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

Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (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 prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin, hyaluronan synthase-3, S100 calcium binding protein A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes—mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, 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 prostate cancer spread. Our RNA-seq and scRNA-seq 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. Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies.


Introduction
Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men (www.cancer.org). The androgen receptor (AR) signaling pathway plays a pivotal role in prostate development and homeostasis, as well as in the progression of prostate cancer (1). Androgen deprivation therapy (ADT), following radical prostatectomy or radiotherapy, remains the main treatment for more advanced cases of castration-sensitive prostate cancer (CSPC). In many patients, ADT effectively suppresses prostate cancer during the first 12-24 months (1-3). However, many patients ultimately develop resistance and show metastatic spread, that is, metastatic castration-resistant prostate cancer (mCRPC) disease (4). The 2020 estimates of transition from non-castrate to mCRPC are approximately 15%, with a mortality rate of 19.5% (4). Neuroendocrine differentiation following ADT to aggressive treatmentresistant neuroendocrine prostate cancer (NEPC) has been estimated at >25% (5). Standard treatment options for CRPC/NEPC include sipuleucel-T, abiraterone acetate plus prednisone (AA/P), or chemotherapy with docetaxel (DTX; refs. [6][7][8]. Cabazitaxel, AA/P, enzalutamide, and radium-223 are approved treatments of CRPC, often following DTX (6,7). These combination treatments can increase median overall survival (OS) by approximately 1 year (9). Resistance development, characterized by increased PSA levels, is almost universal, resulting in a progression-free survival rate of around 0% after 3 years, often accompanied by significant side effects (10)(11)(12). Furthermore, African American (AA) men are more likely to be diagnosed or progress more rapidly to aggressive forms of mCRPC compared with other ethnicities (seer.cancer.gov). Moreover, overall treatment options remain limited, and survival is poor.
Several groups, including ours, have demonstrated that the presence of cancer stem-like cells (CSC) in tumors displaying mesenchymal phenotypes (epithelial-to-mesenchymal transdifferentiation, EMT) and CD44 + /CD133 + cells, with self-renewal and differentiation capacities, play crucial roles in tumor progression and development of mCRPC (13)(14)(15)(16). Furthermore, tumor heterogeneity and host-tumor microenvironments with divergent genetic profiles and molecular signatures represent a challenge for current therapies (17). Therefore, it is necessary to develop new therapeutic approaches to overcome drug resistance in European American (EA) and AA patients with prostate cancer, specifically for mCRPC/NEPC, to improve efficacy and increased OS.
Low-dose continuous drug exposure using metronomic-like (METRO) chemotherapy involves frequent administration of chemotherapeutic agents at low or fractionated doses at close intervals over prolonged periods of time (18,19). METRO is an emerging treatment option that has shown promise for various cancer types, including prostate cancer (20)(21)(22)(23). In our previous study, we reported that metronomic topotecan treatment (METRO-TOPO) was 2.4-to 18-fold more potent (P < 0.05) compared with conventional topotecan (CONV-TOPO) treatment in prostate cancer cell lines (22). We then performed animal study with METRO-TOPO versus CONV-TOPO and demonstrated that by day 17, METRO-TOPO treatment resulted in significantly (P ≤ 0.05) smaller tumor volume (65.4% ± 11.2%) compared with control (136% ± 14%) and CONV-TOPO-treated animals (138% ± 10%) in an aggressive xenograft tumor model of human prostate cancer implanted in male NCr athymic mice (22). This overall antitumor activity of METRO-TOPO was maintained through the end of the study, animals receiving METRO-TOPO dosing regimens had significantly (P ≤ 0.05) smaller tumor volumes (54.8% ± 16.5%) than animals receiving CONV-TOPO dosing (144% ± 11%) or the control group (207% ± 26%; ref. 22). To further investigate the impact of low-dose therapy, the effect of METRO-TOPO, CONV-TOPO intravenous administration on tumor volume was compared with implantation of ALZET micro-osmotic pumps in nude mice after tumor xenografts reached 200-300 mm 3 (22). ALZET pumps were primed to deliver 2.45 mg/kg/day to achieve plasma concentrations at the experimentally determined IC 50 , that is, 4-5 ng/mL and 0.10 mg/kg/day (4% of the IC 50 concentration; ref. 22). After 21 days of treatment, animals receiving "metro-like" dosing at 2.45 and 0.1 mg TOPO/kg/day had significantly (P ≤ 0.05) smaller tumor volumes compared with ALZET control animals with no observable toxicities (22). Molecular analysis revealed METRO-TOPO treatment antitumor activity was associated with the inhibition of major cancer pathway genes, angiogenesis, increasing tumor hypoxia, or normalizing the tumor vasculature to improve blood flow and drug delivery (21,22).
Several other studies have reported that low-dose oral TOPO is potent for patients with cancer (24)(25)(26). However, the clinical benefit of METRO-TOPO and a comprehensive understanding of its mechanism of action are not fully known.
Therefore, we performed pretreatment versus posttreatment bulk and singlecell RNA sequencing (scRNA-seq) to identify differentially expressed genes (DEG) for prostate cancer subtypes of EA and AA origin and potential molecular pathways associated with METRO-TOPO activity in aggressive variant prostate cancer (AVPC; mCRPC, NEPC, and EMT) at the tumor and subclonal levels and to gain molecular insights into METRO-TOPO activity. Furthermore, our RNA-seq and scRNA-seq data showed that METRO-TOPO treatment was potentially effective against the development of prostate cancer subclones with a reduction of treatment-resistant stem-like genes. Using in vitro model systems of treatment-refractory and treatment-emergent AVPC, we demonstrated that the METRO-TOPO showed efficacy as a single agent, as shown previously. We showed synergistic activity in combination with conventional taxanes (CONV-DTX) and METRO-TOPO. A microfluidic chip-based confined cell invasion assay was performed. The assay recapitulates the dimensionality of pores and longitudinal channel-like tracks encountered by cancer cells during migration to investigate the effect of METRO-TOPO on cancer cell invasion and stemness. Flow cytometry and cell sorting with prostate cancer stemness-specific antibodies (CD44, CD133) were accomplished to identify differential percentages of stem-like cell populations (CD44 + , CD133 + , and CD++) in all prostate cancer subtypes, including EA and AA, to evaluate the impact of METRO-TOPO in eroding "stem-like" (CD44 + /CD133 + ) cell subpopulations. The effect of METRO-TOPO treatment on EMT gene expression (GE) and protein expression was determined, and a comparative analysis with whole-genome transcriptomics data and reverse matching DEGs from patients with prostate cancer were used to examine the potential clinical significance of METRO-TOPO treatments.
Therefore, using an innovative approach that integrated advanced molecular techniques [next-generation mRNA sequencing (mRNA-seq) and single-cellomics technologies] with microfluidics, flow cytometry, MoFlo XPD Flow high speed-based cell sorting, cytotoxicity profiling, immunoblotting as in vitro studies, and patient databases (The Cancer Genome Atlas/TCGA for in silico validation), we conclude that METRO-TOPO has the potential to improve the clinical outcome in AVPC chemotherapy by enhancing the therapeutic efficacy of standard-of-care drugs and abrogating the possibilities of development of drug resistance. Such an evidence-based approach promises to minimize the chances of trial failures and improve the probability of clinical success.

