Abstract B009: Isolating human RCC tumor cells details evolutionary relationships between cancer cell subsets and a role of OXPHOS in disease progression
Authors: Sobierajska Ewelina , Medina Christopher , Gregorova Petra , Vo BaoHan , Greenwald Rachel , Jansen Caroline , Larsen Christian , Master Viraj , Kissick Haydn 
Published: August 2023

Abstract
In this study, we aimed to examine cancer-cell intrinsic features that might dictate clear cell renal cell carcinoma (ccRCC) progression. However, cellular heterogeneity within the tumor tissue complicates the analysis of pure kidney cancer cells. Therefore, we performed scRNA-seq of the whole tumor tissue to profile gene expression patterns unique to cancer cells. Next, using this information, we developed a novel FACS sorting strategy and sorted two populations: CD24loENPP3lo (CD24lo) and CD24hiENPP3hi (CD24hi). Whole genome sequencing on the isolated subsets from over 100 patients confirmed &gt;85% tumor purity. Direct comparison with public TCGA data demonstrated that cancer cell enrichment prior to sequencing significantly increased mutation detection sensitivity highlighting the advantage of this approach. Genomic and phenotypic lineage analysis revealed a progressive relationship between CD24lo precursors and CD24hi differentiated cells. Apart from many shared clonal mutations, we detected selected subclonal events in the CD24lo that became clonal in CD24hi. CD24hi cells were characterized by highly malignant properties such as epithelial to mesenchymal transition and an increased proliferative state as measured by Ki67. In accordance with their invasive signature, increased CD24hi frequencies appeared in advanced but not early stages (T1-2) of ccRCC. Importantly, not all late-stage patients with a CD24hi population progressed after surgery. To examine the qualities of CD24hi cells that associate with disease recurrence, we performed RNA-seq on purified cancer cells from progressors or non-progressors. We found a strong link between disease progression and increased oxidative phosphorylation (OXPHOS) in CD24hi cells. Furthermore, to validate our RNA-seq we assessed mitochondrial fitness using the TMRM probe and detected increased mitochondrial potential within CD24hicells from progressors. To explore the intrinsic consequences of increased cancer cell oxidative metabolism, we examined the relationship between proliferation and OXPHOS in CD24hi cells but did not observe any significant correlations. Extrinsically, to evaluate OXPHOS effects on the tumor microenvironment we examined CD8 T cells given that increased infiltration confers a prognostic advantage in ccRCC. We found that elevated tumor oxidative metabolism inversely associated with CD8 T cells numbers and response to immunotherapy, suggesting a role for OXPHOS in anti-tumor immune control. Overall, this work used a novel human cancer cell isolation approach to identify a cancer cell differentiation path from a CD24lo precursor to an aggressive CD24hi cell. Presence alone of the CD24hi does not predict survival, instead, the oxidative dominance within these cells negatively impacts disease control. Interestingly, metabolic focus within ccRCC has overlooked OXPHOS, but we provide insight that it could be leveraged for unexplored treatment avenues in ccRCC.
Citation Format: Ewelina Sobierajska, Christopher Medina, Petra Gregorova, BaoHan Vo, Rachel Greenwald, Caroline Jansen, Christian Larsen, Viraj Master, Haydn Kissick. Isolating human RCC tumor cells details evolutionary relationships between cancer cell subsets and a role of OXPHOS in disease progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B009.