Morphometric analysis of immunoselection against hyperploid cancer cells

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.