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. 2024 Apr 26;10(17):eadl4463.
doi: 10.1126/sciadv.adl4463. Epub 2024 Apr 26.

The role of Piezo1 mechanotransduction in high-grade serous ovarian cancer: Insights from an in vitro model of collective detachment

Hannah M Micek  1 , Ning Yang  2 , Mayuri Dutta  1 , Lauren Rosenstock  1 , Yicheng Ma  1 , Caitlin Hielsberg  1 , Molly McCord  3   4 , Jacob Notbohm  1   3   4   5 , Stephanie McGregor  2   5 , Pamela K Kreeger  1   2   5
Affiliations

The role of Piezo1 mechanotransduction in high-grade serous ovarian cancer: Insights from an in vitro model of collective detachment

Hannah M Micek et al. Sci Adv. .

Abstract

Slowing peritoneal spread in high-grade serous ovarian cancer (HGSOC) would improve patient prognosis and quality of life. HGSOC spreads when single cells and spheroids detach, float through the peritoneal fluid and take over new sites, with spheroids thought to be more aggressive than single cells. Using our in vitro model of spheroid collective detachment, we determine that increased substrate stiffness led to the detachment of more spheroids. We identified a mechanism where Piezo1 activity increased MMP-1/MMP-10, decreased collagen I and fibronectin, and increased spheroid detachment. Piezo1 expression was confirmed in omental masses from patients with stage III/IV HGSOC. Using OV90 and CRISPR-modified PIEZO1-/- OV90 in a mouse xenograft model, we determined that while both genotypes efficiently took over the omentum, loss of Piezo1 significantly decreased ascitic volume, tumor spheroids in the ascites, and the number of macroscopic tumors in the mesentery. These results support that slowing collective detachment may benefit patients and identify Piezo1 as a potential therapeutic target.

