Figures and data
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 cell invasiveness. Representative images show gelatin-Alexa488 conjugates, where dark areas in the green (Gelatin488) background indicate cell invasion through degradation, 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. The number of cell invasion analyses was as follows: MCF10A (ND: 10; OC: 7), MCF10AT1 (ND: 6; OC: 6), MCF10DCIS.com (ND: 9; OC: 9), and MCF10CA1a (ND: 6; OC: 7). 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. The number of single cell volume analyses (technical replicates merged from three independent experimental repeats) was as follows: MCF10A (ND: 44; OC: 14), MCF10AT1 (ND: 16; OC: 16), MCF10DCIS.com (ND: 38; OC: 24), and MCF10CA1a (ND: 29; OC: 31). (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. The number of single cell stiffness measurements (technical replicates merged from two independent experimental repeats) was as follows: MCF10A (ND: 21; OC: 19), MCF10AT1 (ND: 19; OC: 14), MCF10DCIS.com (ND: 19; OC: 21), and MCF10CA1a (ND: 11; OC: 10). (D) Hyperosmotic conditions induced by PEG 300 treatment (light blue and darker blue circles for untreated and 2% PEG 300 = 74.4 mOsm/Kg, respectively; navy circles for 4% PEG 300 = 148.8 mOsm/kg) lead to dose-dependent cell volume reduction. The number of single cell volume analyses (technical replicates merged from three independent experimental repeats) was as follows: MCF10DCIS.com (ND control: 62; ND + 2% PEG: 23; ND + 4% PEG: 10). (E) Treatment with 2% PEG 300 (darker blue circles) for two days significantly increased the invasiveness (mean and SD) of MCF10DCIS.com cells, similar to the OC case (red circles). The number of cell invasion analyses (technical replicates merged from two independent experimental repeats) was as follows: MCF10DCIS.com (ND control: 13; ND + 4% PEG: 12; OC: 10). ****: 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. The number of cells used for line analyses (technical replicates merged from three independent experimental repeats) was as follows: MCF10A (ND: 6; OC: 12), MCF10AT1 (ND: 6; OC: 11), MCF10DCIS.com (ND: 12; OC: 8), and MCF10CA1a (ND: 10; OC: 10). (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 consistent 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. The number of cells used for line analyses (technical replicates merged from two independent experimental repeats) was as follows: MCF10A (ND control: 6; ND + 4% PEG: 15), MCF10AT1 (ND control: 6; ND + 4% PEG: 9), MCF10DCIS.com (ND control: 12; ND + 4% PEG:8), and MCF10CA1a (ND control: 10; ND + 4% PEG: 21). 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 the pathologists disagreed. 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. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05.
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 30 s time interval). (C) Fluo-4 intensity reduction due to cell crowding is significant in MCF10DCIS.com cells. 10 images were used for calculating average Fluo-4 intensities for both ND and Con 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. The number of ΔCa measurements (H) (technical replicates merged from two independent experimental repeats) was follows: MCF10DCIS.com (ND + GSK219 Low: 9; ND + GSK219 High: 12; Con + GSK219 High: 10; Con + GSK219 Low: 14). (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 notably larger 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 a higher GSK101 concentration being strikingly high in Con MCF10DCIS.com cells (0.05 pM: 101L; 0.2 pM: 101H). The number of ΔCa measurements (M) (technical replicates merged from two independent experimental repeats) was follows: MCF10DCIS.com (ND + GSK101 Low: 7; ND + GSK101 High: 9; Con + GSK101 Low: 9; Con + GSK101 High: 9). (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 (Supple. Fig. 13B for all dose cases), 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 (Supple. Fig. 13B for all dose cases), 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. The number of line analyses (P, Q) (technical replicates merged from two independent experimental repeats) was follows: ND and OC MCF10DCIS.com (control: 12 and 8; GSK219 Low: 6 and 7; GSK219 High: 6 and 13; GSK101 Low: 7 and 8; GSK101 High: 7 and 10; 2% PEG: 12 and 12). (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 baseline 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); but a reduced in Fluo-4 level diference 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. The number of Fluo-4 average measurements (technical replicates merged from two independent experimental repeats) was follows: MCF10DCIS.com control and TRPV4 shRNA groups (ND: 19 and 18; Con: 11 and 17). (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. The number of ΔCa measurements under 2% PEG 300 condition was follows: ND MCF10DCIS.com (ND: 19; TRPV4shRNA: 16). (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%. The number of single cell volume measurements (technical replicates merged from two independent experimental repeats) was follows: ND and TRPV4shRNA treated MCF10DCIS.com (Control: 11 and 13; 2% PEG 8 and 11). ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05.
