Editor's evaluation: Defining cellular population dynamics at single-cell resolution during prostate cancer progression (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigate prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in an in vivo murine model. We observe an expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients. Moreover, transformed epithelial cells and associated fibroblasts create a microenvironment conducive to pro-tumorigenic immune infiltration, which is partially androgen responsive. Androgen-independent prostate cancer leads to significant diversification of intermediate luminal cell populations characterized by a range of androgen signaling activity, which is inversely correlated with proliferation and mRNA translation. Accordingly, distinct epithelial populations are exquisitely sensitive to translation inhibition, which leads to epithelial cell death, loss of pro-tumorigenic signaling, and decreased tumor heterogeneity. Our findings reveal a complex tumor environment largely dominated by castration-resistant luminal cells and immunosuppressive infiltrates. Editor's evaluation Prostate cancer cellular heterogeneity is a major problem for disease progression and treatment resistance. This body of work addresses the cellular identity and populations that make up prostate cancer using single-cell sequencing technology, state-of-the-art mouse models and connections to human prostate cancer. The cellular identities, associated signaling networks, and immune complexes accompanying the heterogeneity of the prostate are identified in this work and a resource is provided for scientists in the field. https://doi.org/10.7554/eLife.79076.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Prostate cancer is the most diagnosed malignancy and second leading cause of cancer-related death in men in the United States (Siegel et al., 2021). This is largely due to the development of treatment resistant disease termed castration resistant prostate cancer (CRPC). Historically, CRPC has been considered a singular disease entity. However, this viewpoint has significantly evolved with the advent of next generation sequencing and the characterization of many distinct etiologies (Watson et al., 2015; Bluemn et al., 2017; Labrecque et al., 2019). Intratumoral heterogeneity is also common in prostate cancer, as several studies have highlighted both the complex cellular architecture of the prostate and multiple potential cell-of-origin models (Goldstein et al., 2010; Shen and Abate-Shen, 2010). For instance, while prostate epithelial cells canonically present as either basal cells or highly differentiated luminal cells, a distinct, more stemlike luminal population (Psca+/Krt4+/Tacstd2+) that primarily resides in the proximal prostate has previously been described (Xin et al., 2005; Wang et al., 2006; Korsten et al., 2009; Kwon et al., 2016; Sackmann Sala et al., 2017; McAuley et al., 2019). While the nomenclature for this cell type has varied, most studies have observed a similar set of biomarkers and characteristics, including increased stemlike and inflammatory/immunogenic signatures (Liu et al., 2016). These cells also appear to be castration-resistant; indeed, some studies show canonical prostatic luminal cells may undergo phenotype switching towards a more stemlike Krt4+ cell state in castrate conditions, reverting and helping repopulate the prostate during castration and regeneration cycles (Karthaus et al., 2020; Mevel et al., 2020; Guo et al., 2020; Kwon et al., 2021). This novel cell type was further verified using lineage tracing and single-cell technologies (Liu et al., 2016; Crowley et al., 2020; Joseph et al., 2020; Karthaus et al., 2020; Kwon et al., 2020; Mevel et al., 2020; Guo et al., 2020). Krt4+ cells are also found more rarely in the distal prostate, where they localize to invagination tips (Guo et al., 2020). These cell types remain incompletely characterized, including in their origin; some studies point to proximal luminal cells originating from the urethra, and therefore not being prostatic in nature (Joseph et al., 2020). Others recognize the presence of urethral cells but draw a distinction between them and prostatic Krt4+ proximal cells (Crowley et al., 2020). There is evidence that Krt4 + cells may contribute to tumor initiation in the prostate (Xin et al., 2005; Wang et al., 2006; Korsten et al., 2009; Kwon et al., 2016; Sackmann Sala et al., 2017; Guo et al., 2020). For instance, several