W17 - Modeling the Immunosuppressive Tumor Microenvironment in Glioblastoma Using HA-Based 3D Hydrogels

Introduction: : Glioblastoma (GBM) is an aggressive brain tumor with a poor prognosis and limited treatment options. Despite the promise of CAR T cell therapy in hematologic malignancies, its efficacy in solid tumors like GBM remains low. One major barrier is the tumor microenvironment, which is heavily infiltrated by tumor-associated macrophages (TAMs), comprising up to 40% of the tumor mass. These TAMs contribute to immune suppression and therapeutic resistance, particularly against CAR T cell-based treatments. In this study, we aim to investigate how the GBM tumor microenvironment influences macrophage phenotype and function using engineered 3D hydrogels composed of hyaluronic acid, a key extracellular matrix component in GBM.

Materials and

Methods: : We began by analyzing RNA sequencing data from The Cancer Genome Atlas (TCGA) to compare the expression of hyaluronic acid (HA)-related genes between glioblastoma (GBM) and lower-grade glioma (LGG) patient samples. This analysis confirmed the elevated presence and potential significance of HA in GBM. Based on these findings, we engineered 3D hyaluronic acid-based hydrogels to recapitulate key features of the GBM tumor microenvironment. Human macrophages were cultured within these hydrogels under varying tumor-like conditions. To evaluate changes in macrophage phenotype and function, we performed ELISA to measure cytokine secretion, qPCR to assess gene expression of polarization markers, and RNA sequencing to profile global transcriptional changes.

Results, Conclusions, and Discussions:: Analysis of TCGA RNA sequencing data revealed that hyaluronic acid (HA)-related genes are significantly upregulated in high-grade gliomas (GBM) compared to lower-grade gliomas (LGG), highlighting the relevance of HA in GBM progression. Functional assays using our 3D HA-based hydrogel model demonstrated that increasing HA concentration within the tumor microenvironment leads to immunosuppressive macrophage behavior. ELISA results showed reduced secretion of M1-associated cytokines and elevated levels of M2-associated cytokines. Consistently, qPCR analysis confirmed the downregulation of M1 polarization genes and upregulation of M2 markers in macrophages exposed to higher HA concentrations. Our findings suggest that hyaluronic acid (HA), a major component of the glioblastoma (GBM) extracellular matrix, plays a critical role in shaping the phenotype of tumor-associated macrophages (TAMs). The upregulation of HA-related genes in GBM, combined with our in vitro results, supports the hypothesis that HA-rich tumor microenvironments promote an immunosuppressive, M2-like macrophage phenotype. This shift may contribute to the failure of CAR T cell therapy in GBM by enhancing immune evasion. By mimicking the tumor matrix using HA-based hydrogels, our model provides a controlled platform to dissect the mechanisms of macrophage polarization and identify strategies to reprogram TAMs toward a pro-inflammatory, anti-tumor state.

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