaplan-Meier curves tumor size exceeding 300 mm 2 (nude mice) or 250 mm 2 (C57Bl/6 mice) as an endpoint. (C): n=7 for untreated controls and the groups receiving single treatments, n=10 for the group receiving the combination treatment); (D) n=6 for each group.

Figure 7

 7 Figure 7 Cytofluorometric characterization of tumor-infiltrating lymphocytes (TIL). The number of CD3 + CD8 + cytotoxic T lymphocytes (A), CD4 + FOXP3 + CD25 + regulatory T cells (Treg) (B), the ratio of CD3 + CD8 + T lymphocytes over Treg (C), (compared with CTRL: ***p<0.001; compared with OXA: # p<0.05, NS, not statistically significant; Student's t-test) (n=10 for the untreated controls and the groups receiving single treatments, n=13 for the group receiving the combination treatment).

Fig. 1 DTTFig. 2 Fig. 3 (

 123 Fig.1DTT peptides induce the formation of lipid droplets. Human osteosarcoma U2OS cells were treated with 2.5 µM of either DTT-205 or DTT-304 for 6 h. Cells were fixed and subjected to electron microscopy. The overview micrographs in the upper panel depict necrotic morphologies (N) of some of the treated cells and the high magnification micrographs in the lower panel show the formation of lipid droplets (L) in intact cells. Size bars equals 10 µm (upper panel) and 1 µm (lower panel). Representative images (a) and quantifications (b, c) are depicted (mean ± SD of a minimum of five view fields). The formation of lipid droplets in response to increasing doses from 0.65 to 10 µM DTT peptides was quantified by means of the lipophilic dye Nile Red at 6 h and 24 h post treatment in epifluorescence microscopy. Representative images (d) and quantifications (e, f) are shown (mean ± SD of triplicate assessments, Student's t test, p < 0.5, **p < 0.01). Temperature dependency of the lipid droplet formation was assessed by keeping the cell cultures upon treatment with 0.65 to 10 µM DTT-205 (g) and DTT-304 (h) at the indicated temperature for 6 h before Nile Red staining. Increased number of lipid droplets at physiological temperature is indicative for an underlying active biochemical reaction (mean ± SD of triplicate assessments, Student's t test, *p < 0.5, **p < 0.01)

Fig. 4

 4 Fig.4Necroptotic traits induced by DTT-304 but not by. Human colon carcinoma HT-29 cells knockout for MLKL and stably expressing RIP3 coupled to green fluorescent protein (GFP) were treated with 0.65 to 10 µM DTT peptides for the indicated time and following assessed for the aggregation of RIP3 indicative for necroptosome formation by epifluorescence microscopy. The combination of TNFα (T), SMAC/DIABLO mimetic peptide B6 (S), and the pan-caspase inhibitor z-VAD-fmk (Z) was used as positive control for the induction of necroptosis. Representative images (a, c) and quantifications of RIP3 aggregates (b, d) are depicted (size bar equals 10 µm; mean ± SD of triplicate assessments, Student's t test, *p < 0.5, **p < 0.01, ***p < 0.001). Downstream MLKL activation was visualized with phosphoneoepitope-specific antibody (e, f) Of note, exclusively DTT-304 but not DTT-205 depicted traits of necroptosis. Murine lung cancer TC-1 cells that were CRISPR gene edited in RIP3 and MLKL were treated with 0.65 to 10 µM of DTT-205 or DTT-304 for 6 h and viability was assessed by means of an exclusion dye (g, h; mean ± SD of triplicate assessments, Student's t test, *p < 0.5, **p < 0.01, ***p < 0.001). Knockouts of RIP3 and MLKL were partially resistant to DTT-304 yet not to DTT-205-induced cell death

Fig. 5

 5 Fig.5DAMP release from tumor cells treated with DTT-205 and DTT-304. The exposure of calreticulin (CALR) in human osteosarcoma U2OS cells was measured by flow cytometry using polyclonal anti-CALR antibody while excluding cells that lost cytoplasmic membrane integrity and thus incorporated the exclusion dye propidium iodide (PI) (a, b). Exodus of high mobility group box 1 (HMGB1) from the cells into cell culture supernatants was monitored by HMGB1-specific enzyme-linked immunosorbent assay ELISA. Absorbance was measured and concentrations were calculated based on standards (c, d). Both CALR and HMGB1 were emitted by both DTT-205 and DTT-304 in a dose-dependent fashion. The production of type I interferon (IFN) was measured in by reverse transcription quantitative real time polymerase chain reaction qRT-qPCR from purified mRNA of cells treated with DTT compounds (e, f) (mean ± SD of triplicate assessments; Student's t test, *p < 0.5, **p < 0.01, ***p < 0.001). In summary some ICD DAMPs were release in response to

