Supplemental Figures and Supplemental Methods

Supplementary Tables 10 - 14

Supplementary Tables 15 - 25

In a multi-level "deconstruction" of omental metastases, we previously identified a prognostic matrisome gene expression signature in high-grade serous ovarian cancer (HGSOC) and twelve other malignancies. Here, our aim was to understand how six of these extracellular matrix (ECM) molecules, COL11A1, cartilage oligomeric matrix protein, FN1, versican, cathepsin B, and COL1A1, are upregulated in cancer. Using biopsies, we identified significant associations between TGFβR activity, Hedgehog (Hh) signaling, and these ECM molecules and studied the associations in mono-, co-, and tri-culture. Activated omental fibroblasts (OFs) produced more matrix than malignant cells, directed by TGFβR and Hh signaling cross talk. We "reconstructed" omental metastases in tri-cultures of HGSOC cells, OFs, and adipocytes. This combination was sufficient to generate all six ECM proteins and the matrisome expression signature. TGFβR and Hh inhibitor combinations attenuated fibroblast activation and gel and ECM remodeling in these models. The tri-culture model reproduces key features of omental metastases and allows study of diseased-associated ECM.

Studying the complexity of the tumor microenvironment (TME) still represents a challenge in research. The intricate communication within the stroma - fibroblast, immune cells, endothelial cells and the extracellular matrix (ECM) - and the malignant cell compartment sustains every stage of cancer progression and resistance to cancer therapies, but it is difficult to model this using human cells. Using a multi-level deconstruction of human metastases as a template and validation, we developed multicellular models of high-grade serous ovarian cancer (HGSOC) with increased complexity. The different models - tri-, tetra- and penta-cultures - consist of different combinations of primary adipocytes, fibroblasts, mesothelial cells, HGSOC cells and myeloid cells that mimic the structure and the composition of a human omental metastatic tissue. In HGSOC biopsies, we previously identified a prognostic matrisome signature and a number of pathways correlating ECM deposition and disease progression. This knowledge combined with the versatile nature of our platforms has allowed us to identify the contributions of each cell type in supporting tumor progression and ECM remodeling. For instance, 14-day tri-culture of malignant cells, adipocytes and fibroblasts was sufficient to replicate the deposition of specific ECM proteins and the prognostic matrisome signature seen in human omental metastases. Moreover, we found that TGFβR and Hedgehog signaling pathways act together on the fibroblasts to stimulate ECM production. In addition, in the tetra-culture model, we identified platelets as promoters of early metastatic invasion by activating mesothelial cells. As the immune system plays an important role in HGSOC tumor microenvironment, we have now added myeloid cells to form a more complex penta-culture in which we can analyze the behavior and impact of myeloid cells. In conclusion, the multicellular models presented here could be a new resource to study new and established cancer therapies and provide a useful tool to better understand the molecular mechanisms that drive cancer progression. Citation Format: Beatrice Malacrida, Robin Delaine-Smith, Sam Nichols, Eleni Maniati, Roanne L. Jones, Martin M. Knight, Oliver M. Pearce, Frances R. Balkwill. Using 3-Dimensional in vitro models to unravel the complexity of the metastatic microenvironment in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2642.

Guided by a multi-level "deconstruction" of omental metastases, we developed a tetra (four cell)-culture model of primary human mesothelial cells, fibroblasts, adipocytes, and high-grade serous ovarian cancer (HGSOC) cell lines. This multi-cellular model replicated key elements of human metastases and allowed malignant cell invasion into the artificial omental structure. Prompted by findings in patient biopsies, we used the model to investigate the role of platelets in malignant cell invasion and extracellular matrix, ECM, production. RNA (sequencing and quantitative polymerase-chain reaction), protein (proteomics and immunohistochemistry) and image analysis revealed that platelets stimulated malignant cell invasion and production of ECM molecules associated with poor prognosis. Moreover, we found that platelet activation of mesothelial cells was critical in stimulating malignant cell invasion. Whilst platelets likely activate both malignant cells and mesothelial cells, the tetra-culture model allowed us to dissect the role of both cell types and model the early stages of HGSOC metastases.

Abstract We have profiled, for the first time, an evolving human metastatic microenvironment by measuring gene expression, matrisome proteomics, cytokine and chemokine levels, cellularity, extracellular matrix organization, and biomechanical properties, all on the same sample. Using biopsies of high-grade serous ovarian cancer metastases that ranged from minimal to extensive disease, we show how nonmalignant cell densities and cytokine networks evolve with disease progression. Multivariate integration of the different components allowed us to define, for the first time, gene and protein profiles that predict extent of disease and tissue stiffness, while also revealing the complexity and dynamic nature of matrisome remodeling during development of metastases. Although we studied a single metastatic site from one human malignancy, a pattern of expression of 22 matrisome genes distinguished patients with a shorter overall survival in ovarian and 12 other primary solid cancers, suggesting that there may be a common matrix response to human cancer. Significance: Conducting multilevel analysis with data integration on biopsies with a range of disease involvement identifies important features of the evolving tumor microenvironment. The data suggest that despite the large spectrum of genomic alterations, some human malignancies may have a common and potentially targetable matrix response that influences the course of disease. Cancer Discov; 8(3); 304–19. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 253

Abstract The purpose of this study was to understand the relationships between the molecular mechanisms of disease progression and higher-order features such as tissue stiffness, extent of disease and cellularity in the tumor microenvironment, TME. Using samples of human high-grade serous ovarian cancer metastases, ranging from normal to heavily diseased, we identified molecular components of the TME using transcriptomic and proteomic analysis. We integrated these data against higher-order features of the tissue, namely the biomechanics, cellularity, and disease score. We then used bioinformatics and multivariate statistics to identifying components of the TME that best model the higher-order features. For the first time, we revealed the complexity of extracellular matrix remodeling during metastases development, defining patterns of extracellular-matrix associated genes and proteins that predicted both extent of disease and tissue modulus. This allowed us to identify a core group of twenty-two matrix-associated molecules that modeled the dynamic process of tissue remodeling during tumor progression. We used these data to generate a ‘matrix index’, a quantitative measure of the gene expression of the twenty-two molecules. In cancer transcriptomic databases, this matrix index had prognostic significance in thirteen solid cancers including high-grade serous ovarian cancer, even after multivariate analysis. We conclude that there may be a common host matrix response to human solid cancers. Citation Format: Frances R. Balkwill, Oliver M. Pearce, Robin Delaine-Smith, Eleni Maniati, Sam Nichols, Jun Wang, Conrad Bessant, Martin Knight. Deconstruction of a human tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5558. doi:10.1158/1538-7445.AM2017-5558