studies find these cells enriched in precancerous lesions upon genetic perturbations in murine models (Wang et al., 2006; Korsten et al., 2009). Additionally, proximal cells appear less able to contribute to tumor initiation than those residing in distal prostate lobes (Korsten et al., 2009; Guo et al., 2020). These findings, combined with studies characterizing the role of Krt4+ cells in castration and regeneration, suggest they may contribute to treatment resistance in advanced cancer. However, this has yet to be established in CRPC models. While tumor heterogeneity contributes to treatment resistance, another source of poor clinical outcomes can be found in the consistent lack of response of CRPCs to immunotherapy. Prostate cancer has generally been described as 'immune-cold' due to the presence of multiple immunosuppressive cell types (Stultz and Fong, 2021). For example, tumor infiltration by myeloid-derived suppressor cells (MDSCs) has been implicated as an immunosuppressive phenotype (Garcia et al., 2014; Lopez-Bujanda et al., 2021). Metastatic CRPC also responds poorly to immune checkpoint inhibition and other immunotherapies (Cham et al., 2020). This has been confirmed clinically with autologous active cellular immunotherapy which only demonstrated a small (~4 month) improvement in survival in patients (Kantoff et al., 2010). Therefore, characterizing the immune microenvironment in advanced prostate cancer may be crucial to better understand this disease and inform potential therapeutic vulnerabilities or combinatorial strategies. Here, we have created an atlas of prostate cellular composition and phenotypic evolution through tumor initiation, progression, and hormone independence using a Pten loss murine model as an archetype. We observe a dramatic expansion of an intermediate luminal cell type in cancer and link this epithelial population to treatment resistance in multiple human cohorts. We also characterize increased pro-tumorigenic immune cell recruitment and define cell-cell signaling patterns that contribute to lymphoid and myeloid cell expansion. In addition, using a tissue-specific transgenic model, we demonstrate that cell type specific protein synthesis is essential for the maintenance of tumor heterogeneity in both basal and intermediate cells in the context of AR-low prostate cancer. Finally, we make our data available for further study through an interactive portal, with the aim of providing a broad resource for the cancer research community. Together, our findings highlight the cell type-specific and patient relevant features of prostate cancer progression and demonstrate the utility of single-cell technologies to uncover novel cell-specific paradigms of treatment resistance. Results Characterization of WT and Ptenfl/fl ventral prostates at single cell resolution To determine the cellular architecture of prostate cancer initiation, we conducted single-cell RNA sequencing (scRNAseq) of the ventral prostates of 6-month-old wild-type (WT) and PB-Cre4;Ptenfl/fl;ROSA26-rtTA-IRES-eGFP (herein referred to as Ptenfl/fl, see Methods) prostate cancer mice (Figure 1A). We chose the ventral lobe because it has been well-characterized throughout prostate cancer progression at a deep molecular level and for the abundance of cellular populations (Hsieh et al., 2012; Hsieh et al., 2015; Liu et al., 2019). In the Ptenfl/fl model, exon 5 of the tumor suppressor Pten is deleted within basal and luminal epithelial cells of the prostate and mice uniformly develop prostate cancer (Wang et al., 2003). PTEN is a negative regulator of the oncogenic PI3K-AKT-mTOR signaling pathway, which is deregulated in nearly all advanced prostate cancer patients (Taylor et al., 2010). Figure 1 with 2 supplements see all Download asset Open asset Proliferative split in basal cancer cells enables expansion of intermediate cells. (A) Simplified schematic of single-cell RNA sequencing of WT and Ptenfl/fl ventral prostates. (B) UMAP of WT and Ptenfl/fl epithelial cells. Left, both conditions superposed; middle, WT only; right, Ptenfl/fl only. Epithelial cell types are demarcated by color (red = basal, light blue = luminal progenitor, dark blue = intermediate, green = differentiated). (C) Relative abundance of epithelial cells in WT (n=3) and Ptenfl/fl (n=2) mice. Y-axis shows the % composition of each sample by cell type (***p<0.001, negative binomial test). Data presented as +/-SD. (D) Top GSEA results enriched in Ptenfl/fl compared to WT for each epithelial subtype. Intermediate cells in Ptenfl/fl were compared to luminal progenitor cells in WT. All pathways are enriched with FDR <0.05. (E) Proliferation signature (CCP) composite score in epithelial cells, clustered by condition (Data presented as +/-SD, *p<0.05, n.s.