Subreen A Khatib1,2, Lichun Ma1, Hien Dang1,3, Marshonna Forgues1, Joon-Yong Chung4, Kris Ylaya4, Stephen M Hewitt4, Jittporn Chaisaingmongkol5,6, Mathuros Rucchirawat5,6, Xin Wei Wang1,7. 1. Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. 2. Department of Tumor Biology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA. 3. Division of Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. 4. Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. 5. Laboratory of Chemical Carcinogenesis, Chulabhorn Research Institute, Bangkok, Thailand. 6. Center of Excellence on Environmental Health and Toxicology, Office of the Higher Education Commission, Ministry of Education, Bangkok, Thailand. 7. Liver Cancer Program, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Abstract
BACKGROUND AND AIMS: HCC is a highly aggressive and heterogeneous cancer type with limited treatment options. Identifying drivers of tumor heterogeneity may lead to better therapeutic options and favorable patient outcomes. We investigated whether apoptotic cell death and its spatial architecture is linked to tumor molecular heterogeneity using single-cell in situ hybridization analysis. APPROACH AND RESULTS: We analyzed 254 tumor samples from two HCC cohorts using tissue microarrays. We developed a mathematical model to quantify cellular diversity among HCC samples using two tumor markers, cyclin-dependent kinase inhibitor 3 and protein regulator of cytokinesis 1 as surrogates for heterogeneity and caspase 3 (CASP3) as an apoptotic cell death marker. We further explored the impact of potential dying-cell hubs on tumor cell diversity and patient outcome by density contour mapping and spatial proximity analysis. We also developed a selectively controlled in vitro model of cell death using CRISPR/CRISPR-associated 9 to determine therapy response and growth under hypoxic conditions. We found that increasing levels of CASP3+ tumor cells are associated with higher tumor diversity. Interestingly, we discovered regions of densely populated CASP3+ , which we refer to as CASP3+ cell islands, in which the nearby cellular heterogeneity was found to be the greatest compared to cells farther away from these islands and that this phenomenon was associated with survival. Additionally, cell culture experiments revealed that higher levels of cell death, accompanied by increased CASP3 expression, led to greater therapy resistance and growth under hypoxia. CONCLUSIONS: These results are consistent with the hypothesis that increased apoptotic cell death may lead to greater tumor heterogeneity and thus worse patient outcomes.
BACKGROUND AND AIMS: HCC is a highly aggressive and heterogeneous cancer type with limited treatment options. Identifying drivers of tumor heterogeneity may lead to better therapeutic options and favorable patient outcomes. We investigated whether apoptotic cell death and its spatial architecture is linked to tumor molecular heterogeneity using single-cell in situ hybridization analysis. APPROACH AND RESULTS: We analyzed 254 tumor samples from two HCC cohorts using tissue microarrays. We developed a mathematical model to quantify cellular diversity among HCC samples using two tumor markers, cyclin-dependent kinase inhibitor 3 and protein regulator of cytokinesis 1 as surrogates for heterogeneity and caspase 3 (CASP3) as an apoptotic cell death marker. We further explored the impact of potential dying-cell hubs on tumor cell diversity and patient outcome by density contour mapping and spatial proximity analysis. We also developed a selectively controlled in vitro model of cell death using CRISPR/CRISPR-associated 9 to determine therapy response and growth under hypoxic conditions. We found that increasing levels of CASP3+ tumor cells are associated with higher tumor diversity. Interestingly, we discovered regions of densely populated CASP3+ , which we refer to as CASP3+ cell islands, in which the nearby cellular heterogeneity was found to be the greatest compared to cells farther away from these islands and that this phenomenon was associated with survival. Additionally, cell culture experiments revealed that higher levels of cell death, accompanied by increased CASP3 expression, led to greater therapy resistance and growth under hypoxia. CONCLUSIONS: These results are consistent with the hypothesis that increased apoptotic cell death may lead to greater tumor heterogeneity and thus worse patient outcomes.