Single-cell Biology Uncovers Cell Death as a Potential Driver of Tumor Heterogeneity and Fitness

Abstract

Hepatocellular carcinoma (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. Here, I aimed to investigate whether cell death and its spatial architecture is linked to tumor molecular heterogeneity using single-cell RNAseq and single-cell multiplex immunofluorescence assays. Next, seven single-cell RNAseq datasets were analyzed to compute an Apoptotic Index and tumor diversity score and conducted survival analysis on our apoptotic signature. I then analyzed 254 tumor samples from two HCC cohorts using tissue microarrays stained with the RNAScope assay. I developed a mathematical model to quantify cellular diversity among HCC samples using two tumor markers, CDKN3 and PRC1 as surrogates for heterogeneity and CASP3 as a cell death marker. I further explored the impact of potential dying-cell hubs on tumor cell diversity and patient outcome by density contour mapping and spatial proximity analysis. I also developed a selectively controlled in vitro model of cell death using CRISPR/Cas9 to determine therapy response and growth under hypoxic conditions. I demonstrated that an Apoptotic Index was significantly correlated with a Tumor Shannon Entropy Index, with the strongest association found in primary liver cancer and patients with a high expression of the apoptosis signature displayed worse overall survival. I found that increasing levels of CASP3+ stained tumor cells are associated with higher tumor diversity in our tissue microarrays. Interestingly, I discovered regions of densely populated CASP3+, that I refer to as apoptotic islands, in which the nearby cellular heterogeneity was found to be the greatest compared to cells further away from these apoptotic islands and that this phenomenon was associated with survival. Additionally, cell culture experiments revealed higher levels of cell death led to greater therapy resistance and growth under hypoxia. These results are consistent with the hypothesis that increased cell death may lead to greater tumor heterogeneity and thus worse patient outcomes.