The tissue microenvironment comprises of extracellular matrix, fibroblasts, adipocytes and immune cells, and is known for its role in maintaining the integrity of normal tissue architecture. Disruption of normal balance between epithelial cells and the surrounding stroma leads to tumour development. These early neoplastic changes are highly reflected by the ability of tumour cells to corrupt the surrounding stroma and turn it from restrictive to supportive environment. Defects in tissue microenvironment have been well established in many human cancers. However the signalling alterations in tumour stroma that promotes differentiated to malignant endometrial cancer cell behaviour have not been well investigated.
To study this, we established organotypic three-dimensional (3D) culture system for endometrial cancer cells. This 3D system exhibits a hierarchical structure and cellular heterogeneity and mimics in vivo endometrial cancer cell morphology and function (proliferation, differentiation and gene expression). 3D cultures of different grades of endometrial cancer cell lines revealed two distinct morphologies; (1) round structure with well-organized nuclei and robust cell-cell adhesion (2) stellate shape with disorganized nuclei and invasive processes. We further analysed the proliferation of these two groups of cells in presence of extracellular matrix and found that round colonies showing differentiated cell behaviour have low rates of proliferation as compared to disaggregated colonies having malignant cell behaviour. RNA sequencing of differentiated and malignant endometrial cancer cell line showed distinct gene expression profiling in both two and three dimensional culture. TGFβ1 was one of the top five upstream regulators in both differentiated and malignant cell types in 3D extracellular matrix. However, TGFβ1 expression was more upregulated in malignant cell types than differentiated cells. Analysis of epithelial and mesenchymal markers distinctly differentiated both these cell types. This reveals upregulation of TGFβ1 in 3D microenvironment promotes epithelial to mesenchymal transition that potentiates endometrial cancer progression.