Fig. 6

 6 Fig. 6 In vivo activity of DTT-205 and DTT-304 in immunocompetent animals. Mouse fibrosarcoma cells were inoculated subcutaneously in syngenic C57BL/6 animals and arising tumors were treated when palpable with repeated injections of DTT-205 or DTT-304 in the presence of absence of intraperitoneally injected CD4/CD8 blocking antibody (a). Both DTT-205 and DTT-304-induced efficient oncolysis. Immunocompetent animals depicted long-term effects whereas tumors recurred soon after treatment in immunecompromised animals upon T-cell depletion an effect that is reflected in tumor growth (b-f) and overall survival (g) (Chi 2 test, **p < 0.01, ***p < 0.001). Rechallenge of animals cured from MCA205 fibrosarcoma with MCA205 several weeks after the initial therapy on the contralateral and challenge with syngenic mouse TC-1 lung cancer cells on the ipsilateral side resulted in efficient rejection of MCA205 but aggressive tumor growth of TC-1 (h-k). DTT-205 and DTT-304 caused the generation of immunological memory that sufficed in rejection isogenic tumors

  Cancer, which was first recorded by Edwin Smith in 3000 B.C. and described in a scientific manner by Hippocrates in 400 B.C., refers to a series of diseases due to uncontrollably replicating cells. According to WORLD HEALTH STATISTICS:2018, cancer has become a serious global public health problem, approximately 9 million deaths were caused by cancer in 2016, which accounts for 15.8% of total 57 million deaths all over the world . As a fatal disease, cancer ranks

	IRE1α IFN UPR INTRODUCTION inositol-requiring enzyme 1α interferon unfolded protein response
	IFNAR1 IFN-γ Cancer and anticancer immunity interferon α/β-receptor subunit 1 interferon γ
	IL Cancer epidemiology interleukin
	RIDD	IRE1α-dependent decay
	KS	Kaposi's sarcoma
	LPS	lipopolysaccharides
	LRP1	low density lipoprotein receptor-related protein 1
	MPA	medroxyprogesterone acetate
	MTX	mitoxantrone
	MYD88	myeloid differentiation primary response 88
	NK	natural killer
	NSCLC	non-small cell lung cancers
	NF-κB	nuclear factor-κB
	DAMPs ORR	damage-associated molecular patterns overall response rate
	DC OS	dendritic cell overall survival
	DMBA PERK	dimethylbenzantracene PKR-like ER-resident kinase
	DOXO PD-1	doxorubicin programmed cell death protein 1
	ER PD-L1	endoplasmic reticulum programmed death-ligand 1
	ERAD PKR eIF2α P2RX7	ER-associated degradation protein kinase R eukaryotic translation initiation factor 2 purinergic receptor P2X7
	FDA P2RY2	federal drug administration purinergic receptor P2Y2
	FPR1 ROS	formyl peptide receptor 1 reactive oxygen species
	GCN2 RAGE	general control nonderepressible 2 receptor for advanced glycation end-products
	Hsp70 RAG	heat shock protein 70 recombination Activating
	HCC ROCK1	hepatocellular carcinoma Rho-associated, coiled-coil containing protein kinase 1
	HMGB1 IFNGR1 SCID STAT	high-mobility group box 1 severe combined immunodeficiency IFN-γ receptor signal transducer and activator of transcription
	ICB SCLC	immune checkpoint blockade small-cell lung cancer
	ICD TLR	immunogenic cell death Toll-like receptor

Table 2 (

 2 Galluzzi et al., 2020a).

	Agent	P-

eIF2α DAMPs Main cytokines Immune infiltrate Antagonized by Combination with ICIs a

