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

Abstract

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...

Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG–PE micelles in ovarian cancer cell spheroid model

“…Bcl-2 overexpression in spheroids could increase the resistance against doxorubicin and may reflect the situation in real tumors, thus pointing at the potential usefulness of combination therapy (doxorubicin and an agent down-regulating Bcl-2) [65, 70]. Since spheroids have been detected in peritoneal cavities of ovarian cancer patients [71] and were proposed as metastasis intermediates [32, 33], intraperitoneal administration of 2C5-MDOX could represent a better therapeutic modality against primary and distant ovarian carcinoma as demonstrated previously with tumor necrosis factor α plasmid-loaded micelles for pancreatic cancer in mice [72]. Together our data support the usefulness of cancer cell spheroids for evaluation of targeted drug delivery and suggest the possibility of in vivo efficacy of 2C5-MDOX against ovarian carcinoma.…”

“…Cancer recurrence and metastases are a common occurrence for ovarian cancer with 61% of patients affected with metastatic cancer between 2002-2008 [31]. Since spheroids have been proposed as metastasis intermediates for ovarian cancer [32, 33], evaluation of drug targeting in ovarian carcinoma spheroids may provide useful information for targeting of primary and secondary tumors.…”

Getting to Know Ovarian Cancer Ascites: Opportunities for Targeted Therapy-Based Translational Research

“…Harvested spheroids can be replated onto different solid surfaces or co-cultured with other peritoneal cell populations to model later stages of ovarian cancer metastasis, when multicellular spheroids attach to and invade the peritoneal lining to form a secondary tumor (113–115). In particular, co-cultures of ovarian cancer cells with specific subpopulations of the peritoneal lining and omentum, such as fibroblasts (86, 116), adipocytes (117), and mesothelial cells (115) have provided particular insights into the physical, biomechanical, and chemical interactions between invading tumor cells and the peritoneal environment in the establishment of metastatic nodules within the peritoneum. These models are growing increasingly sophisticated, with the use of primary omental and peritoneal tissue for three-dimensional organotypic models (116, 118).…”

Transient Commensal Clonal Interactions Can Drive Tumor Metastasis

“…Dissemination of ovarian carcinoma cells to distant sites involves attachment to and clearance of the superficial layer of the mesothelium that encloses the organs in the peritoneal cavity [36][37][38] . To investigate whether the induction of CL31 metastatic ability by AREG is due to enhanced mesothelial clearance, we utilized a live-cell microscopybased assay that we previously described 39 . Briefly, CL31 spheroids (from 100 cells each) were formed by overnight suspension culture, treated with either vehicle or recombinant human AREG (100 ng/ml) and clearance of a mesothelial monolayer was recorded over 24 hours.…”