Bart Kolendowski

Transcriptomic Analyses of Ovarian Clear Cell Carcinoma Spheroids Reveal Distinct Proliferative Phenotypes and Therapeutic Vulnerabilities

Cancer cell spheroids autonomously form in the ascites fluid and are considered a conduit for epithelial ovarian cancer metastasis within the peritoneal cavity. Spheroids are homotypic, avascular 3D structures that acquire resistance to anoikis to remain viable after cellular detachment. We used in vitro spheroid model systems to interrogate pathways critical for spheroid cell proliferation, distinct from those driving monolayer cancer cell proliferation. Using the 105C and KOC-7c human ovarian clear cell carcinoma (OCCC) cell lines, which have distinct proliferative phenotypes as spheroids but the same prototypical OCCC gene mutation profile of constitutively activated AKT signaling with the loss of ARID1A, we revealed therapeutic targets that efficiently kill cells in spheroids. RNA-seq analyses compared the transcriptome of 3-day monolayer and spheroid cells from these lines and identified the characteristics of dormant spheroid cell survival, which included the G2/M checkpoint, autophagy, and other stress pathways induced in 105C spheroids, in sharp contrast to the proliferating spheroid cells of the KOC-7c cell line. Next, we assessed levels of various G2/M checkpoint regulators and found a consistent reduction in steady-state levels of checkpoint regulators in dormant spheroid cells, but not proliferative spheroids. Our studies showed that proliferative spheroid cells were sensitive to Wee1 inhibition by AZD1775, but the dormant spheroid cells showed a degree of resistance to AZD1775, both in terms of EC50 values and spheroid reattachment abilities. Thus, we identified biomarkers of dormant spheroids, including the G2/M checkpoint regulators Wee1, Cdc25c, and PLK1, and showed that, when compared to proliferating spheroid cells, the transcriptome of dormant OCCC spheroids is a source of therapeutic targets.

Exploiting Cancer Dormancy Signaling Mechanisms in Epithelial Ovarian Cancer Through Spheroid and Organoid Analysis

Epithelial ovarian cancer (EOC) exhibits a unique mode of metastasis, involving spheroid formation in the peritoneum. Our research on EOC spheroid cell biology has provided valuable insights into the signaling plasticity associated with metastasis. We speculate that EOC cells modify their biology between tumour and spheroid states during cancer dormancy, although the specific mechanisms underlying this transition remain unknown. Here, we present novel findings from direct comparisons between cultured EOC spheroids and organoids. Our results indicated that AMP-activated protein kinase (AMPK) activity was significantly upregulated and protein kinase B (Akt) was downregulated in EOC spheroids compared to organoids, suggesting a clear differential phenotype. Through RNA sequencing analysis, we further supported these phenotypic differences and highlighted the significance of cell cycle regulation in organoids. By inhibiting the G2/M checkpoint via kinase inhibitors, we confirmed that this pathway is essential for organoids. Interestingly, our results suggest that specifically targeting aurora kinase A (AURKA) may represent a promising therapeutic strategy since our cells were equally sensitive to Alisertib treatment as both spheroids and organoids. Our findings emphasize the importance of studying cellular adaptations of EOC cells, as there may be different therapeutic targets depending on the step of EOC disease progression.

Characterization of Mutational Status, Spheroid Formation, and Drug Response of a New Genomically-Stable Human Ovarian Clear Cell Carcinoma Cell Line, 105C

Ovarian clear cell carcinoma (OCCC) is a rare subtype of gynecological cancer for which well-characterized and authenticated model systems are scarce. We provide an extensive characterization of ‘105C’, a cell line generated from an adenocarcinoma of the clear cell histotype using targeted next-generation sequencing, cytogenetic microarrays, along with analyses of AKT/mTOR signaling. We report that that the 105C cell line is a bona fide OCCC cell line, carrying PIK3CA, PTEN, and ARID1A gene mutations, consistent with OCCC, yet maintain a stable genome as reflected by low copy number variation. Unlike KOC-7c, TOV-21G, and RMG-V OCCC lines also mutated for the above genes, the 105C cells do not carry mutations in mismatch repair genes. Importantly, we show that 105C cells exhibit greater resistance to mTOR inhibition and carboplatin treatment compared to 9 other OCCC cell lines in 3D spheroid cultures. This resistance may be attributed to 105C cells remaining dormant in suspension culture which surprisingly, contrasts with several other OCCC lines which continue to proliferate in long-term suspension culture. 105C cells survive xenotransplantation but do not proliferate and metastasize. Collectively, we show that the 105C OCCC cell line exhibits unique properties useful for the pre-clinical investigation of OCCC pathobiology.