	Dinaciclib	ND	CALR, HMGB1, ATP	↑IFNγ	↑DCs, CD8+ CTLs, CD4+ T cells	Rag-/-genotype	Anti-PD-1
	7A7 DTT-205	Yes ND	CALR CALR, HMGB1	↑IFNγ ↑Type I IFNs	↑DCs, CD8+ CTLs, CD4+ T cells ND	CD8+ T cell depletion ND CD8+ and CD4+ T cell depletion ND
	8-MOP PCT DTT-304	ND ND	CALR, ATP, HMGB1 CALR, HMGB1	↑Type I IFNs, IL-6, CCL2 ↑Type I IFNs	↑DCs, monocytes,CD8+ CTLs, NK cells ND	CD8+, CD4+ or NK cell depletion CD8+ and CD4+ T cell depletion	ND ND
	Anthracyclines Electrical nanopulses ND Yes Hypericin-based PDT No	CALR, ATP, HMGB1, ANXA1 CALR, ATP, HMGB1 CALR, ATP, HMGB1	ND ↑IL-1β, IL-6, IL-12, IL-17, CXCL10, type I IFNs, IFNγ ↑IL-1β	↑CD8+ CTLs ↑DCs, CD8+ CTLs, γδ TH17 cells; ↓Treg cells, MDSCs ↑DCs	nu/nu genotype, ND CD8+ T cell depletion, neutralization, IL-17 IFNγ or IL-17 ND	ND Anti-PD-1, anti-CTLA4 ND
	JQ1	Yes	CALR, ATP, HMGB1	ND	↑DCs, CD8+ CTLs; ↓MDSCs	receptor inhibition nu/nu genotype	ND
	Bleomycin LTX-315	Yes No	CALR, ATP, HMGB1 CALR, ATP, HMGB1	↑IFNγ, TGFβ ↑IL-1β, IL-6, type I IFNs	↑CD8+ CTLs;↑Treg cells ↑DCs, macrophages, CD8+ CTLs, TH1 CD4+ cells; ↓MDSCs, Treg cells	CD8+ T cell depletion, IFNγ neutralization ND	ND Anti-PD-1, anti-CTLA4
	Bortezomib LTX-401	Yes No	CALR, HSP70 CALR, ATP, HMGB1	↑IFNγ ↑Type I IFNs	↑DCs, CD8+ CTLs ↑ CD3+ T cells	nu/nu or Rag -/-genotype, CD8+ T cell ND	ND Anti-PD-1, anti-CTLA4
	Cardiac glycosides Lurbinectedin Cetuximab Microwave thermal ablation Clostridium difficile toxin B Newcastle disease virus Crizotinib Non-thermal plasma ND ND Yes ND ND No ND Yes Cyclophosphamide ND Dactinomycin Yes Oxaliplatin Yes	CALR, ATP, HMGB1 CALR, ATP, HMGB1 CALR, HMGB1 CALR, ATP, HMGB1 CALR, HMGB1, ATP CALR, HMGB1 CALR, ATP, HMGB1 CALR, ATP, HMGB1 CALR, HMGB1, ATP CALR, ATP, HMGB1 CALR, ATP, HMGB1, HSP70	↑IFNγ ↑Type I IFNs ND ↑IFNγ, TNF ND ↑IFNγ ↑Type I IFNs, IL-17 ↑ CXCL10, IFNγ, TNF; ↓TGFβ ↑Type I IFNs, IFNγ, IL-17 ↑Type I IFNs, IFNγ, IL-↑IL-1β, type I IFNs, IFNγ	↑CD8+ CTLs, CD4+ T cells, γδ TH17 cells ND ↑DCs, CD8+ CTLs ↑CD8+ CTLs ND ↑DCs, ↑CD8+ CTLs, CD4+ T cells, NK and NKT cells;↓MDSCs ↑DCs, CD8+ CTLs, NKT cells; ↓Treg cells ↑DCs ↑DCs, CD8+ CTLs, NK cells b , NKT cells; ↓MDSCs, Treg cells ↑DCs, CD8+ CTLs, NK cells; ↓Treg cells ↑DCs, CD8+ CTLs, γδ TH17 cells	depletion nu/nu genotype CD8+ and CD4+ T cell depletion ND CD8+ T cell depletion ND Anti-PD-1, anti-CTLA4 ND ND ND Rag2-/-genotype, Anti-CTLA4 CD8+ T cell depletion ND nu/nu genotype ND Anti-PD-L1 Anti-PD-1, anti-PD-L1 nu/nu genotype, ND Anti-PD-1, anti-CTLA4 CD8+ and CD4+ T cell Anti-PD-1 blockage nu/nu genotype, CD8+ T cell depletion, neutralization, IL-17R IFNγ or IL-17 Anti-PD-1, anti-CTLA4
						depletion

Table 2 .

