Ezrin plays a key role in cancer cell spheroid formation in soft environments

Ezrin plays a key role in cancer cell spheroid formation in soft environments | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ezrin plays a key role in cancer cell spheroid formation in soft environments Noelia Lujea, Veronica Lopez, Roberto Pezza, Gabriela Paglini, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6595737/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A unique form of metastasis characterizes ovarian cancer (OC), in which tumors grow either suspended in the peritoneal fluid or attached to peritoneal tissues. Notably, the mechanisms underlying metastasis in ascitic fluid remain poorly understood. A critical step in this process is the formation of cancer cell spheroids. We hypothesize that spheroid generation depends on actin cytoskeleton-associated proteins at the cell cortex. In this context, Ezrin, a member of the ERM (Ezrin-Radixin-Moesin) family protein, emerged as a strong candidate. To investigate, we established a 3D culture model using SKOV3 cells and assess spheroid formation under different conditions. We found that Ezrin-depleted cells, failed to form spheroids in soft agar, a phenotype reversed by reintroducing wild-type Ezrin, ConA treatment, increasing agar concentration, or mimicking the physical interactions of neighboring cells within a spheroid. Complementary experiments in xenograft nude mouse models confirmed the essential role of Ezrin in tumor development. Notably, Intraperitoneal injection of spheroids generated in vitro overcame the effects of Ezrin depletion. Together, these findings underscore the role of Ezrin as a structural component of the cell cortex, crucial for tumor formation in soft environments, and highlight the broader relevance of ERM proteins in intraperitoneal metastasis in OC. Biological sciences/Cancer Biological sciences/Cell biology Ezrin SKOV3 cells ovarian cancer ascitic fluid actin cytoskeleton tumor spheroid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights 1. Ezrin is essential for spheroid formation in soft microenvironments. 2. Ezrin is not needed for spheroid generation in stiffer microenvironments. 3. An increase in cortical cell stiffness or a stiffer environment can compensate for absence of Ezrin. INTRODUCTION Ovarian cancer (OC) metastases present a unique therapeutic challenge as they primarily occurs within the peritoneum, with secondary tumors either suspended in the peritoneal fluid or adhered to the peritoneal surfaces 1 . These metastatic growths take the form either of spheroids or isolated cells. Spheroids may originate from spontanoeous cellular aggregation or from successive mitotic divisions of a single cell 2 , 3 . OC-derived spheroids emerge within a non-structured and soft environment primarily composed of ascitic fluid. While the formation of aggregates supports tumor cell survival through various mechanisms, such as shielding them from anoikis, individual cells face the challenge of surviving and proliferating while suspended in the ascitic fluid, a mechanically unique soft environment 4 , 5 . This is important because the factors and mechanisms underlying cell survival and proliferation in such conditions remain poorly understood. One such factor could be the cortical tension, defined as the mechanical force produced by the cell cortex. This force influences cell survival and proliferation in various environments 6 , 7 . During the generation of cortical tension, actin filaments reorganize and connect to the plasma membrane through associated proteins, such as the Ezrin, Radixin, and Moesin (ERM) proteins 8 , 9 . Thus, to understand how an isolated metastatic cell thrives in a soft, fluid environment requires identifying what proteins enable the interactions between cytoskeletal components and the plasma membrane. In this context, the ERM’s emerge as promising candidates for such a role 10 . In particular Ezrin is involved in migration, invasion, and proliferation of various cancers 11 – 14 . Thus, we hypothesize that Ezrin facilitates spheroid formation within soft environments, favoring OC metastasis. To test this, we carried out experiments in a 3D soft agar culture model with the SKOV3 OC cell line and a xenograft model in nude mice. Our results revealed that Ezrin is critical for cancer cell spheroid generation via supporting cell division in a soft environment reminiscent of peritoneal ascitic fluid. RESULTS Ezrin plays a key role in SKOV3 cell spheroid formation in soft agar. To investigate the involvement of ERM proteins in cell spheroid formation, we utilized a spheroid generation model in a soft agar matrix (Fig. 1 ). We generated Ezrin knockout SKOV3 cells using CRISPR-Cas9 technology. This resulted in two stable cell lines named Ezr6148 and Ezr6059. The presence of mutations in the ezrin gene in Ezr6148 and Ezr6059 cells was confirmed through sequencing and western blot analysis (Suppl. Figure 1a-c). Depletion of Ezrin led to a significant reduction in spheroid formation compared to SKOV3 control cells. Crystal violet staining at 20 days in culture revealed that SKOV3 cells formed spheroids in soft agar with an average of 11.5 (± 2.7) spheroids per field, whereas the depletion of Ezrin protein significantly decreased the spheroid formation compared to control in both cell lines (Ezr6148 = 3.0 ± 0.9 spheroids per field, (n = 4; t(9) = 12,38; p < 0.0001) and Ezr6059 = 1.5 ± 0.5 spheroids per field, (n = 4; t(9) = 11,20; p < 0.0001), respectively) (Figs. 1 a and b). To determine why Ezrin deletion reduced the number of spheroids, we focused on the initial stages of spheroid formation, examining spheroid development at 1, 3, and 5 days. We selected these early times because prolonged culture periods led to larger spheroids that hindered accurate quantification. We found that whereas SKOV3 cells divided and increased in number after 3 days, both Ezr6148 and Ezr6059 mutants remained as individual cells (n = 4; F(1.183, 15.38) = 17.78) (Figs. 1 c and d). Quantitative analysis indicated that SKOV3 cells exhibited a progressive increase in cell count per spheroid over the culture period (1.38 ± 0.03 cell per spheroid; 1.96 ± 0.18 cell per spheroid; 3.11 ± 0.39 cell per spheroid; for 1, 3, and 5 days, respectively (Figs. 1 c and d). In contrast, Ezr6148 and Ezr6059 cells remained at approximately 1 cell per spheroid, with no statistically significant changes observed throughout the culture period (p = 0.0004 and p = 0.0006 for Ezr6148 and Ezr6059, respectively) (Figs. 1 c and d). To assess the ability of cells to complete mitosis and divide in soft agar, we quantified the number of nuclei per cell in each spheroid. SKOV3 cells had an average of 1.08 (± 0.06) nuclei per cell at 5 days (Figs. 1 c and e). In contrast, both Ezr6148 and Ezr6059 cells exhibited more than one nucleus per cell after 1 day, reaching an average of 1.34 (± 0.08) and 1.44 (± 0.13) nuclei per cell by 5 days, respectively (n = 4, F(1.625, 22.75) = 15.55; p < 0.0001) (Figs. 1 c and e). Collectively, these data suggest that Ezrin plays a key role in spheroid formation in soft agar. Restoring Ezrin expression reinstates the ability of the SKOV3 cells to divide in soft agar. To test the direct association of Ezrin and the observed cell growth phenotype, we restored Ezrin expression in both Ezr6148 and Ezr6059 cells (Fig. 2 ). To this end, we transfected SKOV3, Ezr6148, and Ezr6059 cells with the pcDNA3.1-EzrWT plasmid. These modified lines are denoted as SKOV3 EZR , Ezr6148 EZR , and Ezr6059 EZR . Ezrin expression was confirmed by western blot (Suppl. Figure 1e). We induced spheroid formation in 0.5% soft agar using SKOV3, SKOV3 EZR , Ezr6148, and Ezr6148 EZR cells. Quantitative analysis revealed no significant difference in spheroid count between the wild-type SKOV3 and the SKOV3 EZR (10.3 ± 0.6 vs. 11.6 ± 0.5 spheroids per field, respectively) (Fig. 2 a and b). As expected, overexpressing Ezrin in Ezr6148 and Ezr6059 cell lines effectively restored their capacity to form spheroids in soft agar after 20 days in culture (Ezr6148: 0.7 ± 0.3 vs. Ezr6148 EZR : 5.5 ± 0.3 spheroids per field; n = 3; p < 0.0001 and Ezr6059: 0.9 ± 0.3 vs. Ezr6059 EZR : 4.1 ± 0.5 spheroids per field; (one way ANOVA n = 3, F (5, 54) = 101.8; p < 0.001) (Fig. 2 b). Moreover, analysis of spheroid growth by immunofluorescence revealed that both Ezr6148 EZR and Ezr6059 EZR increased the number of cells per spheroid between 1 and 5 days, a value similar to that observed in control (two-way ANOVA, n = 3, F (1.352, 13.52) = 89.38, p < 0.0001) (Ezr6148 EZR : 1.37 ± 0.02 and 2.15 ± 0.05 cells per spheroid for 1 and 5 days respectively (n = 3; p < 0.0001), and Ezr6059 EZR : 1.28 ± 0.04 and 2.43 ± 0.09 spheroids per field for 1 and 5 days in culture respectively (Figs. 2 c and d). More importantly, while Ezr6148 and Ezr6059 cells increased the number of nuclei per cell over time in culture, the Ezr6148 EZR and Ezr6059 EZR cells maintained a single nucleus per cell from 1 to 5 days as the spheroids grew (n = 3, F(1.912, 28.67) = 27.59, p < 0.05) (Figs. 2 c and e). These results indicate that overexpression of Ezrin in the Ezr6148 and Ezr6059 mutants restores spheroid formation, confirming that the observed phenotype is Ezrin-dependent. Enhanced cortical tension induced by ConA treatment reinstates the capability of Ezrin knockout cells to form spheroids in a soft environment. Ezrin plays a crucial role in organizing the cellular cortex, a process essential for cell division 15 – 18 . Prompted by these findings, here we investigate whether the positive effect of Ezrin in cell spheroid formation may be derived from the ability of Ezrin to alter the stiffening of the cellular cortex. In vitro administration of ConA has been shown to induce an increase in cortical tension 16 , 19 . Thus, if stiffening of the cellular cortex due to Ezrin accounts for its role in spheroid formation, we expect that ConA treatment of Ezr6148 cells should restore their ability to form spheroids in soft agar. To test this, both SKOV3 and Ezr6148 cells were incubated for 30 minutes in media supplemented with ConA, while media without ConA was used as control. Then, cells were plated in soft agar and cultured up to 20 days (Fig. 3 a). As predicted, SKOV3 cells, regardless of pre-incubation with or without ConA, formed cell spheroids (9.16 ± 1.72 and 10.17 ± 3.43 spheroids per field), with no differences between them (n = 3, F(3, 20) = 29.72; p = 0.9543, one-way ANOVA) (Fig. 3 a). As expected, Ezr6148 cells did not generate spheroids in 0.5% agarose after 20 days (0.33 ± 0.21 spheroids per field). In sharp contrast, Ezr6148 cells treated with ConA exhibited a significant increase in spheroid formation (4.83 ± 1.16 