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Abstract Glioblastoma multiforme (GBM) is a grade IV astrocytoma and the most common type of adult brain malignancy. The overall survival of patients diagnosed with GBM is less than 5 years. Therefore, it is imperative to understand the mechanisms contributing to GBM progression, invasiveness, and survival. This study aimed to evaluate the effect of the extracellular matrix (ECM) on GBM cell invasion by performing the traditional invasion assay as well as novel in vitro invasion assay strategies. A significant challenge with in vitro cell invasion assays is the use of animal-based ECM: the components of animal-based ECM are not characterized; the batch-to-batch variability of animal-based ECM can influence experimental findings and affect potential clinical applications; and the temperature-sensitive operation protocols make the use of animal-based ECM time-consuming and difficult for automated high-throughput assays. These challenges can be circumvented using VitroGel®, a synthetic xeno-free, bio-functional hydrogel resembling the physiological ECM with tunable biophysical and biochemical properties. In this study, we harnessed the properties of the synthetic xeno-free hydrogel system to examine how different mechanical strengths and functional ligands as well as cytokines and serum within the hydrogel matrices or in the outer well, affect GBM cell invasion. The results indicated that adjusting the mechanical strength of the ECM system affects GBM cell invasion. We further showed that the bio-functional ligands within the hydrogel matrix, such as matrix metalloproteinases, RGD peptide, and others, stimulated cell invasion. This finding exemplifies the importance of using a well-characterized ECM system given that it is difficult to evaluate how specific ECM components contribute to cell invasion using the undefined animal-based ECM. Since the composition of the hydrogel system is chemically defined, we could further control the cytokine milieu within the matrix to understand how the ECM composition affects cell migration. To demonstrate this, we added the cytokine TGF-β1 to the hydrogel and examined GBM cell invasion. Indeed, we showed that adding TGF-β1 to the synthetic VitroGel enhanced GBM cell invasion, thus illustrating that incorporating factors inside the matrix is a powerful method to evaluate chemotaxis. Altogether, this study demonstrates the potency and flexibility of using a xeno-free hydrogel for a myriad of invasion assay applications. Citation Format: Alejandra I. Ferrer Díaz, John Huang. Unraveling the effects of the microenvironment on glioblastoma multiforme invasiveness using a xeno-free 3D hydrogel platform abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4209.
Díaz et al. (Fri,) studied this question.