 2 Confirmed inducers of immunogenic cell death.

	All agents confer enduring immunological memory, excluding dactinomycin,

  21. Peltonen K, Colis L, Liu H, et al. A targeting modality for destruction of RNA polymerase I that possesses anticancer activity. Cancer Cell. 2014;25(1):77-90. doi:10.1016/j.ccr.2013.12.009. 22. Pietrocola F, Pol J, Vacchelli E, et al. Caloric restriction mimetics enhance anticancer immunosurveillance. Cancer Cell. 2016;30 (1):147-160. doi:10.1016/j.ccell.2016.05.016. 23. Martins I, Kepp O, Schlemmer F, et al. Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress. Oncogene. 2011;30(10):1147-1158. doi:10.1038/ onc.2010.500. 24. Iribarren K, Buque A, Mondragon L, et al. Anticancer effects of anti-CD47 immunotherapy in vivo. Oncoimmunology. 2019;8 (3):1550619. doi:10.1080/2162402X.2018.1550619. 25. Zhou H, Mondragon L, Xie W, et al. Oncolysis with DTT-205 and DTT-304 generates immunological memory in cured animals. Cell Death Dis. 2018;9(11):1086. doi:10.1038/s41419-018-1127-3. 26. E notD P,V ac ch ell iE,Jac qu elo tN ,Zi t vo ge lL,T um Gr ow th :KG .An open-access web tool for the statistical analysis of tumor growth curves.

	Oncoimmunology. 2018;7(9):e1462431. doi:10.1080/2162402X.20
	18.1490854.

  Kepp O, Sauvat A, Leduc M, Forveille S, Liu P, Zhao L, Bezu L, Xie W, Zitvogel L, Kroemer G. A fluorescent biosensor-based platform for the discovery of immunogenic cancer cell

	ANNEX 2: PAPERS NOT INCLUDED IN THIS
	death THESIS (INSERTED IN PDF) inducers. Oncoimmunology. 2019	Apr	26;8(8):1606665.	doi:
	10.1080/2162402X.2019.1606665.
	69

Table 1

 1 Peptide design and IC 50 values of DTT peptides against various cell lines human red blood cells, ND not determined Data represents two or more independent experiments conducted in triplicates (IC 50 μM ± SD). Standard concentration gradient 1-100 μg/ml, equaling ∼0.3-31 μM it appears that pro-oxidant and BAX/BAK-dependent processes, but not caspase activation, participate to cell killing by

	Compound	JM1	BEL-7402	HEPG2	HepaRG	CT-26	HT-29	CC531	MRC-5	HUVEC	hRBCs
	DTT-106	17.5 ± 4.2	27.1 ± 0.5	27.05 ± 0.6	24.1	9.7 ± 0.4	25.8 ± 1.2	26.7 ± 4.3	28.6 ± 2.4	29.3 ± 0.2	ND
	DTT-201	26 ± 3.0	> 31	> 31	ND	10.8 ± 1.3	> 31	> 31	> 31	> 31	ND
	DTT-202	15.9 ± 2.3	> 31	> 31	23.3 ± 0.9	7.4 ± 1.6	23.4 ± 0.5	16.5 ± 1.4	23.3 ± 2.2	29.9 ± 1.2	ND
	DTT-203	13.4 ± 5.9	19.0 ± 1.3	23.4 ± 3.1	22.6	8.1 ± 0.6	26.5 ± 0.9	> 31	30.5 ± 1.7	29.5 ± 0.8	ND
	DTT-204	16.4 ± 1.0	22.9 ± 0.8	> 31	ND	8.1 ± 1.2	16.5 ± 3.4	25.2 ± 0.4	24.0 ± 3.0	27.5 ± 2.5	ND
	DTT-205	6.9 ± 1.7	10.3 ± 0.3	23.6 ± 0.7	9.0 ± 0.3	8.2 ± 0.5	9.8 ± 0.9	13.0 ± 0.3	11.2 ± 0.1	10.6 ± 2.6	526-614
	DTT-122	> 31	> 31	> 31	ND	ND	> 31	ND	ND	ND	ND
	DTT-301	26.0 ± 2.9	> 31	> 31	ND	6.7 ± 0.6	> 31	> 31	> 31	> 31	ND
	DTT-302	16.3 ± 1.2	> 31	24.5 ± 3.4	21.1	10.1 ± 1.5	> 31	> 31	> 31	> 31	ND
	DTT-303	13.0 ± 0.8	19.5 ±2.4	20.2 ± 3.6	28.5	6.0 ± 0.5	> 31	25.8 ± 5.8	26.7 ± 3.4	> 31	ND
	DTT-304	13.0 ± 1.3	16.4 ± 0.2	21.4 ± 0.3	15.0 ± 2.1	8.6 ± 2.6	21.1 ± 1.7	24.3 ± 6.7	29.0 ± 2.0	27.9 ± 2.1	> 928
	DTT-305	12.4 ± 2.0	15.8 ± 0.05	20.6 ± 2.4	14.8 ± 0.4	7.8 ± 2.0	9.0 ± 0.8	15.7 ± 0.3	12.4 ± 0.2	15.0 ± 0.3	ND
	DTT-306	8.7 ± 0.7	11.6 ± 0.5	20.1 ± 2.8	12.5	7.5 ± 1.7	4.7 ± 0.9	13.1 ± 2.1	17.5 ± 2.3	16.4 ± 1.5	ND

