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Science 337 (6102): 1678-1684

Copyright © 2012 by the American Association for the Advancement of Science

An Immunosurveillance Mechanism Controls Cancer Cell Ploidy

Laura Senovilla1,2,3, Ilio Vitale1,2,3, Isabelle Martins1,2,3, Maximilien Tailler1,2,3, Claire Pailleret1,2,3, Mickaël Michaud1,2,3, Lorenzo Galluzzi1,2,3, Sandy Adjemian1,2,3, Oliver Kepp1,2,3, Mireia Niso-Santano1,2,3, Shensi Shen1,2,3, Guillermo Mariño1,2,3, Alfredo Criollo1,2,3, Alice Boilève1,2,3, Bastien Job2,4,5, Sylvain Ladoire6,7, François Ghiringhelli6,7, Antonella Sistigu2,3,8, Takahiro Yamazaki2,3,8, Santiago Rello-Varona1,2,3, Clara Locher2,3,8, Vichnou Poirier-Colame2,3,8, Monique Talbot2, Alexander Valent9, Francesco Berardinelli10, Antonio Antoccia10, Fabiola Ciccosanti11, Gian Maria Fimia11, Mauro Piacentini11,12, Antonio Fueyo13, Nicole L. Messina14,15, Ming Li14, Christopher J. Chan14,16, Verena Sigl17, Guillaume Pourcher3,18,19, Christoph Ruckenstuhl20, Didac Carmona-Gutierrez20, Vladimir Lazar2,4,5, Josef M. Penninger17, Frank Madeo20, Carlos López-Otín21, Mark J. Smyth14,16, Laurence Zitvogel2,3,8,22,*, Maria Castedo1,2,3,*, and Guido Kroemer1,23,24,25,26,*

1 INSERM, U848, Villejuif, France.
2 Institut Gustave Roussy, Villejuif, France.
3 Université Paris Sud/Paris 11, Faculté de Médecine, Le Kremlin Bicêtre, France.
4 Unité de Génomique Fonctionnelle et Bioinformatique, Villejuif, France.
5 Genomique Platform, Villejuif, France.
6 Department of Medical Oncology, Georges François Leclerc Center, Dijon, France.
7 Institut National de la Santé et de la Recherche Médicale, Avenir Team INSERM, CRI-866 University of Burgundy, Dijon, France.
8 INSERM, U1015, Villejuif, France.
9 Pathologie Moléculaire, Departement De Biologie et Pathologie Médicales, Villejuif, France.
10 Dipartimento Di Biologia, Università Roma Tre, Rome, Italy.
11 National Institute for Infectious Diseases L. Spallanzani, Rome, Italy.
12 Department of Biology, University of Rome "Tor Vergata," Rome, Italy.
13 Departamento de Biología Funcional, Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain.
14 Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
15 Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia.
16 Department of Immunology, Monash University, Prahran, Victoria, Australia.
17 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria.
18 Department of Minimal Invasive Surgery, Antoine Béclère Hospital, AP-HP, Clamart, France.
19 INSERM, U972, Le Kremlin Bicêtre, France.
20 Institute for Molecular Bioscience, Graz, Austria.
21 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, IUOPA, Universidad de Oviedo, Oviedo, Spain.
22 Center of Clinical Investigations in Biotherapies of Cancer (CICBT) 507, Villejuif, France.
23 Metabolomics Platform, Institut Gustave Roussy, Villejuif, France.
24 Centre de Recherche des Cordeliers, Paris, France.
25 Pôle de Biologie, Hôpital Européen Georges Pompidou, Assistance Publique–Hôpitaux de Paris (AP-HP), Paris, France.
26 Université Paris Descartes/Paris 5, Sorbonne Paris Cité, Paris, France.


