Cell crowding selectively increases invasiveness in high-grade DCIS cells.

(A) We used a collagen-crosslinked polyacrylamide hydrogel matrix-based invasion assay to assess the effect of cell crowding on invasiveness. Representative images show gelatin-Alexa488 conjugates, where dark areas indicate cell invasion through degradation (green; Gelatin488), and DAPI staining marks cell locations (blue; DAPI) in a two-day invasion assay of MCF10DCIS.com cells under normal density (ND; upper panel) and overconfluent (OC; lower panel) conditions. "Masked" images are thresholded to produce positive masks applied to the “DAPI” images. Individual cell locations detected in “DAPI” images are marked with purple circles in “Detected points” images. The total number of cells within the field of view is counted from these points. By overlaying the mask and DAPI images, “Masked DAPI” images are obtained, and invaded cells are detected and represented by purple circles in “Masked points” images. The invasive cell fraction is calculated by the ratio of the number of invaded cells to the total number of cells (0.24 for ND and 0.59 for OC MCF10DCIS.com cells). These data show that cell invasiveness is enhanced by cell crowding. Scale bar = 100 μm. (B) Comparison of “Gelatin488” images of MCF10A (normal breast epithelial cells), MCF10AT1 (ADH-mimicking cells), MCF10DCIS.com (high-grade DCIS mimic), and MCF10CA1a (invasive breast cancer cells) between ND and OC conditions. MCF10DCIS.com cell invasion is significantly higher under cell crowding than under ND conditions. (C) Invasive cell fractions of these cells between ND (blue circles) and OC (red circles) conditions are compared, showing that cell crowding-induced increases in invasiveness are notable only in MCF10DCIS.com cells. We used the two-tailed Mann-Whitney U test, a nonparametric and unpaired statistical method, to compare differences between groups. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05, throughout the manuscript.

Cell Crowding induces significant cell volume reduction and stiffening in High-Grade DCIS Cells.

(A) Cell volume (V; mean and SD) differences between ND (blue circles) and OC (red circles) conditions of MCF10A, MCF10AT1, MCF10DCIS.com, and MCF10CA1a cells are plotted. The high-grade DCIS cell mimic, MCF10DCIS.com, shows a large volume reduction due to cell crowding. (B) Representative confocal microscopy images of RFP-coexpressing cells of the four cell types in ND and OC conditions. The images include x-y (left) and x-z (right) views, with scale bar = 10 μm. The large volume reduction of MCF10DCIS.com cells is evident. (C) Plots showing changes in cortical stiffness (mean and SD) measured by Young’s modulus (Y) using a nanoindenter, displaying significant cell stiffening of MCF10DCIS.com cells due to cell crowding. (D) Hyperosmotic conditions induced by PEG 300 treatment (darker blue circles for 2% = 74.4 mOsm/Kg; navy circles for 4% = 148.8 mOsm/kg) lead to dose-dependent cell volume reduction. (E) Treatment with 2% PEG 300 (darker circles) for two days significantly increased the invasiveness (mean and SD) of MCF10DCIS.com cells, similar to the OC case (red circles). ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05.

Cell crowding induces TRPV4 relocation to plasma membrane in MCF10DCIS.com cells.

