Oncogene (2014), 1–10 © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc
ORIGINAL ARTICLE
Immune-dependent antineoplastic effects of cisplatin plus pyridoxine in non-small-cell lung cancer F Aranda1,2,3, N Bloy1,2,3,4, J Pesquet1,2,3, B Petit5, K Chaba1,2, A Sauvat1,2,3, O Kepp1,2,3, N Khadra1,2,3, D Enot1,2,3, C Pfirschke6, M Pittet6, L Zitvogel4,7,8, G Kroemer1,2,3,9 and L Senovilla1,3,7 cis-Diamminedichloroplatinum(II) (CDDP), which is mostly referred to as cisplatin, is a widely used antineoplastic. The efficacy of cisplatin can be improved by combining it with the vitamin B6 precursor pyridoxine. Here, we evaluated the putative synergistic interaction of CDDP with pyridoxine in the treatment of an orthotopic mouse model of non-small-cell lung cancer (NSCLC). CDDP and pyridoxine exhibited hyperadditive therapeutic effects. However, this synergy was only observed in the context of an intact immune system and disappeared when the otherwise successful drug combination was applied to the same NSCLC cancer implanted in the lungs of athymic mice (which lack T lymphocytes). Immunocompetent mice that had been cured from NSCLC by the combined regimen of CDDP plus pyridoxine became resistant against subcutaneous rechallenge with the same (but not with an unrelated) cancer cell line. In vitro, CDDP and pyridoxine did not only cause synergistic killing of NSCLC cells but also elicited signs of immunogenic cell death including an endoplasmic reticulum stress response and exposure of calreticulin at the surface of the NSCLC cells. NSCLC cells treated with CDDP plus pyridoxine in vitro elicited a protective anticancer immune response upon their injection into immunocompetent mice. Altogether, these results suggest that the combined regimen of cisplatin plus pyridoxine mediates immune-dependent antineoplastic effects against NSCLC. Oncogene advance online publication, 28 July 2014; doi:10.1038/onc.2014.234
INTRODUCTION cis-Diamminedichloroplatinum(II) (CDDP), which is best known as cisplatin, is a prominent anticancer agent that is widely used for the treatment of non-small-cell lung cancer (NSCLC), head and neck cancer or ovarian carcinoma, as well as other neoplasias.1–6 In many cases, CDDP has palliative rather than curative effects (if any), meaning that it causes transient tumor growth arrest or shrinkage that is typically followed by relapse and lethal progression.7,8 Only, in a few malignancies (as exemplified by testicular cancer), CDDP has been shown to mediate consistently long-term cures.9,10 Given the poor long-term outcome of CDDP-based chemotherapies, it appears important to identify the underlying resistance mechanisms.7,11–13 The current literature lists a wide range of possible mechanisms of failing CDDP responses, including pretarget resistance (compromising the bioavailability of CDDP within cells and its interaction with its target, DNA), on-target resistance mechanisms (that undermine CDDP’s capacity to cause durable and lethal DNA lesions; for instance, because of enhanced DNA repair) and off-target resistance mechanisms (that subvert the link between DNA lesions and lethal signaling, usually causing the cell’s active demise via the ignition of the apoptotic death program).7 Chemosensitization to CDDP may be achieved by a series of agents, most of which have cytotoxic side effects. Recently, we observed that the cytocidal activity of CDDP is modulated by the expression level of pyridoxal kinase (PDXK), the enzyme that converts B6 vitameres (such as cell-permeant pyridoxine, PN) into
active vitamin B6, and pyridoxal-50 -phosphate (which is cell impermeant and hence ‘captured’ in cells expressing PDXK), which functions as the prosthetic group of multiple intracellular enzymes. The expression level of PDXK indeed constitutes an independent prognostic marker for disease-free and overall survival of patients with NSCLC.14 More importantly, it is possible to combine CDDP with PN to obtain synergistic anticancer effects, both in vitro, on human cancer cell lines, and in vivo, in immunodeficient mice xenografted with such human cancers.14 We and others have observed that, in multiple instances, chemotherapy is particularly efficient if it succeeds in eliciting anticancer immunosurveillance.15–18 This immunostimulatory effect may be achieved by immune-relevant ‘side’ effects; for instance, the depletion of immunosuppressive cells (including myeloid-derived suppressor cells, M2 macrophages or regulatory T cells) from the tumor bed. In addition, chemotherapies may mediate direct effects on malignant cells that then elevate their immunogenicity and/or their susceptibility to immune control by cytotoxic T and natural killer (NK) cells. Enhanced immunogenicity results either from increased antigenicity (i.e. higher expression of major histocompatibility complex (MHC) class I–peptide complexes at the tumor cells surface) or from augmented adjuvanticity (i.e. improved exposure or release of secondary signals that stimulate the immune response).15,19,20 In this latter category, we observed that several successful chemotherapeutic agents (such as anthracyclines, taxanes and oxaliplatin) are endowed with the capacity of inducing the so-called immunogenic cell death
1 Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, Paris, France; 2Université Paris Descartes, Sorbonne Paris Cité, Paris, France; 3Metabolomics and Cell Biology Platforms, Villejuif, France; 4Université Paris Sud, Villejuif, France; 5INSERM U1030, Villejuif, France; 6Center for Systems Biology, Massachusetts General Hospital/Harvard Medical School, Pittet Lab, Boston, MA, USA; 7INSERM U1015, Villejuif, France; 8Centre d’Investigation Clinique Biothérapie CICBT 507, Villejuif, France and 9Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France. Correspondence: Dr G Kroemer or Dr L Senovilla, Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, 15 rue de l’Ecole de Médecine, 75006 Paris, France. E-mail: [email protected] or [email protected] Received 15 June 2014; revised 24 June 2014; accepted 24 June 2014
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(ICD).21–24 ICD is characterized by a series of changes in the liquid cancer cell microenvironment (with an increase in the chemoattractant ATP and the dendritic cell maturating protein HMGB1 (also known as high-mobility group protein B1), both of which are released from dying and dead tumor cells, respectively) and changes in the surface characteristics of the tumor cells (with the exposure of the endoplasmic reticulum (ER) stress protein calreticulin (CRT) on the external face of the plasma membrane).25 CRT is thought to act as an essential ‘eat-me’ signal that favors the transfer of tumor antigens to dendritic cells,22 as well as the phagocytic removal of stressed and dying tumor cells.26,27 In comparison with well-established ICD inducers, CDDP has turned out to be a relatively poor inducer of CRT surface exposure.
This ‘defect’ has been linked to its incapacity to trigger efficiently an ER stress response, and external supply of ER-stressing agents may indeed improve ICD induction by CDDP, boosting its therapeutic efficacy.22,28,29 Here, we investigated the capacity of PN to improve the efficacy of CDDP in an orthotopic mouse model of NSCLC. We found that CDDP and PN synergistically interact not only to induce tumor cell death but also to stimulate anticancer immune responses in vivo. This latter effect could be related to the capacity of the combination regimen to stimulate ICD. Importantly, it appears that CDDP and PN are much more efficient in vivo if they are used in an immuncompetent, as opposed to an immunodeficient, setting.
