Defining cellular population dynamics at single-cell resolution during prostate cancer progression
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Evaluation Summary:
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 and state-of-the-art mouse models. 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.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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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.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, Germanos et al present preclinical evidence of a dynamic interplay between tumor microenvironmental elements underlying prostate cancer initiation, progression, and emerging therapeutic resistance in the transgenic mouse model. The authors identify an intermediate luminal cell population trans-differentiating from a hypo-proliferative basal cell subset, meanwhile, hyper-proliferative basal cells replenish a non-differentiating basal subpopulation. The meticulous methodologic approach identifies candidate cellular interactions in fibroblasts, MDSCs, and immune cell populations associated with PTEN loss. The generalization of these findings to human data sets is of particular interest and recommended for future studies on this topic. Mechanistic studies with …
Author Response
Reviewer #1 (Public Review):
In this manuscript, Germanos et al present preclinical evidence of a dynamic interplay between tumor microenvironmental elements underlying prostate cancer initiation, progression, and emerging therapeutic resistance in the transgenic mouse model. The authors identify an intermediate luminal cell population trans-differentiating from a hypo-proliferative basal cell subset, meanwhile, hyper-proliferative basal cells replenish a non-differentiating basal subpopulation. The meticulous methodologic approach identifies candidate cellular interactions in fibroblasts, MDSCs, and immune cell populations associated with PTEN loss. The generalization of these findings to human data sets is of particular interest and recommended for future studies on this topic. Mechanistic studies with multi-cellular co-culture models are needed to extend and validate the findings in this report.
We thank the reviewer for finding our research “meticulous” in its approach. We agree that validating our findings in human contexts is a vital next step and have added new orthogonal datasets in the revised manuscript (Figure 4D-E). We also agree that complex molecular studies will be needed to fully evaluate our cell-cell interaction hypotheses. To this end, we have elaborated on appropriate follow-up studies in the discussion (Lines 625-628, 642-643, 657-659, 675-677).
Strengths and Weaknesses:
The study focuses on a clinically highly relevant and timely topic. The strength of this manuscript is the meticulous description of the Methods and model development and the integration of state-of-the-art orthogonal data sets. However, the number of data points across the experiments (n = 2 or 3) with considerable variability in the Ptenfl/fl group limits the interpretation of findings. Additionally, further experiments are needed to validate these observations in human prostate cancer and establish the potential translational relevance of these findings.
We are ecstatic that the reviewer finds our study “clinically highly relevant.” We agree that the low sample size is a potential limitation but believe that our overall results are robust and enable concrete conclusions for both epithelial and immune cell populations. This is in part because we validated our findings in orthogonal human datasets (Figure 4A-C, Figure 5H) in the original manuscript. However, to add rigor to our study, we have conducted new scRNAseq analysis showing that our findings correlate well with both human patient data (Figure 4D-E) and orthogonal mouse models (Figure 4F-G). Furthermore, we conducted additional scRNAseq on castrated WT murine prostate to demonstrate how castration plays an important role in translational heterogeneity in intermediate cells (Figure 4H, Figure 3 – figure supplement 1G).
As such, the report is fairly descriptive, and expanding the discussion on the mechanistic studies needed to identify which of these interactions drives aggressive prostate cancer would improve this report.
We agree with the reviewer that additional discussion of follow-up studies is necessary. As such, we have updated the discussion to highlight the molecular studies needed to fully characterize the cellular phenotypes described in this manuscript (Lines 625-628, 642-643, 657-659, 675-677).
Reviewer #2 (Public Review):
This work provides a thorough characterization of tumor cell and microenvironment dynamics in a castrate Pten null prostate cancer model and details the strength of cellular interactions using single-cell RNA sequencing. The search for a preexisting castrate-resistant prostate progenitor has been upended in recent years with the discovery that prostate luminal cells adapt to low androgen environments by undergoing lineage plasticity rather than an expansion of proximal progenitors. This paper provides indirect evidence that basal epithelia give rise to 'intermediate' epithelia through increased translation in intact and castrate Pten null mice cells, which is validated in a Pten null, 4ebp1 mutant mouse model.