hRBCs

  5, **p < 0.01, ***p < 0.001). Downstream MLKL activation was visualized with phosphoneoepitope-specific antibody (e, f) Of note, exclusively DTT-304 but not DTT-205 depicted traits of necroptosis. Murine lung cancer TC-1 cells that were CRISPR gene edited in RIP3 and MLKL were treated with 0.65 to 10 µM of DTT-205 or DTT-304 for 6 h and viability was assessed by means of an exclusion dye (g, h; mean ± SD of triplicate assessments, Student's t test, *p < 0.5, **p < 0.01, ***p < 0.001). Knockouts of RIP3 and MLKL were partially resistant to DTT-304 yet not to DTT-205-induced cell death

			DTT-205		Ifna1	Ifnab1	DTT-304	Ifna1	Ifnab1
	Rel. mRNA content	9 6 3 0 12	Ctr	0.65	1.25	2.5	5	10		12
	Norway). Plasticware was obtained from Greiner Bio-One	expressing GALT1-GFP, H2B-RFP, CALR-GFP, DIABLO-
	(Monroe, CA, USA), primary antibody (cleaved caspase-3;	GFP or LAMP1-GFP, and HT-29 stably expressing RIP3-
	#9661) came from Cell Signaling (Danvers; MA; USA), and	GFP 28 cells were cultured in Glutamax ® -containing DMEM
	AlexaFluor-coupled secondary antibody from Thermo	medium supplemented with 10% fetal calf serum (FCS),
	Fisher Scientific. Anti-CD4 and anti-CD8 antibodies for	and 10 mM HEPES. Cells were grown in a humidified
	in vivo use were obtained from BioXcell (West Lebanon,	incubator at 37 °C under a 5% CO 2 atmosphere.
	NH, USA). Mouse embryonic fibroblast (MEF), mouse	Murine thymocytes and splenocytes were obtained
	sarcoma cells MCA205, murine lung cancer TC-1,	by dispersing organ extracts in Glutamax ® -containing
	human osteosarcoma U2OS, wild type or stably	DMEM supplemented with 50 µM 2-mercaptoethanol

e1656502-6 W. XIE ET AL.

e1594555-4 W. XIE ET AL.

Medicale. Protected by copyright. on October 19, 2020 at Institut Gustave-Roussy-Bibliotheque http://jitc.bmj.com/ J Immunother Cancer: first published as 10.1136/jitc-2019-000462 on 26 March 2020. Downloaded from

J Immunother Cancer: first published as 10.1136/jitc-2019-000462 on 26 March 2020. Downloaded from

Official journal of the Cell Death Differentiation Association

Acknowledgments

WX and YW were supported by the China Scholarship Council, JH is the recipient of a PhD fellowship from the Foundation Philanthropia; GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) -Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale (FRM); a donation by Elior; European Research Area Network on Cardiovascular Diseases (ERA-CVD, MINOTAUR); Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085 and GDW20181100051), Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology; the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and the SIRIC Cancer Research and Personalized Medicine (CARPEM). Acknowledgements We thank David Rubinstein for the PC12-Q74-GFP cell line. Contributors YW, WX and JH conducted experiments and analyzed data. GC, PL and JP assisted with experimental design. ZZ, OK and GK conceived the study and wrote the manuscript. Funding YW and WX are supported by the China Scholarship Council. YW and ZZ were supported by the National Nature Science Foundation of China (81502049 for YW, 81572955 and 81773357 for ZZ). GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) -Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Association "Le Cancer du Sein, Parlons-en!"; Cancéropôle Ilede-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale (FRM); a donation by Elior; European Research Area Network on Cardiovascular Diseases (ERA-CVD, MINOTAUR); Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085), Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology (ANR-18-IDEX-0001); the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and the SIRIC Cancer Research and Personalized Medicine (CARPEM). Acknowledgements H.Z. and P.L. were supported by the China Scholarship Council, L.Z. is supported by the Ligue contre le Cancer, G.K. and L.Z. are supported by the Ligue contre le Cancer (équipes labelisées); Agence National de la Recherche (ANR) -Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Institut National du Cancer (INCa); Fondation Bettencourt-Schueller; Fondation de France; Fondation pour la Recherche Médicale (FRM); the European Commission (ArtForce); the European Research Council (ERC); the LabEx Immuno-Oncology; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); the Swiss Bridge Foundation, ISREC and the Paris Alliance of Cancer Research Institutes (PACRI). This project was supported by the Norwegian Research Council (254800, 257967). LCGdS is supported by Portuguese Science Foundation (ref. SFRH/BPD/93562/2013).

