Effective Reprogramming of Patient-Derived M2-Polarized Glioblastoma-Associated Microglia/Macrophages by Treatment with GW2580.

PURPOSE: Targeting immunosuppressive and pro-tumorigenic glioblastoma (GBM)-associated macrophages and microglial cells (GAM) has great potential to improve patient outcomes. Colony-stimulating factor-1 receptor (CSF1R) has emerged as a promising target for reprograming anti-inflammatory M2-like GAMs. However, treatment data on patient-derived, tumor-educated GAMs and their influence on the adaptive immunity are lacking. EXPERIMENTAL DESIGN: CD11b+-GAMs freshly isolated from patient tumors were treated with CSF1R-targeting drugs PLX3397, BLZ945, and GW2580. Phenotypical changes upon treatment were assessed using RNA sequencing, flow cytometry, and cytokine quantification. Functional analyses included inducible nitric oxide synthase activity, phagocytosis, transmigration, and autologous tumor cell killing assays. Antitumor effects and changes in GAM activation were confirmed in a complex patient-derived 3D tumor organoid model serving as a tumor avatar. RESULTS: The most effective reprogramming of GAMs was observed upon GW2580 treatment, which led to the downregulation of M2-related markers, IL6, IL10, ERK1/2, and MAPK signaling pathways, while M1-like markers, gene set enrichment indicating activated MHC-II presentation, phagocytosis, and T-cell killing were substantially increased. Moreover, treatment of patient-derived GBM organoids with GW2580 confirmed successful reprogramming, resulting in impaired tumor cell proliferation. In line with its failure in clinical trials, PLX3397 was ineffective in our analysis. CONCLUSIONS: This comparative analysis of CSF1R-targeting drugs on patient-derived GAMs and human GBM avatars identified GW2580 as the most powerful inhibitor with the ability to polarize immunosuppressive GAMs to a proinflammatory phenotype, supporting antitumor T-cell responses while also exerting a direct antitumor effect. These data indicate that GW2580 could be an important pillar in future therapies for GBM.

Molecular Determinants of Calcitriol Signaling and Sensitivity in Glioma Stem-like Cells.

Glioblastoma is the most common primary brain cancer in adults and represents one of the worst cancer diagnoses for patients. Suffering from a poor prognosis and limited treatment options, tumor recurrences are virtually inevitable. Additionally, treatment resistance is very common for this disease and worsens the prognosis. These and other factors are hypothesized to be largely due to the fact that glioblastoma cells are known to be able to obtain stem-like traits, thereby driving these phenotypes. Recently, we have shown that the in vitro and ex vivo treatment of glioblastoma stem-like cells with the hormonally active form of vitamin D3, calcitriol (1α,25(OH)2-vitamin D3) can block stemness in a subset of cell lines and reduce tumor growth. Here, we expanded our cell panel to over 40 different cultures and can show that, while half of the tested cell lines are sensitive, a quarter can be classified as high responders. Using genetic and proteomic analysis, we further determined that treatment success can be partially explained by specific polymorphism of the vitamin D3 receptor and that high responders display a proteome suggestive of blockade of stemness, as well as migratory potential.