Figure 1. Synergistic cytotoxic effects of CDDP plus PN in vitro. LLC cells were cultured in control conditions or exposed for 48 h to PN (0.6, 1.25, 2.5, 5 and 10 mM) and CDDP (1.25, 2.5, 5, 10, 20 and 40 μM) alone or combined with PN and then stained with the vital dye PI and the mitochondrial transmembrane potential (ΔΨm)-sensitive fluorochrome DiOC6(3), to measure apoptosis-associated parameters (a and b). (a) Representative dot plots as obtained at basal condition or upon incubation with PN and CDDP alone or in combination with PN. Numbers indicate the percentage of cells found in each quadrant. In (b), black and white columns illustrate the percentage of dead (PI+) and dying (DiOC6(3)low PI−) cells, respectively. Samples were compared using one-tailed Student’s t-test. Error bars indicate s.e.m. *P o0.05, **Po 0.01, compared with the corresponding CDDP concentration alone. Colorimetric assessment of residual proliferation by means of a WST-1 conversion-based assay. In (c), color-coded surfaces illustrate residual WST-1-converting activity. In (d), combination indexes (CIs), estimated according to the Harbron’s method starting from a data set built on n = 4 independent assessments, are depicted. CI o0.8 is generally viewed as an indicator of bona fide synergistic interactions. Oncogene (2014), 1 – 10
© 2014 Macmillan Publishers Limited
Immune-dependent effects of cisplatin plus pyridoxine F Aranda et al
RESULTS Synergistic cytotoxic effects of CDDP plus PN on NSCLC in vitro Corroborating previously published findings,14 CDDP exerted more pronounced cytotoxic effects if combined with the vitamin B6 precursor PN. Thus, Lewis lung cancer (LLC) cells, which originated spontaneously as an adenocarcinoma of the lung of a C57Bl/6 mouse,30 manifested a significant increase in their apoptotic response if 5 or 10 μM CDDP was combined with 1.25–5 mM of PN. This result (Figures 1a and b) was obtained when cell death was measured by simultaneous staining with the mitochondrial transmembrane potential-sensitive dye 3,3'dihexiloxalocarbocyanine iodide (DiOC6(3)) and the vital exclusion dye propidium iodide (PI), considering cell death as the sum of dying (DiOC6(3)low PI−) and dead (PI+) cells. Isobologram analyses of the loss of viability (measured by assessing the cellular reduction of
3 the tetrazolium salt 4-[3-(4-iodophenyl)-2-(4–nitrophenyl)-2H-5tetrazolio]-1,3-benzene disulfonate (WST-1)) confirmed the synergistic interaction between CDDP and PN (Figures 1c and d). Moreover, synergistic cytotoxic effects could be validated for the combination of CDDP plus PN on additional mouse cancer cell lines including CT26 colorectal carcinoma and MCA205 fibrosarcoma (Supplementary Figures 1 and 2). Synergistic induction of ICD characteristics by CDDP plus PN As compared with anthracyclines and oxaliplatin, CDDP is relatively inefficient in inducing ICD,22 presumably because CDDP is a relatively weak inducer of ER stress response that is required for the exposure of CRT at the cell surface.28,29 Although CDDP alone did cause some degree of eukaryotic initiation factor 2α (eIF2α), serine 51 phosphorylation (detectable with a
Figure 2. CDDP combined with PN induces synergistic ICD characteristics. Effects of CDDP (2.5, 5 and 10 μM) alone or in combination with PN (2.5 and 5 mM) on ER stress (a and b). Twenty-four hours after exposure to the drug combinations, LLC cells were fixed, permeabilized and then were stained for the visualization of phosphorylated eIF2α in the presence of Hoechst 33342, used for nuclear counterstaining. Treatment of tunicamycin (TUN, 1 μM) for 7 h was used as a positive control. Representative fluorescence microphotographs are shown in (a), and their quantitation (as mean fluorescence of phosporylated eIF2α) are shown in (b). Effects of CDDP (2.5 μM) alone or in combination with PN (5 mM) on ICD markers (c–e). CRT exposure (c), HMGB1 release (d) and ATP release (e) were determined. Cells treated with mitoxantrone (MTX) 1 μM for 4 h were used as a positive control. Experiments were performed three times, yielding similar results. Samples were compared using onetailed Student’s t-test. Error bars indicate s.e.m. #Po 0.05, ##P o0.01, compared with untreated cells, and *P o0.05, **Po 0.01, compared with the corresponding CDDP concentration alone. Scale bar, 10 μm. © 2014 Macmillan Publishers Limited
Oncogene (2014), 1 – 10
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4 phospho-neoepitope-specific antibody revealed by indirect immunofluorescence), this effect was exacerbated by combining CDDP with PN (Figures 2a and b). Accordingly, CDDP induced higher levels of CRT exposure when combined with PN (Figure 2c). Moreover, the combination of CDDP plus PN induced higher levels of ATP and HMGB1 release than did CDDP or PN alone (Figures 2d and e). This effect was detected in LLC cells, as well as on three additional cell lines, CT26, MCA205 and LUC lung adenocarcinoma cells (Supplementary Figure 3). Altogether, these results indicate that CDDP and PN induce stronger signs of ICD when combined than if applied separately. To investigate the capacity of CDDP plus PN to induce ICD in functional assays, LLC cells were cultured for 48 h in the presence of CDDP alone or in combination with PN, and the resulting mixture of dead and dying cells were injected subcutaneously into the left flank of syngenic C57Bl/6 mice, which were rechallenged 1 week later with live LLC cells (Figure 3a). As a positive control, we observed that LLC cells treated with the prototypic ICD inducer mitoxantrone, an anthracycline, were able to ‘vaccinate’ the mice and hence to induce a protective anticancer immune response in 70% of the animals. While CCDP-treated LLC cells only protected 1 out of 10 mice, the combination of CDDP plus PN conferred protection to half of the animals and strongly reduced tumor growth (Figures 3b and c). Altogether, these results support the notion that CDDP plus PN can induce ICD. Synergistic antineoplastic effects of CDDP plus PN in a model of orthotopic NSCLC in vivo To investigate the interaction between CDDP and PN in a realistic setting, we evaluated the antineoplastic efficacy of these agents in C57Bl/6 mice carrying orthotopic NSCLCs expressing the firefly luciferase (LUC), monitoring tumor growth by bioluminescence analysis and whole-body imaging (Figures 4a and b). The vast majority (~90%) of the mice treated with vehicle only (phosphatebuffered saline (PBS)) or PN only died within 17 days. CDDP alone reduced tumor growth, yet rarely achieved total cure (15%). In contrast, the combination of CDDP plus PN caused a more pronounced reduction in tumor growth and cured up to 45% of the mice, which remained tumor free (Figures 5a and c). Histologic analyses confirmed that an elevated percentage of mice receiving the combination regimen lacked detectable cancer lesions in the lung (Figure 5d). Altogether, these results underscore the possibility to improve the efficacy of CDDP by combining it with PN. Immune-dependent antineoplastic effects of CDDP plus PN in vivo In an attempt to weight the cell-autonomous versus immunemediated effects of the combination regimen (CDDP plus PN), we implanted LUC tumors in athymic nu/nu mice and treated them with CDDP and/or PN. These experiments were performed at the same time as those carried out on immunocompetent mice (Figures 4 and 5), yet are presented in separate figures (Figures 6 and 7a, c). To our surprise, both CDDP alone and CDDP plus PN failed to avoid tumor growth and to prolong overall survival in nu/ nu mice, although the combination regimen induced a nonsignificant trend toward a reduction in total tumor mass (Figures 6 and 7a, c). Hence, the observed therapeutic effects must rely on the presence of thymus-dependent T lymphocytes (which are deficient in nu/nu mice). Finally, we assessed the presence of a protective anticancer immune response in immunocompetent mice after successful chemotherapy. Mice that had been cured from their orthotopic LUC NSCLC became fully resistant against subcutaneous rechallenge with LUC cells, although they remained susceptible to tumor development after injection of the unrelated MCA205 fibrosarcoma (Figure 7d). These results reveal the induction of a tumor antigen-specific immune response that Oncogene (2014), 1 – 10
Figure 3. Impact of CDDP plus PN on the immunogenicity of cell death. Murine lung adenocarcinoma LLC cells that have been treated in vitro or not with CDDP (2.5 μM), CDDP (2.5 μM) plus PN (5 mM) for 48 h or mitoxantrone (MTX) (1 μM) for 4 h and then subcutaneously injected into C57Bl/6 mice (10 mice/group) were inoculated with live LLC cells 7 days later (a). Tumor growth (b) and tumor incidence (c) were measured. Tumor growth was compared using one-tailed Student’s t-test and tumor incidences (illustrated with Kaplan–Meier curves) were compared by one-tailed Barnard's test. #Po0.05, ##Po 0.01, compared with untreated cells, and *Po 0.05, **Po0.01, compared with CDDP-alone-treated cells.
lasted for several months after discontinuation of the successful chemotherapeutic regimen. DISCUSSION Cytotoxic and targeted anticancer therapeutics have been and are being developed with the idea that they should mediate their cytocidal or cytostatic effects by direct effects on tumor cells. Logically, it is hence possible to predict anticancer effects by testing antineoplastic agents on cultured tumor cells as well as © 2014 Macmillan Publishers Limited
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Figure 4. Effects of CDDP plus PN in a model of orthotopic NSCLC in vivo. Murine lung adenocarcinoma LUC cells were inoculated into the left lung of C57Bl/6 mice (20 mice/group) and then treated with PBS, PN (125 mg/kg) and CDDP (1.5 mg/kg) and alone or in combination with PN, three times per week the 3 first weeks, then two times per week and later stopped the treatment at day 42 (a). Tumor presence was analyzed by luciferase activity. Representative fluorescence photographs of 4 mice/group at different days of analysis are shown in (b). Upper left panels, PBS treated mice; upper right panels, mice treated with PN alone; down left panels, mice treated with CDDP alone; and down right panels, CDDP plus PN combination-treated mice.
