Abstract
<p>Metronomic topotecan treatment as a single agent and in combination with DTX reduces EMT/stemness in AR<sup>Low</sup>/mCRPC/NEPC clonally derived highly metastatic prostate cancer cell lines. <b>A,</b> Single-cell transcriptomics: Identified differential expression of EMT markers in AR<sup>Low</sup>/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. (<b>I</b>) t-SNE plots showing the comparison between the single-cell clusters represented AR<sup>Low</sup>/mCRPC/NEPC (PC-3 vs. PC-3M) cells. (<b>II</b>) Top EMT transdifferentiation markers in PC-3 and PC-3M, included <i>EZH2</i>, <i>Snail</i> (<i>SNAI1</i>), <i>Slug</i> (<i>SNAI2</i>), <i>TWIST1</i> genes. <i>SNAI1</i> and <i>SNAl2</i> are markers for cell invasion. <i>SNSI1</i> and <i>SNSI2</i> increased CD44<sup>+</sup> 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>B,</b> Assessment of posttreated “stem-like” cells (CD44<sup>high</sup>/CD133<sup>high</sup>) population in AR<sup>Low</sup>/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<sup>high</sup> cells 30.5%, 19.5%, and 5.97%, respectively, in AR<sup>Low</sup>/mCRPC (PC-3) cell line. Therefore, METRO-TOPO as a single agent and in combination with DTX reduced higher percentages of “stem-like” CD44<sup>high</sup> cell population compared with CONV-TOPO treatment. Data for PC-3M and DU145 cells are shown in (<a href="#SMF6" target="_blank">Supplementary Fig. S6A</a> and <a href="#SMF6" target="_blank">S6B</a>; <a href="#tbl2" target="_blank">Table 2</a>). <b>C,</b> Baseline ALDH: aldehyde dehydrogenase (ALDH) was assessed using an Aldefluor kit according to the manufacturer's instructions (Stem Cell Technologies).In AR<sup>Low</sup>/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). <b>D,</b> Immunoblot analysis: EMT proteins were significantly downregulated in METRO-TOPO versus CONV-TOPO in AR<sup>Low</sup>/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 (*, <i>P</i> ≤ 0.05). <b>E,</b> PDMS-based microchannel cell migration assay: This assay allows the study of cancer cell invasion into physically restricted spaces. <b>I,</b> Representative images showed that PC-3M cells entered confining microchannels (30 μm<sup>2</sup>) 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 μm<sup>2</sup>) was not statistically significant between PC-3 and PC-3M cells (video). (<b>II</b>) Bar graphs represented the quantification of (<b>I</b>). Figure demonstrated that PC-3M was more invasive compared with PC-3 cells (<i>P</i> < 0.05). (<b>III</b>) 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 <i>P</i> ≤ 0.01).</p>
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