spheroids per field, n = 3, p = 0.0059) (Figs. 3 a and b). Furthermore, immunofluorescence analysis confirmed a significant increase in the number of cells per spheroid (Fig. 3 c and d) in ConA treated Ezr6148 cells from 1 to 5 days in culture (F (2, 6) = 13.74, P = 0.058, two-way ANOVA). Analysis of the average of nuclei per cell revealed that ConA treatment reversed the effect caused by Ezrin silencing (Fig. 3 c and e). We conclude that Ezrin may mediate the stiffening of the cellular cortex, allowing for spheroid formation in soft environments. Increasing the stiffness of the cell culture media reinstates the ability of Ezrin knockout cells to form spheroids in a soft environment. Previous studies have suggested that cells can sense mechanical cues from their surroundings via an actin-dependent mechanism 20 . In order to explore whether this phenomenon explains Ezrin-dependent spheroid formation, we performed an in vitro experiment where we manipulated the agarose concentration to create different levels of environmental rigidity. Advantages of employing agarose are the ability to facilitate the free diffusion of nutrients within the culture media 21 and that rigidity or elasticity of agarose matrices at different densities has been well stablishes 22 – 24 . We utilized various agarose concentrations ranging from 0.5–3%, selected for their resemblance to the reported viscosity levels in human ascites 25 , 26 and because this approach has already been used 27 .We predict that at some agarose rigidity Ezr6148 cells will recover their ability to form cell spheroids. We found that SKOV3 cells formed spheroids after 20 days in all the agar densities we tested but with a noticeably reduced capability to from spheroids in 2% and 3% agarose (F(7, 24) = 51.34, P < 0.0001, n = 3, one-way ANOVA) (Figs. 4 a and b). As expected, Ezr6148 cells failed to form spheroids at 0.5% agarose. Notably, we observed a significant increase in the number of Ezr6148 spheroids at 1% agarose (4.25 ± 0.95 spheroids per field; n = 3, p < 0.0001), with no significant differences compared to SKOV3 cells at 1% agar (Figs. 4 a and b). However, at agar concentrations of 2% and 3%, Ezr6148 exhibited a notable decrease in spheroid formation similar to SKOV3 cells growing in 3% agarose. F-actin immunolabeling and DAPI revealed spheroid formation up to 5 days. Quantitative analysis showed an increased number of cells per spheroid over time in culture in both 0.5% and 1% agarose (n = 3, F(2, 24) = 14.15; p < 0.0001, one-way ANOVA) (Figs. 4 c and d). In addition, SKOV3 spheroids exhibited an average of one nucleus per cell over time in all agarose concentrations (P = 0.2469, Kruskal-Wallis test) (Figs. 4 c and d). More interestingly, whereas the number of cells per spheroid in Ezr6148 significantly increased from 1 to 5 days in culture at 1% agarose (n = 3, F(11, 24) = 10.11; P < 0.0001) (Fig. 4 c and e-f). While the number of nuclei per cell increased in 0.5% agarose over time, cells cultured in 1% soft agar maintained an average of one nucleus, similar to SKOV3 control spheroids (n = 3, F(11, 21) = 9.56; p < 0.0001) (Figs. 4 c, d, g and h). We concluded that increasing the stiffness of the cell media reinstates the ability of Ezrin knockout cells to form spheroids. Spheroids of Ezrin-depleted cells formed by physical aggregation can sustain cell division. The intercellular signals within a spheroid are pivotal for initiating cell aggregation, leading to the formation of a cohesive spheroid structure 28 . Moreover, the neighboring cells surrounding each cell within the spheroid contribute to its structural integrity and stiffness 29 . We tested whether the depletion of Ezrin in Ezr6148 cells affect their ability to undergo proper cell division and maintain one nucleus per cell once they became part of a spheroid. To investigate this, we generated spheroids comprising SKOV3 or Ezr6148 cells using the suspension-based aggregation method of spheroid formation 28 . In this technique, cells are suspended in a liquid medium layered on agar, preventing adhesion to the substrate, and facilitating aggregation into a compact spheroid at the center of the well. Cell division was assessed by labeling against PhosphoHistone3 (Ser-10) (as a mitotic marker), Rhodamine-Phalloidin (an F-actin marker), and DAPI (for nucleus) (Fig. 5 ). Ezr6148 cells were able to form spheroids in suspension (Fig. 5 a). Furthermore, Ezr6148 cells kept only one nucleus on average with the successive cell divisions, suggesting that the spheroid environment sustained cell division despite the absence of Ezrin (P = 0.5390, Kruskal-Wallis test) (Fig. 5 b). Even if the cell had only one nucleus, failure to achieve a complete cell division could be evidenced by an increase in the size of the nucleus. To test this, we measured the nuclear area of each cell within the spheroids for both SKOV3 and Ezr6148 cells. We found no differences in nuclear area between SKOV3 and Ezr6148 cells at any time point (P = 0.5973) (Fig. 5 c). These results suggest that cellular context may restore the ability of Ezrin-depleted cells to multiply. Ezrin is required for tumor formation in a xenograft mouse model. We assessed the ability of SKOV3 or Ezr6148 cells to generate spheroids in vivo using an intraperitoneal metastasis model in nude mice. First, female mice were injected with either SKOV3 or Ezr6148 cells. After 8 weeks, the animals were euthanized, and tumor formation was examined. Table 1 in Suppl. Figure 2 shows the mice weight, the presence of metastases, and the weight of the metastatic masses. We observed that mice injected with SKOV3 cells formed tumors. In contrast, injection with knockout Ezrin cells significantly reduced intraperitoneal tumor formation (Fig. 6 a). Eight out of eleven animals injected with SKOV3 cells formed tumors within 3 months. They displayed various histological tumor subtypes including one endometrioid, one serous, one clear cell, and five undifferentiated tumors. Out of 5 mice injected with the mutated Ezrin, only one formed a tumor. Figure 6 b depicts the HE staining for the tumors obtained under each experimental condition. Next we explored whether spheroids previously formed in vitro could develop tumors in mice. Female mice were injected with spheroids derived from either SKOV3 or Ezr6148 cells. Figure 6 a show that the injection of spheroids generated in vitro resulted in the formation of tumors whereas i.p. administration of isolated individual Ezrin6148 cells resulted in no tumors. These results agree with our in vitro findings that Ezrin-silenced SKOV3 cells are unable to form spheroids in a soft environment. This suggests that Ezrin plays a crucial role in intraperitoneal tumor formation in vivo . DISCUSSION Cells adeptly sense changes in the physical properties of their surroundings and adjust accordingly. Variations in the mechanical characteristics of the environment serve as external cues, impacting the cell cortex and eliciting an elastic deformation response, known as mechanotransduction. In the context of the OC metastasis, tumor cells detach from the primary tumor and proliferate within the peritoneum, often in unstructured or fluid-filled media like ascitic fluid, a hallmark of this cancer subtype. Surviving in such conditions requires adaptive mechanisms. Given the role of the cell cortex and its proteins in mechanotransduction, we focused on Ezrin, a key ERM family member. We propose that Ezrin plays a crucial role in facilitating the transition from the structured environment of the primary tumor to colonizing the unstructured peritoneal milieu. Our findings underscore the indispensability of Ezrin in cellular adaptation, enabling proliferation across diverse environments. Considering Ezrin's role in cortex formation and tension generation, we employed ConA to induce environmental tension in our experiments 10 , 30 . Previous studies have elucidated the effects of ConA on 3D models 31 , 32 . ConA interacts with glycoproteins on the plasma membrane, forming an external mesh that enhances cellular rigidity 33 . Our findings revealed that silencing Ezrin significantly impaired spheroid formation in soft agar, while treatment with ConA reversed this phenotype induced by Ezrin depletion. This underscores Ezrin's pivotal role in adapting to varying mechanical environments and reinforces the importance of cortical rigidity in tumor cell division 16 . Consistent with our results, a recent study demonstrated reduced cortical tension and decreased activation of phosphorylated ERMs in fertilized and cryopreserved oocytes 34 . Consequently, Ezrin-deficient oocytes exhibited defects in spindle positioning and chromosome segregation. Remarkably, supplementing the culture media with ConA restored cortical integrity, ensuring oocyte viability and proper chromosome segregation 34 . Thus, the work by Du and colleagues provide further support for our findings regarding ERMs' function in spheroid formation and, importantly, highlights ConA as an experimental substitute for the structural role of ERMs. Our study adds to the body of knowledge by highlighting the structural role of cortical rigidity mediated by Ezrin in tumor cell spheroid formation. In a complementary approach, we tested how agar matrices can be used to manipulate environmental stiffness. Notably, we observed that precise agar matrix rigidity (1% agarose concentration) can compensate for Ezrin absence and cells are able to complete cell division. Our results clarify the controversial findings regarding the proliferative effect of manipulating the stiffness of the cellular environment 23 35 . Indeed, research using rat prostate cancer cell lines revealed that agar-induced tension promoted spheroid formation 35 . Conversely, increased medium rigidity hindered human colon adenocarcinoma cells spheroid growth 23 . Collectively, our findings point to the importance of Ezrin-mediated cortical rigidity in the ability of cells to divide and, further underscore the potential therapeutic implications of modulating cortical mechanics. We also help to elucidate Ezrin's role in substrate-independent growth within soft environments relevant to the type of peritoneal metastasis associated with OC. This contributes to further understand the intricate interplay between cellular mechanics and environmental cues in cancer progression. Another important aspect of mechanotransduction is the intimate cell-cell contact observed within spheroids, mirroring the physical environment and interactions seen in tissues 36 – 38 . In vitro studies on spheroid formation reveal a transition from loose to compact states, strengthening cohesive forces among constituent cells. This process crucially involves cadherin and integrin proteins 