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Science 334 (6062): 1573-1577

Copyright © 2011 by the American Association for the Advancement of Science

Autophagy-Dependent Anticancer Immune Responses Induced by Chemotherapeutic Agents in Mice

Mickaël Michaud1,2,3,*, Isabelle Martins1,2,3,*, Abdul Qader Sukkurwala1,2,3, Sandy Adjemian1,2,3, Yuting Ma2,3,4,5, Patrizia Pellegatti6, Shensi Shen1,2,3, Oliver Kepp1,2,3, Marie Scoazec2,7, Grégoire Mignot8,9, Santiago Rello-Varona1,2,3, Maximilien Tailler1,2,3, Laurie Menger1,2,3, Erika Vacchelli1,2,3, Lorenzo Galluzzi1,2,3, François Ghiringhelli8,9, Francesco di Virgilio6, Laurence Zitvogel2,3,4,5,{dagger}, and Guido Kroemer1,2,9,10,11,12,{dagger}

1 INSERM, U848, Villejuif, France.
2 Institut Gustave Roussy, Villejuif, France.
3 Université Paris Sud, Faculté de Médecine Paris XI, Le Kremlin Bicêtre, France.
4 INSERM, U1015, Villejuif, France.
5 Center of Clinical Investigations in Biotherapies of Cancer (CICBT) 507, Villejuif, France.
6 Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Ferrara, Italy.
7 Metabolomics Platform, Institut Gustave Roussy, Villejuif, France.
8 Department of Medical Oncology, Georges François Leclerc Center, Dijon, France.
9 INSERM Avenir Team INSERM, CRI-866 University of Burgundy, Dijon, France.
10 Centre de Recherche des Cordeliers, Paris, France.
11 Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France.
12 Université Paris Descartes, Paris 5, Paris, France.

Figure 1
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Fig. 1. Requirement for autophagy in chemotherapy-induced ATP secretion. (A to C) Impact of autophagy on ATP release. CT26 cells stably transfected with a scrambled (SCR) shRNA or an Atg5-specific shRNA (Atg5KD) were cultured with MTX or left untreated (PBS, phosphate-buffered saline), and extracellular ATP was determined by mass spectrometry (A). Alternatively, ATP release induced by MTX or oxaliplatin (OXA) in Atg5KD and Atg7KD CT26 cells (B) or Atg5–/– and Atg7–/– MEFs (C) was measured by enzymatic methods, and autophagy, cell death, CRT exposure, and HMGB1 release were determined. Results are reported as means ± SEM of triplicates, and asterisks indicate significant (P < 0.05, unpaired Student’s t test) differences as compared to autophagy-competent cells cultured in similar conditions. (D) Induction of autophagy by chemotherapy in vivo. Subcutaneous GFP-LC3–expressing CT26-derived tumors were treated by intraperitoneal chemotherapy, and the percentage of tumor cells with cytoplasmic GFP-LC3 dots was determined 48 hours later (means of 10 determinations ± SEM). Asterisks indicate significant (P < 0.01, unpaired Student’s t test) autophagy induction as compared to control conditions (PBS). (E) Extracellular ATP concentration before and after chemotherapy in vivo. Mice bearing palpable luciferase-expressing CT26-derived tumors that had been engineered to express a control shRNA (SCR) or shRNAs targeting Atg5 or Atg7 were treated with PBS or MTX, and D-luciferin–dependent chemoluminescence was measured before treatment (0 hour) or 48 hours later. Results are reported as means ± SEM of triplicates. Asterisks indicate significant (*P < 0.01, unpaired Student’s t test) inhibition of ATP release as compared to autophagy-competent controls.