(A) Mass spectrometry data showing proteins enriched in the plasma membrane (PM) >5-fold (fold changes represented using triangle plots; OC/ND ratio on the right axis) when cells are under OC (red bars) relative to ND conditions (blue bars). Ion channels are marked with red boxes, where TRPV4 shows about a 160-fold increased association with the plasma membrane under OC conditions. (B) Proteins near and on the plasma membrane were pulled down after cell surface biotinylation with streptavidin beads and immunoblotted for TRPV4. TRPV4 is significantly associated with the plasma membrane in OC MCF10DCIS.com cells. In MCF10CA1a cells, TRPV4 appears to be associated with the plasma membrane under both ND and OC conditions, with a slight increase under OC conditions. (C) Immunoblots of whole-protein lysates demonstrate similar overall TRPV4 protein levels across MCF10A cell derivatives, regardless of cell density. This indicates that the differing plasma membrane association of TRPV4 is due to trafficking changes, not expression level changes. GAPDH is used as a loading control. (D) Representative IF images by confocal microscopy show TRPV4 (red) localization compared to the control protein transferrin receptor (TfR; green) in MCF10A, MCF10AT1, MCF10DCIS.com, and MCF10CA1a cells under ND and OC conditions. DAPI (blue) staining was used for visualizing the nuclei. As observed in the biochemical data in (B-C), cell crowding induces the relocation of TRPV4 to the plasma membrane in MCF10DCIS.com cells. TRPV4 is associated with the plasma membrane in ND MCF10CA1a cells, with a clear elevated association in OC cells. Scale bar = 10 μm. (E) Plasma membrane-associated TRPV4 (%) is quantified for the four cell lines under ND and OC conditions by line analysis, showing a significant increase in both MCF10DCIS.com cells and MCF10CA1a cells due to cell crowding. (F) IF images showing that hyperosmotic conditions induced by PEG 300 (74.4 mOsm/Kg) treatment also relocate TRPV4 (red) to the plasma membrane in MCF10DCIS.com cells. TfR localization remains intact under hyperosmotic conditions. Increased relocation is also observed in MCF10CA1a cells. Scale bar = 10 μm. (G) The increased plasma membrane association of TRPV4 due to hyperosmotic stress is quantified by line analysis. Scale bar = 10 μm. (H) Representative regions of interest (ROIs) of TRPV4-stained immunohistochemistry (IHC) images in different pathology phenotypes. High-grade DCIS and invasive ductal cancer (IDC) ROIs clearly exhibit plasma membrane association of TRPV4. Two high-grade DCIS IHC images were acquired by two different people and both show plasma membrane-associated TRPV4. Scale bar = 20 μm. (I) Statistical results from independent histological evaluations of pathologies and TRPV4 distributions of 97 ROIs from 39 patient specimens indicate a high correlation (>70%) of plasma membrane association of TRPV4 with high-grade DCIS or IDC pathologies. Y/N: Yes/no, indicating both pathologists agreed that PM ion channels were present/absent. E: Equivocal, indicating only one pathologist agreed. Significantly high proportions of high-grade DCIS (75%) and IDC (73%) ROIs exhibited plasma membrane TRPV4 association, which was not observed in lower-risk cases.

Cell crowding inhibits ion channels and triggers their plasma membrane relocations.

(A) To compare intracellular calcium (Ca²⁺) levels, we used a Fluo-4 AM assay, where green fluorescence intensity increases with higher intracellular Ca²⁺ levels. Calcium levels are significantly lower in confluent (Con) MCF10DCIS.com cells. (B) The temporal progression of averaged Fluo-4 intensity in ND MCF10DCIS.com cells (blue curve) in the box shown on the left image is compared with that of Con cells (red curve). Fluo-4 intensity is consistently lower in Con cells than in ND cells for approximately 25 minutes (200 ms acquisition time and 1 s time interval). (C) Fluo-4 intensity reduction due to cell crowding is significant in MCF10DCIS.com cells. (D-H) Pharmacological inhibition of TRPV4 with 1 nM GSK219 generates dips in the Fluo-4 signal. Fluo-4 images at the baseline (t1) and the dip (t2) post 1 nM GSK219 are compared in MCF10DCIS.com cells between ND (D) and Con (F) conditions. The Fluo-4 intensity time traces are compared between ND (blue; E) and Con (red; G) conditions, showing that the magnitude of the dip (marked as ΔCa) is significantly lower in Con cells, where TRPV4 activity is largely inhibited under cell crowding conditions. Notably, ΔCa increased with higher GSK219 doses (1 nM vs. 0.2 nM), but remained significantly lower in Con MCF10DCIS.com cells, with smaller changes observed under the 0.2 nM GSK219 condition. (I-M) TRPV4 activation with 0.2 pM GSK101 leads to a small spike in ND cells (I, J). However, in Con cells, the same GSK101 treatment leads to a notable spike in Fluo-4 intensity, indicating that TRPV4 inhibition and subsequent relocation to the plasma membrane by cell crowding primes the ion channels for activation. GSK101 treatment also leads to a dose-dependent increase in the spike magnitude with higher GSK101 concentrations (0.05 pM: 101L; 0.2 pM: 101H), which is strikingly high in Con MCF10DCIS.com cells. (N-Q) TRPV4 activation status-dependent intracellular localization changes. (N) IF images of TRPV4 (red) and TfR (green) in ND MCF10DCIS.com cells show that GSK101 does not increase plasma membrane association of TRPV4. However, GSK219 significantly relocates TRPV4 to the plasma membrane in a dose-dependent manner, similar to ND cells treated with 74.4 mOsm/Kg PEG 300. (O) In OC cells, while GSK219 does not significantly alter TRPV4 association with the plasma membrane, GSK101 depletes plasma membrane TRPV4 in a dose-dependent manner, suggesting that TRPV4 activation status affects its trafficking. Relative plasma membrane associations with different treatments are quantified for ND (P) and OC (Q) cells using line analysis. (R) The magnitudes of Fluo-4 spikes by GSK101 and dips by GSK219 show a linear relationship (R² ∼ 0.69), indicating a negative correlation between them. This reinforces the observation that TRPV4 inhibition increases its association with the plasma membrane, while activation shows the reverse effect. (S-V) Compared to the Fluo-4 intensity in control MCF10DCIS.com cells (S, T), shRNA showed similar Fluo-4 levels (U, V). However, hyperosmotic stress by 74.4 mOsm/Kg PEG 300 (light gray box) led to a noticeable spike only in control ND cells. Additionally, cell crowding conditions (Con) led to a decreased Fluo-4 level (at t1 baseline in the image in S and red time trace in T) and a reduction in Fluo-4 level in shRNA-treated MCF10DCIS.com cells (t1; U) compared to control cases (S, T), as shown in the image and time trace (t1; V). (W) Relative Fluo-4 time-averaged intensities are plotted for individual control ND (blue) vs. Con (red) cells, and shRNA-treated ND (semi-transparent blue) vs. Con (semi-transparent red) cells. Intracellular calcium levels in shRNA ND cells are lower than those in control ND cells, reflecting the reduced number of TRPV4 channels. The decrease in calcium levels by crowding (Con) in shRNA cells is clearly lower than in control cells, reflecting the importance of TRPV4 in mechanosensing cell volume reduction. (X) PEG 300-induced calcium spikes are significantly lower in shRNA cells (semi-transparent gray) than in control cells (gray), reinforcing TRPV4’s crucial role in MCF10DCIS.com mechanotransduction. (Y) TRPV4 silencing significantly reduced the mechanosensing cell volume reduction effect. Control ND cells underwent a 48% volume reduction in response to 74.4 mOsm/Kg PEG 300, whereas TRPV4-silenced cells reduced their volume by only 27%.

