Supplementary Fig. 6 shows Cytotoxicity Profiling of ARLow/mCRPC/NEPC (PC-3 and PC-3M) and Taxane Resistance ARLow/mCRPC (DUTXR): PC-3, PC-3M and DUTXR cell lines were treated with CONV-TOPO, METRO-TOPO, CONV-DTX and combination (CONV-DTX+METRO-TOPO) treatment, and cell cytotoxicity and caspase3/7 levels ware assed A) Cytotoxicity profiling by MTT showed combination (CONV-DTX+METRO-TOPO) reduces highest cell survival compared with other treatments for all PCa cell lines, CONV-TOPO>CONV-DTX>METRO-TOPO B) Caspase3/7 activity showed combination (CONV-DTX+METRO-TOPO) treatment reduced apoptosis the greatest compared to other treatments in all cell lines. C) Cytation5 images showed treatment effects on the cell lines PC-3M and DUTXR. Results showed significantly higher cell death in METRO compared to CONV treatment for both the cell lines and combination (CONV-DTX+METRO-TOPO) treatment reduced cell death greater for both PCa cell lines. ImageJ analysis showed significant differences in cell density for CONV-TOPO, CONV-DTX, METRO-TOPO, and combination (CONV-DTX+METRO-TOPO) treatment in PC-3, PC-3M, and DUTXR cell lines. (*p ≤ 0.05).
ARTICLE ABSTRACT
Prostate cancer (PCa) is the second leading cause of non-cutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiation therapy remain the primary treatment for early-stage PCa patients (castration-sensitive: CSPC). Following ADT, many patients ultimately develop metastatic castration-resistant PCa (mCRPC). Standard chemotherapy options for CRPC are Docetaxel (DTX) and Cabazitaxel (CBZ), which increase median survival, although the development of resistance is common. Cancer-stem-like cells possess mesenchymal phenotypes (EMT) and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits PCa growth by interfering with key cancer-pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis (RNA-sequencing and scRNAseq), and we observed greater expression of several EMT markers, including VIM, HAS3, S100A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant PCa (AVPC) subtypes - mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell sub-clonal populations in mCRPC. Further, metronomic-topotecan single-agent and combinations with DTX downregulated these EMT markers as well as CD44+ and CD44+/CD133+ “stem-like” cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single-agent or in combination with DTX was potentially effective against invasive PCa spread. Our RNAseq and scRNAseq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways.
