Cell crowding induces TRPV4 inhibition and its relocation to plasma membranes, implicating pro-invasive cell volume reduction mechanotransduction pathway
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eLife assessment
In this important study, Bu et al investigate how cell overcrowding triggers a mechano-transduction pathway involving TRPV4 channels, focusing on high-grade ductal carcinoma in situ (DCIS) cells. The authors show that cell crowding in these malignant cells leads to a reduction in cell volume and promotes a pro-invasive phenotype through calcium homeostasis and TRPV4 channel trafficking to the plasma membrane; this phenomenon is specific to invasive cell lines like MCF10CA and DCIS and is corroborated by patient tissue samples. The work suggests the role of TRPV4 in cell motility and mechanical sensing, offering potential therapeutic insights for targeting cancer metastasis. While the study presents robust and convincing data, the absence of TRPV4 genetic ablation is a critical limitation, which would further confirm its role in these processes.
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Abstract
Cell crowding is a common microenvironmental factor that affects various disease processes, but its impact on cell invasiveness into surrounding tissues is not well understood. In this study, we investigated the biomechanical changes induced by cell crowding, focusing on pro-invasive cell volume reduction. We discovered that cell crowding enhanced the invasiveness of high-grade ductal carcinoma in situ (DCIS) cells, which experienced significant cell volume reduction compared to hyperplasia-mimicking or normal cells. Mass spectrometry analyses revealed that cell crowding relocated ion channels, including TRPV4, a calcium-permeant ion channel, to the plasma membrane selectively in high-grade DCIS cells but not in less aggressive or normal cells. Cell crowding inhibited TRPV4 in high-grade DCIS cells, which led to decreased intracellular calcium levels and subsequent volume reduction. TRPV4 inhibition also prompted relocation of TRPV4 to the plasma membrane. This relocation primed inactive TRPV4 for activation, effectively counterbalancing the calcium loss from crowding-induced channel inhibition. Analyses of patient-derived breast cancer tissues validated that TRPV4 selectively associated with the plasma membrane in high-grade DCIS but not in lower-grade DCIS or less aggressive pathologies. The extent of plasma membrane TRPV4 association scaled with cell volume reduction and increased cell invasiveness and motility, suggesting its utility as an active pro-invasive mechanotransduction pathway indicator. Additionally, hyperosmotic conditions and pharmacologic TRPV4 inhibition mimicked the pro-invasive volume reduction observed under cell crowding, while TRPV4 activation reversed this effect by inducing cell volume increase. In summary, our study reveals a previously unrecognized pro-invasive mechanotransduction pathway triggered by cell crowding, which is selective in high-grade DCIS cells. This discovery offers new biophysical perspectives on cell invasiveness, highlighting the critical role of a selective mechanotransduction mechanism in the progression of breast cancer cells that are considered non-invasive but associated with high risk.
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eLife assessment
In this important study, Bu et al investigate how cell overcrowding triggers a mechano-transduction pathway involving TRPV4 channels, focusing on high-grade ductal carcinoma in situ (DCIS) cells. The authors show that cell crowding in these malignant cells leads to a reduction in cell volume and promotes a pro-invasive phenotype through calcium homeostasis and TRPV4 channel trafficking to the plasma membrane; this phenomenon is specific to invasive cell lines like MCF10CA and DCIS and is corroborated by patient tissue samples. The work suggests the role of TRPV4 in cell motility and mechanical sensing, offering potential therapeutic insights for targeting cancer metastasis. While the study presents robust and convincing data, the absence of TRPV4 genetic ablation is a critical limitation, which would further confirm its …
eLife assessment
In this important study, Bu et al investigate how cell overcrowding triggers a mechano-transduction pathway involving TRPV4 channels, focusing on high-grade ductal carcinoma in situ (DCIS) cells. The authors show that cell crowding in these malignant cells leads to a reduction in cell volume and promotes a pro-invasive phenotype through calcium homeostasis and TRPV4 channel trafficking to the plasma membrane; this phenomenon is specific to invasive cell lines like MCF10CA and DCIS and is corroborated by patient tissue samples. The work suggests the role of TRPV4 in cell motility and mechanical sensing, offering potential therapeutic insights for targeting cancer metastasis. While the study presents robust and convincing data, the absence of TRPV4 genetic ablation is a critical limitation, which would further confirm its role in these processes.
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Reviewer #1 (Public review):
Summary:
In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and pro-invasive phenotype.
In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10A) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasma membrane localization correlates with high-grade DCIS cells in patient tissue samples.
Reviewer #1 (Public review):
Summary:
In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and pro-invasive phenotype.