=not significant, permutation test). N/A indicates missing data due to no cells being present in the condition. WT n=3, Ptenfl/fl n=2. (F) UMAP visualization of cell cycle phase assignment per cell, showing % cells in non-G1 (S or G2/M) (black border = WT basal cells, blue border = hypo-proliferative basal cells in Ptenfl/fl, and red border = hyper-proliferative basal cells in Ptenfl/fl. *p<0.05, chi-square test). (G) GSEA between hyper- and hypo-proliferative basal clusters in Ptenfl/fl. All pathways are enriched with FDR <0.05. (H) RNA velocity analysis of Ptenfl/fl epithelial cells; highlighted section shows intersection of basal and intermediate cells. (I) Pseudotime trajectories drawn by Palantir through the basal and intermediate compartments, with hypo-proliferating basal cells as the designated start point. (J) Expression of important cell fate and differentiation regulators along basal-intermediate trajectory. Quality control and filtering (read count thresholds and excluding cells with high mitochondrial content) resulted in transcriptomes from 24,079 total cells across five samples (Supplementary file 1A-C). Using SingleR, we identified epithelial, stromal, and immune cell types (Aran et al., 2019; Figure 1—figure supplement 1A). To confirm that the epithelial cells in the Ptenfl/fl model underwent Cre-mediated recombination, we measured the frequency of rtTA-eGFP fusion mRNA in WT and Ptenfl/fl mice. The rtTA-eGFP transcript was present in 14.5% of Ptenfl/fl epithelial cells (Figure 1—figure supplement 1B). Given that on average ~1200 genes/cell were detected and ~24,000 genes were identified overall, we expect the average transcript to be found in ~5% of cells in the dataset. Therefore, our finding suggests widespread recombination, and therefore loss of Pten, within the epithelial compartment. Importantly, recombination was not observed in WT mice or non-epithelial cell types (Figure 1—figure supplement 1B). We further defined the epithelial population in WT mice via signatures and biomarkers obtained from the Strand (Basal, Urethral, and VP), Sawyers (Basal, L1, and L2), and Xin (Sca-1+) groups (Joseph et al., 2020; Karthaus et al., 2020; Liu et al., 2016). We found canonical basal (Krt5+/Sox4+) and luminal (Nkx3-1+/Sbp+) cells, as well as two distinct clusters expressing luminal progenitor markers (Psca+/Tactsd2+/Krt4+) (Figure 1—figure supplement 1C–D). These clusters were mainly distinguished by different basal, L2, and luminal (VP/L1) signature scores. In addition, one cluster clearly expressed previously identified urethral markers Aqp3 and Ly6d, and was negative for other progenitor markers (Ppp1r1b, Clu; Figure 1—figure supplement 1C; Crowley et al., 2020). Accordingly, we designated this cluster as urethral. The other Krt4+ cluster likely represents progenitor prostatic cells, either from the proximal or distal prostate (Crowley et al., 2020; Guo et al., 2020). Therefore, we refer to this cluster as luminal progenitor hereafter (Figure 1—figure supplement 1D). We then examined epithelial cell subtypes in Ptenfl/fl mice. We identified basal, urethral, and canonical luminal cells, as well as a large cluster of cells similar to WT luminal progenitor cells, characterized by expression of Krt4/Tacstd2/Ppp1r1b but not Psca (Figure 1—figure supplement 1E–F). Clustering urethral cells with luminal progenitor and this new Ptenfl/fl cluster, we find that the urethral cells cluster apart from the other cell types and express significantly lower levels of prostate luminal or pan-epithelial markers (Krt8, Krt18, Epcam) (Figure 1—figure supplement 1G–H). This supports the hypothesis that we have identified both urethral and prostate cells, and likely indicates a prostatic origin for the Ptenfl/fl cluster. We designated Krt4+/Tacstd2+/Ppp1r1b+ cells in Ptenfl/fl mice as 'intermediate' cells (Figure 1—figure supplement 1E). Pten loss generates differentially proliferating populations and a dominance of intermediate luminal cell states from multiple cellular origins Next, we sought to determine how the epithelial compartment is remodeled in the context of tumor initiation. We excluded the urethral cluster because it is not prostatic in origin