Figure 1
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Fig. 1. Functional linkage of hyperploidization, CRT exposure, and immunosurveillance. (A and B) CT26 cells were treated for 48 hours with cytochalasin D (CytD), nocodazole (Noco), epothilone B (EpoB), or taxotere (TXT) and then either subjected to the cytofluorometric detection of CRT exposure (A) or analyzed by immunoblotting for eIF2α phosphorylation (B). R.U. indicates relative units. (C) BALB/c mice were injected subcutaneously with wild-type (WT) CT26 cells that had been treated or not with 100 nM Noco for 48 hours or with CT26 cells stably expressing a CRT-specific short hairpin RNA (shRNA) that had been preincubated or not with recombinant CRT (recCRT) and treated with 100 nM Noco for 48 hours. In all cases, mice were injected 1 week later with live WT CT26 cells, and tumor incidence was monitored. These experiments were done three times (15 to 20 mice per group). (D) Parental (P) and hyperploid (H) CT26 cells were stained to measure CRT exposure on the cell surface. (E) Constitutive eIF2α phosphorylation in H CT26 clones (two representative clones of at least n = 5), as determined by immunoblotting. Clonogenicity of hyperploid mouse embryonic fibroblasts (MEFs) (F) or CT26 cells (G) upon disruption of the CRT exposure pathway. The cloning efficiency of nocodazole-treated, FACS-purified WT cells or cells transfected with a scrambled control with 8n DNA content was considered as 100%. In vitro data were compared with one-tailed Student’s t tests, tumor incidence with the log rank test. *P < 0.05; **P < 0.01; n.s., nonsignificant, as compared to untreated P CT26 cells [(A), (B), (D), and (E)], untreated (F), or shCo-transfected H (G) CT26 cells, or mice challenged with phosphate-buffered saline (PBS) only (C). (H to M) Evidence for an immune response to hyperploid cancer cells. (H) P or H CT26 cell clones were injected into Rag {gamma} or BALB/c mice. (I) Tumor growth of P or H CT26 cells treated with a shRNA (shCRT) to CRT was monitored in Rag {gamma} or BALB/c mice. (J) Growth of P CT26 cells in naïve mice and in tumor-free BALB/c mice previously (2 months before) inoculated with H CT26 cells [as in (H)]. (K) H CT26 cells were inoculated into BALB/c mice that were depleted of CD4+ or CD8+ T lymphocytes by injection of suitable antibodies. (L and M) Contribution of IFN-{gamma} and Ifnar1 to the immunosurveillance of hyperploid cells. H LLC (L) and MCA205 (M) cells were inoculated into wild-type [(L) and (M)], Ifng–/– (L), or Ifnar1–/– (M) C57Bl/6 mice. Tumor growth curves [(H) to (M), top graphs] were analyzed with one-tailed Student’s t test, whereas tumor incidence [(C) and (H) to (M), bottom graphs, illustrated with Kaplan-Meier curves] was compared by log rank test. Error bars indicate SEM. *P < 0.05, **P < 0.01, as compared with P cells (H), PBS-challenged mice [(J) and (K)], or immunocompetent C57Bl/6 mice (M).

 

Figure 2
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Fig. 2. Reduced DNA content and diminished CRT exposure in initially hyperploid cancers grown on immunocompetent mice. (A) Reduction in the nuclear diameter of immunoselected hyperploid cancer cells, as determined by hematoxylin and eosin staining of CT26 tumors growing in Rag {gamma} and BALB/c mice. (B) Chromosome numbers of hyperploid CT26 cells immunoselected (in BALB/c mice) or grown in vivo without immunoselection (in Rag {gamma} mice). (C) DNA loss in immunoselected hyperploids. CT26 hyperploids were immunoselected or grown without immunoselection [as in (A) and (B)], and the DNA content of isolated cancer cells was determined by cytofluorometry. The mean ploidy of the G0/G1 peak was determined for multiple tumors. (D to G) Immune response effect on CRT exposure. Hyperploid clones were either cultured in vitro (1), grown in vivo without immunoselection (in Rag {gamma} mice) (2), or sequentially transferred into immunocompetent mice for immunoselection (3 to 5) (D), recovered and then subjected to the determination of ploidy (E), CRT exposure (F), or the phosphorylation of PERK and eIF2α, normalized to controls (G). In (G), representative results and quantitative data (obtained by densitometry on multiple tumors) are shown. One-tailed Student’s t test was used for statistical comparisons. Error bars indicate SEM. *P < 0.05, **P < 0.01, as compared to tumors untreated Rag {gamma} mice (A), parental (P) CT26 cells cultured in vitro [(B), (C), (E), (F)], or hyperploid CT26 cells cultured in vitro (G).