Cell crowding-induced plasma membrane TRPV4 association scales with cell volume reduction and increases in invasiveness and motility

A-C. MCF10DCIS.com cell volume changes with TRPV4 inhibition and activation. (A) In ND MCF10DCIS.com cells, GSK101, which did not alter plasma membrane association of TRPV4, did not affect cell volume. Conversely, GSK219, which increased such association in a dose-dependent manner, reduced cell volume, with the effect of 1 nM GSK219 (219H) being similar to that of 74.4 mOsm/Kg PEG 300. (B) Under OC conditions, GSK101, which leads to significant Fluo-4 spikes, increased cell volume in a dose-dependent manner, while GSK219 and PEG only mildly reduced cell volume. (C) Cell volume changes in MCF10DCIS.com cells show an inverse relationship with plasma membrane association of TRPV4, reflecting the activation status of the channel (R² = 0.59). (D-F) Cell invasiveness increases with cell volume reduction and greater plasma membrane association of TRPV4. (D) Cell invasiveness significantly increases with GSK219 under ND conditions. (E) GSK101 under OC conditions shows a notable decrease in cell invasiveness in a dose-dependent manner. (F) Plasma membrane association of TRPV4 predictably reports cell invasiveness (R² ∼ 0.69), while cell invasiveness and cell volume are inversely related, reinforcing our observation that cell volume reduction promotes cell invasiveness. (H-M) To assess if cell motility also follows the trend of cell invasiveness, we performed a single-cell motility assay by tracking nuclear WGA in individual live cells every 5 s for 25 min. (H) Representative trajectories of individual cells are color-coded to reflect displacement at each time interval. Compared to untreated ND cells, 0.2 pM GSK101 treatment slowed overall cell diffusion, while 1 nM GSK219 and 74.4 mOsm/Kg PEG 300 treatments increased cell diffusion. ShRNA TRPV4 (Sh-ctrl) had no effect on cell motility under ND conditions. Furthermore, treatment with 74.4 mOsm/Kg PEG 300 failed to increase cell diffusivity (D) in shRNA-treated cells (Sh-PEG). Scale bar = 200 μm. Using single-cell analysis, we quantified cell diffusivity (D) and speed (v; movement directionality). (I) GSK101 treatment significantly reduced D, while GSK219 and PEG 300 notably increased D. ShRNA TRPV4 did not alter ND cell D. (J) GSK101 treatment increased v, while GSK219 and PEG treatments significantly increased D but those did not affect directionality. ShRNA ND cell directionality remained similar to that of control cells and was unchanged by PEG treatment. (K) Like cell invasiveness, cell motility (D) positively scales with plasma membrane association of TRPV4 (R² ∼ 0.73). (L) Cell motility (D) inversely relates to cell volume (R² ∼ 0.89). (M) Cell motility (D) and cell invasiveness show a strong linear relationship (R² ∼ 0.85), enabling the use of cell motility measurements to assess overall cell invasiveness.