Treatment Schedules for mRNA-seq and scRNA-seq
Three-dimensional (3D) spheroids were harvested and transferred into a 96well plate in 100 μL of recommended media. After 2 days of acclimation, 100 μL of media ± drug/well were added to each well containing a spheroid. On days 3 and 5, 100 μL of media/well were removed and replaced with 100 μL of fresh media with/without (±) drug/well. On off-media exchange days, 10 μL of media/well was removed and replaced with 10 μL of media ± drug. METRO-TOPO dosing was simulated by using 20x TOPO directly spiked into the wells at 10 μL in 190 μL of media. The CONV-TOPO was added as a bolus dose on day 0. METRO/extended exposure (EE)-TOPO treatment was given daily as a fractionated dose at 1/7th the CONV-TOPO. Total topotecan dosing was 100 nmol/L during each week of therapy. Control and treated spheroid were collected and stored in RNAlaterer (Qiagen) for RNA-seq. Fresh/live samples were used for scRNA-seq.

Pretreatment and Posttreatment Tumor mRNA-seq
GE of all prostate cancer and normal prostate cell lines at baseline (no treatment) was assessed by RNA-seq. Furthermore, the effects of METRO-TOPO and CONV-TOPO exposure for 6 weeks on AR Low mCRPC/NEPC PC-3 tumor model (3D spheroid) were assessed using RNA-seq. Pre-and post-drug exposure, as described above, tumor cells were harvested, and high-quality RNA was extracted using QIAshredder and RNeasy kit (Qiagen). RNA concentration and integrity were assessed using a Nanodrop-8000 spectrophotometer and Agilent 2100 Bioanalyzer. An RNA integrity number threshold >8 was applied, and RNA-seq libraries were constructed using Illumina TruSeq RNA Sample Preparation kit v2. Libraries were then size-selected to generate inserts of approximately 200 bp. RNA-seq was performed on Illumina's NovaSeq platform using a 150 bp paired-end protocol with a depth of >20 million reads per sample. Average quality scores were above Q30 for all libraries in both R1 and R2 (29). AA cell line RNA was isolated from cultured cells using TRIzol Reagent (Sigma Life Sciences) following the manufacturer's protocol (27). Library preparation, quality control, and sequencing of extracted RNA were performed by the Center for Pharmacogenomics and Single-Cell Omics (AUPharmGX).

RNA-seq Data Analysis
RNA-seq data analysis was performed using a command line-based analysis pipeline (DEseq2 and edgeR) and Partek Flow software. Briefly, reads were preprocessed and mapped to the hg38 human genome build using the STAR Aligner tool. Next, mapped read counts were counts per million (CPM) normalized, and differential Gene Expression Profile (GEP) analysis was performed. Genes with mean fold change >|1| and P < 0.05 were considered the threshold for reporting significant DEGs. Heat maps were generated using unsupervised hierarchical clustering (HC) analysis based on top DEGs. Sequencing data on AA cell lines were compiled as FastQ files for downstream analysis (27).

scRNA-seq
scRNA-seq analysis was assessed for prostate cancer cell lines at baseline and TOPO-METRO (EE vs. CONV for 6 weeks, as described above) in the PC-3 tumor model (3D spheroid). Automated single-cell capture and cDNA synthesis were performed using the 10X Genomics Chromium platform. scRNA-seqbased GE analysis was performed on an Illumina HiSeq 2500 next-generation sequencing (NGS) platform by paired-end sequencing technique at 2*125 bp and 100 cycles using v3 chemistry.

scRNA-seq Data Analysis
scRNA-seq datasets were obtained as matrices in the Hierarchical Data Format (HDF5 or H5). CellRanger, Seurat, and Partek Flow software packages were used to preprocess the data analysis. Highly variable genes were selected for clustering analysis based on a graph-based cluster approach. The visualization of cell populations was performed by t-distributed stochastic neighbor embedding (t-SNE) and Uniform Manifold Approximation and Projection (UMAP) for dimension reduction analysis for biomarker-based identification of subclones representing CSC-EMT, taxane-resistant cells, potential METRO-TOPO targeted subclones, and METRO-TOPO treatment-induced erosion of these subclones.

Ingenuity Pathway Analysis
Ingenuity Pathway Analysis (IPA; Qiagen) was performed using top DEGs to reveal molecular pathways/mechanisms, upstream regulator molecules, downstream effects, biological processes, and predicted causal networks governing prostate cancer subtypes and METRO-TOPO functions in AVPC (30).

Patient Samples
In this study, the interactive web portals UALCAN and Gene Expression Profiling Interactive Analysis (GEPIA) were used to compare transcriptome data on target candidate pathway genes with tumor metastasis and patient survival from the prostate expression data matrix (31,32 Flow Cytometry Detection of CD44 + , CD133 + , and CD44 + /CD133 + Cells Prostate cancer cells were labeled with a binding buffer containing stemness markers CD44, CD133, or both antibodies. Cells were collected and quantified as CD44 + , CD133 + , and double-positive cells using a Beckman Coulter Analytical Flow Cytometer-a CytoFLEX LX flow cytometer at 50,000 events/measurement. "Stem-like" cell populations were measured by assessing the shift in the mean fluorescence intensity by flow cytometry. Data were analyzed (gates were set), normalizing to unstained cells of each prostate cancer subtype. Ghost Dye Red 780 was used to detect live cells in each cell population. Furthermore, AR Low cells were seeded in 6-well plates and exposed to CONV-TOPO, METRO-TOPO, and CONV-DTX+METRO-TOPO as combination treatments calculated on the basis of our earlier studies (22,29). We have determined IC 50 for MERO-TOPO, CONV-TOPO, and CONV-DTX as single agents for 48 and 72 hours for all prostate cancer cell lines (21,22,28). Plated cells were exposed to TOPO and DTX at the estimated 3-  Table   S5). After 72 hours, cells were labeled and quantified for CD44 + , CD133 + , and double-positive cells with the protocol above. All Flow Cytometry data were analyzed by using FlowJo software (https://www.flowjo.com/).