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, TRPV4 agonist GSK101, which did not alter plasma membrane association of TRPV4, did not affect cell volume. Conversely, TRPV4 inhibitor GSK219, which increased plasma membrane 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 (2%) PEG 300. (B) Under OC conditions, GSK101, which led 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 (R² = 0.59) with plasma membrane association of TRPV4, reflecting the activation status of the channel. The number of single cell volume measurements (technical replicates merged from three independent experimental repeats): ND (A) and OC (B) MCF10DCIS.com cells (Control: 33 and 43; GSK101 0.05 pM: 19 and 15; GSK101 0.2 pM: 9 and 22; GSK219 0.1 nM: 10 and 36; GSK101 1 nM: 15 and 9; 2% PEG 300: 23 and 8). (D-F) Cell invasiveness increases with greater cell volume reduction and plasma membrane association of TRPV4. (D) Cell invasiveness significantly increased with higher GSK219 concentrations under ND conditions. (E) GSK101 under OC conditions caused a notable decrease in cell invasiveness in a dose-dependent manner. (F-G) Plasma membrane association of TRPV4 predictably reports cell invasiveness (R² ∼ 0.69; F), while cell invasiveness and cell volume are inversely related (R² ∼ 0.69; G), reinforcing our observation that cell volume reduction promotes cell invasiveness. The number of invasive cell fraction measurements (technical replicates merged from two independent experimental repeats) : ND (D) and OC (E) MCF10DCIS.com cells (Control: 6 and 4; GSK101 0.05 pM: 4 and 4; GSK101 0.2 pM: 4 and 4; GSK219 0.1 nM: 4 and 4; GSK101 1 nM: 4 and 4; 2% PEG 300: 4 and 7). (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 60 s for 25 min. (H) Representative trajectories of individual cells were 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) increased cell motility under ND conditions. However, with TRPV4 depletion, treatment with 74.4 mOsm/Kg PEG 300 failed to increase cell diffusivity (D) in shRNA-treated cells (Sh-PEG), unlike in the untreatec cells. 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 it. shRNA TRPV4 also increased ND cell D, but PEG treatment did not change D in the shRNA-treated cells. (J) GSK101, GSK219, PEG 300, and shRNA treatments increased v, with GSK101 causing the most significant increase. The directionality of shRNA-treated ND cells was unaffected by PEG treatment. The number of single-cell motility measurements of MCF10DCIS.com cells (technical replicates merged from two independent experimental repeats): Control: 81; GSK101: 100; GSK219: 100, PEG: 100; shRNA: 102; shRNA + PEG: 104. (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. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05.
Pro-invasive cell volume mechanotransduction pathway indicators.
(A) In MCF10CA1a cells, plasma membrane relocation of TRPV4 was induced by a 15-minute treatment with 2% PEG 300 (74.4 mOsm/kg) or by OC conditions. IF images show a largely intracellular distribution of TRPV4 (red) in ND control cells, whereas cells treated with PEG 300 or subjected to OC exhibit a significant increase in plasma membrane-associated TRPV4. Nuclei are stained with DAPI (blue). Scale bars in all panels of Fig. 6 represent 10 μm. (B) Line analysis quantifying plasma membrane-associated TRPV4 (%) reveals significant increases following a 1-hour treatment with GSK219 (1 nM), a 15-minute exposure to 74.4 mOsm/kg PEG 300, or under OC conditions in MCF10CA1a cells. In contrast, no significant increase in plasma membrane TRPV4 is observed with GSK101 treatment. (C) In MCF10CA1a cells, cell movement diffusivity (D) increased following GSK219 or PEG 300 treatments, whereas it decreased with GSK101. Conversely, movement directionality (v) increased significantly with GSK101 (0.2 pM) but remained unchanged with GSK219 or PEG treatments. (D-O) No plasma membrane relocation of TRPV4 was observed in response to inhibition by GSK219, hyperosmotic stress induced by PEG 300, or cell crowding (OC) in MCF10AT1 (D, E, F), MDA-MB-231 (G, H, I), ETCC-006 (J, K, L), and ETCC-010 (M, N, O) cells. Similarly, GSK219 or PEG 300 did not increase single-cell motility in these cells. This is demonstrated by IF images (TRPV4: red; DAPI: blue) (D, G, J, M), line analysis results for plasma membrane-associated TRPV4 (E, H, K, N), and single-cell motility analyses for diffusivity (D) and directionality (v) (F, I, L, O). Notably, none of these cell lines showed motility changes in response to PEG 300 treatment. However, responses to TRPV4 activation (GSK101) and inhibition (GSK219) varied across cell types, suggesting distinct roles of TRPV4 