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Abstract
Introduction/Background Targeted therapeutics, including bevacizumab and PARPi, gained traction for the maintenance treatment of high-grade serous ovarian cancer (HGSOC) following platinum-based chemotherapy. However, there are still limited methods to predict patient-specific responses to chemotherapy. In this aim, we investigate cellular changes elicited by the carboplatin in vitro in HGSOC patient-derived organoids (PDOs). Furthermore, in the context of drug response, we explore the stemness potential and putative mechanisms of the emergence of resistance to carboplatin.
Carboplatin treatment triggers acute DNA damage responses and causes change in stemness regulation in patient derived organoids a) t-sne plot (Loupe browser) ilustrates patient specific single cell clustering from sensitive and resistant organoid lines (as determined by clinical disease progression) that remains intact also in carboplated treated samples Core analysis by ingenuity IPA software of the platinum resistant line 48 h post tretment reveals activation of DNA replication changes in cell cycle control and downregulation of protective mechanisms (senescence) c) WB and FACS plots showing upregulation of stemness marker CD133 in the long term organoid culture of the organoids preexposed ta carboplatin d) Subsorting of the CD133+/- populations confirms organoid forming capacity is defined by CD133+ marker expression and indicates morphological changes in progeny derived from platinum pre treated cultures (arrow=
Methodology Phenotypically and genotypically validated PDOs from primary tumor deposits of HGSOC were used to study biological changes after carboplatin exposure by combining single-cell RNA sequencing, and functional in vitro assays. Organoid forming efficiency assay (OFE) was developed to quantify the growth capacity of PDOs following 48 hours of carboplatin and analyse the long-term expansion of platinum pretreated lines. Changes in organoid cellular architecture were investigated by FACS sorting of progenitor populations, confocal imaging, and qPCR of key developmental regulators.
Results Overall, 4 organoid lines with known clinical response to carboplatin was investigated by sc RNA sequencing providing insight into tumor heterogeneity and key cellular pathways activated in PDOs resistant to carboplatin. The expression profile of the acute response (48 h) revealed candidate pathways behind incomplete drug response and tumor survival, among which activation of DNA replication and changes in cell cycle regulation were notable. Organoids which retained regeneration capacity after carboplatin exposure, showed a sustained shift in expression of stemness associated surface marker CD133+ in the subsequent passages as showed by fluorescence-activated cell sorting and Western blot
Conclusion We hereby propose OFE assay as a novel functional readout for carboplatin sensitivity. Furthermore, expression profile changes in organoids during acute response to carboplatin provide insights into specific signaling hallmarks that are associated with resistance to carboplatin. and could help identify the cellular mechanism behind the process.