and therefore is less likely to significantly impact cellular dynamics in prostate cancer. We observed a dramatic increase in the proportion of intermediate cells in Ptenfl/fl mice compared to luminal progenitor abundance in the WT setting (Figure 1B–C). To investigate potential mechanisms of this expansion of the intermediate population, we identified differentially expressed genes (DEG) and performed gene set enrichment analysis (GSEA) on each epithelial subtype, comparing the WT and Ptenfl/fl epithelial compartments (Ptenfl/fl basal:WT basal, Ptenfl/fl intermediate:WT luminal progenitor, Ptenfl/fl luminal:WT luminal) (Supplementary file 1D). We noted enrichment of oncogene and tumor suppressor pathways in the Ptenfl/fl mice, including upregulation of AKT and mTOR, which are expected in this model. We also observed increased Wnt signaling and metabolic processes across all epithelial cell types. Interestingly, both epithelial cell migration and proliferation were enriched in most epithelial cells (Figure 1D). These findings suggest that intermediate cell expansion could be mediated by increased proliferation of luminal progenitors, or by increased transdifferentiation of other cell types, which has been widely characterized in WT settings (Karthaus et al., 2020; Mevel et al., 2020; Kwon et al., 2021). To evaluate the possibility of increased proliferation of different epithelial cell types, we used the Cell Cycle Progression (CCP) score, a proliferation gene signature developed and validated in human prostate cancer patients (Cuzick et al., 2011), and generated a composite score (see Materials and methods) in our dataset. Surprisingly, while there was a significant increase in CCP score in basal cells in Ptenfl/fl mice compared to WT, we observed no significant change in intermediate cells compared to luminal progenitor cells (Figure 1E). To further characterize epithelial proliferation, we performed cell cycle scoring, assigning one of three phases (G1, G2/M, or S) to each cell in the dataset, and found a striking split in the basal compartment of Ptenfl/fl mice, but not WT mice (Supplementary file 1E). In Ptenfl/fl mouse prostates, 18.6% of basal cells were hyper-proliferative, with 99.7% of these cells in a proliferative phase (G2/M or S phases). The rest of the basal compartment only had 52.2% of cells in a G2/M or S phase, lower than WT basal cells (68.5%) (Figure 1F, Figure 1—figure supplement 2A). We also verified that the increase in basal cell CCP score was specifically due to this hyper-proliferative cluster (Figure 1—figure supplement 2B). One possibility for this differential increase in basal cell proliferation is higher Cre-mediated recombination efficiency in some clusters over others. We analyzed transgene abundance in our basal subclusters and found that in the Ptenfl/fl mouse, 13.6% (12.0%–15.7%) of hypo-proliferating and 19.6% (19.1%–20.3%) of hyper-proliferating basal cells express the rtTA-eGFP transgene. This <1.5-fold difference does not account for the >twofold increase in cycling cells, or the ~threefold increase in CCP score observed between the subclusters. As such, we conclude that Pten loss in the murine prostate promotes a dual phenotype in basal cells, with most cells displaying decreased proliferation while a subset becomes hyper-proliferative. We further characterized the basal subclusters in Ptenfl/fl mice by performing DEG analysis followed by GSEA (Supplementary file 1F). As expected, we observed several cell-cycle-related pathways enriched in the hyper-proliferative cluster. However, the hypo-proliferative basal cluster was enriched for several migration, development, and differentiation signatures (Figure 1G). Transdifferentiation of basal cells to luminal cells in the context of malignant transformation has previously been reported in prostate cancer (Goldstein et al., 2010; Choi et al., 2012; Lu et al., 2013; Wang et al., 2013). As such, we hypothesized that the hypo-proliferative basal subcluster might transition into intermediate luminal cells, thus providing one potential mechanism for the expansion of this cell type in the absence of increased proliferation. To verify this, we generated pseudotime trajectories using Monocle3 (Cao et al., 2019), which confirmed a direct path from hypo-proliferative basal cells to intermediate luminal cells (Figure 1—figure supplement 2C). We also performed RNA velocity, a technique to visualize differentiation dynamics on a per cell basis (La Manno