cancers established in immunodeficient mice (as this is usually done when preclinical tests involve xenotransplanted human cancer cell lines or primary tumors).31,32 Following this intellectual scheme, we show here that cisplatin can be combined with PN to kill synergistically NSCLC cells in vitro and to treat successfully mice with NSCLC (in an orthotopic location) in vivo. To our surprise, however, the linear link between the in vitro and the in vivo data was only apparent. Indeed, the synergistic combination extended the life of a significant percentage of tumor-bearing mice, only if such tumors developed in an immunocompetent environment. Treatment of the same tumors with the same combined regimen was totally inefficient in an immunodeficient context, indicating that the therapeutic success was entirely dependent on the presence of an intact immune system (and, in particular, T lymphocytes, which are absent in nu/nu mice). These results underscore the contribution of anticancer immune © 2014 Macmillan Publishers Limited
responses to the therapeutic efficacy of the treatment with CDDP plus PN. It may appear counterintuitive that the combination treatment with CDDP plus PN effectively reduces the cellularity of NSCLC cultures in vitro, yet fail to mediate any major antineoplastic effects in vivo, on NSCLC established in nu/nu mice, at least at the doses that were used in this study. To observe antitumor efficacy, both agents had to be administered to immunocompetent mice, underscoring the cardinal importance of the anticancer immune response to the long-term effects of this particular combination regimen. Previous clinical studies using prototypic ICD inducers such as oxaliplatin or doxorubicin have failed to show superior efficacy in NSCLC if compared with or combined with CDDP.33–36 At this point, it is difficult to understand whether these negative data must be attributed to the advanced stage of NSCLC (when the immune system may be overwhelmed and hence intrinsically inefficient) or Oncogene (2014), 1 – 10
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Figure 5. Synergistic effects of CDDP plus PN in the orthotopic model in immunocompetent mice. C57Bl/6 mice were injected orthotopically into the left lung with LUC cells and then treated as described in Figure 4. Tumor incidence (a), tumor growth (b) and tumor survival (c) were monitorized. Representative pictures (d) of hematoxylin–eosin-safran (HES) stainings from normal versus tumor-bearing lungs treated or not with PN and CDDP alone or in combination with PN are shown. Tumor incidences (a) and tumor survival (c) (illustrated with Kaplan–Meier curves) curves were compared by the one-tailed Barnard's test. Tumor growth was compared using one-tailed Student’s t-test. ##Po 0.01, compared with untreated cells, and *P o0.05, **P o0.01, compared with CDDP alone. Scales bar, 500 μm (upper panels) and 50 μm (down panels).
suboptimal dosing and scheduling.37,38 A recent study performed on a mouse model of KRAS-induced NSCLC revealed that oxaliplatin was particularly efficient if combined with cyclophosphamide,39 and that this antineoplastic effect was entirely dependent on the immune system, as it disappeared in Rag2− / − mice that lack B and T lymphocytes.39 It will be interesting to evaluate this type of combination therapy in future clinical trials. At the mechanistic level, it appears that CDDP and PN do not only mediate a synergistic cytotoxic effect but that they also cooperate in the induction of ICD hallmarks. Thus, CDDP plus PN induced more CRT exposure, HMGB1 exodus and ATP release than did either of the two agents alone. With regard to CRT exposure, both agents interacted synergistically to elicit ER stress at the level of the phosphorylation of eIF2α, which constitutes a stringent requirement for CRT to be exposed.40 Indeed, CDDP and PN induced a higher level of eIF2α phosphorylation, if combined rather than used separately. The detailed mechanisms of this synergistic interaction are elusive. However, it is tempting to speculate that the capacity of CDDP plus PN to mediate a hyperadditive redox stress, as this is shown experimentally at the level of glutathione depletion,14 might facilitate the activation of the ER stress kinase PERK (official name: eukaryotic translation initiation factor 2-α kinase 3) that is required for chemotherapyinduced eIF2α phosphorylation, upstream of CRT exposure.41,42 Indeed, the antioxidants N-acetyl cysteine and glutathione ethyl ester can prevent cell killing by CDDP plus PN,14,41 as they prevent the induction of CRT exposure by classical ICD inducers.40 The fact that CDDP plus PN elicit the mitochondrial (intrinsic) pathway of apoptosis is underscored by the observations that cell death is Oncogene (2014), 1 – 10
consistently accompanied by an early decrease of the mitochondrial transmembrane potential (ΔΨm), modulated by knockdown of proteins of the Bcl-2 family, and delayed by the addition of caspase inhibitors.14 Altogether, it appears that CDDP plus PN are efficient in inducing anticancer immune responses in vivo, both in vaccination assays and in the context of chemotherapeutic treatments of established cancers. As a proof in favor of this hypothesis, mice that have been cured from NSCLC by means of the combination regimen exhibit a specific, long-term protection against rechallenge with cancer cells. In synthesis, our data delineate a strategy to improve the efficacy of CDDP-based chemotherapy by combining it with the chemosensitizer PN. This strategy may increase the immunogenic properties of dying NSCLC cells, thereby triggering an efficient and therapeutically relevant antitumor immune response. MATERIALS AND METHODS Unless otherwise indicated, media and supplements for cell culture were purchased from Gibco-Invitrogen (Carlsbad, CA, USA), plasticware from Corning BV Life Sciences (Schiphol-Rijk, The Netherlands) and chemicals from Sigma-Aldrich (St Louis, MO, USA).
Antibodies Rabbit polyclonal antibody against CRT (ab2907) was purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibody against anti-phosphoeIF2α (Ser51) (3597) was from Cell Signaling Technology (Danvers, MA, USA). © 2014 Macmillan Publishers Limited
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Figure 6. Absence of effects with CDDP plus PN treatment in the orthotopic model in immunodeficient mice. Murine lung adenocarcinoma LUC cells were inoculated into the left lung of nu/nu mice (10 mice/group) as described in Figure 4. Tumor presence was analyzed by luciferase activity. Representative fluorescence photographs of 4 mice/group at different days of analysis are shown. Upper left panels, PBStreated mice; upper right panels, mice treated with PN alone; down left panels, mice treated with CDDP alone; and down right panels, CDDP plus PN combination-treated mice.
Cell lines and culture conditions All cell lines were cultured at 37 °C under 5% of CO2 in medium containing 10% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin. Cell type-specific culture conditions include: Dulbecco’s modified Eagle’s medium supplemented as above plus 1 mM sodium pyruvate for murine LLC cells; RPMI 1640 medium supplemented as above plus 1 mM sodium pyruvate and 1 mM HEPES buffer for murine colon carcinoma CT26, murine fibrosarcoma MCA205 cells and non-small-cell lung carcinoma TC-1 expressing the firefly LUC.43
(ΔΨm), plus 1 μg/ml PI, for the identification of plasma membrane breakdown.44 Surface exposure of CRT was detected by immunofluorescence staining as described previously41 after 48 h of treatment. Samples were then analyzed by means of a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA) or FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). Statistical analyses were carried out by using the CellQuest software (BD Biosciences), upon gating on the events characterized by normal forwardand side-scatter values.
Immunofluorescence microscopy and automated image analysis Treatments Cells were seeded in 6-, 12-, 96-well plates, 25, 75 or 175 cm2 flasks and allowed to attach for at least 24 h before experiments. Cells were challenged with the following pharmacologic agents: CDDP (1.25, 2.5, 5, 10, 20, 40 or 80 μM) and PN (⩾98% purity; 0.6, 1.25, 2.5, 5 and 10 mM) and 1 μM mitoxantrone and 1 μM tunicamycin as positive controls when corresponding.
Cytofluorometry Cell death was measured after 48 h of treatment; unfixed cells were co-stained with 40 nM DiOC6(3) (from Molecular Probes-Invitrogen, Eugene, OR, USA), which quantifies the mitochondrial transmembrane potential © 2014 Macmillan Publishers Limited
Cells were treated for 24 h and then washed with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 10 min and finally rinsed three times with PBS. Nonspecific binding sites were blocked with blocking buffer for 30 min and cells were incubated with primary anti-phospho-eIF2α (Ser51) antibody for 1 h. Subsequently, cells were washed three times with PBS and incubated for 30 min in AlexaFluor 488-conjugated secondary antibodies (Molecular ProbesInvitrogen). Hoechst 33342 (10 μM) was used for nuclear counterstaining. Fluorescence microscopic assessments were performed by automated image analysis. Micrographs have been acquired using an IXM XL (Molecular Devices, Sunnyvale, CA, USA) equipped with a × 20 PlanApo objective (Nikon, Tokyo, Japan) and a laser-based autofocus. Nine view fields within each well have been sequentially imaged using 4′,6Oncogene (2014), 1 – 10
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Figure 7. Immune-dependent antineoplastic effects of CDDP plus PN in vivo. Immunodeficient nu/nu mice were injected orthotopically in the left lung with LUC cells and then treated as described in Figure 4. Tumor incidence (a), tumor growth (b) and tumor survival (c) were measured. Anticancer immune response was observed in immunocompetent mice after successful chemotherapy (d). C57Bl/6 control (upper panels, 5 mice/group) and mice previously cured from their orthotopic LUC tumors after chemotherapy with CDDP plus PN, as shown in Figure 5 (lower panels, 3 mice/group), were subjected to subcutaneous injection with unrelated MCA 205 cells (left panels) or LUC cells (right panels) at the same time. Each line represents the growth of a tumor in one individual mouse.
diamidino-2-phenylindole (DAPI) and GFP standard filter sets at a binning of 1. The images obtained were segmented into nuclear and cytoplasmic regions by means of the MetaXpress image analysis, and the cytoplasmic fluorescence intensity of GFP was evaluated, while debris were omitted by size exclusion. Mean fluorescence intensities were calculated and depicted as heat map by means of the freely available statistics program ‘R’ (http:// www.r-project.org).