Figure 2
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Fig. 2. Impact of autophagy on the immunogenicity of cell death. (A) Requirement for autophagy in immunogenic cell death. Murine colon carcinoma CT26 cells transfected with the indicated siRNAs were treated with MTX in vitro and then subcutaneously injected into BALB/c mice (20 mice per group) that were inoculated with living CT26 cells 7 days later. (B) Requirement for autophagy in T cell priming. MTX-treated autophagy-competent (SCR) or deficient (Atg5KD or Atg7KD) CT26 cells were injected into the footpad, followed by restimulation of popliteal lymph node cells by CT26 lysates in vitro and quantification of IFN-{gamma} secretion. Results are reported as means ± SEM of triplicates. (C) Implication of ATP and purinergic receptors in the reduced immunogenicity of autophagy-deficient cells. Murine fibrosarcoma MCA205 cells were transfected with the indicated siRNAs and then treated with MTX, followed by subcutaneous injection in the presence or absence of the ecto-ATPase inhibitor ARL67156 (ARL) and/or purinergic receptor antagonists (oxidized ATP or suramin). One week later, mice were rechallenged with live MCA205 cells, and the absence of tumor growth was scored 60 days later as the indication of an anticancer immune response. (D and E) Recombinant IL-1β restores the defective immunogenicity of autophagy-deficient cells succumbing to chemotherapy. CT26 (D) or MCA205 cells (E) were transfected with the indicated siRNAs, cultured with MTX, and subcutaneously injected, either alone or in the presence of recombinant IL-1β (rIL-1β), into syngenic mice. Seven days later, mice were subcutaneously inoculated with the same tumor cell type. Tumor incidence was monitored for 60 days. Kaplan-Meier curves were statistically evaluated by the logrank test (*P < 0.05, as compared to the same siRNA-transfected MTX-treated cells without rIL-1β). [*P < 0.05, unpaired Student’s t test in (B); logrank test in (A) and (D); {chi}2 test in (C) and (E) to indicate significant vaccination; n = 10 unless otherwise indicated.]


Figure 3
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Fig. 3. Failure of autophagy-deficient tumor cells to induce a therapeutic immune response. (A and B). Reduced T cell–dependent therapeutic response of autophagy-incompetent tumors. Palpable CT26-derived tumors stably transfected with the indicated constucts growing on immunocompetent BALB/c mice (A) or immunodeficient nu/nu hosts (B) (n = 10 per group) were treated with one intraperitoneal injection of MTX or vehicle (PBS) (day 0), and tumor growth was monitored. (C) Failure of autophagy-deficient tumors to recruit DCs upon chemotherapy. Control (SCR) or autophagy-deficient (Atg5KD) tumors were implanted on transgenic mice expressing GFP under the control of the CD11c promoter. Once tumors became palpable, mice were treated with MTX or vehicle (PBS), and 48 hours later tumors were subjected to immunofluorescence detection of apoptotic cells (that stain positively for active caspase-3, Casp3a) and GFP-positive cells. (D) Failure of autophagy-deficient tumors to recruit T lymphocytes. Control (SCR) or autophagy-deficient (Atg5KD) CT26-derived tumors were recovered 7 days after MTX or PBS injection, and the frequency of cells with the indicated immunophenotypes was determined by cytofluorometry. Results are reported as means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t test; ns, not significant).


Figure 4
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Fig. 4. Restoration of therapeutic immune responses by injection of ecto-ATPase inhibitors into autophagy-deficient tumors. (A) Restoration of ATP levels. Extracellular ATP was measured in Atg5-deficient (Atg5KD) and Atg7-deficient (Atg7KD) CT26 cell-derived tumors (n = 3 per group) engineered to express luciferase, 48 hours after the intraperitoneal injection of MTX in combination with the intratumoral injection of PBS or of the ecto-ATPase inhibitor ARL67156 (ARL). (B to E) Restoration of DC and lymphocyte recruitment by ARL. Atg5KD tumors were treated with MTX as they became palpable and injected with either PBS or ARL. Then, the percentage of infiltrating dendritic or T cells among all cells was determined on day 9, as in Fig. 3, C and D. Results are means ± SEM (n = 10 mice per group). (F) ARL-mediated restoration of T cell priming by Atg5-deficient tumor cells, determined as in Fig. 2B (n = 5 per group). (G and H) Effect of ecto-ATPase inhibition on immune-dependent chemotherapy responses in vivo. Autophagy-competent or -deficient CT26-derived tumors (n = 10 per group) growing on immunocompetent BALB/c (G) or athymic nu/nu mice (H) were treated with MTX or PBS (day 0) alone or in combination with intratumoral ARL (on days 0 and 3), followed by tumor growth monitoring. Results are reported as means ± SEM (*P < 0.05, unpaired Student’s t test).


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