et al., 2018). This analysis revealed significant movement from hypo-proliferative, but not hyper-proliferative basal cells to intermediate cells (Figure 1H). Finally, we used a pseudotime algorithm (Setty et al., 2019) to identify two potential trajectories starting from the hypo-proliferative basal cells and ending either in the hyper-proliferative basal cluster or in the intermediate luminal cell compartment (Figure 1I). We generated clusters of genes with similar expression patterns across these pseudotime axes (Figure 1—figure supplement 2D) and performed GSEA on the clusters. We found that several genes involved in cell fate decisions including Pdgfb, Trp63, and Shh are highly expressed early but rapidly decrease over the trajectories. Conversely, genes implicated in differentiation such as Notch1 increased over the basal-intermediate path, but not the basal-basal path (Figure 1J). This suggests that hypo-proliferating basal cells strongly express development markers associated with cell fate choice, but these genes are not expressed in hyper-proliferative basal cells or intermediate cells. Moreover, intermediate cells express higher levels of differentiation-associated genes than either basal subcluster. Together, these findings support the idea that hypo-proliferative basal cells transdifferentiate into intermediate luminal cells during tumorigenesis, while the basal compartment may be replenished by a hyper-proliferative, non-differentiating population. Multiple studies have suggested that prostate luminal cells can phenotype switch to intermediate-like states under conditions such as castration (Karthaus et al., 2020; Guo et al., 2020). To verify whether luminal populations may contribute to the intermediate cell state, we plotted the expression of genes canonically expressed in prostate luminal cells and found that while luminal cells did express these genes most strongly, there was overlap with a population of intermediate cells immediately adjacent to the luminal cells in Ptenfl/fl mice (Figure 1—figure supplement 2E). This suggests that luminal cells may phenotype switch into an intermediate state and retain some identity markers in the context of Pten loss. It is also possible that luminal progenitor cells contribute to the intermediate population, as Krt4+ cells within the proximal and distal prostate have been described as tumor initiating cells after Pten loss (Guo et al., 2020). Overall, our findings support multiple cells of origin for Krt4+ intermediate cells in Ptenfl/fl mice, including phenotype switching of basal and luminal cells, as well as potential expansion of luminal progenitor cells. In vivo lineage tracing studies using highly precise Cre-drivers will likely be necessary to fully disentangle the cellular origins of intermediate cell states in the Ptenfl/fl prostate cancer model. Immune infiltration increases in Ptenfl/fl mice and is pro-tumorigenic Our GSEA of all three epithelial cell types showed an enrichment for immune-related pathways in Ptenfl/fl mice, suggesting increased immunogenic signaling relative to WT mice (Figure 2A). To determine the impact of epithelial Pten loss on immune cells, we calculated the relative abundance of immune cells in WT and Ptenfl/fl mice. We found that T cells, macrophages, neutrophils, and dendritic cells were all significantly expanded in the Ptenfl/fl mouse; in fact, only B cells were not expanded compared to WT (Figure 2—figure supplement 1A–B). Immune infiltration has previously been characterized as immunosuppressive in PTEN null prostate tumors (Garcia et al., 2014). Therefore, we expected these expanding immune populations to be immunosuppressive and therefore pro-tumorigenic. To test this hypothesis, we assigned activation states or subtypes to immune cells based on known biomarkers (Figure 2B, Figure 2—figure supplement 1C–E). First, we characterized the neutrophil population as myeloid-derived suppressor cells (MDSCs) based on published biomarkers (Alshetaiwi et al., 2020; Figure 2—figure supplement 1C). We also found three macrophage cell states: tumor-associated macrophages (TAMs), M2-activated macrophages, and M1-activated macrophages. Interestingly, the M1 cells expressed the markers and and as a we termed them macrophages Figure 2—figure supplement 1D). Finally, we characterized three T cell T cells, T cells, and T cells. and T cells expressed the markers of and and suggesting their may be in the context of Pten loss (Figure 2—figure supplement 