HBMG1 release was measured using the cell supernatants collected after 48 h of treatment and cleared from dying tumor cells by centrifugation (800 g, 5 min), and then immediately analyzed for HMGB1 abundance. HMGB1 quantification was performed by means of an Enzyme-Linked Immunosorbent Assay (ELISA) Kit (HMBG1 ELISA Kit II; Gentaur Europe, Kampenhout, Belgium), according to the manufacturer's instructions.
Mice Colorimetric assays In vitro assessments of the synergistics effects between CDDP and PN after 48 h of treatment were performed by means of a colorimetric assay based on the reduction of the colorless tetrazolium salt WST-1 (from Roche Diagnostics GmbH, Mannheim, Germany) to formazan (which exhibit an absorbance peak around 450 nm), following conventional procedures.14 Absorbance at 450 nm was measured on a FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany) and—following background subtraction—WST-1 conversion data were normalized to the readings of untreated cells included in the same test plate. Extracellular ATP levels were measured after 24 h of treatment by the luciferin-based ENLITEN ATP Assay Kit (Promega Corporation, Madison, WI, USA), in excess of luciferin and LUC, as indicated by the manufacturer. ATP-driven chemiluminescence was recorded on a FLUOstar multiwell plate luminometer (BMG Labtech, Ortenberg, Germany) as described previously.45 Oncogene (2014), 1 – 10
Mice were maintained in specific pathogen-free conditions, and experiments followed the Federation of European Laboratory Animal Science Association (FELASA). Animal experiments were in compliance with the EU Directive 63/2010 and protocols (protocols 2012_138 and 2012_156) were approved by the Ethical Committee of the Gustave Roussy Campus Cancer (GRCC, Villejuif, France) (CEEA IRCIV/IGR no. 26, registered at the French Ministry of Research). Wild-type C57Bl/6 mice were obtained from Harlan France (Gannat, France) and nu/nu mice from the GRCC animal facility.
Antitumor vaccination A total of 3 × 106 LLC tumor cells were left untreated or treated with CDDP (2.5 μM) alone or combined with PN (5 mM) for 48 h, and then were inoculated subcutaneously in 200 μl PBS into the lower flank of C57Bl/6 mice. As a positive control, the cells were treated with mitoxantrone (1 μM) for 7 h and then cultured in the fresh medium and inoculated after 24 h as © 2014 Macmillan Publishers Limited
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9 described above. A total of 5 × 105 untreated cells were inoculated into the contralateral flank 1 week later. Tumor growth was routinely monitored with a common caliper regardless of the presence or absence of tumor after the first injection. Animals bearing tumors that exceeded 20–25% body mass were killed. C57Bl/6 mice that had previously been submitted to lung orthotopic model but were cured from their developed tumors were reinjected with 5 × 105 LUC cells to establish the possible vaccination in this model. The unrelated MCA205 fibrosarcoma cell line was used as a positive control.
Lung orthotopic model A total of 4× 105 LUC tumor cells were injected into the left lung by intercostal injection at the median axillary line of C57Bl/6 or nu/nu mice as described previously.46 Mice were treated with PBS, PN (125 mg/kg) and CDDP (1.5 mg/kg) and alone or in combination with PN, three times per week in the 3 first weeks, then two times per week and later stopped the treatment at day 42. Tumor growth was analyzed by in vivo photonic imaging of tumor cells luciferase activity. Mice were injected intraperitoneally once per week (starting at day 3 after tumor cell inoculation) with the substrate of luciferase, D-luciferin potassium salt (Promega), at a dose of 10 μm/kg. In vivo imaging was performed with Xenogen IVIS 50 bioluminiscence imaging system (Caliper Life Sciences Inc., Hopkinton, MA, USA). Photons were acquired after 5 min of luciferin inoculation for maximum of 3 min, and then 1 min or 30 s if saturation occurred. Mice showing photon saturation levels at 30 s were killed.
Lung histology and hematoxylin, eosin and safran staining Recovered lungs were fixed with 4% paraformaldehyde for 4 h and then embedded into paraffin. Sections of 4 μm paraffin were fixed and stained with hematoxylin, eosin and safran according to standard protocols. Representative pictures were acquired using ZEISS Primo Star AxioCAM ERc 5s microscope and AxioVision LE software.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We are indepted to Dr Gonin (Gustave Roussy) for help in mouse experiments. CP is supported by the Deutsche Forschungsgemeinschaft (DFG) PF809/1-1. LS is supported by the Fondation Tourre, the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE) and the SIRIC Cancer Research and Personalized Medicine (CARPEM). GK is supported by the Ligue contre le Cancer (équipe labelisée); Agence National de la Recherche (ANR); 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); and the Paris Alliance of Cancer Research Institutes (PACRI).
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8 Koberle B, Tomicic MT, Usanova S, Kaina B. Cisplatin resistance: preclinical findings and clinical implications. Biochim Biophys Acta 2010; 1806: 172–182. 9 Winter C, Albers P. Testicular germ cell tumors: pathogenesis, diagnosis and treatment. Nat Rev Endocrinol 2011; 7: 43–53. 10 Feldman DR, Bosl GJ, Sheinfeld J, Motzer RJ. Medical treatment of advanced testicular cancer. JAMA 2008; 299: 672–684. 11 Kelman AD, Peresie HJ. Mode of DNA binding of cis-platinum(II) antitumor drugs: a base sequence-dependent mechanism is proposed. Cancer Treat Rep 1979; 63: 1445–1452. 12 Sanderson BJ, Ferguson LR, Denny WA. Mutagenic and carcinogenic properties of platinum-based anticancer drugs. Mutat Res. 1996; 355: 59–70. 13 Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003; 22: 7265–7279. 14 Galluzzi L, Vitale I, Senovilla L, Olaussen KA, Pinna G, Eisenberg T et al. Prognostic impact of vitamin B6 metabolism in lung cancer. Cell Rep 2012; 2: 257–269. 15 Zitvogel L, Galluzzi L, Smyth MJ, Kroemer G. Mechanism of action of conventional and targeted anticancer therapies: reinstating immunosurveillance. Immunity 2013; 39: 74–88. 16 Finn OJ. Immuno-oncology: understanding the function and dysfunction of the immune system in cancer. Ann Oncol 2012; 23: viii6–viii9. 17 McDonnell AM, Nowak AK, Lake RA. Contribution of the immune system to the chemotherapeutic response. Semin Immunopathol 2011; 33: 353–367. 18 Menard C, Martin F, Apetoh L, Bouyer F, Ghiringhelli F. Cancer chemotherapy: not only a direct cytotoxic effect, but also an adjuvant for antitumor immunity. Cancer Immunol Immunother 2008; 57: 1579–1587. 19 Finn OJ. Cancer immunology. New Engl J Med 2008; 358: 2704–2715. 20 Coussens LM, Zitvogel L, Palucka AK. Neutralizing tumor-promoting chronic inflammation: a magic bullet? Science 2013; 339: 286–291. 21 Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, Chaput N et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med 2005; 202: 1691–1701. 22 Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 2007; 13: 54–61. 23 Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 2007; 13: 1050–1059. 24 Senovilla L, Vitale I, Martins I, Tailler M, Pailleret C, Michaud M et al. An immunosurveillance mechanism controls cancer cell ploidy. Science 2012; 337: 1678–1684. 25 Kepp O, Galluzzi L, Martins I, Schlemmer F, Adjemian S, Michaud M et al. Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy. Cancer Metast Rev 2011; 30: 61–69. 26 Chao MP, Jaiswal S, Weissman-Tsukamoto R, Alizadeh AA, Gentles AJ, Volkmer J et al. Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47. Sci Transl Med 2010; 2: 63ra94. 27 Chao MP, Majeti R, Weissman IL. Programmed cell removal: a new obstacle in the road to developing cancer. Nat Rev Cancer 2012; 12: 58–67. 28 Tesniere A, Schlemmer F, Boige V, Kepp O, Martins I, Ghiringhelli F et al. Immunogenic death of colon cancer cells treated with oxaliplatin. Oncogene 2010; 29: 482–491. 29 Martins I, Kepp O, Schlemmer F, Adjemian S, Tailler M, Shen S et al. Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress. Oncogene 2011; 30: 1147–1158. 30 Mayo JG. Biologic characterization of the subcutaneously implanted Lewis lung tumor. Cancer Chemother Rep 2 1972; 3: 325–330. 31 Talmadge JE, Singh RK, Fidler IJ, Raz A. Murine models to evaluate novel and conventional therapeutic strategies for cancer. Am J Pathol 2007; 170: 793–804. 32 Kelland LR. Of mice and men: values and liabilities of the athymic nude mouse model in anticancer drug development. Eur J Cancer 2004; 40: 827–836. 33 Phernambucq EC, Biesma B, Smit EF, Paul MA, vd Tol A, Schramel FM et al. Multicenter phase II trial of accelerated cisplatin and high-dose epirubicin followed by surgery or radiotherapy in patients with stage IIIa non-small-cell lung cancer with mediastinal lymph node involvement (N2-disease). Br J Cancer 2006; 95: 470–474. 34 Hsu C, Kuo SH, Hu FC, Cheng AL, Shih JY, Yu CJ et al. Gemcitabine plus conventional-dose epirubicin versus gemcitabine plus cisplatin as first-line chemotherapy for stage IIIB/IV non-small cell lung carcinoma--a randomized phase II trial. Lung Cancer 2008; 62: 334–343. 35 Otterson GA, Villalona-Calero MA, Hicks W, Pan X, Ellerton JA, Gettinger SN, et al. Phase I/II study of inhaled doxorubicin combined with platinum-based therapy for advanced non-small cell lung cancer. Clin Cancer Res 2010; 16: 2466–2473. 36 Atmaca A, Al-Batran SE, Werner D, Pauligk C, Güner T, Koepke A et al. A randomised multicentre phase II study with cisplatin/docetaxel vs oxaliplatin/docetaxel
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as first-line therapy in patients with advanced or metastatic non-small cell lung cancer. Br J Cancer 2013; 108: 265–270. Zitvogel L, Kepp O, Kroemer G. Immune parameters affecting the efficacy of chemotherapeutic regimens. Nat Rev Clin Oncol 2011; 8: 151–160. Galluzzi L, Senovilla L, Zitvogel L, Kroemer G. The secret ally: immunostimulation by anticancer drugs. Nat Rev Drug Discov 2012; 11: 215–233. Viaud S, Saccheri F, Mignot G, Yamazaki T, Daillère R, Hannani D et al. The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Science 2013; 342: 971–976. Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC et al. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J. 2009; 28: 578–590. Galluzzi L, Marsili S, Vitale I, Senovilla L, Michels J, Garcia P et al. Vitamin B6 metabolism influences the intracellular accumulation of cisplatin. Cell Cycle 2013; 12: 417–421.
