Acquired resistance to carboplatin is a major obstacle to the cure of ovarian cancer, but its molecular underpinnings are still poorly defined or inconsistently reported. Multiple cellular functions have been identified as being capable of modulating sensitivity to the platinum drugs; however, results from single-gene or transcriptome-wide studies have failed to disclose any one actionable gene or set of genes that are consistently altered across different cell lines or studies. We hypothesized that heterogeneity within the treated cell populations is responsible for this situation and that cloning and molecular analysis of single cells can capture the heterogeneity and dynamics of resistance acquisition, revealing cellular and molecular principles driving the phenotype. Following a rigorous experimental design, starting with a single ovarian cancer cell, we selected 8 subclones for resistance to carboplatin using repeated cycles of drug exposure. An additional 4 clones were left untreated for comparison. The resistant clones did not show significant mutations or gene copy number variations in common or reduced drug uptake, but displayed important phenotypic and gene expression heterogeneity. A gene set enrichment analysis revealed that each clone altered different molecular processes to become resistant. Suspecting that clonal heterogeneity was also present within each experimental replicate, we measured mRNA expression from 26,892 single cells from both sensitive and resistant clones, which resulted in the identification of 7 transcriptional states associated with resistance evolution and clone identity. Interestingly, the resistant states were not stable and continued to evolve after removal of the drug, without noticeable change in resistance level. Reduced proliferation and active interferon signaling were identified as two processes shared by most resistant states, becoming apparent early in the course of the resistance acquisition. Up to 12% of untreated cells were found to already be in a resistant state and this fraction diminished with each round of sub-cloning. Up to 18% of the cells from resistant clones showed extreme levels of key resistance genes and pathways, suggesting the importance of cellular heterogeneity in the development of resistance. Finally, six expression signatures derived from the in vitro resistant states distinguished primary from recurrent high-grade serous ovarian cancers and predicted both response to primary therapy and overall survival. The analysis of the biologic processes associated with each signature suggested functional differences between intrinsic and acquired resistance. This multidimensional, single-cell analysis decomposed a multigenic phenotype into tangible resistant states, offering new insights into the dynamics of the acquisition of resistance to carboplatin, a drug of major importance to the treatment of ovarian and other cancers.
Citation Format: Devora Champa, Si Sun, Cheng-Yu Tsai, Stephen Howell, Olivier Harismendy. Evolution and characterization of carboplatin resistance at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2860A.