1E). All these cell types for cells and are expanded in Ptenfl/fl mice, a potential role for these immune cells in or a pro-tumorigenic prostate tumor microenvironment (Figure 2C). Figure 2 with 1 supplement see all Download asset Open asset Immune recruitment in Ptenfl/fl prostates is mediated by both epithelial and immune cell (A) Top immune-related GSEA results enriched in Ptenfl/fl compared to WT mice for each epithelial subtype. All pathways are enriched with FDR <0.05. (B) UMAP visualization of immune cells by cell or (C) Relative abundance of immune cells in WT (n=3) and Ptenfl/fl (n=2) mice. Y-axis shows the % composition of each sample by cell type (Data presented as +/-SD, n.s.=not significant, negative binomial test). (D) of signaling between macrophages and epithelial cells. from cell-cell are the gene in a is expressed in the cell in the cell-cell (E) of epithelial and macrophage gene expression in WT and Ptenfl/fl mice. (F) of and expression in and macrophages in WT and Ptenfl/fl ventral prostates. are only present in Ptenfl/fl and therefore not have a WT by (G) of signaling between cells and epithelial cells and macrophages. (H) of T cell and epithelial and macrophage gene expression in WT and Ptenfl/fl ventral prostates. macrophages, and are generally pro-tumorigenic et al., et al., 2014; et al., 2014). with T cells, this suggests a pro-tumorigenic immune We hypothesized that cell-cell signaling originating from tumor epithelial cells may an important role in immune cells to the To cell-cell in our we used a and algorithm that the of specific between cell groups et 2020). We found between epithelial subtypes and macrophages and such as the that point to active recruitment (Figure file in the epithelial basal, intermediate, and differentiated cell populations were more highly expressed in Ptenfl/fl mice than in WT mice, which to the increased macrophage abundance in Ptenfl/fl mice (Figure 2E). Additionally, we observed increased between fibroblasts and upon Pten suggesting that fibroblasts in prostate cancer may also an active role in macrophage recruitment (Figure 2—figure supplement 1F). are all significantly in Ptenfl/fl fibroblasts (Figure 2—figure supplement 1G). Interestingly, is expressed highly in and in cancer, but not in (Figure 2—figure supplement 1H). The lack of signaling to a possible cellular for the lower abundance of this macrophage in Ptenfl/fl mice compared to WT. possibility is that and transition into macrophages, in response to epithelial Overall, these findings highlight a role for epithelial cells and fibroblasts in pro-tumorigenic macrophages to the prostate in the context of Pten loss. While we observed significant epithelial to macrophage signaling, the between epithelial cells and were characterized by expressed in epithelial cells and expressed in As such, it is that epithelial cells are for the expansion of in the prostate (Figure 2—figure supplement 1I). we noted increased expression of the in and a increase in expression in macrophages. These genes are known et al., which suggests that are by M2-activated macrophages (Figure Interestingly, are not expressed in or the of Finally, we examined between epithelial cells and T cells. We found both and between T cells and epithelial cells. We found these between macrophages and cells, as well as (Figure and to the of T cell has a while is and both are by the and and and 2009). These findings a signaling with both tumor and macrophages and the of T cells. We noted that expression of and suggesting that similar to may specifically be for abundance and (Figure Overall, our data suggests that pro-tumorigenic macrophages are by epithelial and signaling in Pten null These macrophages then tumor signaling in the immune including and T cells and pro-tumorigenic Given expression macrophages may be mainly for recruitment and for T cell These findings reveal a complex signaling with multiple of expression in to a microenvironment to tumor from intermediate cell heterogeneity resistance to androgen examined epithelial and immune populations in prostate cancer initiation, we sought to determine how these cells over the of castration resistance. It has been that castration of Ptenfl/fl mice leads to the of AR-low tumors (Liu et al., 2019). As such, we conducted in Ptenfl/fl mice with and castration at of and the that in the epithelial compartment (Figure the intermediate luminal cell population to while the differentiated luminal cells (Figure We