42 Zitvogel L, Kepp O, Senovilla L, Menger L, Chaput N, Kroemer G. Immunogenic tumor cell death for optimal anticancer therapy: the calreticulin exposure pathway. Clin Cancer Res 2010; 16: 3100–3104. 43 Kim D, Hung CF, Wu TC. Monitoring the trafficking of adoptively transferred antigen- specific CD8-positive T cells in vivo, using noninvasive luminescence imaging. Hum Gene Ther 2007; 18: 575–588. 44 Castedo M, Ferri K, Roumier T, Metivier D, Zamzami N, Kroemer G. Quantitation of mitochondrial alterations associated with apoptosis. J Immunol Methods 2002; 265: 39–47. 45 Martins I, Tesniere A, Kepp O, Michaud M, Schlemmer F, Senovilla L et al. Chemotherapy induces ATP release from tumor cells. Cell Cycle 2009; 8: 3723–3728. 46 Mordant P, Loriot Y, Lahon B, Castier Y, Leseche G, Soria JC et al. Bioluminescent orthotopic mouse models of human localized non-small cell lung cancer: feasibility and identification of circulating tumour cells. PLoS ONE 2011; 6: e26073.
Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
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ORIGINAL ARTICLE
Immune-dependent antineoplastic effects of cisplatin plus pyridoxine in non-small-cell lung cancer F Aranda1,2,3, N Bloy1,2,3,4, J Pesquet1,2,3, B Petit5, K Chaba1,2, A Sauvat1,2,3, O Kepp1,2,3, N Khadra1,2,3, D Enot1,2,3, C Pfirschke6, M Pittet6, L Zitvogel4,7,8, G Kroemer1,2,3,9 and L Senovilla1,3,7 cis-Diamminedichloroplatinum(II) (CDDP), which is mostly referred to as cisplatin, is a widely used antineoplastic. The efficacy of cisplatin can be improved by combining it with the vitamin B6 precursor pyridoxine. Here, we evaluated the putative synergistic interaction of CDDP with pyridoxine in the treatment of an orthotopic mouse model of non-small-cell lung cancer (NSCLC). CDDP and pyridoxine exhibited hyperadditive therapeutic effects. However, this synergy was only observed in the context of an intact immune system and disappeared when the otherwise successful drug combination was applied to the same NSCLC cancer implanted in the lungs of athymic mice (which lack T lymphocytes). Immunocompetent mice that had been cured from NSCLC by the combined regimen of CDDP plus pyridoxine became resistant against subcutaneous rechallenge with the same (but not with an unrelated) cancer cell line. In vitro, CDDP and pyridoxine did not only cause synergistic killing of NSCLC cells but also elicited signs of immunogenic cell death including an endoplasmic reticulum stress response and exposure of calreticulin at the surface of the NSCLC cells. NSCLC cells treated with CDDP plus pyridoxine in vitro elicited a protective anticancer immune response upon their injection into immunocompetent mice. Altogether, these results suggest that the combined regimen of cisplatin plus pyridoxine mediates immune-dependent antineoplastic effects against NSCLC. Oncogene advance online publication, 28 July 2014; doi:10.1038/onc.2014.234
INTRODUCTION cis-Diamminedichloroplatinum(II) (CDDP), which is best known as cisplatin, is a prominent anticancer agent that is widely used for the treatment of non-small-cell lung cancer (NSCLC), head and neck cancer or ovarian carcinoma, as well as other neoplasias.1–6 In many cases, CDDP has palliative rather than curative effects (if any), meaning that it causes transient tumor growth arrest or shrinkage that is typically followed by relapse and lethal progression.7,8 Only, in a few malignancies (as exemplified by testicular cancer), CDDP has been shown to mediate consistently long-term cures.9,10 Given the poor long-term outcome of CDDP-based chemotherapies, it appears important to identify the underlying resistance mechanisms.7,11–13 The current literature lists a wide range of possible mechanisms of failing CDDP responses, including pretarget resistance (compromising the bioavailability of CDDP within cells and its interaction with its target, DNA), on-target resistance mechanisms (that undermine CDDP’s capacity to cause durable and lethal DNA lesions; for instance, because of enhanced DNA repair) and off-target resistance mechanisms (that subvert the link between DNA lesions and lethal signaling, usually causing the cell’s active demise via the ignition of the apoptotic death program).7 Chemosensitization to CDDP may be achieved by a series of agents, most of which have cytotoxic side effects. Recently, we observed that the cytocidal activity of CDDP is modulated by the expression level of pyridoxal kinase (PDXK), the enzyme that converts B6 vitameres (such as cell-permeant pyridoxine, PN) into
active vitamin B6, and pyridoxal-50 -phosphate (which is cell impermeant and hence ‘captured’ in cells expressing PDXK), which functions as the prosthetic group of multiple intracellular enzymes. The expression level of PDXK indeed constitutes an independent prognostic marker for disease-free and overall survival of patients with NSCLC.14 More importantly, it is possible to combine CDDP with PN to obtain synergistic anticancer effects, both in vitro, on human cancer cell lines, and in vivo, in immunodeficient mice xenografted with such human cancers.14 We and others have observed that, in multiple instances, chemotherapy is particularly efficient if it succeeds in eliciting anticancer immunosurveillance.15–18 This immunostimulatory effect may be achieved by immune-relevant ‘side’ effects; for instance, the depletion of immunosuppressive cells (including myeloid-derived suppressor cells, M2 macrophages or regulatory T cells) from the tumor bed. In addition, chemotherapies may mediate direct effects on malignant cells that then elevate their immunogenicity and/or their susceptibility to immune control by cytotoxic T and natural killer (NK) cells. Enhanced immunogenicity results either from increased antigenicity (i.e. higher expression of major histocompatibility complex (MHC) class I–peptide complexes at the tumor cells surface) or from augmented adjuvanticity (i.e. improved exposure or release of secondary signals that stimulate the immune response).15,19,20 In this latter category, we observed that several successful chemotherapeutic agents (such as anthracyclines, taxanes and oxaliplatin) are endowed with the capacity of inducing the so-called immunogenic cell death
1 Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, Paris, France; 2Université Paris Descartes, Sorbonne Paris Cité, Paris, France; 3Metabolomics and Cell Biology Platforms, Villejuif, France; 4Université Paris Sud, Villejuif, France; 5INSERM U1030, Villejuif, France; 6Center for Systems Biology, Massachusetts General Hospital/Harvard Medical School, Pittet Lab, Boston, MA, USA; 7INSERM U1015, Villejuif, France; 8Centre d’Investigation Clinique Biothérapie CICBT 507, Villejuif, France and 9Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France. Correspondence: Dr G Kroemer or Dr L Senovilla, Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, 15 rue de l’Ecole de Médecine, 75006 Paris, France. E-mail: [email protected] or [email protected] Received 15 June 2014; revised 24 June 2014; accepted 24 June 2014
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(ICD).21–24 ICD is characterized by a series of changes in the liquid cancer cell microenvironment (with an increase in the chemoattractant ATP and the dendritic cell maturating protein HMGB1 (also known as high-mobility group protein B1), both of which are released from dying and dead tumor cells, respectively) and changes in the surface characteristics of the tumor cells (with the exposure of the endoplasmic reticulum (ER) stress protein calreticulin (CRT) on the external face of the plasma membrane).25 CRT is thought to act as an essential ‘eat-me’ signal that favors the transfer of tumor antigens to dendritic cells,22 as well as the phagocytic removal of stressed and dying tumor cells.26,27 In comparison with well-established ICD inducers, CDDP has turned out to be a relatively poor inducer of CRT surface exposure.