In Vitro Cytotoxicity (MTT) Assay
In vitro cytotoxicity assays were performed using the MTT assay. Briefly, cells were plated in a 96-well culture plate at 2 × 10 3 cells/well and incubated for 24 hours at 37°C with 5% CO 2 . We have determined IC 50 for MERO-TOPO, CONV-TOPO, and CONV-DTX for 48 and 72 hours for all prostate cancer cell lines (21,28). The specific IC 50

Caspase 3/7 Activity Assay
Cell death by apoptosis was measured using a Caspase-Glo 3/7 kit. Briefly, cells were seeded and treated according to the MTT protocol (described above; Supplementary Table S5; refs. 22,29). Following 72 hours incubation, Caspase-Glo 3/7 reagent was added, and luminescence was measured using a Synergy Neo2 Microplate Reader. The apoptotic level of each treatment group was normalized to the no drug treatment (baseline) caspase 3/7 for each cell line.

Assessment of Cellular Morphology
Cellular morphology was assessed after cells were seeded at 2.5 × 10 4 cells (mL/well) in 6-well plates and exposed to TOPO-CONV, TOPO-METRO, CONV-DTX as a single agent and in combination with CONV-DTX+ METRO-TOPO according to MTT protocol (described above; Supplementary  Table S5; refs. 22,29). After 72 hours, cells were stained with crystal violet and washed with PBS. Three areas with approximately equal cell densities were identified in each well. Images were captured with an Agilent Cytation5 digital cell imaging system using a 4X objective and Texas Red filter with 559-34 Excitation, 630-69 Emission, and 585 DM. Images were analyzed using ImageJ software (https://imagej.nih.gov/ij/) in a double-blind manner.

Aldefluor Activity (Aldehyde Dehydrogenase) Assay
Aldehyde dehydrogenase (ALDH1) activity was assessed using an Aldefluor assay kit. Briefly, 1 × 10 4 prostate cancer cells were harvested and resuspended in Aldefluor assay buffer containing the ALDH substrate, BODIPY-amino acetaldehyde (BAAA). Negative control samples were treated with diethylamino-benzaldehyde (DEAB), an inhibitor of ALDH1 enzymatic activity. Cells were incubated at 37°C and suspended in Aldefluor assay buffer. The brightly fluorescent ALDH + cells were detected by BD LSR II flow cytometry.

Pretreatment and Posttreatment cells were lysed in RIPA Lysis and Extraction
Buffer (Thermo Fisher Scientific). Briefly, cells were seeded and treated according to the MTT protocol (described above; Supplementary Table S5; refs. 21,22,28). Quantification of proteins was performed using a Bradford assay with a BSA protein standard kit (Bio-Rad). Equal amounts of protein were loaded onto 4%-15% Criterion TGX Stain-Free Precast Gels. Proteins were separated under reducing conditions and then transferred to a polyvinylidene difluoride membrane using a Bio-Rad Semi-dry Blotting Apparatus. Nonspecific binding was limited by incubating the membrane in blocking buffer [2.5% (w/v) casein, pH 7.6, 150 mmol/L NaCl, 10 mmol/L TRIS-HCl, and 0.02% sodium azide]. Membranes were then incubated with primary antibodies for the targeted gene/protein (1:1,000), followed by secondary antibodies (1:10,000). Immunoreactivity was detected using enhanced chemiluminescence Western Blotting substrate. Images were captured on a Gel Doc EZ Gel Documentation System using ImageLab software (Azure Biosystems). Densitometry analysis was performed using ImageJ software (https://imagej.nih.gov/ij/).

Microfluidic Cell Migration Assay
The fabrication of the polydimethylsiloxane (PDMS)-based microchannel device using standard multilayer photolithography and replica molding has been reported earlier (33). Briefly, cells were seeded and treated according to the MTT protocol (described above; Supplementary Table S5;

Colony Formation Assay
DUTXR cells were seeded in a 6-well plate at 2.5 × 10 4 cells (mL/well), incubated overnight , and treated with CONV-TOPO, METRO-TOPO, CONV-DTX as a single agent, and CONV-DTX+METRO-TOPO in combination according to the MTT protocol (describe above; Supplementary Table S5; refs. 22,29). The cells were harvested from a 24-well plate at 1,000 cells/well and incubated for 1-2 weeks. The colonies were fixed with 100% methanol and stained with crystal violet. Images were taken of cell colonies using an EVOS FL digital cell imaging system (Thermo Fisher Scientific). Images were recorded in brightfield and phase-contrast modes at 1X magnification and analyzed using ImageJ software https://imagej.nih.gov/ij/.