In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10A) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasma membrane localization correlates with high-grade DCIS cells in patient tissue samples.
Specifically in non-invasive MCF10DCIS.com cells, they showed that overcrowding or over-confluence leads to a decrease in cell volume and intracellular calcium levels. This condition also triggers the trafficking of TRPV4 channels from intracellular stores (nucleus and potentially endosomes), to the plasma membrane (PM). When these over-confluent cells are incubated with a TRPV4 activator, there is an acute and substantial influx of calcium, attesting to the fact that there are a high number of TRPV4 channels present on the PM. Long-term incubation of these over-confluent cells with the TRPV4 activator results in the internalization of the PM-localized TRPV4 channels.In contrast, cells plated at lower confluence primarily have TRPV4 channels localized in the nucleus and cytosol. Long-term incubation of these cells at lower confluence with a TRPV4 inhibitor leads to the relocation of TRPV4 channels to the plasma membrane from intracellular stores and a subsequent reduction in cell volume. Similarly, incubation of these cells at low confluence with PEG 3000 (a hyperosmotic agent) promotes the trafficking of TRPV4 channels from intracellular stores to the plasma membrane.
Strengths:
The study is elegantly designed and the findings are novel. Their findings on this mechano-transduction pathway involving TRPV4 channels, calcium homeostasis, cell volume plasticity, motility, and invasiveness will have a great impact in the cancer field and are potentially applicable to other fields as well. Experiments are well-planned and executed, and the data is convincing. The authors investigated TRVP4 dynamics using multiple different strategies- overcrowding, hyperosmotic stress, and pharmacological means, and showed a good correlation between different phenomena.
Weaknesses:
A major emphasis in the study is on pharmacological means to relate TRPV4 channel function to the phenotype. I believe the use of genetic means would greatly enhance the impact and provide compelling proof for the involvement of TRPV4 channels in the associated phenotype. In this regard, I wonder if siRNA-mediated knockdown of TRPV4 in over-confluent cells (or knockout) would lead to an increase in cell volume and normalize the intracellular calcium levels back to normal, thus ultimately leading to a decrease in cell invasiveness.
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Reviewer #2 (Public review):
Summary:
The metastasis poses a significant challenge in cancer treatment. During the transition from non-invasive cells to invasive metastasis cells, cancer cells usually experience mechanical stress due to a crowded cellular environment. The molecular mechanisms underlying mechanical signaling during this transition remain largely elusive. In this work, the authors utilize an in vitro cell culture system and advanced imaging techniques to investigate how non-invasive and invasive cells respond to cell crowding, respectively.
Strengths:
The results clearly show that pre-malignant cells exhibit a more pronounced reduction in cell volume and are more prone to spreading compared to non-invasive cells. Furthermore, the study identifies that TRPV4, a calcium channel, relocates to the plasma membrane both in …
Reviewer #2 (Public review):
Summary:
The metastasis poses a significant challenge in cancer treatment. During the transition from non-invasive cells to invasive metastasis cells, cancer cells usually experience mechanical stress due to a crowded cellular environment. The molecular mechanisms underlying mechanical signaling during this transition remain largely elusive. In this work, the authors utilize an in vitro cell culture system and advanced imaging techniques to investigate how non-invasive and invasive cells respond to cell crowding, respectively.
Strengths:
The results clearly show that pre-malignant cells exhibit a more pronounced reduction in cell volume and are more prone to spreading compared to non-invasive cells. Furthermore, the study identifies that TRPV4, a calcium channel, relocates to the plasma membrane both in vitro and in vivo (patient samples). Activation and inhibition of the TRPV4 channel can modulate the cell volume and cell mobility. These results unveil a novel mechanism of mechanical sensing in cancer cells, potentially offering new avenues for therapeutic intervention targeting cancer metastasis by modulating TRPV4 activity. This is a very comprehensive study, and the data presented in the paper are clear and convincing. The study represents a very important advance in our understanding of the mechanical biology of cancer.
Weaknesses:
However, I do think that there are several additional experiments that could strengthen the conclusions of this work. A critical limitation is the absence of genetic ablation of the TRPV4 gene to confirm its essential role in the response to cell crowding.
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Author response:
Reviewer #1 (Public review):
Summary:
In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and pro-invasive phenotype.
In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10CA) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasma membrane localization correlates with high-grade DCIS cells in patient …
Author response:
Reviewer #1 (Public review):
Summary:
In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and pro-invasive phenotype.
In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10CA) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasma membrane localization correlates with high-grade DCIS cells in patient tissue samples.