Pro-invasive cell volume reduction mechanotransduction pathway is indicated by TRPV4 inhibition and hyperosmotic stress-driven increases in TRPV4 association with the plasma membrane and cell motility, as well as TRPV4 activation-induced decreases in cell motility.

(A) MCF10CA1a cells respond to 15 minutes of PEG 300 (74.4 mOsm/kg) and OC conditions by relocating TRPV4 to the plasma membrane, increasing the channel’s association with the membrane. IF images show a predominantly intracellular TRPV4 (red) distribution in the ND control MCF10CA1a cells, whereas plasma membrane association of TRPV4 occurs in response to PEG 300 and OC. DAPI (blue) staining indicates the nucleus. Scale bars throughout Fig. 6 = 10 μm. (B) Line analysis results of plasma membrane-associated TRPV4 (%) show significant increases in plasma membrane TRPV4 (PM TRPV4) with 1 hour of GSK219 (1 nM), 15 minutes of 74.4 mOsm/kg PEG 300, and OC conditions. (C) Cell movement diffusivity (D) increases with GSK219 and PEG treatments, while movement directionality (v) increases with GSK101 (0.2 pM) but shows no other effects. (D-O) MCF10AT1 (D, E), MDA-MB-231 (G, H), ETCC-06 (J, K), and ETCC-10 (M, N) do not relocate TRPV4 to the plasma membrane in response to TRPV4 inhibition by GSK219, hyperosmotic conditions by PEG-300, or OC conditions, as evidenced by IF images (TRPV4: red; DAPI: blue) and line analysis results for plasma membrane associations of TRPV4. (F) None of these cells’ motility responded to PEG 300. However, their responses to TRPV4 activation (GSK101) and inhibition (GSK219) varied, suggesting different roles of TRPV4 in their cancer biology. MCF10AT1 diffusivity (D) significantly reduced with GSK219, while the rest of the conditions did not affect D. Movement directionality (v) was not affected by any treatment conditions. (G) MDA-MB-231 cell D or v did not alter with any treatment conditions, suggesting an insignificant role of TRPV4 in their cell motility. (L, M) Both ETCC-06 and ETCC-10 D increased with GSK101, but GSK219 also increased ETCC-06 diffusivity, while not altering ETCC-10 diffusivity. ETCC-06 directionality (v) increased with GSK101, but ETCC-10 directionality remained unchanged. (P) Plasma membrane association of TRPV4 (% PM TRPV4) positively scalex with cell movement D over a larger range for MCF10DCIS.com cells than for MCF10CA1a, reflecting the high cell volume plasticity observed in MCF10DCIS.com cells. This result suggests that both cell types have a pro-invasive mechanotransduction pathway. (Q) Such scaling is absent in MCF10AT1, MDA-MB-231, ETCC-06, and ETCC-10 cells. (R) The presence of such a mechanotransduction pathway is observed for MCF10CA1a and MCF10DCIS.com cells from the plot of greater than 2-fold increase in plasma membrane association of TRPV4 (x-axis; PM TRPV4_peg/PM TRPV4_ctrl) and greater than 1-fold increase in diffusivity (y-axis; Dpeg/Dctrl) by PEG-300. (S) Cell volume reduction-mediated mechanotransduction pathway can be clearly observed from plotting plasma membrane association of TRPV4 (x-axis) and relative increase in diffusivity with GSK101 versus GSK219 (y-axis), which are significantly greater than 2 for MCF10CA1a and MCF10DCIS.com cells.

Cell crowding triggers the activation of a pro-invasive mechanotransduction pathway in high-grade DCIS cells but not in less aggressive or normal cells.

This pro-invasive mechanotransduction pathway involves cell volume reduction and cortical stiffening driven by ion channel inhibition. Ion channel inhibition leads to the relocation of ion channels to the plasma membrane (PM). Under ND conditions, ion channels are mainly cytoplasmic in both high-grade DCIS and less aggressive/normal cells. However, high-grade DCIS cells have a larger cell volume under ND conditions. Cell crowding induces ion channel inhibition, leading to decreased intracellular calcium and subsequent cell volume reduction, which correlates with increased invasiveness. In contrast, less aggressive and normal cells do not exhibit this response. The inset image shows that mechanosensitive plasma membrane relocation of ion channels (orange) scales with increased invasiveness due to the activation of the cell volume reduction mechanotransduction pathway, while the absence of relocation indicates no mechanosensitive invasive increase.

Quantifying the Invasive cell fraction using a 2D polyacrylamide hydrogel-based invasion assay.