28 , 39 . Hence, when analyzing spheroids, it is imperative to consider the mechanical forces within the cellular milieu and the interactions among neighboring cells as observed in living tissues 39 . These cellular interaction forces profoundly impact various cellular processes such as division. In light of these dynamics, we generated spheroids with Ezrin knockout cells via aggregation in suspension. Notably, observations within these spheroids revealed normal mitotic activity, highlighting the crucial role of the cellular environment in restoring tumor cells capacity for division. Again, these findings underscore Ezrin significance in governing cell division, particularly in scenarios lacking structured environmental support. Our studies utilized SKOV3 cells, which were originated from the ascitic fluid of a patient with ovarian papillary cystadenocarcinoma 40 . This cell line closely mimics specific cellular and molecular processes anticipated within the peritoneal environment occurring in patients, thus offering valuable insights into the potential role of Ezrin in this context. Importantly, to address limitations in using in vitro systems, we employed a xenograft model using nude mice 41 . Our results in this model mirrored the observations made in vitro using the soft agar model, and more importantly, injecting previously cultured spheroids reversed the effect of deleting Ezrin. Integrating in vitro and in vivo observations allowed us to better understand the role of Ezrin role in OC metastasis. The new insights we provide regarding the factors that facilitate spheroid formation in a soft environment is significant, as the mechanical properties of the environment can promote malignancy in various types of cancers, particularly in OC, which carries a high mortality rate 42 – 44 . OC is notorious for its late diagnosis, limited treatment options for advanced stages, high recurrence rates (85%), and a modest 46% 5-year survival rate 45 , 46 . The challenge in treating metastatic OC over the past five decades stems from minimal advancements in understanding the disease, delayed detection, chemoresistance, and the complexity of the tumor microenvironment 47 , 48 . Hence, identifying and understanding mechanisms of action of key proteins such as Ezrin involved in spheroid formation within the peritoneum can significantly impact future therapeutic strategies associated with OC. METHODS Cell Culture We cultured the human epithelial OC cell line SKOV3 as described 49 . The SKOV3 cells were generously provided by Dr. Osvaldo Podjhajcer (Leloir Institute, Buenos Aires, Argentina). This cell line was at passage 3 from an originally certified cell line obtained from ATCC (ATCC HTB77) and was screened for Mycoplasma sp. contamination using PCR, following standard protocols 50 . Soft agar assay Spheroids were generated through successive cell divisions from a single cell utilizing the modified soft agar technique 51 . Briefly, 1 ml of 1% low melting point agarose (Bethesda Research Laboratories, USA) was applied to coat the base of a 6-multiwell plate. Cells were trypsinized and diluted to a concentration of 8,000 cells per well. Subsequently, a layer containing 3 ml of 0.5% agar mixed with culture medium containing the cells was added to each well and allowed to solidify at room temperature. The culture medium was replenished on the agar surface every 3 days until used. Crystal violet staining Spheroids on agar were fixed in methanol/acetic acid (3:1) for 30 minutes, incubated for 1 hour with 0.5% crystal violet in 20% methanol. Images were captured using a Zeiss SZX7 microscope, and the numbers of spheroids per field were counted from 10 fields per condition in each experiment. Spheroids were stained after 20 days of culture and their area measured 52 , at which point they reached a spherical shape and an average size of 317 µm² (± 146 µm²) for SKOV3 cells. At this stage, spheroids were optimal in size and clearly visible. Cells within the spheroids showed high viability and low apoptosis, similar to monolayer cultures. Immunofluorescence For mitosis quantification and multinuclearity assessment, cells were fixed in 4% PFA/sucrose for 20 minutes, permeabilized with 0.2% Triton-X100, and blocked with 5% BSA in PBS for 30 minutes 16 , 49 . Phospho-Histone-3, (Ser-10) (clone 6G3) mouse monoclonal antibody (Cell Signaling Technology Cat# 9706, RRID: AB_331748) was used at 1:1,000, incubated overnight at 4°C, followed by incubation for 2 hours at room temperature with a secondary antibody (Alexa Fluor 488, Donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Invitrogen Cat# A21-202, RRID: AB_141607), at 1:500. Rhodamine-conjugated phalloidin (Sigma, used at 0.1 mg/ml) and DAPI (Invitrogen; 1:10,000) were also used 53 . Immunostained spheroids were mounted with Mowiol media, and images were acquired using Olympus FV-1200, LSM800 confocal, or Olympus IX81 microscopes. Image analysis was performed using ImageJ software ( https://imagej.net/Fiji , RRID:SCR_002285) 49 . Ezrin CRISPR-Cas9 stable cell lines Two Ezrin-targeting plasmids were designed using CRISPR-Cas9 (VectorBuilder) with gRNAs #6148 (exon 9) and #6059 (exon 8), based on on-target and off-target ratios of 53.3/90.3 and 55.6/49.7, respectively. The Ezrin protein map (Suppl. Figure 1A) shows gRNA recognition sites within the α-helical domain. SKOV3 cells were co-transfected with CRISPR constructs and PEGFP-Puro and then selected with puromycin for 15 days. The mutations (Ezr6148 and Ezr6059) were confirmed by DNA sequencing. The sequences have been submitted to GenBank under accession numbers Seq1 PV703827 and Seq3 PV703829 for the mutants Ezr6148 and Ezr6059 respectively and their respective SKOV3 control WT (seq2 PV703828 and Seq4 PV703830). The clones with mutations were also confirmed by Western blot analysis using an ERM antibody (Cell Signaling Technology #3141), with tubulin (Anti-α-Tubulin, clone DM1A, Sigma) as a loading control 49 . To evaluate Ezrin silencing effects on cell survival, proliferation, and apoptosis, SKOV3, Ezr6148, and Ezr6059 cells were compared. Viability was 99% in all groups (determined by resazurin and trypan blue). Proliferation was assessed with anti-phospho-Histone-3 and DAPI, and apoptosis was quantified by apoptotic body count (Suppl. Figure 1D) 54 . No significant differences were found in survival, proliferation, or apoptosis between CRISPR clones and SKOV3 control (one-way ANOVA: p = 0.6349 for mitosis; p = 0.7612 for apoptosis). Genetic rescue Stable cell lines were generated using SKOV3 and two CRISPR-modified forms of Ezrin (Ezr6148 and Ezr6059). These cell lines were transfected with the pcDNA3.1-topo-GFP-Ezrin wild-type plasmid (provided by Dr. Guillaume Charras) and selected with G418 (500 µg/ml). Clones were selected by dilution in a 96-well plate. A positive clone was chosen for each line: SKOV3EZR, Ezr6148EZR, and Ezr6059EZR. Western blot analysis (as described in 2.5) showed a significant increase in ERM protein expression in all clones (Suppl. Figure 1E). Concanavalin A treatment A phenotype rescue experiment was conducted using Concanavalin A (Sigma, #C2010) (ConA). Ezr6148 or SKOV3 cells (8,000 cells) were incubated with 100 µg/ml ConA at 37°C for 30 minutes, then plated in soft agar. Control cells were incubated with media alone. Samples were collected at 1, 3, and 5 days, fixed with 4% PFA/sucrose for immunofluorescence staining, or at 20 days and stained with 0.5% crystal violet. Formation of spheroids in suspension SKOV3 and Ezr6148 cells were trypsinized and adjusted to 5,000 cells/ml. Next, 100 µl of the cell suspension was added to a 96-well plate coated with 1% agar. The plates were incubated for 24, 48, 72, and 96 hours. After incubation, spheroids were fixed in 4% PFA/sucrose for 20 minutes and processed for immunofluorescence. Immunostaining with DAPI and phalloidin was performed as described previously, and 3D images were captured using the Olympus LSM800 confocal microscope Soft agar rescue assay For the soft agar rescue assay, agar solutions were prepared at final concentrations of 0.5%, 1%, 2%, and 3%. Subsequently, 8,000 cells (SKOV3 or Ezr6148) were plated in each well of a 6-well plate containing 1% agar at the base, with each well containing varying agar concentrations. Spheroids were fixed using 4% PFA/sucrose at 1, 3, and 5 days in culture and subsequently processed for immunofluorescence analysis. Crystal violet staining at 0.5% was conducted at day 20 in culture. In vivo studies Four female athymic N:NIH(S)-nu mice (5–6 weeks old, from the University of La Plata, Argentina) were intraperitoneally injected with 6 × 10^6 SKOV3-luc or 6 × 10^6 EZR6148-luc cells, as described 41 . Cell viability was 99%, as confirmed by a Countess cell counter (Invitrogen). For the rescue phenotype assay, SKOV3 and EZR6148 spheroids were generated on a p150 plate with 10 ml of solidified agar and 20 ml of medium containing 2 × 10^6 cells. After 72 hours, spheroids were collected, washed in PBS 1X, and resuspended in 200 µl of PBS. Seven female nude mice (4–6 weeks old) were intraperitoneally injected with this spheroid suspension. Each assay was performed twice, and mice were sacrificed at 7–8 weeks post-injection. Ethical considerations All experiments involving mice were carried out in accordance with the relevant guidelines and regulations. This study was approved by the Ethics Committee at the Leloir Institute, under protocol number CICUAL-FIL 92. All methods are reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Animals were housed and cared for following the institutional and national guidelines for the care and use of laboratory animals. No procedures causing undue stress or pain were conducted without appropriate anesthesia (isoflurane, induction at 5%, maintenance at 3%). Thus, mice were euthanized using carbon dioxide inhalation displacement rate from 30–70% of the chamber volume/min in accordance with the AVMA Guidelines for the Euthanasia of Animals, 2020 Edition ( https://www.avma.org/resources-tools/avma-policies/avma-guidelines-euthanasia-animals ). Hematoxylin/Eosin staining (HE) Sections of 10 µm thickness were de-paraffinized and stained with Mayer's Hematoxylin for 10 minutes, followed by 0.2% Eosin for 30 seconds. After washing with ethanol and xylene, the sections were mounted with DPX medium. Images were captured with a NIKON T2000U microscope. Statistical analysis All data are presented as mean ± SEM from 3 or 4 independent experiments. Statistical analysis and graphs were performed using GraphPad Prism 6.0 software. The assumption of normality for each dataset was assessed using the D’Agostino-Pearson and Shapiro-Wilk normality tests. To compare 2 conditions (Control