Funding WX,HZ and YW were supported by the China Scholarship Council, GK and LZ are supported by the Ligue contre le Cancer (équipes labelisées); Agence National de la Recherche (ANR) -Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Institut National du Cancer (INCa); Fondation Bettencourt-Schueller; Fondation de France; Fondation pour la Recherche Médicale (FRM); the European Commission (ArtForce); the European Research Council (ERC); the LabEx Immuno-Oncology; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); the Seerave foundation; the Swiss Bridge Foundation, ISREC and the Paris Alliance of Cancer Research Institutes (PACRI). This project was supported by the Norwegian Research Council (254800, 257967).

data availability statement Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information.

Abbreviations

DAMP danger-associated molecular pattern ICD immunogenic cell death

Conflict of interest disclosure

This study was supported by Pharmamar. JFM-L is a full-time employee of Pharmamar.

Abbreviations

DAMP danger-associated molecular pattern ICD immunogenic cell death

CONCLUDING REMARKS

The main conclusions are: To optimise lurbinectedin mediated protecting effects, different drug concentrations and time points should be tested to find a proper condition. Given that the sensitivity of cancer cells to lurbinectedin may varies depends on genetic background, tumour heterogeneity or other factors, it's also an important issue to identify drug sensitive cancer types before clinical usage.

ACKNOWLEDGEMENT

I would like to give biggest thanks to all of you who helps me, supports me and companies me in the past years. I cannot enjoy a so inspiring and fantastic doctoral journey without your presence.

To my supervisor Guido, thanks for your kind supervision during my Ph.D. study, your insightful perspectives and passions on science always inspire me to think big scientific questions.

To my co-supervisor Oliver, thanks for welcoming to the lab. You are always reliable and available whenever I need guidance or help, your supports and encouragements mean a lot to me.

To Dr. Éric Tartour and Dr. François Ghiringhelli, thanks for joining my jury as rapporteurs.

To Dr. Donnadieu Emmanuel, thanks for serving as the president of the jury and examine my study.

To Dr. Catherine Brenner and Dr. Sébastien Apcher, thanks for accepting my invitation to examine this work.

To Guo, Yan and Shuai, thanks for helping me in finalizing experiments and your useful advices.

To Peng, Liwei and Chen, thanks for the happiness you brought to me, I do enjoy the time we spent together both in and out of the lab.

To Giulia and Juliette H, thanks for your patience and kindness, I cannot adapted to the French life smoothly without your help.

To all the members in our lab, I do appreciate your generous help and advices in my Ph.D projects and future careers.

To Chinese Scholarship Council, thanks for financial support during my study.

To my beloved parents and little sister, I cannot appreciate more for your consistent love and support, you give me the courage to explore, to extend my limitation without worry.

To my girlfriend Yanli, thanks for your unswerving love, your company, your tolerance, your comfort and inspiration. You lightened my life and make me better and better. Love you so much. Tesniere, A., Schlemmer, F., Boige, V., Kepp, O., Martins, I., Ghiringhelli, F., Aymeric, L., Michaud, M., Apetoh, L., Barault, L., et al. (2010). Immunogenic death of colon cancer cells treated with oxaliplatin. Oncogene 29, 482-491. Thomas, L. (1982). On immunosurveillance in human cancer. The Yale journal of biology and medicine 55, 329-333.

Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S. Competing interests OK and GK are scientific co-founders of Samsara Therapeutics.

Author details

Conflict of interest

The authors declare that they have no conflict of interest.

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-018-1127-3).