FIGURE 1 from Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial–mesenchymal Transition in Aggressive Variant Prostate Cancers
Authors: Mitra Ghosh Taraswi , Mazumder Suman , Davis Joshua , Yadav Jyoti , Akinpelu Ayuba , Alnaim Ahmed , Kumar Harish , Waliagha Razan , Church Bird Allison E. , Rais-Bahrami Soroush , Bird R. Curtis , Mistriotis Panagiotis , Mishra Amarjit , Yates Clayton C. , Mitra Amit K. , Arnold Robert D. 
Published: July 2023

<p>NGS and single-cell transcriptomics identified signatures of EMT markers in AR<sup>High</sup>/mCSPC, AR<sup>Low</sup>/mCRPC/NEPC, AR<sup>Low</sup>/mCSPC/NEPC<sup>taxane resistant</sup> EA/Caucasian and AA prostate cancer subtypes. <b>A,</b> Heat map representing DEG signature from bulk RNA-seq. (<b>I</b>) DEGs among AR<sup>High</sup>/mCSPC (LNCaP, VCAP, 22RV1) versus AR<sup>Low</sup>/mCRPC/NEPC (PC-3, PC-3M, DU145) prostate cancer and normal prostate cell lines (RWPE1, RWPE2; <i>P</i> < 0.05). (<b>II</b>) DEGs among top 100 genes in AR<sup>High</sup>/mCSPC (EA-LNCaP, VCAP, 22RV1 vs. AA-MDA-Pca-2b, RC77T/E, RC165T) and AR<sup>Low</sup>/mCRPC (EA-DU145, PC-3, PC-3M vs. AA-RC43T; <i>P</i> < 0.05). (<b>III</b>) DEGs among top EMT markers in AR<sup>High</sup>/mCSPC (EA-LNCaP, VCAP, 22RV1 vs. AA-MDA-Pca-2b, RC77T/E, RC165T) and AR<sup>Low</sup>/mCRPC/NEPC (EA-DU145, PC-3, PC-3M vs. AA-RC43T) prostate cancer cell lines (<i>P</i> < 0.05). <b>B,</b> EMT markers in AR<sup>High</sup>/mCSPC versus AR<sup>Low</sup>/mCRPC (scRNA-seq): Single-cell RNA sequencing using the droplet sequencing method (10X Genomics) was performed on the prostate cancer cell lines. Each dot represents a single cell. Contaminated (doublet) cells were not included. t-SNE plots showing the comparison between the single-cell clusters. (<b>I</b>) Subclonal distribution in all cell lines AR<sup>High</sup>/mCSPC (LNCaP, 22RV1) and AR<sup>Low</sup>/mCRPC/NEPC (PC-3, PC-3M, DU145). (<b>II</b>) Subclonal distribution of top two EMT markers (<i>CD44</i> and <i>HAS3</i>) in all prostate cancer cell lines. (<b>III</b> and <b>IV</b>) Subclonal distribution of major EMT markers (<i>AHNAK2</i>, <i>LOXL2</i>, <i>S1000A6</i>, <i>TGFB1</i>, <i>TGFBR2</i>, <i>UCHL1</i>, <i>VIM</i>, <i>ANXA2</i>, <i>ANXA2P2</i>, <i>ANXA3</i>, <i>CD55</i>, <i>CD109</i>) in all prostate cancer cell lines. (<b>V</b>) Subclonal distribution of top taxane resistance marker <i>FSCN1</i> in all prostate cancer cell lines. Results exhibiting EMT and taxane resistance marker expression were higher in AR<sup>Low</sup>/mCRPC/NEPC compared with AR<sup>High</sup>/mCSPC. (<b>VI</b>) Subclonal populations for all prostate cancer subtypes. The relative representation of each subclone between the prostate cancer cell lines was also provided using comparative pie charts. Results showed the intratumor heterogeneity and several clusters which are unique to each prostate cancer cell line, in addition to the shared subclusters possibly representing gene signatures common to the biology of prostate cancer. <b>C,</b> EMT markers in AR<sup>Low</sup>/mCRPC/NEPC versus AR<sup>Low</sup>/mCSPC/NEPC<sup>taxane-resistant</sup> prostate cancer: UMAP-distributed stochastic neighbor embedded plots showing the comparison between the single-cell clusters. (<b>I</b>) Subclonal distribution between AR<sup>Low</sup>/mCRPC/NEPC (DU145) and AR<sup>Low</sup>/mCSPC/NEPC<sup>taxane-resistant</sup> (DUTXR). (<b>II</b>) Subclonal distribution of top two EMT markers <i>CD44</i> and <i>HAS3</i> in DU145 versus DUTXR. Pie charts represent percentages of subclonal distribution of EMT markers. <i>CD44</i> expression is higher in DUTXR and <i>HAS-3</i> expression higher in DU145. (<b>III</b>) Subclonal distribution of major EMT markers (<i>AHNAK2</i>, <i>LOXL2</i>, <i>S1000A6</i>, <i>TGFB1</i>, <i>TGFBR2</i>, <i>UCHL1</i>, <i>VIM</i>. (<b>IV</b>) <i>ANXA2</i>, <i>ANXA2P2</i>, <i>ANXA3</i>, <i>CD55</i>, <i>CD109</i>) in DU145 versus DUTXR. Pie chart represented majority of EMT markers (<i>LOXL2</i>, <i>TGFB1</i>, <i>UCHL1</i>, <i>VIM</i>, <i>ANXA2P2</i>, <i>CD55</i>, <i>CD109</i>) expressed higher in DUTXR cell line. Results exhibiting EMT marker expression were high (EMT markets nonexpressing cells were low) in the AR<sup>Low</sup>/mCSPC/NEPC<sup>taxane-resistant</sup> prostate cancer cell line (DUTXR). <b>V,</b> Top genes representing single-cell clusters that were shared and which were unique between DU145 and DUTXR. Common and unique DEGs between RNA-seq and scRNA-seq were also identified for all prostate cancer subtypes. Common genes were EMT markers, for example, <i>VIM</i>, <i>AHNAK2</i>, <i>ANXA1</i>, <i>ANXA2</i>, <i>CD109</i>, <i>CD44</i>, <i>CD59</i>, <i>GPRC5A</i>, <i>HAS</i>, <i>S100A6</i>, <i>TGFBR2</i> (<a href="#SMF1" target="_blank">Supplementary Figs. S4</a> and <a href="#SMF5" target="_blank">S5</a>; <a href="#SMT1" target="_blank">Supplementary Table S7</a>).</p>