Strengths:
The single-cell data are robust and expertly presented in the figures. The methods are largely appropriate and the delineation of experimental protocols is straightforward. The analysis is comprehensive and well described in relation to biological questions of interest to the community. The validation of the effect of translation on prostate epithelial viability in relation to initial findings advances our understanding of how cells survive in low androgen environments. The addition of a public portal for the data is highly useful.
We thank the reviewer for evaluating our work as “robust and expertly presented,” “comprehensive,” and “highly useful.”
In response to the reviewer’s in-depth comments, we have revised our nomenclature of WT epithelial cell subtypes to specifically distinguish between Krt4+/Tacstd2+ urethral, prostatic, and cancer-derived cells (Lines 163-185). We now find urethral and luminal progenitor groups in WT intact mice, which are distinct from “intermediate” cells arising from Pten loss (Figure 1 – figure supplement 1D-F). We have accordingly revised our interpretation of the potential origins of these intermediate cells in cancer (Lines 256-275).
Weaknesses:
The PB-Cre4 promoter seems to be promiscuously inactivating Pten in basal, intermediate, and luminal cells, which is problematic as this confounds the ability to differentiate between cells that are undergoing lineage plasticity vs. expansion of a pre-existing progenitor cell type. Much recent evidence points to lineage plasticity of prostate luminal tumor cells under androgen deprivation rather than survival and expansion of a pre-existing castrate-resistant basal or intermediate cell type. Accordingly, the observation that basal epithelia may transdifferentiate to intermediate epithelia or that a pre-existing intermediate luminal cell state is expanded under castration may be artifacts of the model without reproduction in human prostate cancer. The use of trajectory analysis of single-cell data to demonstrate basal or intermediate cell lineage transdifferentiation is a weaker type of evidence than the lineage tracing of individual cell types provided by other groups, which argue against transdifferentiation and for lineage plasticity.
This is a very thoughtful and nuanced comment. We agree that the PB-Cre4 promoter is promiscuously inactivating Pten in basal, luminal progenitor cells, and luminal cells which does confound the ability to differentiate between cells that are undergoing lineage plasticity versus expansion of pre-exisiting progenitor cell types. As such, we now expand our results section to include non-basal routes to the expansion of the Pten intermediate cell population (Lines 261-275). Furthermore, we also comprehensively discuss the limitations of our models in the discussion section highlighting the need to validate our findings using lineage tracing or newer techniques such as DNA Typewriter (Lines 616-628) (Choi et al., Nature 2022).
Currently it is not possible to conduct lineage tracing within the human prostate making it impossible to determine if basal epithelia may transdifferentiate to intermediate epithelia or if a pre-existing intermediate luminal cell state is expanded under castration. However, we do present new human scRNAseq data that the intermediate cell state, as reflected by the 5-gene castration signature, is enriched specifically in metastatic, but not localized prostate cancer (Figure 4D-E). Furthermore, we show that this gene signature is also relevant in a completely different progression model of murine prostate cancer (Figure 4F-G). Thus, while not perfect, our model does have potential human relevance despite the limitations which we address in the manuscript (Lines 261-275, 616-628).