This ‘defect’ has been linked to its incapacity to trigger efficiently an ER stress response, and external supply of ER-stressing agents may indeed improve ICD induction by CDDP, boosting its therapeutic efficacy.22,28,29 Here, we investigated the capacity of PN to improve the efficacy of CDDP in an orthotopic mouse model of NSCLC. We found that CDDP and PN synergistically interact not only to induce tumor cell death but also to stimulate anticancer immune responses in vivo. This latter effect could be related to the capacity of the combination regimen to stimulate ICD. Importantly, it appears that CDDP and PN are much more efficient in vivo if they are used in an immuncompetent, as opposed to an immunodeficient, setting.
Figure 1. Synergistic cytotoxic effects of CDDP plus PN in vitro. LLC cells were cultured in control conditions or exposed for 48 h to PN (0.6, 1.25, 2.5, 5 and 10 mM) and CDDP (1.25, 2.5, 5, 10, 20 and 40 μM) alone or combined with PN and then stained with the vital dye PI and the mitochondrial transmembrane potential (ΔΨm)-sensitive fluorochrome DiOC6(3), to measure apoptosis-associated parameters (a and b). (a) Representative dot plots as obtained at basal condition or upon incubation with PN and CDDP alone or in combination with PN. Numbers indicate the percentage of cells found in each quadrant. In (b), black and white columns illustrate the percentage of dead (PI+) and dying (DiOC6(3)low PI−) cells, respectively. Samples were compared using one-tailed Student’s t-test. Error bars indicate s.e.m. *P o0.05, **Po 0.01, compared with the corresponding CDDP concentration alone. Colorimetric assessment of residual proliferation by means of a WST-1 conversion-based assay. In (c), color-coded surfaces illustrate residual WST-1-converting activity. In (d), combination indexes (CIs), estimated according to the Harbron’s method starting from a data set built on n = 4 independent assessments, are depicted. CI o0.8 is generally viewed as an indicator of bona fide synergistic interactions. Oncogene (2014), 1 – 10
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RESULTS Synergistic cytotoxic effects of CDDP plus PN on NSCLC in vitro Corroborating previously published findings,14 CDDP exerted more pronounced cytotoxic effects if combined with the vitamin B6 precursor PN. Thus, Lewis lung cancer (LLC) cells, which originated spontaneously as an adenocarcinoma of the lung of a C57Bl/6 mouse,30 manifested a significant increase in their apoptotic response if 5 or 10 μM CDDP was combined with 1.25–5 mM of PN. This result (Figures 1a and b) was obtained when cell death was measured by simultaneous staining with the mitochondrial transmembrane potential-sensitive dye 3,3'dihexiloxalocarbocyanine iodide (DiOC6(3)) and the vital exclusion dye propidium iodide (PI), considering cell death as the sum of dying (DiOC6(3)low PI−) and dead (PI+) cells. Isobologram analyses of the loss of viability (measured by assessing the cellular reduction of
3 the tetrazolium salt 4-[3-(4-iodophenyl)-2-(4–nitrophenyl)-2H-5tetrazolio]-1,3-benzene disulfonate (WST-1)) confirmed the synergistic interaction between CDDP and PN (Figures 1c and d). Moreover, synergistic cytotoxic effects could be validated for the combination of CDDP plus PN on additional mouse cancer cell lines including CT26 colorectal carcinoma and MCA205 fibrosarcoma (Supplementary Figures 1 and 2). Synergistic induction of ICD characteristics by CDDP plus PN As compared with anthracyclines and oxaliplatin, CDDP is relatively inefficient in inducing ICD,22 presumably because CDDP is a relatively weak inducer of ER stress response that is required for the exposure of CRT at the cell surface.28,29 Although CDDP alone did cause some degree of eukaryotic initiation factor 2α (eIF2α), serine 51 phosphorylation (detectable with a
Figure 2. CDDP combined with PN induces synergistic ICD characteristics. Effects of CDDP (2.5, 5 and 10 μM) alone or in combination with PN (2.5 and 5 mM) on ER stress (a and b). Twenty-four hours after exposure to the drug combinations, LLC cells were fixed, permeabilized and then were stained for the visualization of phosphorylated eIF2α in the presence of Hoechst 33342, used for nuclear counterstaining. Treatment of tunicamycin (TUN, 1 μM) for 7 h was used as a positive control. Representative fluorescence microphotographs are shown in (a), and their quantitation (as mean fluorescence of phosporylated eIF2α) are shown in (b). Effects of CDDP (2.5 μM) alone or in combination with PN (5 mM) on ICD markers (c–e). CRT exposure (c), HMGB1 release (d) and ATP release (e) were determined. Cells treated with mitoxantrone (MTX) 1 μM for 4 h were used as a positive control. Experiments were performed three times, yielding similar results. Samples were compared using onetailed Student’s t-test. Error bars indicate s.e.m. #Po 0.05, ##P o0.01, compared with untreated cells, and *P o0.05, **Po 0.01, compared with the corresponding CDDP concentration alone. Scale bar, 10 μm. © 2014 Macmillan Publishers Limited
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4 phospho-neoepitope-specific antibody revealed by indirect immunofluorescence), this effect was exacerbated by combining CDDP with PN (Figures 2a and b). Accordingly, CDDP induced higher levels of CRT exposure when combined with PN (Figure 2c). Moreover, the combination of CDDP plus PN induced higher levels of ATP and HMGB1 release than did CDDP or PN alone (Figures 2d and e). This effect was detected in LLC cells, as well as on three additional cell lines, CT26, MCA205 and LUC lung adenocarcinoma cells (Supplementary Figure 3). Altogether, these results indicate that CDDP and PN induce stronger signs of ICD when combined than if applied separately. To investigate the capacity of CDDP plus PN to induce ICD in functional assays, LLC cells were cultured for 48 h in the presence of CDDP alone or in combination with PN, and the resulting mixture of dead and dying cells were injected subcutaneously into the left flank of syngenic C57Bl/6 mice, which were rechallenged 1 week later with live LLC cells (Figure 3a). As a positive control, we observed that LLC cells treated with the prototypic ICD inducer mitoxantrone, an anthracycline, were able to ‘vaccinate’ the mice and hence to induce a protective anticancer immune response in 70% of the animals. While CCDP-treated LLC cells only protected 1 out of 10 mice, the combination of CDDP plus PN conferred protection to half of the animals and strongly reduced tumor growth (Figures 3b and c). Altogether, these results support the notion that CDDP plus PN can induce ICD. Synergistic antineoplastic effects of CDDP plus PN in a model of orthotopic NSCLC in vivo To investigate the interaction between CDDP and PN in a realistic setting, we evaluated the antineoplastic efficacy of these agents in C57Bl/6 mice carrying orthotopic NSCLCs expressing the firefly luciferase (LUC), monitoring tumor growth by bioluminescence analysis and whole-body imaging (Figures 4a and b). The vast majority (~90%) of the mice treated with vehicle only (phosphatebuffered saline (PBS)) or PN only died within 17 days. CDDP alone reduced tumor growth, yet rarely achieved total cure (15%). In contrast, the combination of CDDP plus PN caused a more pronounced reduction in tumor growth and cured up to 45% of the mice, which remained tumor free (Figures 5a and c). Histologic analyses confirmed that an elevated percentage of mice receiving the combination regimen lacked detectable cancer lesions in the lung (Figure 5d). Altogether, these results underscore the possibility to improve the efficacy of CDDP by combining it with PN. Immune-dependent antineoplastic effects of CDDP plus PN in vivo In an attempt to weight the cell-autonomous versus immunemediated effects of the combination regimen (CDDP plus PN), we implanted LUC tumors in athymic nu/nu mice and treated them with CDDP and/or PN. These experiments were performed at the same time as those carried out on immunocompetent mice (Figures 4 and 5), yet are presented in separate figures (Figures 6 and 7a, c). To our surprise, both CDDP alone and CDDP plus PN failed to avoid tumor growth and to prolong overall survival in nu/ nu mice, although the combination regimen induced a nonsignificant trend toward a reduction in total tumor mass (Figures 6 and 7a, c). Hence, the observed therapeutic effects must rely on the presence of thymus-dependent T lymphocytes (which are deficient in nu/nu mice). Finally, we assessed the presence of a protective anticancer immune response in immunocompetent mice after successful chemotherapy. Mice that had been cured from their orthotopic LUC NSCLC became fully resistant against subcutaneous rechallenge with LUC cells, although they remained susceptible to tumor development after injection of the unrelated MCA205 fibrosarcoma (Figure 7d). These results reveal the induction of a tumor antigen-specific immune response that Oncogene (2014), 1 – 10
Figure 3. Impact of CDDP plus PN on the immunogenicity of cell death. Murine lung adenocarcinoma LLC cells that have been treated in vitro or not with CDDP (2.5 μM), CDDP (2.5 μM) plus PN (5 mM) for 48 h or mitoxantrone (MTX) (1 μM) for 4 h and then subcutaneously injected into C57Bl/6 mice (10 mice/group) were inoculated with live LLC cells 7 days later (a). Tumor growth (b) and tumor incidence (c) were measured. Tumor growth was compared using one-tailed Student’s t-test and tumor incidences (illustrated with Kaplan–Meier curves) were compared by one-tailed Barnard's test. #Po0.05, ##Po 0.01, compared with untreated cells, and *Po 0.05, **Po0.01, compared with CDDP-alone-treated cells.
