Mesothelial Clearance Assay: An In Vitro Technique to Quantify the Invasion Ability of Ovarian Cancer Spheroids into Mesothelial Cell Monolayers
JoVE Encyclopedia of Experiments
Cancer Research
0 views • 4:34 min • April 30th, 2023
During ovarian cancer metastasis, malignant cells detach from the primary tumor site and start circulating within the peritoneal cavity. These cells can then form clusters or spheroids that adhere to the mesothelial cell monolayer lining the peritoneal cavity. As the tumor grows, it clears the underlying mesothelial cells to facilitate invasion.
To model mesothelial clearance, begin by seeding mesothelial cells expressing green fluorescent protein or GFP to a fibronectin-coated glass-bottom culture dish. The fibronectin coating provides a matrix that promotes cell attachment. Incubate the dish to allow mesothelial cells to spread and form a monolayer.
Next, transfer a suspension of ovarian cancer cell spheroids expressing red fluorescent protein onto the mesothelial monolayer. Image the culture for an extended duration using time-lapse fluorescence microscopy. The red-fluorescent spheroids will settle and attach to the green-fluorescent mesothelial monolayer.
The cancer cells exert force on the mesothelial cells, disrupting cell junctions and altering the mesothelial layer integrity. The force also displaces the underlying mesothelial cells, generating a hole. Choose an appropriate GFP filter setting to visualize the resultant hole as a black space in the image. Measure the space to quantify the extent of mesothelial clearance.
To begin this procedure, pre-coat the wells of a 6-well glass-bottom MatTek dish with fibronectin. Add 2 milliliters of fibronectin/PBS solution to each well of the dish and incubate at room temperature for 30 minutes. The green fluorescent protein-expressing mesothelial cells, for forming a mesothelial cell monolayer, are cultured in 10% base medium. Trypsinize a plate of mesothelial cells and spin down in a tabletop centrifuge at 900 RPM for three minutes.
Aspirate the supernatant and resuspend cells in 10% base medium. Adjust to the desired concentration with 10% base medium. When the 30-minute fibronectin incubation of the MatTek dish is complete, wash the wells with 2 milliliters of PBS. Aspirate the PBS and plate 6 times 10 to the fifth mesothelial cells per well in each well of the 6-well MatTek dish. Incubate the MatTek dish in a 37 degrees Celsius cell culture incubator overnight to allow the mesothelial cells to attach to the dish and form a monolayer.
Use a pipette to collect the ovarian cancer spheroids from the 96-well poly-HEMA coated plate. Aspirate the medium from one well of the 6-well MatTek dish containing a mesothelial cell monolayer. Wash once with 2 milliliters of PBS. Add all of the spheroids from the 96-well plate to one well of the MatTek dish. This is approximately three times the number of spheroids that are going to be imaged to account for spheroids landing on the part of the dish that cannot be imaged.
Since we want to image only one spheroid per field of view, aggregation of the spheroids must be avoided. Rock the dish gently from side to side to distribute the spheroids evenly across the monolayer before they attach.
Place the MatTek dish on the stage of an inverted wide-field fluorescence microscope capable of performing time-lapse imaging for the duration of at least eight hours. Use a motorized stage to image multiple positions in the dish, with multiple spheroid intercalation events, in a single experiment. The ovarian cancer cell spheroids will settle to the bottom of the dish and attach to the mesothelial cell monolayer. Collect GFP, RFP, and phase images of 20 plus spheroid/monolayer interactions every ten minutes for eight hours.
Overview
This study investigates the interaction between ovarian cancer spheroids and mesothelial cells during metastasis. The methodology involves imaging these interactions to understand the mechanisms of mesothelial clearance.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Cancer Research
Background
- Ovarian cancer cells can detach from primary tumors and circulate in the peritoneal cavity.
- These cells can form clusters that adhere to mesothelial cell layers.
- Understanding mesothelial clearance is crucial for insights into cancer metastasis.
- Time-lapse imaging techniques are employed to visualize these cellular interactions.