Statistical Analysis
All tests were two sided. Differences with P values <0.05 were considered significant. ANOVA was performed for continuous outcomes, and the Benjamini-Hochberg multiple testing methods were used as a post hoc test. The two-group t test was used to perform differential GE analysis between groups and detect the DEGs. Genes with mean fold changes>|1| and P < 0.05 were considered as the threshold for reporting significant differential GE. Finally, heat maps were generated using unsupervised HC analysis based on the top DEGs. Kaplan-Meier curves were generated using survival time and censored data to plot survival against high versus low expression of significantly associated genes. All statistical analyses were performed using R v4.1.0 and GraphPad Prism v9.0.

Data Availability Statement
The datasets generated during or analyzed during the current study are available at the NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/ geo/), accession number GSE233341 or from the corresponding author upon reasonable request.

GEP Revealed Signatures for Each Prostate Cancer Subtype in EA versus AA Cell Lines
Baseline GEP identified DEGs for each prostate cancer subtype AR High /mCSPC   Fig. S2B). IPA also predicted HIF1α (P < 9.43E-06) and EMT (P < 5.63E-06) as key pathways associated with prostate cancer development in AA ( Supplementary Fig. S2C).

FIGURE 1 (Continued on the following page)
CD expression was also higher in PC-3, whereas CD was greater in PC-3M ( Fig. 1BII-IV). A comparison of other major EMT markers between PC3 and PC-3M revealed EZH, Snail/SNAI, Slug/SNAI, and TWIST, were expressed highly in PC-3M compared with PC-3, yet both are AR Low /mCRPC/NEPC prostate cancer subtypes (Fig. 5A). Minute levels of EMT markers were also expressed in AR High /mCSPC cell lines (22RV1 and LNCaP; Fig. 1BII-IV).
Notably, FSCN associated with taxane-resistant expressed highly in single-cell subclonal populations in AR Low /mCRPC/NEPC prostate cancer (PC3, PC3M, and DU145; Fig. 1BV). In addition, the intratumor heterogeneity within various cell lines is revealed by single-cell clusters (representing subclonal populations) determined by t-SNE/UMAP analysis based on the expression of top biomarkers for each subcluster. The relative representation of each subclone between the prostate cancer cell lines was also provided using comparative pie charts. Our results showed 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 (Fig. IBVI).
Next, we compared the EMT markers between taxane-sensitive (DU145) and the clonally derived acquired taxane-resistant mCRPC cell line DUTXR. Some common overlap in GE was observed between the DUTXR and DU145 subclones, as expected (Fig.1CI). However, the subclonal analysis showed a greater percentage of cells not expressing EMT markers in taxane-sensitive DU145 cells compared with the taxane-resistant DUTXR cell line ( Fig. 1CII-IV). This finding demonstrated that a greater percentage of DUTXR cells exhibited top EMT markers compared with DU145. The expression of VIM, LOXL, TGFB, TGFBR, UCHL, ANXAP, CD, and CD was greater in taxane-resistant DUTXR subclonal populations compared with taxane-sensitive DU145 ( Fig. 1CII-IV). While the expression of other prostate cancer EMT markers HAS-, AHNAK, TGFBR, ANXA, and ANXA was higher in DU145 compared with DUTXR ( Fig. 1CII-IV). Overall, the expression of EMT markers in subclonal cell populations was greatest in DUTXR.

RNA-seq versus scRNA-seq Analysis Identified Common and Unique DEGs Among all Prostate Cancer Subtypes
The analysis of RNA-seq and scRNA-seq data revealed 70 genes common to all prostate cancer subtypes, while scRNA-seq and RNA-seq analysis identified 582 and 73 unique DEGs, respectively (Supplementary Fig. S4; Supplementary Table S7). Among the common genes were EMT markers, for  Table S7).
The IPA based on the common DEGs between RNA-seq and scRNA-seq predicted cell invasion, movement, neoplasia, migration, transformation, metastatic, and growth as key pathway-associated prostate cancer aggressiveness ( Supplementary Fig. S5A). Furthermore, causal network pathway for  Fig. S5C). The S100 family, hepatic fibrosis, Rho family, RHOGDI, and integrin singling pathway signaling were identified as top canonical pathways for prostate cancer ( Supplementary Fig. S5D).

