Morphometric analysis of immunoselection against hyperploid cancer cells

// Norma Bloy 1, 2, 3, 4, * , Allan Sauvat 1, 3, 4, 5, * , Kariman Chaba 1, 3, 4 , Aitziber Buqué 1, 3, 4 , Juliette Humeau 1, 2, 3, 4 , José Manuel Bravo-San Pedro 1, 3, 4 , Jack Bui 6 , Oliver Kepp 1, 3, 4, 5 , Guido Kroemer 1, 4, 5, 7, 8, # Laura Senovilla 1, 3, 4, # 1 Equipe 11 Labellisée Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM U 1138, 75006 Paris, France 2 Université Paris Sud, Faculté de Médecine, 94270 Kremlin Bicêtre, France 3 Université Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France 4 Université Pierre et Marie Curie, 75006 Paris, France 5 Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, 94805 Villejuif, France 6 Department of Pathology, University of California San Diego, CA 92093, USA 7 Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, 75015 Paris, France 8 Karolinska Institute, Department of Women’s and Children’s Health, Karolinska University Hospital, 17176 Stockholm, Sweden * These authors have contributed equally to this work # share senior co-authorship Correspondence to: Laura Senovilla, e-mail: laurasenovilla@hotmail.com Guido Kroemer, e-mail: kroemer@orange.fr Keywords: morphometric analysis, ploidy, ER stress, immunoselection Received: August 08, 2015      Accepted: August 29, 2015      Published: October 15, 2015 ABSTRACT An at least transient increase of ploidy, usually by whole genome duplication, is a frequent event in oncogenesis, explaining the cytogenetic features of at least 40% of solid cancers. Here, we show that fibrosarcomas induced by the carcinogen methylcholanthrene (MCA) are distinct with respect to their ploidy status when they arise in immunocompetent wild type versus severely immunodeficient Rag2 −/− γc −/− mice. MCA-induced fibrosarcomas are particularly hyperploid if they develop in an immunodeficient setting, correlating with higher DNA content, increased nuclear surface, as well as hyperphosphorylation of eukaryotic initiation factor 2α (eIF2α), a biomarker indicating endoplasmic reticulum (ER) stress. Upon transfer of such cells into wild type mice, such hyperploid, ER-stressed cells (that originated in Rag2 −/− γc −/− mice) fail to proliferate and actually induce a protective anticancer immune response. In contrast, such cells do form tumors in Rag2 −/− γc −/− recipients (which lack T, B and NK cells) as well as in Rag2 −/− recipients (which only lack T and B lymphocytes) and conserve their hyperploidy as well as eIF2α hyperphosphorylation. To measure these parameters, we developed a morphometric analysis tool that is applicable to immunohistochemistry of formaldehyde-fixed, paraffin-embedded tissues. This software automatically identifies and quantifies the surface of nuclei and determines the intensity of eIF2α phosphorylation within a perinuclear region of interest. Comparative analyses performed on cultured cells and tissue sections validated the accuracy of this method, which can be used to investigate ploidy and ER stress in cancers in situ .