vs. Treatment) Student’s t-test was performed for data that passed the normality test, reported as t(df) = t-statistic, p = significance value. Mann-Whitney was used for datasets that failed the normality test. One-way ANOVA or two-way ANOVA was used for comparing data sets with one or more degrees of freedom, respectively, reported as F(between groups df, within groups df) = [F-value], degrees of freedom, p = [p-value]. Kruskal-Wallis was used as nonparametric test. A p-value < 0.05 was considered statistically significant. Significance is denoted on all graphs as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Declarations Acknowledgments CA, VL, GP and PK are researchers of CONICET. This research was financially supported by INC (project designation number 30-54666038-5), to PK. NL received a studentship from CONICET. We gratefully acknowledge CEMINCO, CIBICI, and the Virology Institute at UNC for granting us access to their microscopy facilities. Author contributions NL and VL performed experiments and data analysis, VL performed xenograph experiments, RP and GP writing and editing, CA data analysis, writing and editing, PK paper conception, analysis, and writing. All authors reviewed and approved the manuscript. Data availability All datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Sequence data that support the findings of this study have been deposited in the GenBank from the National Center for Biotechnology Information (NCBI) with the following primary accession codes: PV703827; PV703828; PV703829; PV703830. Conflict of interest All the authors declare no competing interests. References Tan, D. S., Agarwal, R. & Kaye, S. B. Mechanisms of transcoelomic metastasis in ovarian cancer. 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Oncol. 138 , 181–189. 10.1016/j.ygyno.2015.04.014 (2015). Kunda, P. E., Cavicchia, J. C. & Acosta, C. G. Lipopolysaccharides and trophic factors regulate the LPS receptor complex in nodose and trigeminal neurons. Neuroscience 280 , 60–72. 10.1016/j.neuroscience.2014.08.053 (2014). Mandelkow, R. et al. Detection and Quantification of Nuclear Morphology Changes in Apoptotic Cells by Fluorescence Microscopy and Subsequent Analysis of Visualized Fluorescent Signals. Anticancer Res. 37 , 2239–2244. 10.21873/anticanres.11560 (2017). Additional Declarations No competing interests reported. Supplementary Files SupplFiguresLunea2025SciRep.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6595737","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":468043177,"identity":"f69b2d8a-8e3a-4b03-827d-f86a1479a08b","order_by":0,"name":"Noelia Lujea","email":"","orcid":"","institution":"Instituto Universitario Ciencias Biomédicas Córdoba (IUCBC)","correspondingAuthor":false,"prefix":"","firstName":"Noelia","middleName":"","lastName":"Lujea","suffix":""},{"id":468043178,"identity":"fe4c7b25-a531-4ee2-908d-ed0743225a24","order_by":1,"name":"Veronica Lopez","email":"","orcid":"","institution":"Fundación Instituto Leloir, IIBBA-CONICET","correspondingAuthor":false,"prefix":"","firstName":"Veronica","middleName":"","lastName":"Lopez","suffix":""},{"id":468043179,"identity":"a3db09fc-2feb-4acd-8d59-eb602140f2d7","order_by":2,"name":"Roberto Pezza","email":"","orcid":"","institution":"Oklahoma Medical Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Roberto","middleName":"","lastName":"Pezza","suffix":""},{"id":468043180,"identity":"1eac3b48-094c-4e8e-b060-efcf1701875d","order_by":3,"name":"Gabriela Paglini","email":"","orcid":"","institution":"Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC-CONICET-Universidad Nacional de Córdoba","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"","lastName":"Paglini","suffix":""},{"id":468043181,"identity":"de62c461-2ed5-40a1-8c49-a19b71a0bf0f","order_by":4,"name":"Cristian Acosta","email":"","orcid":"","institution":"Universidad Nacional de Cuyo. 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Crystal violet staining of spheroids formed in soft agar. The black dots in control are SKOV3 spheroids. In the samples where Ezrin was silenced, the cells formed smaller (barely visible) and fewer spheroids, indicated by yellow arrows. b. Plot of the number of spheroids of SKOV3, Ezr6148 and Ezr6059. c. Representative images of spheroids in 0.5% agarose at 1, 3 and 5 days, labeled with Rhodamine-Phalloidin as F-actin marker, and DAPI for nucleus. d. Plot of the number of cells per spheroid. e. Plot of the number of nuclei per cell. Data are presented as mean ± SEM, *p\u0026lt;0.05, **p\u0026lt;0.01; ****p\u0026lt;0.00001.\u003c/p\u003e","description":"","filename":"Figure1Lujeaetal2025SciRep.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6595737/v1/16602833b2abb8eda5fc867d.jpg"},{"id":84228196,"identity":"195ccd0f-81d4-4f4e-b1ff-b9f5363dcd93","added_by":"auto","created_at":"2025-06-09 13:22:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":466481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRestoring Ezrin expression reinstates the ability of SKOV3 cells to divide in soft agar\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003ea. Crystal violet staining from SKOV3, SKOV3\u003csup\u003eEZR\u003c/sup\u003e, Ezr6148, Ezr6148\u003csup\u003eEZR\u003c/sup\u003e, Ezr6059, and Ezr6059\u003csup\u003eEZR\u003c/sup\u003e spheroids formed in soft agar. b. Quantitation of spheroid formation by SKOV3, Ezr6148, and Ezr6059 cells. The Y axis denotes the number of spheroids formed per field. c. Representative images of soft agar spheroids at 1, 3 and 5-days culture for SKOV3, SKOV3\u003csup\u003eEZR\u003c/sup\u003e, Ezr6148, Ezr6148\u003csup\u003eEZR\u003c/sup\u003e, Ezr6059, and Ezr6059\u003csup\u003eEZR\u003c/sup\u003e with Rhodamine-Phalloidin (red) and DAPI (blue) labeling. d. Bar plot of spheroid formation of SKOV3, Ezr6148, Ezr6148\u003csup\u003eEZR\u003c/sup\u003e, Ezr6059, and Ezr6059\u003csup\u003eEZR\u003c/sup\u003e over time. SKOV3 spheroids are represented in blue, Ezr6148 in red and Ezr6059 in gray. The y-axis represents the number of cells per spheroid. e. Bar plot of the number of nuclei per cell. Data are presented as mean ± SEM, *p\u0026lt;0.05, ***p\u0026lt;0.001; ****p\u0026lt;0.00001, ns: no significant differences.\u003c/p\u003e","description":"","filename":"Figure2Lujeaetal2025SciRep.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6595737/v1/28ba860ceed985e2e2b783b4.jpg"},{"id":84226855,"identity":"d82e7bb5-b4fb-4dca-a709-08b8ecb1e87f","added_by":"auto","created_at":"2025-06-09 13:06:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":396078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRescue of the Ezr6148 impaired spheroid phenotype by ConA. \u003c/strong\u003ea. Crystal violet staining of SKOV3 and Ezr6148 spheroids incubated with or without ConA. b. Bar plot depicting spheroid formation with and without ConA treatment. The Y-axis indicates the number of spheroids formed per field. c. Immunofluorescence (IF) images displaying soft agar spheroid formation at 1, 3, and 5 days in culture. The upper section shows SKOV3 spheroids formed in the presence of ConA along with their respective control. The lower part displays Ezr6148 spheroids stained with Rhodamine-Phalloidin and DAPI. d. Bar plot of spheroid formation of SKOV3, Ezr6148, with and without ConA over time. e. Plot of number of nuclei per cell. The data is presented as mean ± SEM, ** p\u0026lt;0.005, *** p\u0026lt;0.0005. ns: no significant differences.\u003c/p\u003e","description":"","filename":"Figure3Lujeaetal2025SciRep.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6595737/v1/c5b7fc7fc4f15cab6710b6a0.jpg"},{"id":84226863,"identity":"77cf241d-6616-4dab-a48e-74af2f0f8d78","added_by":"auto","created_at":"2025-06-09 13:06:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":520614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA definite stiffness of the cell culture media was capable of reversing the inability of Ezrin depleted cells to form spheroids.\u003c/strong\u003e a. Crystal violet staining revealing SKOV3 and Ezr6148 spheroids formed on different agar concentrations (0.5, 1, 2 and 3%). b. Quantitation of SKOV3 and Ezr6148 spheroids formation shown in a. c and d. Examples of individual SKOV3 or Ezr6148 cells and spheroid formation (µm\u003csup\u003e2\u003c/sup\u003e) on different agarose concentrations stained with Phalloidin and DAPI. The data is presented as mean ± SEM, *** P\u0026lt;0.0005, ns: no significant differences. e and f. Formation of SKOV3 and Ezr6148 spheroids in different agarose concentrations at 1, 3 and 5 days in culture. Spheroids SKOV3 represented in blue and Ezr6148 in red. g and h. Number of nuclei per cell.\u003c/p\u003e","description":"","filename":"Figure4Lujea2025SciRep.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6595737/v1/b4aca973a56083516e13c5d7.jpg"},{"id":84227759,"identity":"067dee6d-042c-43fe-b3f8-ad181b206ac4","added_by":"auto","created_at":"2025-06-09 13:14:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":338404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpheroids provide the microenvironment required for Ezrin-depleted cells to divide accurately.\u003c/strong\u003e a. 3D reconstruction of a confocal images of a representative SKOV3 spheroid (left) and Ezr6148 (right), both formed by aggregation in liquid medium on an agar surface. Images of 24, 48, 72 and 96 hours of formation. Rhodamine-Phalloidin (red, as a F-actin marker), PhosphoHistone3 (green, a mitotic cell marker) and DAPI (blue, for nucleus). b. Graphs of the number of nuclei per cell and c, area of nuclei over time. Data are presented as mean ± SEM, ns: no difference significant.\u003c/p\u003e","description":"","filename":"Figure5Lujea2025SciRep.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6595737/v1/cbdf5fc83ec6452015218648.jpg"},{"id":84226866,"identity":"e55f743f-5307-4964-9357-48c2f16e50f3","added_by":"auto","created_at":"2025-06-09 13:06:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":515231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor formation in mice injected intraperitoneally with SKOV3, Ezr6148 cells, SKOV3 or Ezr6148 spheroids.\u003c/strong\u003e a. Mouse intraperitoneal tumor formation originated by injection of SKOV3 and Ezr6148 cells. The SKOV3 controls were set to a value of 1 and Ezr6148 was relativized in each case to its corresponding control. b. H \u0026amp; E staining of tumors formed in the peritoneum seven weeks after injection of SKOV3 or Ezr6148 cells and SKOV3 or Ezr6148 spheroids.\u003c/p\u003e","description":"","filename":"Figure6Lujea2025SciRep.