In Silico Analysis Using Prostate Cancer Patient Transcriptomes Validated the Clinical Relevance of Top EMT Markers
An in silico analysis using TCGA dataset showed that the top gene, FSCN, was significantly associated with disease-free survival (DFS), Kaplan-Meier curves (Fig. 2AI), with HR 2.2 (P = 0.00051). Low expression of FSCN was associated with longer DFS. In contrast, high expression of FSCN was associated with poor DFS. Another top EMT marker, TGFB, showed similar trends.
Low expression was associated with longer DFS, whereas high expression was associated with poor DFS (P = 0.00019, HR = 2.3; Fig. 2AII).

Immunoblotting Showed Higher EMT Protein Expression in AR Low /mCRPC/NEPC and Taxane-resistant AR Low /mCRPC/NEPC Compared with AR High /mCSPC Prostate Cancer Subtypes
Our RNA-seq DEGs, scRNA-seq, in silico, and in vitro flow cytometry results indicated that aggressive AR Low /mCRPC/NEPC and taxane-resistant

Metronomic Topotecan in Combination with DTX Reduced Cell Growth and Cell Density in AR Low /mCRPC/NEPC and Acquired Taxane-resistant AR Low /mCRPC/NEPC Prostate Cancer Subtypes
Previously we reported the cytotoxicity of CONV-TOPO, CONV-DTX, and METRO-TOPO for all cell lines (22,29). In this study, the effect of CONV-

Metronomic Topotecan in Combination with DTX Induced Apoptosis in AR Low /mCRPC/NEPC and Acquired Taxane-resistant AR Low /mCRPC/NEPC Prostate Cancer Subtypes
The    Table S1).

Metronomic Topotecan Treatment Reduced "Stem-like" Cell Load in AR Low /mCRPC/NEPC Prostate Cancer Subtypes
Flow cytometry analysis identified ALDH in PC-3 and PC-3M, indicating the presence of a "stem-like phenotype" (Fig. 5C). We also showed ALDH ex-  Table S2).

A Microfluidic Screen Showed Metronomic Topotecan Treatment is Potentially Effective Against Cell Invasion in AR Low /mCRPC/NEPC Prostate Cancer Subtypes
This experiment allowed us to study the effect of drug and dosing schedule as a single agent and in combination on tumor cell motility through microchannels of dimensions that mimic the size of confining pores and channel-like tracks encountered by migrating cells in vivo (36). Figure 5EI-

II (and Supplementary
Videos) showed that cell entry into confining (W × H = 3 × 10 μm 2 ) microchannels was greater in PC-3M compared with PC-3, suggesting that PC3-M cells are more invasive into mechanically challenging microenvironments. For confined (W × H = 10 × 10 μm 2 ) microchannels, cell entry was greater for PC-3M compared with PC-3 but the difference was not significant.
The effect of drug treatment on PC-3M migration was accessed. PC3-M entry into confining microchannels was markedly suppressed upon TOPO-METRO single-agent and TOPO-METRO+CONV-DTX combination treatment. Of note, combination therapy resulted in a higher reduction in the invasiveness of PC-3M compared with other treatments (Fig. 5EIII).

Metronomic Topotecan in Combination with DTX Reduced Decreased "Stem-like" Cell Load in Acquired AR Low /mCRPC/NEPC taxane-resistant Prostate Cancer Subtypes
The DUTXR cell line was treated with CONV-TOPO, METRO-TOPO, and combination CONV-DTX+METRO-TOPO. Pretreated and posttreated cells were stained with stemness marker antibodies CD44 and CD133 individually, and together (as described above), and analyzed by flow cytometry. These results showed a greater reduction in CD44 high ("stem-like") cell populations by METRO-TOPO (81.0%) compared with CONV-TOPO treatments (71.5%). Furthermore, combination treatment showed the greatest reduction (63.4%) of the "stem-like" CD44 high cell populations ( Fig. 6A; Table 2).
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 (

Metronomic Topotecan in Combination with DTX Reduced Proliferative Capacity in Acquired AR Low /mCRPC/NEPC taxane-resistant Prostate Cancer Subtypes
Next, we evaluated the potential effect of CONV-TOPO, CONV-DTX, METRO-TOPO, and combination (METRO-TOPO+CONV-DTX) treatment on the proliferative capacity of the DUTXR cells using the colony-forming assay. TOPO-METRO treatment alone significantly reduced colony number (16.4%) as well as colony size when compared with control or CONV-TOPO (67.0%) or CONV-DTX (33.5%) treatment. Furthermore, colony-forming assay results also showed the combination of METRO-TOPO+CONV-DTX treatments further reduced the colony numbers (9.53%) and size (Fig. 6B).