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Figures

Fig. 1.
Fig. 1.. Stiffness increases collective detachment of spheroids.
(A) Overview of culture system consisting of a PAA gel on top of a filter. At the completion of culture, the cover slip and PAA gel were removed for analysis. To isolate the Sph-CD (#1), the filter was inverted and captured spheroids were washed out. To isolate single cells (#2), the media was collected from the bottom well and passed through a cell strainer. Last, to isolate Sph-SC (#3), the cell strainer used to isolate single cells was inverted and washed to remove captured spheroids. All three fractions were then transferred to cell culture dishes to image and quantify. (B) Quantification of OV90, OVCAR3, and OVCAR8 Sph-CD from soft (5 kPa) or stiff (44 kPa) PAA gels functionalized with a low (100 μg/ml) or high (2000 μg/ml) Col I. (C) Quantification of OV90, OVCAR3, and OVCAR8 buds on soft or stiff PAA gels functionalized with Col I (100 μg/ml), five fields of view were averaged per gel. Data in (B) and (C) are the average ± SD of N = 3 to 4 individual gels. Statistical tests are two-way analysis of variance (ANOVA) with Tukey’s (B) and unpaired t test (C). ns indicates not statistically significant. *P < 0.05, ***P < 0.001, and ****P < 0.0001.
Fig. 2.
Fig. 2.. Stiffness-induced proliferation and YAP signaling are not responsible for increased collective detachment.
(A) Quantification of OV90 Sph-CD from soft or stiff gels treated with vehicle [dimethyl sulfoxide (DMSO)] or aphidicolin (4 μg/ml) to inhibit proliferation. (B) Representative images from Click-iT EdU staining of OV90 cells seeded on soft or stiff PAA gels. Spheroid buds are circled in white. (C) Quantification of proliferating OV90 cells within buds on the gel or in the monolayer (bulk) for soft and stiff gels, three fields of view were averaged per gel. (D) Representative images of YAP immunostaining (green) for OV90 on soft and stiff gels. (E) Total YAP signal and percentage of YAP localized to the nucleus quantified from immunofluorescent images of OV90 cells seeded on soft or stiff substrates, three fields of view were averaged per gel. (F) Quantification of OV90 Sph-CD from soft or stiff PAA gels treated with vehicle (DMSO) or 2 μM verteporfin to inhibit YAP. For (A), (C), (E), and (F), data shown are the average ± SD of N = 3 to 6 individual gels per condition. Statistical tests are two-way ANOVA with Tukey’s [(A), (C), and (F)], unpaired t test (E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 100 μm (B) and 10 μm (D). DAPI, 4′,6-diamidino-2-phenylindole; A.U., arbitrary units.
Fig. 3.
Fig. 3.. Piezo1 activity is needed for stiffness-induced collective detachment.
(A) Quantification of Sph-CD from OV90 cells on soft or stiff gels treated with vehicle (DMSO) or 10 μM GsMTx-4 to inhibit Piezo1. (B) Quantification of Sph-CD from wild-type (WT) OV90 or OV90 with PIEZO1 knocked out (KO) by CRISPR on soft or stiff gels. (C) Quantification of Sph-CD from OV90 cells on soft or stiff gels treated with vehicle (DMSO) or 10 μM Yoda1 to activate Piezo1. (D) qRT-PCR for PIEZO1 for OV90 cells on soft and stiff gels at 72 hours. Data are average ΔΔCt ± SD relative to GAPDH and soft gels, technical replicates. (E) Representative Piezo1 staining of OV90 on soft and stiff gels. (F) Piezo1 signal from OV90 cells seeded on soft or stiff gels, at least five fields of view were averaged per gel. (G) Root mean square (RMS) traction of OV-90 cells on soft and stiff substrates at 24 hours, two to five fields of view were averaged per gel. (H) Quantification of Sph-CD from OV90 cells seeded on soft or stiff gels treated with vehicle (DMSO) or 0.5 μM ML-7 to inhibit myosin light chain kinase. (I) Representative images of immunofluorescent staining of benign and HGSOC omenta for Piezo1. Pan-cytokeratin (PanCK) was used to mark tumor cells. (J) Integrated intensity of Piezo1 staining within the tissue, three fields of view were averaged per patient. For (A) to (H), data shown are the average ± SD of N = 3 to 5 individual gels per condition. In (J), data shown are the average ± SD of N = 7 to 10 patients per group. Statistical tests are two-way ANOVA with Tukey’s [(A) to (C) and (H)], unpaired t test [(D) and (F)], unpaired t test with Welch’s correction (G), Mann-Whitney test (J). *P < 0.05, **P < 0.01, and ****P < 0.0001. Scale bars, 100 μm.
Fig. 4.
Fig. 4.. Stiffness increases MMPs, leading to decreased ECM.
(A) Quantification of Sph-CD from OV90 cells on soft or stiff gels treated with vehicle (DMSO) or 10 μM batimastat. (B) MMP concentrations for OV90 cells on soft or stiff gels. (C) MMP-1 and MMP-10 concentrations for WT OV90 [same data in (B)] or OV90 with PIEZO1 knocked out by CRISPR on stiff gels. (D) Representative images and quantification of immunofluorescent staining of OV90 cells seeded on soft or stiff gels for 72 hours stained with CNA35-EGFP (to visualize deposited Col I) or antifibronectin. For (A) to (D), data shown are the average ± SD of N = 3 to 5 individual gels per condition. Statistical tests are two-way ANOVA with Tukey’s (A), Sidak’s multiple comparisons [(B) and (C)], and unpaired t test (D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Scale bars, 50 μm. FN, fibronectin.
Fig. 5.
Fig. 5.. Loss of PIEZO1 slows metastasis to the mesentery.
(A) Tumors were initiated by intraperitoneal injection of WT and PIEZO−/− OV90 cells. After 13 or 35 days, the omentum and mesentery were examined for tumor burden. (B) By 35 days, all mice developed ascites, with the volume significantly less in mice injected with OV90 with PIEZO1 knocked out by CRISPR. Quantification of spheroids isolated from ascites fluid, data shown are average spheroids per field of view (FOV). N = 6 to 8 mice per group, with at least three fields of view averaged per mouse. ***P < 0.001 by unpaired t test. (C) Mice were categorized by the presence or absence of macroscopic tumors. N = 12 to 13 mice per group per time point. P values are for Fisher’s exact test. (D) The total volume of all macroscopic tumors on the two tissue sites. Because of the low number of macroscopic tumors in the mesentery, statistics were not possible. (E) Hematoxylin and eosin (H&E)–stained sections of mesentery were imaged, and the percentage of tissue that was tumor was quantified. N = 9 to 11 mice per group, not significant by Mann-Whitney test. Data from day 13 were collected from a single trial. The day 35 data are a concatenation of two separate experiments; ascites collection was only conducted on the second trial.
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