in their cancer biology. In MCF10AT1 cells (F), GSK219 significantly reduced diffusivity (D), while no other treatment affected D or v. In MDA-MB-231 cells (I), neither D nor v was altered by any treatment, indicating that TRPV4 has an insignificant role in their motility. Both ETCC-006 and ETCC-010 cells exhibited increased diffusivity with GSK101; however, GSK219 also increased diffusivity in ETCC-006 cells (L), while having no effect on ETCC-010 cells (O). Directionality (v) increased with GSK101 in ETCC-006 cells (L), whereas ETCC-010 cells showed no change in v across all conditions (O). The number of line analyses for plasma membrane-associated TRPV4 under ND control, ND + 0.2 pM GSK101, ND + 1 nM GSK219, ND + 2% PEG 300, and OC conditions (technical replicates merged from three independent experimental repeats) were: MCF10CA1a (B): 11, 6, 8, 12, 10; MCF10AT1 (E): 13, 9, 8, 9, 19; MDA-MB-231 (H): 13, 9, 8, 9, 19; ETCC-006 (K): 12, 10, 10, 5, 10; and ETCC-010 (N): 5, 5, 7, 5, 6. The number of single-cell motility analyses under ND control, ND + 0.2 pM GSK101, ND + 1 nM GSK219, and ND + 2% PEG 300 conditions were: MCF10CA1a (C): 100, 100, 100, 100; MCF10AT1 (F): 130, 161, 582, 183; MDA-MB-231 (I): 57, 6, 21, 442; ETCC-006 (L): 65, 66, 24, 100; and ETCC-010 (O): 317, 1136, 43, 71. (P) Plasma membrane association of TRPV4 (% PM TRPV4) scaled positively with cell diffusivity (D) over a broader range in MCF10DCIS.com cells compared to MCF10CA1a cells, consistent with the higher cell volume plasticity observed in MCF10DCIS.com cells. This finding suggests that both cell types engage a pro-invasive mechanotransduction pathway. (Q) In contrast, this scaling relationship is absent in MCF10AT1, MDA-MB-231, ETCC-006, and ETCC-010 cells, indicating a lack of the mechanotransduction response. (R) The presence of this pathway in MCF10CA1a and MCF10DCIS.com cells is further supported by the observed >2-fold increase in TRPV4 plasma membrane association (x-axis; PM TRPV4_peg/PM TRPV4_ctrl) and >1-fold increase in diffusivity (y-axis; Dpeg/Dctrl) following PEG-300 treatment. (S) The cell volume reduction-driven mechanotransduction pathway is further demonstrated by plotting PEG-300-induced changes in TRPV4 plasma membrane association (x-axis; PM TRPV4_peg/PM TRPV4_ctrl) against the diffusivity ratio with GSK219 versus GSK101 (y-axis), where both cell types show a significantly greater than 2-fold increase, highlighting the activation of this pathway in MCF10CA1a and MCF10DCIS.com cells.****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: p > 0.05.
Cell crowding activates a pro-invasive mechanotransduction pathway in high-grade DCIS cells but not in less aggressive or normal cells.
This cell-crowding-induced pro-invasive pathway involves a cascade of events, including ion channel inhibition, intracellular calcium reduction, cell volume reduction and cortical stiffening, and increased cell invasiveness and motility. In high-grade DCIS cells, calcium-permeable ion channels such as TRPV4 relocate to the plasma membrane upon inhibition, compensating for reduced intracellular calcium levels by priming the channels for later activation under mechanical stress. The pro-invasive mechanotransduction pathway is selectively triggered by cell crowding or hyperosmotic stress in high-grade DCIS cells, which exhibit significant TRPV4 plasma membrane relocation, pronounced cell volume reduction, and increased motility and invasiveness. In contrast, less aggressive or normal cells remain significantly less or non-responsive to these stimuli. Notably, MCF10DCIS.com cells exhibit greater cell volume reduction compared to other cells, likely due to their larger baseline cell volume at normal density, demonstrating their high cell volume plasticity that correlates with crowding-induced invasiveness. The extent of TRPV4 plasma membrane relocation, cell volume reduction, and increased invasiveness and motility scales with each other, where the increased TRPV4 association with the plasma membrane can robustly serve as a marker of pro-invasive mechanotransduction activation. This mechanotransduction capability sets high-grade DCIS cells apart from less aggressive cells, providing a critical criterion that may help identify high-risk cells with invasive potential. This mechanotransduction capability was also validated in patient specimens, suggesting its relevance in clinical settings. Future investigations will include utilizing TRPV4 localization patterns as a diagnostic tool to assist in pathological grading and as a prognostic marker to identify high-risk DCIS cells likely to activate this pathway under mechanical stress.