(A) Green fluorescent gelatin images were thresholded to highlight invasion areas (in red) due to degradation (visible as dark regions in the original image). Here, 4.34% of the total area indicated cell invasion. (B) DAPI images (in blue, showing nuclei locations) were processed with the Trackmate plugin in Image J to detect individual DAPI spots (displayed in purple). The total cell count in this instance was estimated at 315. (C) The highlighted invasive areas from (A) served as masks for the DAPI locations, selecting only the invasive cells (those in the white regions resulting from the overlay between cell positions in purple and invasive zones in cyan). (D) The resultant images display the locations of invasive cells in purple. The invasive cell count from this image was 80. (E) The fraction of invasive cells was determined by comparing the number of invasive cells (from D) with the overall cell count (from B). Thus, the invasive cell fraction was 25.4% (80 out of 315). Scale bar = 100 μm.

MCF10DCIS.com cells exhibited comparable viability under OC conditions to those under ND conditions.

(A-B) Live cells seeded at ND (∼70%) (n=2) were cultured for 1 (n=4), 3 (n=4), 5 (n=8), 7 (n=8), and 10 (n=8) days post-confluence (denoted as "d"). Cells were stained with propidium iodide (PI, red) to mark dead cells, followed by WGA-488 (green) to label nuclei. After staining, cells were fixed and imaged using confocal microscopy (4X), with red and green signals quantified via the TrackMate plugin in ImageJ. The percentage of dead cells, based on PI-positive signals, is presented in A. Both confluent (day 1 and day 3) and OC (day 5–10) MCF10DCIS.com cells showed similar viability to ND cells, with less than 1% cell death (0.85 ± 0.25%). On average, the fraction of dead cells was 1.58 ± 0.98%, confirming that cell crowding does not additionally induce cell death. Representative PI/WGA-488/merged images from days 1, 3, 5, 7, and 10 are shown in B. Scale bar = 100 μm. For the t-test, we employed a nonparametric approach using the Mann-Whitney test with a two-tailed p-value, which was used throughout the manuscript. The statistical significance levels are denoted as follows: ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05.

Time window for cell crowding conditions in vitro.

(A) MCF10DCIS.com cells were grown to: normal density (ND; 40%–70%), confluence (100%), and overconfluence (OC). The time points for these growth stages were two days before confluence (D-2) for ND, day 0 (D0) for confluence, and days 5–10 after confluence (D5) for OC. We selected the cell crowding condition (OC conditions) during days 5–10 as cell morphology (B) and invasiveness (C) reached equilibrium after day 5. (B) Brightfield microscopy images of MCF10DCIS.com cells on the indicated days. Cells exhibited significant compaction starting from day 5. Scale bar = 100 μm. (C-D) Time-dependent invasiveness of MCF10DCIS.com cells as they progressed to OC. The cell invasiveness increased until day 5 and plateaued between days 5 and 10. When these cells were reseeded at normal density on day 2 (reseeded D-2), their invasiveness decreased to levels similar to the original ND cells. (E) Formerly OC cells reseeded at ND showed a slight reduction in the fraction of invasive cells (light teal circles) to approximately 15%, comparable to ND cells (blue circles) before exposure to OC conditions. These results suggest that the OC-induced increase in invasiveness is largely reversible. (F) To test if acidity of OC cell media, despite frequent changing, contributed to increased invasiveness of DCIS.com cells, we used acidic OC media (day 7) to treat ND DCIS.com cells for two days. Conditioned media did not alter invasiveness of ND DCIS.com cells. For the t-test, we employed a nonparametric approach using the Mann-Whitney test with a two-tailed p-value, which was used throughout the manuscript. The statistical significance levels are denoted as follows: ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05.

Increased invasiveness of MCF10DCIS.com cells correlates with cell volume plasticity, not the acidity of cell media.

(A) Cell crowding induced cell volume and stiffness changes assessed by our confocal microscope, which captures 3D volume of single RFP-expressing cells (right image) and includes a nanoindenter device (Chiaro, Optics11life) that can indent a single cell (load vs indentation curve) to extract Young’s modulus in the elastic regime (red dashed box) using a Hertzian model. The indentation probe has a spring constant and tip diameter of ∼0.24 N/m and 10 μm, respectively. We confirmed that RFP expression did not alter cell volume. (B-C) Cell volume and stiffness differences between ND and OC conditions calculated using the cell volume and stiffness data in Fig. 2A and 2C were normalized to the ND cell volume (B) or stiffness (C), with the highest changes observed in high-grade MCF10DCIS.com cells. (D) Normalized volume change (Nor. ΔV) linearly scaled with ND cell volume (mean VND), with an R² value of approximately 0.97, signifying a highly linear relationship. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05.