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6595737/v1/93d20065be72d98b648afc89.jpg"},{"id":85268292,"identity":"d2287131-92b0-4e2d-96c5-ec303ac314df","added_by":"auto","created_at":"2025-06-24 06:02:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3819953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6595737/v1/6134b4cf-7926-4f9f-933e-1ff6984d963e.pdf"},{"id":84226857,"identity":"7e8c5c7d-b0b6-4217-8b2c-3fa34e8a2a40","added_by":"auto","created_at":"2025-06-09 13:06:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2667073,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFiguresLunea2025SciRep.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6595737/v1/73c888b80a0f66f89debd17e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ezrin plays a key role in cancer cell spheroid formation in soft environments","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. Ezrin is essential for spheroid formation in soft microenvironments.\u003c/p\u003e\u003cp\u003e2. Ezrin is not needed for spheroid generation in stiffer microenvironments.\u003c/p\u003e\u003cp\u003e3. An increase in cortical cell stiffness or a stiffer environment can compensate for absence of Ezrin.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eOvarian cancer (OC) metastases present a unique therapeutic challenge as they primarily occurs within the peritoneum, with secondary tumors either suspended in the peritoneal fluid or adhered to the peritoneal surfaces\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. These metastatic growths take the form either of spheroids or isolated cells. Spheroids may originate from spontanoeous cellular aggregation or from successive mitotic divisions of a single cell\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOC-derived spheroids emerge within a non-structured and soft environment primarily composed of ascitic fluid. While the formation of aggregates supports tumor cell survival through various mechanisms, such as shielding them from anoikis, individual cells face the challenge of surviving and proliferating while suspended in the ascitic fluid, a mechanically unique soft environment\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This is important because the factors and mechanisms underlying cell survival and proliferation in such conditions remain poorly understood. One such factor could be the cortical tension, defined as the mechanical force produced by the cell cortex. This force influences cell survival and proliferation in various environments\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. During the generation of cortical tension, actin filaments reorganize and connect to the plasma membrane through associated proteins, such as the Ezrin, Radixin, and Moesin (ERM) proteins\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Thus, to understand how an isolated metastatic cell thrives in a soft, fluid environment requires identifying what proteins enable the interactions between cytoskeletal components and the plasma membrane.\u003c/p\u003e \u003cp\u003eIn this context, the ERM\u0026rsquo;s emerge as promising candidates for such a role \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In particular Ezrin is involved in migration, invasion, and proliferation of various cancers\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Thus, we hypothesize that Ezrin facilitates spheroid formation within soft environments, favoring OC metastasis. To test this, we carried out experiments in a 3D soft agar culture model with the SKOV3 OC cell line and a xenograft model in nude mice. Our results revealed that Ezrin is critical for cancer cell spheroid generation via supporting cell division in a soft environment reminiscent of peritoneal ascitic fluid.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eEzrin plays a key role in SKOV3 cell spheroid formation in soft agar.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the involvement of ERM proteins in cell spheroid formation, we utilized a spheroid generation model in a soft agar matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We generated Ezrin knockout SKOV3 cells using CRISPR-Cas9 technology. This resulted in two stable cell lines named Ezr6148 and Ezr6059. The presence of mutations in the ezrin gene in Ezr6148 and Ezr6059 cells was confirmed through sequencing and western blot analysis (Suppl. Figure\u0026nbsp;1a-c). Depletion of Ezrin led to a significant reduction in spheroid formation compared to SKOV3 control cells. Crystal violet staining at 20 days in culture revealed that SKOV3 cells formed spheroids in soft agar with an average of 11.5 (\u0026plusmn;\u0026thinsp;2.7) spheroids per field, whereas the depletion of Ezrin protein significantly decreased the spheroid formation compared to control in both cell lines (Ezr6148\u0026thinsp;=\u0026thinsp;3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 spheroids per field, (n\u0026thinsp;=\u0026thinsp;4; t(9)\u0026thinsp;=\u0026thinsp;12,38; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and Ezr6059\u0026thinsp;=\u0026thinsp;1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 spheroids per field, (n\u0026thinsp;=\u0026thinsp;4; t(9)\u0026thinsp;=\u0026thinsp;11,20; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), respectively) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b).\u003c/p\u003e \u003cp\u003eTo determine why Ezrin deletion reduced the number of spheroids, we focused on the initial stages of spheroid formation, examining spheroid development at 1, 3, and 5 days. We selected these early times because prolonged culture periods led to larger spheroids that hindered accurate quantification. We found that whereas SKOV3 cells divided and increased in number after 3 days, both Ezr6148 and Ezr6059 mutants remained as individual cells (n\u0026thinsp;=\u0026thinsp;4; F(1.183, 15.38)\u0026thinsp;=\u0026thinsp;17.78) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d). Quantitative analysis indicated that SKOV3 cells exhibited a progressive increase in cell count per spheroid over the culture period (1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 cell per spheroid; 1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 cell per spheroid; 3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 cell per spheroid; for 1, 3, and 5 days, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d). In contrast, Ezr6148 and Ezr6059 cells remained at approximately 1 cell per spheroid, with no statistically significant changes observed throughout the culture period (p\u0026thinsp;=\u0026thinsp;0.0004 and p\u0026thinsp;=\u0026thinsp;0.0006 for Ezr6148 and Ezr6059, respectively) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d).\u003c/p\u003e \u003cp\u003eTo assess the ability of cells to complete mitosis and divide in soft agar, we quantified the number of nuclei per cell in each spheroid. SKOV3 cells had an average of 1.08 (\u0026plusmn;\u0026thinsp;0.06) nuclei per cell at 5 days (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and e). In contrast, both Ezr6148 and Ezr6059 cells exhibited more than one nucleus per cell after 1 day, reaching an average of 1.34 (\u0026plusmn;\u0026thinsp;0.08) and 1.44 (\u0026plusmn;\u0026thinsp;0.13) nuclei per cell by 5 days, respectively (n\u0026thinsp;=\u0026thinsp;4, F(1.625, 22.75)\u0026thinsp;=\u0026thinsp;15.55; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and e).\u003c/p\u003e \u003cp\u003eCollectively, these data suggest that Ezrin plays a key role in spheroid formation in soft agar.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRestoring Ezrin expression reinstates the ability of the SKOV3 cells to divide in soft agar.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo test the direct association of Ezrin and the observed cell growth phenotype, we restored Ezrin expression in both Ezr6148 and Ezr6059 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To this end, we transfected SKOV3, Ezr6148, and Ezr6059 cells with the pcDNA3.1-EzrWT plasmid. These modified lines are denoted as SKOV3\u003csup\u003eEZR\u003c/sup\u003e, Ezr6148\u003csup\u003eEZR\u003c/sup\u003e, and Ezr6059\u003csup\u003eEZR\u003c/sup\u003e. Ezrin expression was confirmed by western blot (Suppl. Figure\u0026nbsp;1e). We induced spheroid formation in 0.5% soft agar using SKOV3, SKOV3\u003csup\u003eEZR\u003c/sup\u003e, Ezr6148, and Ezr6148\u003csup\u003eEZR\u003c/sup\u003e cells. Quantitative analysis revealed no significant difference in spheroid count between the wild-type SKOV3 and the SKOV3\u003csup\u003eEZR\u003c/sup\u003e (10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 vs. 11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 spheroids per field, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). As expected, overexpressing Ezrin in Ezr6148 and Ezr6059 cell lines effectively restored their capacity to form spheroids in soft agar after 20 days in culture (Ezr6148: 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 vs. Ezr6148\u003csup\u003eEZR\u003c/sup\u003e: 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 spheroids per field; n\u0026thinsp;=\u0026thinsp;3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and Ezr6059: 0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 vs. Ezr6059\u003csup\u003eEZR\u003c/sup\u003e: 4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 spheroids per field; (one way ANOVA n\u0026thinsp;=\u0026thinsp;3, F (5, 54)\u0026thinsp;=\u0026thinsp;101.8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Moreover, analysis of spheroid growth by immunofluorescence revealed that both Ezr6148\u003csup\u003eEZR\u003c/sup\u003e and Ezr6059\u003csup\u003eEZR\u003c/sup\u003e increased the number of cells per spheroid between 1 and 5 days, a value similar to that observed in control (two-way ANOVA, n\u0026thinsp;=\u0026thinsp;3, F (1.352, 13.52)\u0026thinsp;=\u0026thinsp;89.38, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Ezr6148\u003csup\u003eEZR\u003c/sup\u003e: 1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 and 2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 cells per spheroid for 1 and 5 days respectively (n\u0026thinsp;=\u0026thinsp;3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and Ezr6059\u003csup\u003eEZR\u003c/sup\u003e: 1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and 2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 spheroids per field for 1 and 5 days in culture respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and d). More importantly, while Ezr6148 and Ezr6059 cells increased the number of nuclei per cell over time in culture, the Ezr6148\u003csup\u003eEZR\u003c/sup\u003e and Ezr6059\u003csup\u003eEZR\u003c/sup\u003e cells maintained a single nucleus per cell from 1 to 5 days as the spheroids grew (n\u0026thinsp;=\u0026thinsp;3, F(1.912, 28.67)\u0026thinsp;=\u0026thinsp;27.59, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and e).