Establishing Microglia-DMG Co-Cultures to Study GD2-CAR-T Cell Sensitivity

Abstract

Diffuse midline glioma (DMG) is one of the most deadly pediatric–type diffuse high-grade gliomas and there is currently no treatment for it. All DMGs are hallmarked by an alteration on Histone 3 Lysine 27 (H3K27), mostly resulting from a H3K27M mutation. This histone modification causes a widespread disbalance in the tumor cell expression signature, making it difficult to pinpoint the responsible genes for tumor onset, and thus, drug targeting. DMG is located in the brainstem and characterized by a diffuse growth profile, which impedes surgical resection. Furthermore, DMG harbour an intact blood- brain barrier, impeding the delivery of many therapeutic agents. Additionally, DMG conceal a ‘cold’ immune-microenvironment, highlighted by a low infiltration of T-cells and NK cells. On the contrary, myeloid cells make up at least a quarter of the tumor mass, suggesting that they play an important role in tumor growth. Microglia are the resident myeloid cells of the central nervous system and play an important role in the surveillance, development, and maintenance of the brain. Recent studies support the hypothesisthat pediatric brain tumors use microglia for their benefit, creating an anti-inflammatory and immune- cold environment by mechanisms still unknown. The recent revolution and evolution withing the immunotherapuy field has created new therapeutic strategies that might change prognosis of brain tumor patients dramatically. One of the most successful immunotherapeutic strategies includes CAR-T cell therapy, which has rendered impressive preclinical results in brain tumor models. However, the far majority does not contain a fully functional immune system, which could potentially explain the limited efficacy of clinical CAR-T therapies in pediatric brain tumor patients. Because of this, there is a need fo in vitro models that mimic the microglia-tumor interaction. We analyzed the tumor microenvironment of immune-competent DMG mice models via CITE-Seq and identified tumor-associated microglia-specific markers that can be used as potential drug target in DMG. To efficiently test such drug targets, we have set-up a microglia-DMG co-culture system. Addiotionally, these co-cultures were used to assess the GD2-CAR-T cell efficacy against DMG cells with and without tumor-associated microglia cells. This project allowed us enhance our comprehension of the role of microglia on tumor aggressiveness and progression. Moreover, in this study we addressed various inquiries concerning the efficacy of immunotherapy against DMG patients. Eventually, this project may contrubute to find a promising and efficient therapy for DMG.