Discussion
Drug development for aggressive, lethal treatment-resistant prostate cancer poses a significant challenge with few therapeutic successes (4,12). We used scRNA-seq and bulk RNA-seq as an approach to demonstrate that EMT and cancer "stemness" signatures are key pathways to developing metastatically aggressive prostate cancer, including castration-resistant and taxane-resistant tumors in EA and AA. As shown in Fig. 1BVI  Earlier, we demonstrated that METRO-TOPO single-agent treatment is effective against prostate cancer animal xenograft model and cell lines (22). Here, we have extended our previous findings and have shown that METRO-TOPO is highly synergistic in combination with CONV-DTX. We performed pretreatment versus posttreatment scRNA-seq and RNA-seq analysis, which revealed that METRO-TOPO treatment abrogates stem-like cell types (representing NEPC and EMT phenotypes) in lethal prostate cancer. METRO-TOPO also reduced acquired taxane resistance by downregulating EMT gene and protein and by reducing the "stem-like" CD44 + cell population. Furthermore, in silico validation with TCGA prostate cancer patient cohort databases established the relevance of top DEGs in patient survival. Using comparative analysis with whole-genome transcriptomics data from patients with prostate cancer, we concluded that METRO-TOPO has the potential to be clinically effective based on the reverse matching of DEGs.
Importantly, we identified HAS as one of the top EMT markers in RNAseq and scRNA-seq for aggressive prostate cancer that is downregulated by TOPO-METRO treatment. Hyaluronan (HA) is an important constituent of the stem cell niche (34,35). CD44 is the major HA receptor and EMT marker in prostate cancer. High expression of HAS (4.8-fold upregulation) resulted in the secretion of large amounts of HA bound to CD44 + in the "stemlike" cell population and plays a critical role in the development CSCs by regulating cell adhesion, migration, proliferation, differentiation, cancer metastasis, and multidrug-resistant (37,38). In our study, HA was upregulated 4.8-fold in lethal prostate cancer subtypes. We also identified high levels of expression (4.07-fold upregulation) of EMP in our aggressive prostate cancer subtypes which regulates the expression of CD44 to promote stemness (39).  resistance (41). Some studies also suggested that CD44 plays a vital role in cancer stemness, specifically in prostate cancer (42,43). We observed that CD44 + cells (96%) frequency and protein expression were greatest in taxane-resistant mCRPC cell lines. Furthermore, we identified a higher percentage of CD44overexpressed subclonal population (scRNA-seq) in aggressive taxane-resistant DUTXR compared with taxane-sensitive DU145 prostate cancer subtypes. The ability to reduce stemness offers a potential strategy to treat aggressive and resistant prostate cancer. In our study, METRO-TOPO and combination (METRO-TOPO+CONV-DTX) treatment downregulated CD44 + (71.7% and 63.4%) cells along with protein expression (55% and 29%). We also observed high percentages of CD44 + cell populations in mCRPC cell lines and METRO-TOPO following single agent and in combination therapy with DTX reduced the amount of CD44 + cells significantly after treatment. Furthermore, METRO-TOPO treatment significantly decreased cell invasion and colony formation in aggressive and taxane-resistant forms of prostate cancer. Further studies employing CRISPR knockdown of HAS (and other top genes) are needed to further explore the significance of these novel mechanisms and their impact on cancer stemness and its association with METRO-TOPO treatment-related efficacy.
Here, we have identified VIM as a top common EMT marker from RNA-seq and scRNA-seq for aggressive prostate cancer subtypes. Earlier studies also identified its role in invasion and metastasis in prostate cancer (16,(44)(45)(46). We also showed that METRO-TOPO (6-week EE) treatment reduces VIM expression in subclonal populations.
Furthermore, SNAI plays a critical role in the aggressiveness of prostate cancer by increasing the expression of CD and vimentin (47). In our study, we detected greater expression of these markers in the subclonal population (scRNA-seq analysis) and RNA-seq GE (5.42-fold upregulation) of mCRPC/NEPC. SNAl is associated with cell proliferation and cell invasion in prostate cancer (41). We observed high expression (scRNA-seq) of SNAl and SNAl in mCRPC (PC-3 and PC-3M) prostate cancer subtypes. Our microfluidbased migration assay showed high cell invasion for these prostate cancer cell lines. Furthermore, METRO-TOPO as a single agent and in combination with DTX significantly reduced cell invasion in the mCRPC cell line model.
Furthermore, CD also promotes prostate cancer cell survival and metastatic lesion formation (48,49). Recent studies have identified CD as a new marker for invasive breast and prostate carcinoma (50). Our scRNA-seq and RNA-seq analysis agree with this finding. We observed greater expression of CD and CD (4.46-and 3.43-fold upregulation, respectively) in aggressive mCRPC/NEPC and taxane-resistant mCRPC/NEPC subtypes. We also identified a higher percentage of subclonal cell populations in aggressive taxane resistance prostate cancer subtypes overexpressing CD and