\u003c/p\u003e \u003cp\u003eThese results indicate that overexpression of Ezrin in the Ezr6148 and Ezr6059 mutants restores spheroid formation, confirming that the observed phenotype is Ezrin-dependent.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnhanced cortical tension induced by ConA treatment reinstates the capability of Ezrin knockout cells to form spheroids in a soft environment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEzrin plays a crucial role in organizing the cellular cortex, a process essential for cell division\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Prompted by these findings, here we investigate whether the positive effect of Ezrin in cell spheroid formation may be derived from the ability of Ezrin to alter the stiffening of the cellular cortex. \u003cem\u003eIn vitro\u003c/em\u003e administration of ConA has been shown to induce an increase in cortical tension\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Thus, if stiffening of the cellular cortex due to Ezrin accounts for its role in spheroid formation, we expect that ConA treatment of Ezr6148 cells should restore their ability to form spheroids in soft agar. To test this, both SKOV3 and Ezr6148 cells were incubated for 30 minutes in media supplemented with ConA, while media without ConA was used as control. Then, cells were plated in soft agar and cultured up to 20 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). As predicted, SKOV3 cells, regardless of pre-incubation with or without ConA, formed cell spheroids (9.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72 and 10.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43 spheroids per field), with no differences between them (n\u0026thinsp;=\u0026thinsp;3, F(3, 20)\u0026thinsp;=\u0026thinsp;29.72; p\u0026thinsp;=\u0026thinsp;0.9543, one-way ANOVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). As expected, Ezr6148 cells did not generate spheroids in 0.5% agarose after 20 days (0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 spheroids per field). In sharp contrast, Ezr6148 cells treated with ConA exhibited a significant increase in spheroid formation (4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 spheroids per field, n\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.0059) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). Furthermore, immunofluorescence analysis confirmed a significant increase in the number of cells per spheroid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and d) in ConA treated Ezr6148 cells from 1 to 5 days in culture (F (2, 6)\u0026thinsp;=\u0026thinsp;13.74, P\u0026thinsp;=\u0026thinsp;0.058, two-way ANOVA). Analysis of the average of nuclei per cell revealed that ConA treatment reversed the effect caused by Ezrin silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and e). We conclude that Ezrin may mediate the stiffening of the cellular cortex, allowing for spheroid formation in soft environments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreasing the stiffness of the cell culture media reinstates the ability of Ezrin knockout cells to form spheroids in a soft environment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have suggested that cells can sense mechanical cues from their surroundings via an actin-dependent mechanism\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In order to explore whether this phenomenon explains Ezrin-dependent spheroid formation, we performed an \u003cem\u003ein vitro\u003c/em\u003e experiment where we manipulated the agarose concentration to create different levels of environmental rigidity. Advantages of employing agarose are the ability to facilitate the free diffusion of nutrients within the culture media\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and that rigidity or elasticity of agarose matrices at different densities has been well stablishes \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe utilized various agarose concentrations ranging from 0.5\u0026ndash;3%, selected for their resemblance to the reported viscosity levels in human ascites\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and because this approach has already been used\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.We predict that at some agarose rigidity Ezr6148 cells will recover their ability to form cell spheroids. We found that SKOV3 cells formed spheroids after 20 days in all the agar densities we tested but with a noticeably reduced capability to from spheroids in 2% and 3% agarose (F(7, 24)\u0026thinsp;=\u0026thinsp;51.34, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, n\u0026thinsp;=\u0026thinsp;3, one-way ANOVA) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b). As expected, Ezr6148 cells failed to form spheroids at 0.5% agarose. Notably, we observed a significant increase in the number of Ezr6148 spheroids at 1% agarose (4.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 spheroids per field; n\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with no significant differences compared to SKOV3 cells at 1% agar (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b). However, at agar concentrations of 2% and 3%, Ezr6148 exhibited a notable decrease in spheroid formation similar to SKOV3 cells growing in 3% agarose.\u003c/p\u003e \u003cp\u003eF-actin immunolabeling and DAPI revealed spheroid formation up to 5 days. Quantitative analysis showed an increased number of cells per spheroid over time in culture in both 0.5% and 1% agarose (n\u0026thinsp;=\u0026thinsp;3, F(2, 24)\u0026thinsp;=\u0026thinsp;14.15; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, one-way ANOVA) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and d). In addition, SKOV3 spheroids exhibited an average of one nucleus per cell over time in all agarose concentrations (P\u0026thinsp;=\u0026thinsp;0.2469, Kruskal-Wallis test) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and d). More interestingly, whereas the number of cells per spheroid in Ezr6148 significantly increased from 1 to 5 days in culture at 1% agarose (n\u0026thinsp;=\u0026thinsp;3, F(11, 24)\u0026thinsp;=\u0026thinsp;10.11; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and e-f). While the number of nuclei per cell increased in 0.5% agarose over time, cells cultured in 1% soft agar maintained an average of one nucleus, similar to SKOV3 control spheroids (n\u0026thinsp;=\u0026thinsp;3, F(11, 21)\u0026thinsp;=\u0026thinsp;9.56; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d, g and h).\u003c/p\u003e \u003cp\u003eWe concluded that increasing the stiffness of the cell media reinstates the ability of Ezrin knockout cells to form spheroids.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpheroids of Ezrin-depleted cells formed by physical aggregation can sustain cell division.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe intercellular signals within a spheroid are pivotal for initiating cell aggregation, leading to the formation of a cohesive spheroid structure\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, the neighboring cells surrounding each cell within the spheroid contribute to its structural integrity and stiffness\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We tested whether the depletion of Ezrin in Ezr6148 cells affect their ability to undergo proper cell division and maintain one nucleus per cell once they became part of a spheroid. To investigate this, we generated spheroids comprising SKOV3 or Ezr6148 cells using the suspension-based aggregation method of spheroid formation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In this technique, cells are suspended in a liquid medium layered on agar, preventing adhesion to the substrate, and facilitating aggregation into a compact spheroid at the center of the well. Cell division was assessed by labeling against PhosphoHistone3 (Ser-10) (as a mitotic marker), Rhodamine-Phalloidin (an F-actin marker), and DAPI (for nucleus) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Ezr6148 cells were able to form spheroids in suspension (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Furthermore, Ezr6148 cells kept only one nucleus on average with the successive cell divisions, suggesting that the spheroid environment sustained cell division despite the absence of Ezrin (P\u0026thinsp;=\u0026thinsp;0.5390, Kruskal-Wallis test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Even if the cell had only one nucleus, failure to achieve a complete cell division could be evidenced by an increase in the size of the nucleus. To test this, we measured the nuclear area of each cell within the spheroids for both SKOV3 and Ezr6148 cells. We found no differences in nuclear area between SKOV3 and Ezr6148 cells at any time point (P\u0026thinsp;=\u0026thinsp;0.5973) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThese results suggest that cellular context may restore the ability of Ezrin-depleted cells to multiply.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEzrin is required for tumor formation in a xenograft mouse model.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe assessed the ability of SKOV3 or Ezr6148 cells to generate spheroids \u003cem\u003ein vivo\u003c/em\u003e using an intraperitoneal metastasis model in nude mice.\u003c/p\u003e \u003cp\u003eFirst, female mice were injected with either SKOV3 or Ezr6148 cells. After 8 weeks, the animals were euthanized, and tumor formation was examined. Table\u0026nbsp;1 in Suppl. Figure\u0026nbsp;2 shows the mice weight, the presence of metastases, and the weight of the metastatic masses. We observed that mice injected with SKOV3 cells formed tumors. In contrast, injection with knockout Ezrin cells significantly reduced intraperitoneal tumor formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Eight out of eleven animals injected with SKOV3 cells formed tumors within 3 months. They displayed various histological tumor subtypes including one endometrioid, one serous, one clear cell, and five undifferentiated tumors. Out of 5 mice injected with the mutated Ezrin, only one formed a tumor. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb depicts the HE staining for the tumors obtained under each experimental condition.\u003c/p\u003e \u003cp\u003eNext we explored whether spheroids previously formed \u003cem\u003ein vitro\u003c/em\u003e could develop tumors in mice. Female mice were injected with spheroids derived from either SKOV3 or Ezr6148 cells. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea show that the injection of spheroids generated \u003cem\u003ein vitro\u003c/em\u003e resulted in the formation of tumors whereas i.p. administration of isolated individual Ezrin6148 cells resulted in no tumors.