Developing Advanced Tumoroid Models Driven by Epithelial-to-Mesenchymal Transition with a Novel Xeno-Free and Biofunctional Hydrogel System

Abstract:

Epithelial-to-mesenchymal transition (EMT) is a key biological process that facilitates cancer metastasis to distant organs. Most preclinical studies investigating EMT are conducted using traditional two-dimensional (2D) culture models, which fail to recapitulate the in vivo tumor microenvironment, limiting the translational relevance of their findings to clinical settings. Consequently, three-dimensional (3D) culture systems have emerged as a superior approach for modeling metastatic processes and evaluating therapeutic candidates.

However, these models typically rely on animal-derived extracellular matrices like Matrigel, which are poorly defined, exhibit batch-to-batch variability, and lack mechanical and biochemical stability, thereby compromising experimental reproducibility and clinical translation.

In this study, we developed tumoroids from two cancer types known to undergo EMT, such as glioblastoma multiforme (GBM) and breast cancer, using VitroGel® hydrogel, a fully synthetic and biocompatible hydrogel system. The VitroGel® hydrogel is tunable in both mechanical and biofunctional properties, making it a powerful tool for generating advanced tumoroid models that might require distinct microenvironments. The tumoroids were generated from a single spheroid formed in ultra-low-attachment, U-shaped 96-well plates, which were then embedded in VitroGel® hydrogel diluted with our xeno-free supplement.

Within the first three days after adding the hydrogel, the cells began migrating from the spheroid into the matrix, developing a tube-like structure with rapidly proliferating cells. By day seven, the tumoroids exhibited an outer layer enriched with EMT marker-positive cells expressing vimentin and N-cadherin, while the spheroid core and tube-like structure were mostly composed of cells expressing the cancer stem cell–associated markers Oct4 and Sox2. The tumoroids were susceptible to standard chemotherapies, including temozolomide and 5-fluorouracil, indicating that these structures developed in the xeno-free hydrogel are a suitable system for drug screening studies. Additionally, to enhance the physiological relevance of the tumoroid model, we aimed to mimic tumor-vasculature interactions by performing co-cultures of tumoroids with endothelial cells. The endothelial cells penetrated the matrix and migrated towards the tumoroids, enhancing their long-term survival.

Altogether, these findings demonstrate that the xeno-free hydrogel system supports the development of robust 3D cancer models, providing a platform for the evaluation of therapeutic drug candidates, which sends a significant advancement for precision oncology and drug discovery.

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