Gene and protein names that showed more than a 5-fold increase in plasma membrane association under OC conditions relative to ND conditions in MCF10DCIS.com cells were identified by mass spectrometry. Ion channels among these are highlighted in yellow.

Top 25 gene and protein names that showed more than a 100-fold increase (MCF10A; light blue header) and 5-fold increases (MCF10AT1; blue header and MCF10CA1a; violet header) in plasma membrane association under OC conditions relative to ND conditions in MCF10A (left), MCF10AT1 (middle), and MCF10CA1a (right) cells were identified by mass spectrometry. One ion transporter demonstrating this behavior in MCF10CA1a cells is highlighted in yellow.

Original immunoprecipitation and western blot images.

(A) Left: Plasma membrane proteins pulled down after cell surface biotinylating with streptavidin beads and immunoblotted for TRPV4 and GAPDH (loading control) for ND vs OC cells of MCF10A (10A), MCF10AT1 (AT1), and MCF10DCIS.com (DCIS.com). Right: The same procedure was performed to compare PM TRPV4 between ND vs OC MCF10CA1a cells. (B) Overall TRPV4 protein levels from whole-cell lysates from four 10A cell derivatives. GAPDH was a loading control.

Binding specificity of TRPV4 antibody.

Immunofluorescence images (A) and immunoblots (B) verified the binding specificity of TRPV4 antibodies in control ND DCIS.com cells and TRPV4-depleted cells treated with either 2 μM siRNA (Dharmacon On Target Plus SMART Pool L-004195-00-0005) or 1 and 2 μg shRNA (shRNA pool, Santa Cruz sc-61726-SH) for 36 hr. (A) Compared to invariant transferrin receptor (TfR) staining (green), TRPV4 (red) depletion was 40% with 1 µg shRNA and 80% with 2 µg shRNA, as quantified by intensity measurements. DAPI (blue) is also shown in the merged images. All images were visualized using the same intensity settings. Scale bar = 20 µm. (B) Immunoblot results confirmed this dose-responsive depletion of TRPV4, with 33% reduction observed at 1 µg shRNA and 51% at 2 µg.

Various ion channels are relocated to the plasma membrane under cell crowding conditions. (A) We used the plasma membrane marker DiIC18(3) to confirm the association of TRPV4 with the plasma membrane in MCF10DCIS.com and MCF10CA1a cells under OC conditions. As described in the Methods section, we stained DiIC18(3) in live cells and co-stained TRPV4 and DAPI in fixed and permeabilized cells. The IF images show TRPV4 (red), DiIC18(3) (DiI, green), and DAPI (blue). The line profile plots on the right demonstrate colocalization of TRPV4 with DiIC18(3) at the plasma membrane (PM), marked by the green DiIC18(3) signal, which overlaps with the red TRPV4 signal. The nucleus location (NUC) is indicated by the blue DAPI signal. Scale bar = 20 μm. (B) We examined the relocation of KCNN4 and PIEZO1 to the plasma membrane in response to cell crowding. Mass spectrometry showed a slight increase in KCNN4 at the plasma membrane under OC conditions. In ND MCF10DCIS.com cells, KCNN4 was predominantly cytosolic, whereas PIEZO1 showed some plasma membrane association. Under OC conditions, both KCNN4 and PIEZO1 showed a modest relocation to the plasma membrane. (C) Line analysis confirmed a slight increase in plasma membrane association for both KCNN4 and PIEZO1 under OC conditions compared to ND conditions. Scale bar = 20 μm. For the statistical analysis, we employed a nonparametric approach using the Mann-Whitney test with a two-tailed p-value. The levels of statistical significance are denoted as follows: **** indicates p < 0.0001, *** indicates p < 0.001, * indicates p < 0.1, and "ns" indicates p > 0.05.

Plots of the relative intracellular TRPV4 associations between ND and OC or hyperosmotic conditions in all four cell types.

(A) Relative TRPV4 associations with the plasma membrane (PM), cytoplasm (Cyt), and nucleus (Nuc) are plotted for ND versus OC conditions in MCF10A (10A), MCF10AT1 (10AT1), MCF10DCIS.com (10DCIS.com), and MCF10CA1a (10CA1a) cells. (B) Similar analyses were performed to compare the intracellular TRPV4 associations in PM, Cyt, and Nuc between control ND and 74.4 or 148.8 mOsm/kg PEG300 treatment groups. We employed a nonparametric approach using the Mann-Whitney test with a two-tailed p-value for the statistical analysis. The levels of statistical significance are denoted as follows: **** indicates p < 0.0001, *** indicates p < 0.001, * indicates p < 0.1, and "ns" indicates p > 0.05.