\u003c/p\u003e \u003cp\u003eThese results agree with our \u003cem\u003ein vitro\u003c/em\u003e findings that Ezrin-silenced SKOV3 cells are unable to form spheroids in a soft environment. This suggests that Ezrin plays a crucial role in intraperitoneal tumor formation \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCells adeptly sense changes in the physical properties of their surroundings and adjust accordingly. Variations in the mechanical characteristics of the environment serve as external cues, impacting the cell cortex and eliciting an elastic deformation response, known as mechanotransduction. In the context of the OC metastasis, tumor cells detach from the primary tumor and proliferate within the peritoneum, often in unstructured or fluid-filled media like ascitic fluid, a hallmark of this cancer subtype. Surviving in such conditions requires adaptive mechanisms. Given the role of the cell cortex and its proteins in mechanotransduction, we focused on Ezrin, a key ERM family member. We propose that Ezrin plays a crucial role in facilitating the transition from the structured environment of the primary tumor to colonizing the unstructured peritoneal milieu. Our findings underscore the indispensability of Ezrin in cellular adaptation, enabling proliferation across diverse environments.\u003c/p\u003e \u003cp\u003eConsidering Ezrin's role in cortex formation and tension generation, we employed ConA to induce environmental tension in our experiments\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Previous studies have elucidated the effects of ConA on 3D models \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. ConA interacts with glycoproteins on the plasma membrane, forming an external mesh that enhances cellular rigidity\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our findings revealed that silencing Ezrin significantly impaired spheroid formation in soft agar, while treatment with ConA reversed this phenotype induced by Ezrin depletion. This underscores Ezrin's pivotal role in adapting to varying mechanical environments and reinforces the importance of cortical rigidity in tumor cell division\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsistent with our results, a recent study demonstrated reduced cortical tension and decreased activation of phosphorylated ERMs in fertilized and cryopreserved oocytes\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Consequently, Ezrin-deficient oocytes exhibited defects in spindle positioning and chromosome segregation. Remarkably, supplementing the culture media with ConA restored cortical integrity, ensuring oocyte viability and proper chromosome segregation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Thus, the work by Du and colleagues provide further support for our findings regarding ERMs' function in spheroid formation and, importantly, highlights ConA as an experimental substitute for the structural role of ERMs. Our study adds to the body of knowledge by highlighting the structural role of cortical rigidity mediated by Ezrin in tumor cell spheroid formation.\u003c/p\u003e \u003cp\u003eIn a complementary approach, we tested how agar matrices can be used to manipulate environmental stiffness. Notably, we observed that precise agar matrix rigidity (1% agarose concentration) can compensate for Ezrin absence and cells are able to complete cell division. Our results clarify the controversial findings regarding the proliferative effect of manipulating the stiffness of the cellular environment\u003csup\u003e23 35\u003c/sup\u003e. Indeed, research using rat prostate cancer cell lines revealed that agar-induced tension promoted spheroid formation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Conversely, increased medium rigidity hindered human colon adenocarcinoma cells spheroid growth\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCollectively, our findings point to the importance of Ezrin-mediated cortical rigidity in the ability of cells to divide and, further underscore the potential therapeutic implications of modulating cortical mechanics. We also help to elucidate Ezrin's role in substrate-independent growth within soft environments relevant to the type of peritoneal metastasis associated with OC. This contributes to further understand the intricate interplay between cellular mechanics and environmental cues in cancer progression.\u003c/p\u003e \u003cp\u003eAnother important aspect of mechanotransduction is the intimate cell-cell contact observed within spheroids, mirroring the physical environment and interactions seen in tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e studies on spheroid formation reveal a transition from loose to compact states, strengthening cohesive forces among constituent cells. This process crucially involves cadherin and integrin proteins \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Hence, when analyzing spheroids, it is imperative to consider the mechanical forces within the cellular milieu and the interactions among neighboring cells as observed in living tissues\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These cellular interaction forces profoundly impact various cellular processes such as division. In light of these dynamics, we generated spheroids with Ezrin knockout cells via aggregation in suspension. Notably, observations within these spheroids revealed normal mitotic activity, highlighting the crucial role of the cellular environment in restoring tumor cells capacity for division. Again, these findings underscore Ezrin significance in governing cell division, particularly in scenarios lacking structured environmental support.\u003c/p\u003e \u003cp\u003eOur studies utilized SKOV3 cells, which were originated from the ascitic fluid of a patient with ovarian papillary cystadenocarcinoma\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This cell line closely mimics specific cellular and molecular processes anticipated within the peritoneal environment occurring in patients, thus offering valuable insights into the potential role of Ezrin in this context. Importantly, to address limitations in using \u003cem\u003ein vitro\u003c/em\u003e systems, we employed a xenograft model using nude mice\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our results in this model mirrored the observations made \u003cem\u003ein vitro\u003c/em\u003e using the soft agar model, and more importantly, injecting previously cultured spheroids reversed the effect of deleting Ezrin. Integrating \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e observations allowed us to better understand the role of Ezrin role in OC metastasis.\u003c/p\u003e \u003cp\u003eThe new insights we provide regarding the factors that facilitate spheroid formation in a soft environment is significant, as the mechanical properties of the environment can promote malignancy in various types of cancers, particularly in OC, which carries a high mortality rate\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. OC is notorious for its late diagnosis, limited treatment options for advanced stages, high recurrence rates (85%), and a modest 46% 5-year survival rate\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The challenge in treating metastatic OC over the past five decades stems from minimal advancements in understanding the disease, delayed detection, chemoresistance, and the complexity of the tumor microenvironment\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Hence, identifying and understanding mechanisms of action of key proteins such as Ezrin involved in spheroid formation within the peritoneum can significantly impact future therapeutic strategies associated with OC.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eWe cultured the human epithelial OC cell line SKOV3 as described\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The SKOV3 cells were generously provided by Dr. Osvaldo Podjhajcer (Leloir Institute, Buenos Aires, Argentina). This cell line was at passage 3 from an originally certified cell line obtained from ATCC (ATCC HTB77) and was screened for Mycoplasma sp. contamination using PCR, following standard protocols\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSoft agar assay\u003c/h3\u003e\n\u003cp\u003eSpheroids were generated through successive cell divisions from a single cell utilizing the modified soft agar technique\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Briefly, 1 ml of 1% low melting point agarose (Bethesda Research Laboratories, USA) was applied to coat the base of a 6-multiwell plate. Cells were trypsinized and diluted to a concentration of 8,000 cells per well. Subsequently, a layer containing 3 ml of 0.5% agar mixed with culture medium containing the cells was added to each well and allowed to solidify at room temperature. The culture medium was replenished on the agar surface every 3 days until used.\u003c/p\u003e\n\u003ch3\u003eCrystal violet staining\u003c/h3\u003e\n\u003cp\u003eSpheroids on agar were fixed in methanol/acetic acid (3:1) for 30 minutes, incubated for 1 hour with 0.5% crystal violet in 20% methanol. Images were captured using a Zeiss SZX7 microscope, and the numbers of spheroids per field were counted from 10 fields per condition in each experiment. Spheroids were stained after 20 days of culture and their area measured\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, at which point they reached a spherical shape and an average size of 317 \u0026micro;m\u0026sup2; (\u0026plusmn;\u0026thinsp;146 \u0026micro;m\u0026sup2;) for SKOV3 cells. At this stage, spheroids were optimal in size and clearly visible. Cells within the spheroids showed high viability and low apoptosis, similar to monolayer cultures.