Purpose of Study
- To model the process of mesothelial clearance by ovarian cancer cells.
- To quantify the extent of mesothelial layer disruption caused by cancer cell spheroids.
- To utilize fluorescence microscopy for real-time observation of cell interactions.
Methods Used
- Mesothelial cells expressing GFP are cultured on fibronectin-coated dishes.
- Ovarian cancer spheroids expressing RFP are introduced to the mesothelial monolayer.
- Time-lapse fluorescence microscopy is used to capture interactions over eight hours.
- Cell junction integrity and mesothelial clearance are assessed through imaging.
Main Results
- Ovarian cancer spheroids successfully attach to the mesothelial monolayer.
- Force exerted by cancer cells disrupts mesothelial cell junctions.
- Clearance of mesothelial cells is visualized as black spaces in images.
- Quantification of mesothelial clearance is achieved through image analysis.
Conclusions
- The study provides insights into the metastatic behavior of ovarian cancer cells.
- Understanding mesothelial clearance may inform therapeutic strategies.
- Time-lapse imaging is an effective method for studying cell interactions in cancer research.
Frequently Asked Questions
Executive Industry Relevance
Quantitative assessment of ovarian cancer spheroid invasion into mesothelial monolayers addresses a critical early step in metastatic progression, directly informing target validation and mechanistic de-risking in oncology pipelines. This assay enables high-content, reproducible measurement of cell-cell interactions relevant to metastatic dissemination, supporting predictive confidence at the discovery-to-preclinical inflection point. Integration of quantitative imaging outputs strengthens portfolio triage and prioritization for anti-metastatic therapeutic strategies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of metastatic invasion mechanisms in a controlled, disease-relevant system.
- Supports functional validation of candidate targets involved in cell adhesion, migration, and clearance.
- Provides mechanistic de-risking by quantifying the impact of genetic or pharmacological perturbations on invasion.
- Facilitates predictive confidence in target selection for anti-metastatic drug discovery.
Screening & Assay Development
- Establishes a standardized, quantitative assay platform for evaluating compound effects on spheroid invasion.
- Delivers reproducible, image-based readouts suitable for comparative analysis across conditions.
- Enables scalability for medium-throughput screening of inhibitors targeting metastatic processes.
- Supports assay transferability and platform reuse in oncology research programs.
Translational & Preclinical Research
- Models early metastatic events with translational relevance to ovarian cancer progression.
- Aligns in vitro findings with preclinical models for continuity in biomarker and efficacy validation.
- Informs risk-adjusted advancement decisions for anti-metastatic candidates.
- Provides a mechanistic bridge between discovery biology and preclinical evaluation.
Pipeline & Workflow Integration
This mesothelial clearance assay positions within the discovery-to-preclinical continuum, enabling hypothesis testing, target validation, and early compound screening for metastatic inhibition.
- Discovery Biology: Quantifies cell-cell interaction dynamics and pathway involvement in metastatic invasion.
- Screening: Delivers standardized, quantitative outputs for compound and genetic perturbation assessment.
- Analytics: Provides image-based measurements of mesothelial clearance for robust statistical comparison.
- Translational Research: Bridges in vitro mechanistic insights with in vivo metastatic models when aligned with disease context.
- Enterprise Reuse: Functions as a reusable assay platform for diverse oncology R&D initiatives targeting metastasis.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in metastatic target validation.
- Operational Value: Promotes assay standardization, reproducibility, and scalability for cross-program deployment.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient prioritization of anti-metastatic strategies.
- Portfolio Impact: Supports risk-adjusted advancement and triage of oncology assets targeting metastatic progression.
Implementation Considerations
- Requires expertise in cell culture, fluorescence microscopy, and quantitative image analysis.
- Demands access to time-lapse imaging platforms and analytical software for data quantification.
- Necessitates cross-team standardization of assay setup and imaging protocols for reproducibility.
- May require adaptation for different cancer cell types or mesothelial sources based on research focus.
- Throughput and scalability are limited by imaging capacity and manual handling steps.
Biopharma FAQs
Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time
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Last updated: 22 August 2026