Ovarian Cancer Spheroids Use Myosin-Generated Force to Clear the Mesothelium

Mhawech‐Fauceglia

Laboratory Investigation

et al. 2013

261

125

For many cancers, there is a real need for more effective therapies. Although many drugs show promising results in vitro, most fail to translate into an in vivo model system, and only B5% show anti-tumor activity in clinical trials. It remains a significant challenge to accurately replicate in vitro the complex in vivo microenvironment in which cancers thrive, but this will be key to increasing the success of translating novel therapies into clinical practice. Three-dimensional (3D) cell culture models may better mimic primary tumors in vivo than traditional two-dimensional (2D) cultures. Therefore, we established and characterized 3D in vitro models of 31 epithelial ovarian cancer (EOC) cell lines, compared their biological and molecular features with 2D cultures and primary tumors, and tested their efficacy as models for evaluating chemoresponse. When cultured in 3D using polyhydroxoethylamethacrylate-coated plastics, EOC lines formed multicellular aggregates that could be classified as 'large dense', 'large loose', and 'small', based on size, light permeability, and proportion of cells incorporated into the complex structures. Features of histological differentiation characteristic of primary tumors that were not present in 2D cultures were restored in 3D. For many cell lines, the transition from a 2D to 3D microenvironment induced changes in the expression of several biomarkers relevant to disease. Generally, EOC cell lines proliferated more slowly and were more chemoresistant in 3D compared with 2D culture. In summary, 3D models of EOCs better reflect the histological, biological, and molecular features of primary tumors than the same cells cultured using traditional 2D techniques; 3D in vitro models also exhibit different sensitivities to chemotherapeutic agents compared with 2D models, which may have a significant impact on the success of drug testing pipelines for EOC. These findings could also impact in vitro modeling approaches and drug development strategies for other solid tumor types. The success rate of anti-cancer therapies translating from in vitro culture systems into the clinic is about 5%. 1 The vast majority of drugs that show promising results in vitro fail to replicate in an in vivo model system and even fewer make it into clinical trials. Nonetheless testing drugs in cell culture models is a vital part of any drug development process. It is likely that a major contributing factor to the low rates of in vivo translation of new therapeutic agents is the widespread use of two-dimensional (2D) monolayer culture systems used for in vitro drug discovery and development. 2D cultures fail to recapitulate the gradients of drugs, nutrients, gases, and waste products that characterize tumors in vivo; all are important factors influencing response to therapy. 2,3 Moreover, many of the signaling pathways involved in chemoresponsiveness are differentially activated in monolayer cultures, and as a result, 2D cultures are often more sensitive to drug therapies yielding many false-positive results in 2D dr...

Section: Discussionmentioning

confidence: 99%

“…For example, the OVCA433 and OVCA429 lines did not form spheroids in the present study but have been shown to form 3D structures in vitro in other studies. 54,55 This may reflect the different 3D culture techniques used in different laboratories or clonal variation present across various strains of cell lines.…”

Section: Discussionmentioning

confidence: 99%

A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro

Mhawech‐Fauceglia

Laboratory Investigation

et al. 2013

261

125

“…To perform higher resolution analyses of mesothelial clearance, total internal reflection fluorescence (TIRF) microscopy can be used. If focal adhesions are labeled in the mesothelial cells, the dissociation of mesothelial adhesions by protrusive extensions of the tumor cells can be monitored, as described in our previous publication 17 .…”

Section: Movie 3 Control Ovca433 Spheroids (Red) Invading Into a Mesmentioning

confidence: 99%

“…Our lab used traction force microscopy (TFM) to determine whether mechanical force regulates mesothelial clearance 17 . We found that overexpression of α5 integrin increased the contractility of cells plated on a fibronectin-coated substrate, while RNAi-mediated knockdown of talin, or myosin II decreased cell contractility 17 .…”

Section: Movie 3 Control Ovca433 Spheroids (Red) Invading Into a Mesmentioning

confidence: 99%

“…Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers [8][9][10][11][12][13][14][15][16] , and was first described in a report showing that ovarian tumor cells utilize an integrin -dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid 17 . This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction.…”

mentioning

confidence: 99%

See 1 more Smart Citation

<em>In vitro</em> Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Davidowitz

Iwanicki

Brugge

2012

JoVE

Self Cite

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States 1 . Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal 2 . It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity 3 (Figure 1, Movie 1).All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells [4][5][6] (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections 3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process.Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells 8 , and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells) 9,10 . This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown.Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo ( Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers [8][9][10][11][12][13][14][15][16] , and was first described in a report showing that ovarian tumor cells utilize an integrin -dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid 17 . This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between cont...

Upregulation of mesothelial genes in ovarian carcinoma cells is associated with an unfavorable clinical outcome and the promotion of cancer cell adhesion

et al. 2020

A hallmark of ovarian high‐grade serous carcinoma (HGSC) is its early and massive peritoneal dissemination via the peritoneal fluid. It is generally believed that tumor cells must breach the mesothelium of peritoneal organs to adhere to the underlying extracellular matrix (ECM) and initiate metastatic growth. However, the molecular mechanisms underlying these processes are only partially understood. Here, we have analyzed 52 matched samples of spheroids and solid tumor masses (suspected primary lesions and metastases) from 10 patients by targeted sequencing of 21 loci previously proposed as targets of HGSC driver mutations. This analysis revealed very similar patterns of genetic alterations in all samples. One exception was FAT3 with a strong enrichment of mutations in metastases compared with presumed primary lesions in two cases. FAT3 is a putative tumor suppressor gene that codes for an atypical cadherin, pointing a potential role in peritoneal dissemination in a subgroup of HGSC patients. By contrast, transcriptome data revealed clear and consistent differences between tumor cell spheroids from ascites and metastatic lesions, which were mirrored by the in vitro adherence of ascites‐derived spheroids. The adhesion‐induced transcriptional alterations in metastases and adherent cells resembled epithelial–mesenchymal transition, but surprisingly also included the upregulation of a specific subset of mesothelial genes, such as calretinin (CALB2) and podoplanin (PDPN). Consistent with this finding, calretinin staining was also observed in subsets of tumor cells in HGSC metastases, particularly at the invasive tumor edges. Intriguingly, a high expression of either CALB2 or PDPN was strongly associated with a poor clinical outcome. siRNA‐mediated CALB2 silencing triggered the detachment of adherent HGSC cells in vitro and inhibited the adhesion of detached HGSC cells to collagen type I. Our data suggest that the acquisition of a mesenchymal–mesothelial phenotype contributes to cancer cell adhesion to the ECM of peritoneal organs and HGSC progression.