lasted for several months after discontinuation of the successful chemotherapeutic regimen. DISCUSSION Cytotoxic and targeted anticancer therapeutics have been and are being developed with the idea that they should mediate their cytocidal or cytostatic effects by direct effects on tumor cells. Logically, it is hence possible to predict anticancer effects by testing antineoplastic agents on cultured tumor cells as well as © 2014 Macmillan Publishers Limited
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Figure 4. Effects of CDDP plus PN in a model of orthotopic NSCLC in vivo. Murine lung adenocarcinoma LUC cells were inoculated into the left lung of C57Bl/6 mice (20 mice/group) and then treated with PBS, PN (125 mg/kg) and CDDP (1.5 mg/kg) and alone or in combination with PN, three times per week the 3 first weeks, then two times per week and later stopped the treatment at day 42 (a). Tumor presence was analyzed by luciferase activity. Representative fluorescence photographs of 4 mice/group at different days of analysis are shown in (b). Upper left panels, PBS treated mice; upper right panels, mice treated with PN alone; down left panels, mice treated with CDDP alone; and down right panels, CDDP plus PN combination-treated mice.
cancers established in immunodeficient mice (as this is usually done when preclinical tests involve xenotransplanted human cancer cell lines or primary tumors).31,32 Following this intellectual scheme, we show here that cisplatin can be combined with PN to kill synergistically NSCLC cells in vitro and to treat successfully mice with NSCLC (in an orthotopic location) in vivo. To our surprise, however, the linear link between the in vitro and the in vivo data was only apparent. Indeed, the synergistic combination extended the life of a significant percentage of tumor-bearing mice, only if such tumors developed in an immunocompetent environment. Treatment of the same tumors with the same combined regimen was totally inefficient in an immunodeficient context, indicating that the therapeutic success was entirely dependent on the presence of an intact immune system (and, in particular, T lymphocytes, which are absent in nu/nu mice). These results underscore the contribution of anticancer immune © 2014 Macmillan Publishers Limited
responses to the therapeutic efficacy of the treatment with CDDP plus PN. It may appear counterintuitive that the combination treatment with CDDP plus PN effectively reduces the cellularity of NSCLC cultures in vitro, yet fail to mediate any major antineoplastic effects in vivo, on NSCLC established in nu/nu mice, at least at the doses that were used in this study. To observe antitumor efficacy, both agents had to be administered to immunocompetent mice, underscoring the cardinal importance of the anticancer immune response to the long-term effects of this particular combination regimen. Previous clinical studies using prototypic ICD inducers such as oxaliplatin or doxorubicin have failed to show superior efficacy in NSCLC if compared with or combined with CDDP.33–36 At this point, it is difficult to understand whether these negative data must be attributed to the advanced stage of NSCLC (when the immune system may be overwhelmed and hence intrinsically inefficient) or Oncogene (2014), 1 – 10
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Figure 5. Synergistic effects of CDDP plus PN in the orthotopic model in immunocompetent mice. C57Bl/6 mice were injected orthotopically into the left lung with LUC cells and then treated as described in Figure 4. Tumor incidence (a), tumor growth (b) and tumor survival (c) were monitorized. Representative pictures (d) of hematoxylin–eosin-safran (HES) stainings from normal versus tumor-bearing lungs treated or not with PN and CDDP alone or in combination with PN are shown. Tumor incidences (a) and tumor survival (c) (illustrated with Kaplan–Meier curves) curves were compared by the one-tailed Barnard's test. Tumor growth was compared using one-tailed Student’s t-test. ##Po 0.01, compared with untreated cells, and *P o0.05, **P o0.01, compared with CDDP alone. Scales bar, 500 μm (upper panels) and 50 μm (down panels).
suboptimal dosing and scheduling.37,38 A recent study performed on a mouse model of KRAS-induced NSCLC revealed that oxaliplatin was particularly efficient if combined with cyclophosphamide,39 and that this antineoplastic effect was entirely dependent on the immune system, as it disappeared in Rag2− / − mice that lack B and T lymphocytes.39 It will be interesting to evaluate this type of combination therapy in future clinical trials. At the mechanistic level, it appears that CDDP and PN do not only mediate a synergistic cytotoxic effect but that they also cooperate in the induction of ICD hallmarks. Thus, CDDP plus PN induced more CRT exposure, HMGB1 exodus and ATP release than did either of the two agents alone. With regard to CRT exposure, both agents interacted synergistically to elicit ER stress at the level of the phosphorylation of eIF2α, which constitutes a stringent requirement for CRT to be exposed.40 Indeed, CDDP and PN induced a higher level of eIF2α phosphorylation, if combined rather than used separately. The detailed mechanisms of this synergistic interaction are elusive. However, it is tempting to speculate that the capacity of CDDP plus PN to mediate a hyperadditive redox stress, as this is shown experimentally at the level of glutathione depletion,14 might facilitate the activation of the ER stress kinase PERK (official name: eukaryotic translation initiation factor 2-α kinase 3) that is required for chemotherapyinduced eIF2α phosphorylation, upstream of CRT exposure.41,42 Indeed, the antioxidants N-acetyl cysteine and glutathione ethyl ester can prevent cell killing by CDDP plus PN,14,41 as they prevent the induction of CRT exposure by classical ICD inducers.40 The fact that CDDP plus PN elicit the mitochondrial (intrinsic) pathway of apoptosis is underscored by the observations that cell death is Oncogene (2014), 1 – 10
consistently accompanied by an early decrease of the mitochondrial transmembrane potential (ΔΨm), modulated by knockdown of proteins of the Bcl-2 family, and delayed by the addition of caspase inhibitors.14 Altogether, it appears that CDDP plus PN are efficient in inducing anticancer immune responses in vivo, both in vaccination assays and in the context of chemotherapeutic treatments of established cancers. As a proof in favor of this hypothesis, mice that have been cured from NSCLC by means of the combination regimen exhibit a specific, long-term protection against rechallenge with cancer cells. In synthesis, our data delineate a strategy to improve the efficacy of CDDP-based chemotherapy by combining it with the chemosensitizer PN. This strategy may increase the immunogenic properties of dying NSCLC cells, thereby triggering an efficient and therapeutically relevant antitumor immune response. MATERIALS AND METHODS Unless otherwise indicated, media and supplements for cell culture were purchased from Gibco-Invitrogen (Carlsbad, CA, USA), plasticware from Corning BV Life Sciences (Schiphol-Rijk, The Netherlands) and chemicals from Sigma-Aldrich (St Louis, MO, USA).