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eFor mitosis quantification and multinuclearity assessment, cells were fixed in 4% PFA/sucrose for 20 minutes, permeabilized with 0.2% Triton-X100, and blocked with 5% BSA in PBS for 30 minutes\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e Phospho-Histone-3, (Ser-10) (clone 6G3) mouse monoclonal antibody (Cell Signaling Technology Cat# 9706, RRID: AB_331748) was used at 1:1,000, incubated overnight at 4\u0026deg;C, followed by incubation for 2 hours at room temperature with a secondary antibody (Alexa Fluor 488, Donkey anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) highly cross-adsorbed secondary antibody, Invitrogen Cat# A21-202, RRID: AB_141607), at 1:500. Rhodamine-conjugated phalloidin (Sigma, used at 0.1 mg/ml) and DAPI (Invitrogen; 1:10,000) were also used\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImmunostained spheroids were mounted with Mowiol media, and images were acquired using Olympus FV-1200, LSM800 confocal, or Olympus IX81 microscopes. Image analysis was performed using ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/Fiji\u003c/span\u003e\u003cspan address=\"https://imagej.net/Fiji\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, RRID:SCR_002285)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEzrin CRISPR-Cas9 stable cell lines\u003c/h3\u003e\n\u003cp\u003eTwo Ezrin-targeting plasmids were designed using CRISPR-Cas9 (VectorBuilder) with gRNAs #6148 (exon 9) and #6059 (exon 8), based on on-target and off-target ratios of 53.3/90.3 and 55.6/49.7, respectively. The Ezrin protein map (Suppl. Figure\u0026nbsp;1A) shows gRNA recognition sites within the α-helical domain. SKOV3 cells were co-transfected with CRISPR constructs and PEGFP-Puro and then selected with puromycin for 15 days. The mutations (Ezr6148 and Ezr6059) were confirmed by DNA sequencing. The sequences have been submitted to GenBank under accession numbers Seq1 PV703827 and Seq3 PV703829 for the mutants Ezr6148 and Ezr6059 respectively and their respective SKOV3 control WT (seq2 PV703828 and Seq4 PV703830). The clones with mutations were also confirmed by Western blot analysis using an ERM antibody (Cell Signaling Technology #3141), with tubulin (Anti-α-Tubulin, clone DM1A, Sigma) as a loading control\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo evaluate Ezrin silencing effects on cell survival, proliferation, and apoptosis, SKOV3, Ezr6148, and Ezr6059 cells were compared. Viability was 99% in all groups (determined by resazurin and trypan blue). Proliferation was assessed with anti-phospho-Histone-3 and DAPI, and apoptosis was quantified by apoptotic body count (Suppl. Figure\u0026nbsp;1D)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. No significant differences were found in survival, proliferation, or apoptosis between CRISPR clones and SKOV3 control (one-way ANOVA: p\u0026thinsp;=\u0026thinsp;0.6349 for mitosis; p\u0026thinsp;=\u0026thinsp;0.7612 for apoptosis).\u003c/p\u003e\n\u003ch3\u003eGenetic rescue\u003c/h3\u003e\n\u003cp\u003eStable cell lines were generated using SKOV3 and two CRISPR-modified forms of Ezrin (Ezr6148 and Ezr6059). These cell lines were transfected with the pcDNA3.1-topo-GFP-Ezrin wild-type plasmid (provided by Dr. Guillaume Charras) and selected with G418 (500 \u0026micro;g/ml). Clones were selected by dilution in a 96-well plate. A positive clone was chosen for each line: SKOV3EZR, Ezr6148EZR, and Ezr6059EZR. Western blot analysis (as described in 2.5) showed a significant increase in ERM protein expression in all clones (Suppl. Figure\u0026nbsp;1E).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eConcanavalin A treatment\u003c/h2\u003e \u003cp\u003eA phenotype rescue experiment was conducted using Concanavalin A (Sigma, #C2010) (ConA). Ezr6148 or SKOV3 cells (8,000 cells) were incubated with 100 \u0026micro;g/ml ConA at 37\u0026deg;C for 30 minutes, then plated in soft agar. Control cells were incubated with media alone. Samples were collected at 1, 3, and 5 days, fixed with 4% PFA/sucrose for immunofluorescence staining, or at 20 days and stained with 0.5% crystal violet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFormation of spheroids in suspension\u003c/h2\u003e \u003cp\u003eSKOV3 and Ezr6148 cells were trypsinized and adjusted to 5,000 cells/ml. Next, 100 \u0026micro;l of the cell suspension was added to a 96-well plate coated with 1% agar. The plates were incubated for 24, 48, 72, and 96 hours. After incubation, spheroids were fixed in 4% PFA/sucrose for 20 minutes and processed for immunofluorescence. Immunostaining with DAPI and phalloidin was performed as described previously, and 3D images were captured using the Olympus LSM800 confocal microscope\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSoft agar rescue assay\u003c/h2\u003e \u003cp\u003eFor the soft agar rescue assay, agar solutions were prepared at final concentrations of 0.5%, 1%, 2%, and 3%. Subsequently, 8,000 cells (SKOV3 or Ezr6148) were plated in each well of a 6-well plate containing 1% agar at the base, with each well containing varying agar concentrations. Spheroids were fixed using 4% PFA/sucrose at 1, 3, and 5 days in culture and subsequently processed for immunofluorescence analysis. Crystal violet staining at 0.5% was conducted at day 20 in culture.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003estudies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFour female athymic N:NIH(S)-nu mice (5\u0026ndash;6 weeks old, from the University of La Plata, Argentina) were intraperitoneally injected with 6 \u0026times; 10^6 SKOV3-luc or 6 \u0026times; 10^6 EZR6148-luc cells, as described\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Cell viability was 99%, as confirmed by a Countess cell counter (Invitrogen). For the rescue phenotype assay, SKOV3 and EZR6148 spheroids were generated on a p150 plate with 10 ml of solidified agar and 20 ml of medium containing 2 \u0026times; 10^6 cells. After 72 hours, spheroids were collected, washed in PBS 1X, and resuspended in 200 \u0026micro;l of PBS. Seven female nude mice (4\u0026ndash;6 weeks old) were intraperitoneally injected with this spheroid suspension. Each assay was performed twice, and mice were sacrificed at 7\u0026ndash;8 weeks post-injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEthical considerations\u003c/h2\u003e \u003cp\u003e All experiments involving mice were carried out in accordance with the relevant guidelines and regulations. This study was approved by the Ethics Committee at the Leloir Institute, under protocol number CICUAL-FIL 92. All methods are reported in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Animals were housed and cared for following the institutional and national guidelines for the care and use of laboratory animals. No procedures causing undue stress or pain were conducted without appropriate anesthesia (isoflurane, induction at 5%, maintenance at 3%). Thus, mice were euthanized using carbon dioxide inhalation displacement rate from 30\u0026ndash;70% of the chamber volume/min in accordance with the AVMA Guidelines for the Euthanasia of Animals, 2020 Edition (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.avma.org/resources-tools/avma-policies/avma-guidelines-euthanasia-animals\u003c/span\u003e\u003cspan address=\"https://www.avma.org/resources-tools/avma-policies/avma-guidelines-euthanasia-animals\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin/Eosin staining (HE)\u003c/h2\u003e \u003cp\u003eSections of 10 \u0026micro;m thickness were de-paraffinized and stained with Mayer's Hematoxylin for 10 minutes, followed by 0.2% Eosin for 30 seconds. After washing with ethanol and xylene, the sections were mounted with DPX medium. Images were captured with a NIKON T2000U microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from 3 or 4 independent experiments. Statistical analysis and graphs were performed using GraphPad Prism 6.0 software. The assumption of normality for each dataset was assessed using the D\u0026rsquo;Agostino-Pearson and Shapiro-Wilk normality tests. To compare 2 conditions (Control vs. Treatment) Student\u0026rsquo;s t-test was performed for data that passed the normality test, reported as t(df)\u0026thinsp;=\u0026thinsp;t-statistic, p\u0026thinsp;=\u0026thinsp;significance value. Mann-Whitney was used for datasets that failed the normality test. One-way ANOVA or two-way ANOVA was used for comparing data sets with one or more degrees of freedom, respectively, reported as F(between groups df, within groups df) = [F-value], degrees of freedom, p = [p-value]. Kruskal-Wallis was used as nonparametric test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Significance is denoted on all graphs as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCA, VL, GP and PK are researchers of CONICET. This research was financially supported by INC (project designation number 30-54666038-5), to PK. NL received a studentship from CONICET. We gratefully acknowledge CEMINCO, CIBICI, and the Virology Institute at UNC for granting us access to their microscopy facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNL and VL performed experiments and data analysis, VL performed xenograph experiments, RP and GP writing and editing, CA data analysis, writing and editing, PK paper conception, analysis, and writing. All authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;Sequence data that support the findings of this study have been deposited in the\u0026nbsp;GenBank from the National Center for Biotechnology Information (NCBI) with the following primary accession codes: PV703827; PV703828; PV703829; PV703830.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTan, D. S., Agarwal, R. \u0026amp; Kaye, S. B. 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Notably, the mechanisms underlying metastasis in ascitic fluid remain poorly understood. A critical step in this process is the formation of cancer cell spheroids. We hypothesize that spheroid generation depends on actin cytoskeleton-associated proteins at the cell cortex. In this context, Ezrin, a member of the ERM (Ezrin-Radixin-Moesin) family protein, emerged as a strong candidate. To investigate, we established a 3D culture model using SKOV3 cells and assess spheroid formation under different conditions. We found that Ezrin-depleted cells, failed to form spheroids in soft agar, a phenotype reversed by reintroducing wild-type Ezrin, ConA treatment, increasing agar concentration, or mimicking the physical interactions of neighboring cells within a spheroid. Complementary experiments in xenograft nude mouse models confirmed the essential role of Ezrin in tumor development. Notably, Intraperitoneal injection of spheroids generated in vitro overcame the effects of Ezrin depletion. Together, these findings underscore the role of Ezrin as a structural component of the cell cortex, crucial for tumor formation in soft environments, and highlight the broader relevance of ERM proteins in intraperitoneal metastasis in OC.\u003c/p\u003e","manuscriptTitle":"Ezrin plays a key role in cancer cell spheroid formation in soft environments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 13:06:00","doi":"10.21203/rs.3.rs-6595737/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba2460a7-d2ed-4679-b936-519a3e0e1b8f","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49693721,"name":"Biological sciences/Cancer"},{"id":49693722,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-06-24T05:54:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-09 13:06:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6595737","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6595737","identity":"rs-6595737","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}