A Collagen-Based 3D Microtissue Platform Preserves Structural, Molecular, and Therapeutic Heterogeneity in Ovarian Cancer

Background: Ovarian cancer is comprised of biologically diverse tumour histotypes that are difficult to represent using conventional two-dimensional (2D) culture. These systems lack the tissue organization and extracellular matrix interactions found in native tumours. We developed a type I collagen-based three-dimensional (3D) microtissue model to determine whether a tumor supportive matrix environment could maintain key characteristics of different ovarian cancer histotypes. Methods: 3D collagen microtissues were generated using five ovarian cancer cell lines: OVCAR3, SNU119, KURAMOCHI, OVTOKO, and SKOV3. Long-term growth, viability, and morphology were assessed using quantitative growth analysis, confocal microscopy, and longitudinal bright-field imaging. Tumour architecture and histotype-associated phenotypes were evaluated by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Therapeutic responses to carboplatin, paclitaxel, olaparib, and niraparib were compared between 3D microtissues and corresponding 2D monolayer cultures. Results: All five models remained viable and showed sustained growth within the collagen scaffold, supported by growth-curve analysis and confocal imaging. Despite being maintained in the same matrix environment, the models developed distinct 3D architectures. OVCAR3, KURAMOCHI and SNU119 formed prominent papillary-like structures of high-grade serous carcinoma (HGSC), while OVTOKO and SKOV3 displayed distinct surface-associated growth patterns of clear cell and endometrioid ovarian carcinomas, respectively. H&E analysis confirmed these architectural differences, and IHC profiling demonstrated preservation of selected histotype-associated features, including distinct PAX8, WT1, and p53 expression patterns. Therapeutic responses varied across cell lines and treatments, with 3D microtissues generally maintaining greater viability following treatment than corresponding 2D cultures. These findings indicate that the collagen environment supports long-term tumour growth while preserving ovarian cancer histotype specific structural, molecular, and therapeutic heterogeneity. Conclusions: The collagen-based microtissue platform preserved structural, molecular, and therapeutic heterogeneity across distinct ovarian cancer models within a shared 3D environment. By maintaining tumour-associated architecture, distinct immunophenotypic features, and model-specific therapeutic responses, the platform captures key aspects of ovarian cancer biology that are not adequately represented in conventional 2D culture. These findings support its use as a physiologically relevant system for investigating ovarian cancer heterogeneity, tumour-matrix interactions, and therapeutic response.