 

Figure 3
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Fig. 3. Immunoselection against hyperploidy and eIF2α phosphorylation. (A to D) Nuclear diameter and eIF2α phosphorylation in methylcholanthrene-induced fibrosarcomas (A and B), Eμ-myc–driven B cell lymphomas (C), or medroxyprogesterone acetate–induced mammary carcinomas (D) from immunodeficient (Rag {gamma}, Stat1–/– or Dnam1–/–) versus immunocompetent mice. (E) Immunoselection in human mammary adenocarcinomas treated with neo-adjuvant chemotherapy. Tumor specimens obtained by surgery from 18 responders and 42 nonresponders before (pre) and after (post) treatment were stained to determine nuclear diameter, ratio of the number of tumor-infiltrating CD8+ to FOXP3+ lymphocytes, and eIF2α phosphorylation. One-tailed Student’s t test was used for statistical comparisons. Error bars indicate SEM. *P < 0.05, **P < 0.01, compared to tumor cells with the same nuclear diameter obtained from immunocompetent mice (A to D) or pretreatment (E). #P < 0.05, ##P < 0.01, compared to cells from immunodeficient mice with nuclear diameter < 9 μm (A), or to responders (E). {dagger}P < 0.05, compared to cells with a nuclear diameter < 10 μm from nonresponders (E). A.U., arbitrary units.

 

Figure 4
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Fig. 4. Ecto-CRT underlies the immunogenicity of hyperploid cancer cells. (A to D) Immunosurveillance and immunoselection mechanisms activated by membrane-bound CRT on CT26 cells. Parental (P) CT26 cells were transduced with a construct encoding mCRT or vector only and subjected to immunostaining for the quantification of externalized CRT or MHC class I molecules (A). Alternatively, mCRT-expressing P cells were injected into Rag {gamma} or BALB/c mice, followed by monitoring of tumor growth and incidence (B). Ecto-CRT was measured by immunostaining on recovered cancer cells (C). (D) Tumor growth and incidence of mCRT-expressing P cells sequentially injected into Rag {gamma} (1) or BALB/c (2 and 3) mice. (E) Restoration of immunosurveillance on immunoselected (IS) hyperploid (H) cells by mCRT. H clones were injected into BALB/c mice for immunoselection (1). Then, tumor cells were recovered, transduced with a mCRT-encoding construct (3) or the empty vector (2), analyzed for CRT exposure, and re-injected into BALB/c mice, followed by monitoring of tumor growth and incidence. Experiments were performed three times, yielding comparable results. One-tailed Student’s t tests were used for statistical comparisons of in vitro data. Tumor growth and incidence (the latter being illustrated with Kaplan-Meier curves) were compared by one-tailed Student’s t and log rank tests, respectively. Error bars indicate SEM. *P < 0.05; **P < 0.01; n.s., nonsignificant compared with P CT26 cells [(A) to (C)], mCRT-expressing cells (D), or H cells (E) growing for the first time in BALB/c mice. ##P < 0.01, compared with mCRT-expressing CT26 cells grown in vitro (C) or P CT26 cells (E). {dagger}{dagger}P < 0.01, compared with mCRT-expressing CT26 cells grown in Rag {gamma} mice (C).

 


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