VI. Immune-dependent antineoplastic effects of CDDP plus PN in vivo Immune-dependent antineoplastic effects of cisplatin plus pyridoxine in non-small-cell lung cancer Aranda F1, Bloy N2, Pesquet J1, Petit B3, Chaba K4, Sauvat A1, Kepp O1, Khadra N1, Enot D1, Pfirschke C5,Pittet M5, Zitvogel L6, Kroemer G7, Senovilla L8 11] Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, Paris, France [2] Université Paris Descartes, Sorbonne Paris Cité, Paris, France [3] Metabolomics and Cell Biology Platforms, Villejuif, France. 21] Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, Paris, France [2] Université Paris Descartes, Sorbonne Paris Cité, Paris, France [3] Metabolomics and Cell Biology Platforms, Villejuif, France [4] Université Paris Sud, Villejuif, France. 3INSERM U1030, Villejuif, France. 41] Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, Paris, France [2] Université Paris Descartes, Sorbonne Paris Cité, Paris, France. 5Center for Systems Biology, Massachusetts General Hospital/Harvard Medical School, Pittet Lab, Boston, MA, USA. 61] Université Paris Sud, Villejuif, France [2] INSERM U1015, Villejuif, France [3] Centre d'Investigation Clinique Biothérapie CICBT 507, Villejuif, France. 71] Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, Paris, France [2] Université Paris Descartes, Sorbonne Paris Cité, Paris, France [3] Metabolomics and Cell Biology Platforms, Villejuif, France [4] Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France. 81] Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, INSERM U1138, Paris, France [2] Metabolomics and Cell Biology Platforms, Villejuif, France [3] INSERM U1015, Villejuif, France. cis-Diamminedichloroplatinum(II) (CDDP), which is mostly referred to as cisplatin, is a widely used antineoplastic. The efficacy of CDDP can be improved by combining it with the vitamin B6 precursor pyridoxine (PN). This combination, CDDP plus PN, increases the immunogenicity of non-small cell lung carcinoma (NSCLC) cells succumbing to CDDP. Accordingly, PN promoted the antineoplastic activity of CDDP in NSCLC-bearing mice only in the presence of an intact immune system. These findings may have implications for the development of novel strategies to circumvent CDDP resistance. Altogether, these results suggest that the combined regimen of cisplatin plus pyridoxine mediates immune-dependent antineoplastic effects against NSCLC. RESULTS IV. Lung orthotopic model & Treatment Protocol I. Synergistic citotoxic effects of CDDP plus PN in vitro IV. 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 b V. Synergistic effects of CDDP plus PN in the orthotopic model in immunocompetent mice a b c I. LLC cells were cultured in control conditions or exposed for 48 h to PN and CDDP alone or combined with PN and then stained with the vital dye PI and the mitocondrial transmembrane potential (ΔΨm)-sensitive fluorochrome DiOC6(3), to measure apoptosis-associated parameters (a). a. Representative dot plots as obtain at basal condition or upon incubation with PN and CDDP alone or in combination with PN. Numbers indicate the percentade of cells found in each quadrant. b. Colorimetric assessment of residual proliferation by means of a WST-1 conversion-based assay. Color-coded surfaces illustrate residual WST-1-converting activity. II. CDDP combined with PN induces synergistic ICD characteristics V. C57Bl/6 mice were injected orthotopically into the left lung with LUC cells and then treated as described in Figure IV. Tumor incidence (a), tumor growth (b) and tumor survival (c) were monitorized. Tumor incides and tumor survival (illustrated Kaplan-Meier curves) curves were compared by the one-tailed Barnard´s test. Tumor growth was compared using one-tailed Student´s t-test. #p<0.01, compared with untreated cells, and *p<0.05, **p<0.01, compared with CDDP alone. a VI. Immune-dependent antineoplastic effects of CDDP plus PN in vivo II. a) Effects of CDDP alone or in combination with PN on ER stress. Twenty-four hours after exposure to the drug combinations, LLC cells were fixed, permeabilized and then were stained for the quantification of phosphorylated eIF2α (as mean fluorescence). Effects of CDDP alone or in combination with PN on ICD markers: b) CRT exposure, c) HMGB1 release and d) ATP release were determined. Cells trated with mitoxantrone (MTX) for 4h were used as a positive control. Error bars indicate SEM #p<0.05, ##p<0.01, compared with untreated cells, and *p<0.05, **p<0.01, compared with the corresponding CDDP concentration alone. a b c b c d VI. Immunodeficient nu/nu mice were injected orthotopically into the left lung with LUC cells and then treated as described in Figure IV. Tumor incidence (a), tumor growth (b) and tumor survival (c) were monitorized. CONCLUSION III. Impact of CDDP plus PN on the immunogenicity of cells death Impact of vitamin B6 on the immunogenicity of cisplatin-induced cell death a) In most circumstances, CDDP employed as a standalone therapeutic intervention kills neoplastic cells in an immunologically silent a III. a) Murine lung adenocarcinoma LLC cells that have been treated in vitro or not with CDDP, CDDP plus PN for 48 h or mitoxantrone (MTX) for 4 h and then subcutaneosly injected into C57Bl/6 mice (10 mice/group) were inoculated with live LLC cells 7 days later. b) Tumor grwth and c) tumor incidencewere measured. Tumor growth was compared using one-tailed Student´s t-test and tumor indicences (illustrated with Kaplan-Meier curves) were compared by one-tailed Barnard´s test. #p<0.05, ##p<0.01, compared with untreated cells, and *p<0.05, **p<0.01, compared with CDDP-alone-treated cells. fashion. In other words, cancer cells dying upon exposure to CCDP fail to elicit a therapeutically relevant adaptive immune response against dead cell-associated antigens. b) In the presence of PN, malignant cells succumb to CDDP while emitting a spatiotemporally defined combination of damage associated molecular patterns with potent immunostimulatory effects. Thus, PN appears to efficiently convert the immunologically silent demise of cancer cells exposed to CDDP into bona fide immunogenic cell death (ICD). b c 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