CD.
Earlier studies reported that ANXA, ANXA, and its pseudogene ANXAP are overexpressed in various cancers, including prostate cancer, and facilitated transcription of the stemness genes Nanog, Sox, and Oct (51)(52)(53). AHNAK upregulation is correlated significantly with advanced grades of various cancers and was associated with EMT (54). Our scRNA-seq analysis detected greater expression of ANXA, ANXA, ANXAP, and AHNAK in aggressive prostate cancer. Our RNA-seq data also corroborated with scRNA-seq finding Interestingly, RNA-seq and flowcytometry data revealed that EMT markers are enriched in EA compared with AA prostate cancer cell lines. An earlier study showed KRT, RUNX, GATA, SERPINB, and SLCa are important markers for prostate cancer progression (27), whereas we also identified KRT, RUNX as well as GATA, GATA-AS in our study. We also found KRT is upregulated in EA compared with AA cell lines, although in future, we need further explore its significance in prostate cancer progression.
Recent studies identified FSCN as a taxane-resistant marker in several solid tumors, including prostate cancer (56). We identified FSCN as a key upregulated gene (4.71-fold) for taxane resistance in aggressive prostate cancer. Our results also showed that the treatment of METRO-TOPO (6-weeks EE) downregulates FSCN expression in subclonal populations in aggressive mCRPC/NEPC 3D tumor models, whereas CONV-TOPO resulted in increased expression (P < 0.05). Furthermore, TCGA patient cohort also revealed lower expression of FSCN associated with better DFS (P = 0.00051).
We also identified Corf, TUBBB are common genes in taxane-resistant prostate cancer subtypes. Corf is a novel EMT biomarker for prostate cancer, and our study showed 17-fold and 24-fold downregulation of this gene in taxane-resistant prostate cancer subtypes DUTXR and PC-3TXR, respectively. Contrastingly, TUBBB was upregulated in both taxane-resistant prostate cancer cell lines, approximately 12-fold in DUTXR and approximately 43-fold in PC-3TXR. An earlier study revealed downregulation of Corf is associated with poor prognosis in patients with lung and prostate cancer (57). TUBBB is the major constituent of microtubules, the primary target for taxane-based drugs. TUBBB has also been associated with DTX resistance in prostate cancer (58,59).
Cell invasion is one of the major characteristics of EMT transition and cancer progression (60). Our microfluidic-based invasion assay supported the reduction in cell invasion potential, followed by METRO-TOPO treatments. Earlier, we demonstrated the increased potency of METRO-TOPO in animal models. However, in this study, our approach focused on in vitro model systems to better identify the mechanistic underpinnings of metronomic administration (two-dimensional and 3D tumor models) prostate cancer. Further preclinical validation and single-cell multi-omics strategies using CRISPR-based knockout xenograft models and patient-derived organoids/xenografts will be necessary. Our approach in this study promotes the understanding of subclonal molecular pathways underlying the differential patterns of prostate cancer aggressiveness and drug response among various prostate cancer subtypes. Overall, our study identified novel mechanisms of action that can serve as a pipeline to advance METRO-TOPO as a potent clinical-trial-ready therapeutic option for the management of lethal prostate cancer with stem-like features. These mechanisms will be key in identifying patients with molecular signatures that are sensitive to treatment, and, most importantly, can identify biomarkers that can be used to monitor treatment effectiveness and disease progression.