Specifically in invasive MCF10DCIS.com cells, they showed that overcrowding or over-confluence leads to a decrease in cell volume and intracellular calcium levels. This condition also triggers the trafficking of TRPV4 channels from intracellular stores (nucleus and potentially endosomes), to the plasma membrane (PM). When these over-confluent cells are incubated with a TRPV4 activator, there is an acute and substantial influx of calcium, attesting to the fact that there are a high number of TRPV4 channels present on the PM. Long-term incubation of these over-confluent cells with the TRPV4 activator results in the internalization of the PM-localized TRPV4 channels.
In contrast, cells plated at lower confluence primarily have TRPV4 channels localized in the nucleus and cytosol. Long-term incubation of these cells at lower confluence with a TRPV4 inhibitor leads to the relocation of TRPV4 channels to the plasma membrane from intracellular stores and a subsequent reduction in cell volume. Similarly, incubation of these cells at low confluence with PEG 3000 (a hyperosmotic agent) promotes the trafficking of TRPV4 channels from intracellular stores to the plasma membrane.
Strengths:
The study is elegantly designed and the findings are novel. Their findings on this mechano-transduction pathway involving TRPV4 channels, calcium homeostasis, cell volume plasticity, motility, and invasiveness will have a great impact in the cancer field and are potentially applicable to other fields as well. Experiments are well-planned and executed, and the data is convincing. The authors investigated TRVP4 dynamics using multiple different strategies- overcrowding, hyperosmotic stress, and pharmacological means, and showed a good correlation between different phenomena.
Weaknesses:
A major emphasis in the study is on pharmacological means to relate TRPV4 channel function to the phenotype. I believe the use of genetic means would greatly enhance the impact and provide compelling proof for the involvement of TRPV4 channels in the associated phenotype. In this regard, I wonder if siRNA-mediated knockdown of TRPV4 in over-confluent cells (or knockout) would lead to an increase in cell volume and normalize the intracellular calcium levels back to normal, thus ultimately leading to a decrease in cell invasiveness.
We greatly appreciate the positive feedback regarding the design of our study and the novelty of our findings. We also acknowledge the constructive suggestion to complement our pharmacological approaches with genetic manipulation of TRPV4.
In response to the comment regarding siRNA-mediated knockdown or knockout of TRPV4, we fully agree that this would further substantiate our findings. We will use shRNA targeting TRPV4 approaches to further explore the functional effects of TRPV4 knockdown on cell volume plasticity, intracellular calcium level changes, and invasiveness phenotypes through motility assays at the single cell level under cell crowding or hyperosmotic stress and will include these results in our revised manuscript.
Reviewer #2 (Public review):
Summary:
The metastasis poses a significant challenge in cancer treatment. During the transition from non-invasive cells to invasive metastasis cells, cancer cells usually experience mechanical stress due to a crowded cellular environment. The molecular mechanisms underlying mechanical signaling during this transition remain largely elusive. In this work, the authors utilize an in vitro cell culture system and advanced imaging techniques to investigate how non-invasive and invasive cells respond to cell crowding, respectively.
Strengths:
The results clearly show that pre-malignant cells exhibit a more pronounced reduction in cell volume and are more prone to spreading compared to non-invasive cells. Furthermore, the study identifies that TRPV4, a calcium channel, relocates to the plasma membrane both in vitro and in vivo (patient samples). Activation and inhibition of the TRPV4 channel can modulate the cell volume and cell mobility. These results unveil a novel mechanism of mechanical sensing in cancer cells, potentially offering new avenues for therapeutic intervention targeting cancer metastasis by modulating TRPV4 activity. This is a very comprehensive study, and the data presented in the paper are clear and convincing. The study represents a very important advance in our understanding of the mechanical biology of cancer.
Weaknesses:
However, I do think that there are several additional experiments that could strengthen the conclusions of this work. A critical limitation is the absence of genetic ablation of the TRPV4 gene to confirm its essential role in the response to cell crowding.
We are grateful for the positive assessment of our study and the acknowledgment of the impact of our findings on the understanding of mechanical signaling in cancer progression. We also appreciate the suggestion to include genetic ablation experiments to confirm the role of TRPV4 in cell crowding responses. As noted in our response to reviewer #1, we plan to use shRNA TRPV4 to examine the functional effects of TRPV4 knockdown on cell volume plasticity, changes in intracellular calcium levels, and invasive phenotypes through motility assays at the single-cell level under conditions of cell crowding or hyperosmotic stress. We will include these results in our revised manuscript.
Once again, we thank the reviewers for their valuable feedback, which will help us further improve our manuscript.
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