Background: Ovarian cancer is the most common cause of death from gynaecological cancer worldwide (1). Unlike other solid cancers, ovarian cancer cells rarely spread via the vasculature, instead disseminating within the peritoneal fluid or ascites (2). Invading ovarian cancer cells interact with mesothelial cells lining the peritoneum, attach to, and invade the basement membrane to establish secondary lesions (3). The molecular events at this ‘invasive interface’ have been poorly studied. Therefore, we aimed to: i) develop an innovative method to co-culture ascites-derived ovarian cancer cells and measure invasion through peritoneal mesothelial cell layers in real time (4); ii) identify the unique protein expression profile at the ovarian spheroid-peritoneal interface during early invasion using proteomics.
Methods: Real Time Cell Analysis technology was adapted to establish a three-dimensional co-culture model of the tumor microenvironment of the peritoneum (4). Multicellular spheroids were generated from primary ovarian cancer lines or benign ascites derived cells cultured under non-adherent conditions. Real time measurements were conducted to determine the precise period when invasion commenced. Parallel cultures were histologically preserved at invasion initiation and processed for assessment by Matrix-Assisted Laser Desorption/Ionization (MALDI) imaging.
Results: Continuous real time measurements revealed that individual ovarian cancer samples exhibited differences in the timing of the onset of invasion. High-grade serous carcinoma samples initiated invasion rapidly after addition, some in under 30 min. MALDI imaging identified peptides expressed at the tumor-mesothelial interface during early invasion. These molecules represent novel molecules involved in the initiation of the metastatic process and are the subject of further characterisation.
Conclusion: This method represents a high-throughput quantitative analysis of ovarian cancer spheroid invasion of mesothelial and ECM barriers. The study has provided an unprecedented insight into the molecular mechanisms involved in the initiation of ovarian cancer invasion of the mesothelium, identifying new therapeutic targets.