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Evaluation Summary:
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 and state-of-the-art mouse models. 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.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Germanos et al present preclinical evidence of a dynamic interplay between tumor microenvironmental elements underlying prostate cancer initiation, progression, and emerging therapeutic resistance in the transgenic mouse model. The authors identify an intermediate luminal cell population trans-differentiating from a hypo-proliferative basal cell subset, meanwhile, hyper-proliferative basal cells replenish a non-differentiating basal subpopulation. The meticulous methodologic approach identifies candidate cellular interactions in fibroblasts, MDSCs, and immune cell populations associated with PTEN loss. The generalization of these findings to human data sets is of particular interest and recommended for future studies on this topic. Mechanistic studies with multi-cellular co-culture models …
Reviewer #1 (Public Review):
In this manuscript, Germanos et al present preclinical evidence of a dynamic interplay between tumor microenvironmental elements underlying prostate cancer initiation, progression, and emerging therapeutic resistance in the transgenic mouse model. The authors identify an intermediate luminal cell population trans-differentiating from a hypo-proliferative basal cell subset, meanwhile, hyper-proliferative basal cells replenish a non-differentiating basal subpopulation. The meticulous methodologic approach identifies candidate cellular interactions in fibroblasts, MDSCs, and immune cell populations associated with PTEN loss. The generalization of these findings to human data sets is of particular interest and recommended for future studies on this topic. Mechanistic studies with multi-cellular co-culture models are needed to extend and validate the findings in this report.
Strengths and Weaknesses:
The study focuses on a clinically highly relevant and timely topic. The strength of this manuscript is the meticulous description of the Methods and model development and the integration of state-of-the-art orthogonal data sets. However, the number of data points across the experiments (n = 2 or 3) with considerable variability in the Ptenfl/fl group limits the interpretation of findings. Additionally, further experiments are needed to validate these observations in human prostate cancer and establish the potential translational relevance of these findings.As such, the report is fairly descriptive, and expanding the discussion on the mechanistic studies needed to identify which of these interactions drives aggressive prostate cancer would improve this report.
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Reviewer #2 (Public Review):
This work provides a thorough characterization of tumor cell and microenvironment dynamics in a castrate Pten null prostate cancer model and details the strength of cellular interactions using single-cell RNA sequencing. The search for a preexisting castrate-resistant prostate progenitor has been upended in recent years with the discovery that prostate luminal cells adapt to low androgen environments by undergoing lineage plasticity rather than an expansion of proximal progenitors. This paper provides indirect evidence that basal epithelia give rise to 'intermediate' epithelia through increased translation in intact and castrate Pten null mice cells, which is validated in a Pten null, 4ebp1 mutant mouse model.
Strengths:
The single-cell data are robust and expertly presented in the figures. The methods are …Reviewer #2 (Public Review):
This work provides a thorough characterization of tumor cell and microenvironment dynamics in a castrate Pten null prostate cancer model and details the strength of cellular interactions using single-cell RNA sequencing. The search for a preexisting castrate-resistant prostate progenitor has been upended in recent years with the discovery that prostate luminal cells adapt to low androgen environments by undergoing lineage plasticity rather than an expansion of proximal progenitors. This paper provides indirect evidence that basal epithelia give rise to 'intermediate' epithelia through increased translation in intact and castrate Pten null mice cells, which is validated in a Pten null, 4ebp1 mutant mouse model.
Strengths:
The single-cell data are robust and expertly presented in the figures. The methods are largely appropriate and the delineation of experimental protocols is straightforward. The analysis is comprehensive and well described in relation to biological questions of interest to the community. The validation of the effect of translation on prostate epithelial viability in relation to initial findings advances our understanding of how cells survive in low androgen environments. The addition of a public portal for the data is highly useful.Weaknesses:
The PB-Cre4 promoter seems to be promiscuously inactivating Pten in basal, intermediate, and luminal cells, which is problematic as this confounds the ability to differentiate between cells that are undergoing lineage plasticity vs. expansion of a pre-existing progenitor cell type. Much recent evidence points to lineage plasticity of prostate luminal tumor cells under androgen deprivation rather than survival and expansion of a pre-existing castrate-resistant basal or intermediate cell type. Accordingly, the observation that basal epithelia may transdifferentiate to intermediate epithelia or that a pre-existing intermediate luminal cell state is expanded under castration may be artifacts of the model without reproduction in human prostate cancer. The use of trajectory analysis of single-cell data to demonstrate basal or intermediate cell lineage transdifferentiation is a weaker type of evidence than the lineage tracing of individual cell types provided by other groups, which argue against transdifferentiation and for lineage plasticity. -