Validation of mechanosensitive relocation of TRPV4 to the plasma membrane in MCF10DCIS.com cells.

(A) To determine whether the seemingly plasma membrane-associated TRPV4 under hyperosmotic conditions was indeed localized at the plasma membrane of live cells, we used an extracellular domain (ECD) TRPV4 antibody. See the Methods section for sample preparation details. Live-cell imaging with 60X confocal microscopy revealed distinct binding of the ECD TRPV4 antibodies (red) exclusively in MCF10DCIS.com cells treated with 74.4 mOsm/kg PEG 300 for 15 minutes. No binding was observed in the untreated control group. Brightfield images are shown below the fluorescence images. (B) Line analysis demonstrated that ∼60% of TRPV4 was plasma membrane-associated under 2% (74.4 mOsm/kg) PEG 300 conditions, compared to ∼0% in untreated cells. Statistical analysis was conducted using a nonparametric Mann-Whitney test with two-tailed p-values. Statistical significance levels are indicated as follows: **** p < 0.0001, *** p < 0.001, * p < 0.1, and "ns" denotes p > 0.05.

Reversibility of cell-crowding-driven mechanosensing effects in MCF10DCIS.com cells.

(A) Previously OC MCF10DCIS.com cells were trypsinized and reseeded under ND conditions. These cells were fixed and stained for TRPV4 (red) and DAPI (blue) for IF imaging. While TRPV4 was elevated at the plasma membrane under OC conditions, it redistributed to the cytoplasm after the cells were reseeded under ND conditions. (B) The reseeded ND cells responded similarly to hyperosmotic stress (74.4 mOsm/Kg PEG 300 for 15 min), with TRPV4 relocating to the plasma membrane. (C) Line analysis demonstrated that the reseeded ND cells exhibited a TRPV4 distribution pattern comparable to their initial ND counterparts without (blue circles) and with (red circles) hyperosmotic conditions. Statistical analysis was performed using a nonparametric Mann-Whitney test with two-tailed p-values. Statistical significance levels are denoted as follows: **** p < 0.0001, *** p < 0.001, * p < 0.1, and "ns" indicates p > 0.05.

Pathology-dependent differential TRPV4 distributions in patients’ IHC images.

Representative IHC images for each pathology from different patients are displayed. A selective presence of TRPV4 pools in the plasma membrane was mainly observed in high-grade DCIS and IDC lesions. Two pathologists independently conducted annotations. When a consensus was not reached, the case was labeled “equivocal.” The cases were categorized according to the subsequent criteria:

Case 1: Absence of TRPV4.

Case 2: Intracellular TRPV4 localization.

Case 3: Presence of TRPV4 in the plasma membrane, with or without intracellular TRPV4.

A. Benign cases.

IHC images (Top): Pathology: UDH: usual ductal hyperplasia; protein distribution: case 2;

(Middle): Pathology: benign (columna); protein distribution: case 2;

(Bottom): Pathology: benign (papilloma); protein distribution: case 2. Scale bars = 30 μm.

B. Atypical ductal hyperplasia (ADH) cases.

IHC images (Top): Pathology: ADH: usual ductal hyperplasia; protein distribution: case 2;

(Middle): Pathology: ADH: usual ductal hyperplasia; protein distribution: case 2. Scale bars = 50 μm.

C. Low-grade (LG) DCIS cases.

IHC images (Top): Pathology: low-grade DCIS; protein distribution: case 2;

(Middle): Pathology: low-grade DCIS; protein distribution: case 2. Scale bars = 50 μm.

D. Intermediate-grade (IMG) DCIS cases.

IHC images (Top): Pathology: intermediate-grade DCIS; protein distribution: case 2;

(Middle): Pathology: intermediate-grade DCIS; protein distribution: case 2. Scale bars = 50 μm.

E. High-grade (HG) DCIS cases.

IHC images (Top): Pathology: high-grade DCIS; protein distribution: case 3;

(Middle): Pathology: high-grade DCIS; protein distribution: the distinction between case 2 and case 3 is equivocal;

(Bottom): Pathology: high-grade DCIS; protein distribution: case 3. Scale bars = 50 μm.

F. Invasive ductal carcinoma (IDC) cases.

IHC images (Top): Pathology: IDC; protein distribution: case 3;

(Middle): Pathology: IDC; protein distribution: case 2.

(Bottom): Pathology: IDC; protein distribution: case 3. Scale bars = 50 μm.