Antibodies Rabbit polyclonal antibody against CRT (ab2907) was purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibody against anti-phosphoeIF2α (Ser51) (3597) was from Cell Signaling Technology (Danvers, MA, USA). © 2014 Macmillan Publishers Limited
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Figure 6. Absence of effects with CDDP plus PN treatment in the orthotopic model in immunodeficient mice. Murine lung adenocarcinoma LUC cells were inoculated into the left lung of nu/nu mice (10 mice/group) as described in Figure 4. Tumor presence was analyzed by luciferase activity. Representative fluorescence photographs of 4 mice/group at different days of analysis are shown. Upper left panels, PBStreated mice; upper right panels, mice treated with PN alone; down left panels, mice treated with CDDP alone; and down right panels, CDDP plus PN combination-treated mice.
Cell lines and culture conditions All cell lines were cultured at 37 °C under 5% of CO2 in medium containing 10% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin. Cell type-specific culture conditions include: Dulbecco’s modified Eagle’s medium supplemented as above plus 1 mM sodium pyruvate for murine LLC cells; RPMI 1640 medium supplemented as above plus 1 mM sodium pyruvate and 1 mM HEPES buffer for murine colon carcinoma CT26, murine fibrosarcoma MCA205 cells and non-small-cell lung carcinoma TC-1 expressing the firefly LUC.43
(ΔΨm), plus 1 μg/ml PI, for the identification of plasma membrane breakdown.44 Surface exposure of CRT was detected by immunofluorescence staining as described previously41 after 48 h of treatment. Samples were then analyzed by means of a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA) or FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). Statistical analyses were carried out by using the CellQuest software (BD Biosciences), upon gating on the events characterized by normal forwardand side-scatter values.
Immunofluorescence microscopy and automated image analysis Treatments Cells were seeded in 6-, 12-, 96-well plates, 25, 75 or 175 cm2 flasks and allowed to attach for at least 24 h before experiments. Cells were challenged with the following pharmacologic agents: CDDP (1.25, 2.5, 5, 10, 20, 40 or 80 μM) and PN (⩾98% purity; 0.6, 1.25, 2.5, 5 and 10 mM) and 1 μM mitoxantrone and 1 μM tunicamycin as positive controls when corresponding.
Cytofluorometry Cell death was measured after 48 h of treatment; unfixed cells were co-stained with 40 nM DiOC6(3) (from Molecular Probes-Invitrogen, Eugene, OR, USA), which quantifies the mitochondrial transmembrane potential © 2014 Macmillan Publishers Limited
Cells were treated for 24 h and then washed with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 10 min and finally rinsed three times with PBS. Nonspecific binding sites were blocked with blocking buffer for 30 min and cells were incubated with primary anti-phospho-eIF2α (Ser51) antibody for 1 h. Subsequently, cells were washed three times with PBS and incubated for 30 min in AlexaFluor 488-conjugated secondary antibodies (Molecular ProbesInvitrogen). Hoechst 33342 (10 μM) was used for nuclear counterstaining. Fluorescence microscopic assessments were performed by automated image analysis. Micrographs have been acquired using an IXM XL (Molecular Devices, Sunnyvale, CA, USA) equipped with a × 20 PlanApo objective (Nikon, Tokyo, Japan) and a laser-based autofocus. Nine view fields within each well have been sequentially imaged using 4′,6Oncogene (2014), 1 – 10
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Figure 7. Immune-dependent antineoplastic effects of CDDP plus PN in vivo. Immunodeficient nu/nu mice were injected orthotopically in the left lung with LUC cells and then treated as described in Figure 4. Tumor incidence (a), tumor growth (b) and tumor survival (c) were measured. Anticancer immune response was observed in immunocompetent mice after successful chemotherapy (d). C57Bl/6 control (upper panels, 5 mice/group) and mice previously cured from their orthotopic LUC tumors after chemotherapy with CDDP plus PN, as shown in Figure 5 (lower panels, 3 mice/group), were subjected to subcutaneous injection with unrelated MCA 205 cells (left panels) or LUC cells (right panels) at the same time. Each line represents the growth of a tumor in one individual mouse.
diamidino-2-phenylindole (DAPI) and GFP standard filter sets at a binning of 1. The images obtained were segmented into nuclear and cytoplasmic regions by means of the MetaXpress image analysis, and the cytoplasmic fluorescence intensity of GFP was evaluated, while debris were omitted by size exclusion. Mean fluorescence intensities were calculated and depicted as heat map by means of the freely available statistics program ‘R’ (http:// www.r-project.org).
HBMG1 release was measured using the cell supernatants collected after 48 h of treatment and cleared from dying tumor cells by centrifugation (800 g, 5 min), and then immediately analyzed for HMGB1 abundance. HMGB1 quantification was performed by means of an Enzyme-Linked Immunosorbent Assay (ELISA) Kit (HMBG1 ELISA Kit II; Gentaur Europe, Kampenhout, Belgium), according to the manufacturer's instructions.
Mice Colorimetric assays In vitro assessments of the synergistics effects between CDDP and PN after 48 h of treatment were performed by means of a colorimetric assay based on the reduction of the colorless tetrazolium salt WST-1 (from Roche Diagnostics GmbH, Mannheim, Germany) to formazan (which exhibit an absorbance peak around 450 nm), following conventional procedures.14 Absorbance at 450 nm was measured on a FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany) and—following background subtraction—WST-1 conversion data were normalized to the readings of untreated cells included in the same test plate. Extracellular ATP levels were measured after 24 h of treatment by the luciferin-based ENLITEN ATP Assay Kit (Promega Corporation, Madison, WI, USA), in excess of luciferin and LUC, as indicated by the manufacturer. ATP-driven chemiluminescence was recorded on a FLUOstar multiwell plate luminometer (BMG Labtech, Ortenberg, Germany) as described previously.45 Oncogene (2014), 1 – 10
Mice were maintained in specific pathogen-free conditions, and experiments followed the Federation of European Laboratory Animal Science Association (FELASA). Animal experiments were in compliance with the EU Directive 63/2010 and protocols (protocols 2012_138 and 2012_156) were approved by the Ethical Committee of the Gustave Roussy Campus Cancer (GRCC, Villejuif, France) (CEEA IRCIV/IGR no. 26, registered at the French Ministry of Research). Wild-type C57Bl/6 mice were obtained from Harlan France (Gannat, France) and nu/nu mice from the GRCC animal facility.
Antitumor vaccination A total of 3 × 106 LLC tumor cells were left untreated or treated with CDDP (2.5 μM) alone or combined with PN (5 mM) for 48 h, and then were inoculated subcutaneously in 200 μl PBS into the lower flank of C57Bl/6 mice. As a positive control, the cells were treated with mitoxantrone (1 μM) for 7 h and then cultured in the fresh medium and inoculated after 24 h as © 2014 Macmillan Publishers Limited
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9 described above. A total of 5 × 105 untreated cells were inoculated into the contralateral flank 1 week later. Tumor growth was routinely monitored with a common caliper regardless of the presence or absence of tumor after the first injection. Animals bearing tumors that exceeded 20–25% body mass were killed. C57Bl/6 mice that had previously been submitted to lung orthotopic model but were cured from their developed tumors were reinjected with 5 × 105 LUC cells to establish the possible vaccination in this model. The unrelated MCA205 fibrosarcoma cell line was used as a positive control.
Lung orthotopic model A total of 4× 105 LUC tumor cells were injected into the left lung by intercostal injection at the median axillary line of C57Bl/6 or nu/nu mice as described previously.46 Mice were treated with PBS, PN (125 mg/kg) and CDDP (1.5 mg/kg) and alone or in combination with PN, three times per week in the 3 first weeks, then two times per week and later stopped the treatment at day 42. Tumor growth was analyzed by in vivo photonic imaging of tumor cells luciferase activity. Mice were injected intraperitoneally once per week (starting at day 3 after tumor cell inoculation) with the substrate of luciferase, D-luciferin potassium salt (Promega), at a dose of 10 μm/kg. In vivo imaging was performed with Xenogen IVIS 50 bioluminiscence imaging system (Caliper Life Sciences Inc., Hopkinton, MA, USA). Photons were acquired after 5 min of luciferin inoculation for maximum of 3 min, and then 1 min or 30 s if saturation occurred. Mice showing photon saturation levels at 30 s were killed.
Lung histology and hematoxylin, eosin and safran staining Recovered lungs were fixed with 4% paraformaldehyde for 4 h and then embedded into paraffin. Sections of 4 μm paraffin were fixed and stained with hematoxylin, eosin and safran according to standard protocols. Representative pictures were acquired using ZEISS Primo Star AxioCAM ERc 5s microscope and AxioVision LE software.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We are indepted to Dr Gonin (Gustave Roussy) for help in mouse experiments. CP is supported by the Deutsche Forschungsgemeinschaft (DFG) PF809/1-1. LS is supported by the Fondation Tourre, the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE) and the SIRIC Cancer Research and Personalized Medicine (CARPEM). GK is supported by the Ligue contre le Cancer (équipe labelisée); Agence National de la Recherche (ANR); 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); and the Paris Alliance of Cancer Research Institutes (PACRI).
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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
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