FIGURE 5 from Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial–mesenchymal Transition in Aggressive Variant Prostate Cancers

Metronomic topotecan treatment as a single agent and in combination with DTX reduces EMT/stemness in AR^Low/mCRPC/NEPC clonally derived highly metastatic prostate cancer cell lines. A, Single-cell transcriptomics: Identified differential expression of EMT markers in AR^Low/mCRPC/NEPC (PC-3 vs. PC-3M) cells. scRNA-seq using the Droplet sequencing method (10X Genomics) was performed on the prostate cancer cell lines. Each dot represents a single cell. (I) t-SNE plots showing the comparison between the single-cell clusters represented AR^Low/mCRPC/NEPC (PC-3 vs. PC-3M) cells. (II) Top EMT transdifferentiation markers in PC-3 and PC-3M, included EZH2, Snail (SNAI1), Slug (SNAI2), TWIST1 genes. SNAI1 and SNAl2 are markers for cell invasion. SNSI1 and SNSI2 increased CD44⁺ expressing cells in prostate cancer populations. All EMT markers were expressed at higher levels in PC-3M compared with PC-3 cells, which indicated more stemness and cell invasion character for PC-3M cells. B, Assessment of posttreated “stem-like” cells (CD44^high/CD133^high) population in AR^Low/mCRPC cell lines by Flowcytometry: prostate cancer cell lines stained with stemness markers (CD44, CD133, and both CD44/133) after CONV-TOPO, METRO-TOPO, and combination (CONV-DTX+METRO-TOPO) treatment. CONV-TOPO, METRO-TOPO and combination (CONV-DTX+METRO-TOPO) treatment reduces CD44^high cells 30.5%, 19.5%, and 5.97%, respectively, in AR^Low/mCRPC (PC-3) cell line. Therefore, METRO-TOPO as a single agent and in combination with DTX reduced higher percentages of “stem-like” CD44^high cell population compared with CONV-TOPO treatment. Data for PC-3M and DU145 cells are shown in (Supplementary Fig. S6A and S6B; Table 2). C, Baseline ALDH: aldehyde dehydrogenase (ALDH) was assessed using an Aldefluor kit according to the manufacturer's instructions (Stem Cell Technologies).In AR^Low/mCRPC/NEPCP prostate cancer cell lines (PC-3 vs. PC-3M), ALDH was marginally higher in PC-3M compared with PC-3, indicating the presence of a “stem-like phenotype.” The ALDH inhibitor DEAB was used as a negative control. The cells without inhibitor shifted to the right were considered ALDH+ cells (right). D, Immunoblot analysis: EMT proteins were significantly downregulated in METRO-TOPO versus CONV-TOPO in AR^Low/mCSPC/NEPC (PC-3M) prostate cancer cells. Combination treatment, CONV-DTX+METRO-TOPO exhibited the highest downregulation of EMT proteins compared with other treatments. Posttreatment protein expression downregulation was following orders: CONV-TOPO>CONV-DTX>METRO-TOPO. Beta-actin was used as a control housekeeping gene to normalize the protein expression of other genes (*, P ≤ 0.05). E, PDMS-based microchannel cell migration assay: This assay allows the study of cancer cell invasion into physically restricted spaces. I, Representative images showed that PC-3M cells entered confining microchannels (30 μm²) more effectively compared with PC-3 cells, suggesting that PC-3M cells were more invasive. However, the differential percentage of cell entry into partially confined microchannels (100 μm²) was not statistically significant between PC-3 and PC-3M cells (video). (II) Bar graphs represented the quantification of (I). Figure demonstrated that PC-3M was more invasive compared with PC-3 cells (P < 0.05). (III) Assessed the entry of post-drug exposure PC-3M cells into confining microchannels. Experiments showed that treatment with METRO-TOPO (lower dose) reduced cell invasion more compared with treatment with CONV-TOPO (high dose). Combination (CONV-DTX+METRO-TOPO) treatment showed highest reduction of posttreatment cell invasion compared with other treatments. ANOVA followed by multiple comparisons (**, Bonferroni P ≤ 0.01).