Peripheral cells within MCF10DCIS.com cell clusters exhibit higher calcium levels due to reduced cell crowding effects.

(A) Confluent cell density (Con) also triggered the relocation of TRPV4 (red) to the plasma membrane, similar to OC conditions, as shown in IF image (left). We used DiIC18(3) to indicate the plasma membrane location (green; DiI; middle image), and the merged image (right) of TRPV4 and DiIC18(3) shows excellent overlay at the plasma membrane (PM), as illustrated in the line profile plots from our line analysis. DAPI (blue) staining was used to locate the nucleus (NUC). Scale bar = 20 μm. (B) Confluent cell density resulted in lower intracellular calcium levels compared to less confluent cells. Live MCF10DCIS.com cells stained with Fluo-4 were imaged using confocal microscopy at 488 nm. Two line profiles (1, 2) crossing peripheral cells (less confluent than confluent cells) and adjacent confluent cells within the clusters clearly showed that the peripheral cells have a higher Fluo-4 signal (700 au) compared to the confluent cells within the cluster (200 au), highlighting the crowding-induced intracellular calcium reduction. Background regions (bg) were noted in cyan in the fluorescent image and in the plots. Scale bar = 100 μm.

Determination of treatment concentration ranges for TRPV4 activator (GSK101) and inhibitor (GSK219).

(A) Cell viability assays in which viable cells were counted based on trypan blue staining after two days of GSK101 or GSK219 treatment in the specified concentration ranges of DCIS in ND (white bars) and OC (gray bars) conditions. Treatment ranges were selected so that cell viability was >90%. Concentrations used for dose-dependent assays were 0.05 and 0.2 pM for GSK101, and 0.2 and 1 nM for GSK219 (marked by dotted red boxes). (B) Representative confocal microscopy immunofluorescence images showed effects of GSK101 (0.05 and 0.2 pM) or GSK219 (0.2 and 1 nM) treatment for two days on TRPV4 (red) and control transferrin receptor (TfR; green) distributions in ND and OC cells in a dose-dependent manner. DAPI (blue) signal is shown in the merged images. Scale bar = 20 μm.

Hyperosmotic stress also induces plasma membrane relocation of ion channels, similar to cell crowding.

(A) Using the Fluo-4 assay, we observed an initial calcium spike (marked as "Rise") in ND MCF10DCIS.com cells in response to 74.4 mOsmol/kg PEG 300, due to osmotic water outflow. This was followed by a homeostatic relaxation, aimed at restoring calcium levels, which likely involved the inhibition of ion channels like TRPV4, leading to their plasma membrane relocation. Scale bars = 20 μm. (B) The same hyperosmotic condition (74.4 mOsm/Kg PEG 300 for 15 min) led to the relocation of KCNN4 and PIEZO1 to the plasma membrane, similar to the relocations observed under OC conditions. Line analysis results showed the relative relocations of each channel in response to hyperosmotic (PEG) and cell crowding (OC) stresses, compared to ND conditions.

Mechanical stresses and TRPV4 activation status affect MCF10DCIS.com cell invasiveness.

(A) The effects of 2 days of treatment with GSK101 (0.05 and 0.2 pM), GSK219 (0.2 and 1 nM), and PEG 300 (74.4 mOsm/kg) on the invasiveness of MCF10DCIS.com cells under ND and OC conditions. Gelatin488 images demonstrated the dose-dependent negative effects of GSK101 and positive effects of GSK219 on cell invasiveness. Similar to GSK219, PEG 300 also increased cell invasiveness. Scale bar = 100 μm. (B) IF images of βactin (green) and DAPI (blue) in MCF10DCIS.com cells under ND (left) and OC (right) conditions. Strong stress fiber formation was observed in OC cells, while it was absent in ND cells, which was reflected by the increased stiffness of OC cells (Fig. 2C). This suggests that cell crowding may enhance cell motility. Scale bar = 20 μm.

Cells lacking the capability for activating pro-invasive mechanotransduction pathway via TRPV4 inhibition-induced cell volume reduction do not relocate TRPV4 to the plasma membrane under TRPV4 inhibition and mechanical stresses.

The effects of 2 days of treatment with GSK101 (0.2 pM), GSK219 (1 nM), and PEG 300 (74.4 mOsm/kg) on cells under ND or OC conditions revealed differences in TRPV4 localization (red) in the immunofluorescence (IF) images (cyan: DAPI). Only MCF10CA1a cells show GSK219, PEG 300, and OC-induced TRPV4 relocation to the plasma membrane. Other cell types, including MCF10AT1, MDA-MB-231, ETCC-06, and ETCC-10, did not exhibit this translocation. Scale bar = 20 μm.