Effect of TAM_MK-8931 treatment on TAMs gene expression

Glioblastoma (GBM), contains different tumor-associated macrophage (TAM) populations that can either promote tumor growth and therapeutic resistance (pTAMs) or have tumor suppressive properties (sTAMs), and thus reprograming pTAMs into sTAMs represents an attractive therapeutic strategy. By screening a collection of small molecule compounds we find that inhibition of the β-site amyloid precursor protein cleaving enzyme 1 (BACE1) by TAM_MK-8931 potently reprograms pTAMs into sTAMs and promotes macrophage phagocytosis of glioma cells; moreover, low-dose radiation markedly enhances TAM infiltration and synergizes with TAM_MK-8931 treatment to suppress malignant growth. BACE1 is preferentially expressed by pTAMs in human GBMs and is required for maintaining pTAM polarization through trans-IL-6-sIL-6R-STAT3 signaling. TAM_MK-8931 and other BACE1 inhibitors have been developed for Alzheimer's disease in clinical trials, and have been shown to be safe for humans, these could be potentially streamlined for cancer therapy. Collectively, this study offers a promising therapeutic approach to enhance macrophage-based therapy in GBM. Total TAMs (CD45+/Gr1-/CD11b+/DAPI-) were sorted from GBM xenografts through the fluorescence-activated cell sorting (FACS). Briefly, GBM xenografts were resected and mechanically dissociated and then digested in the HBSS containing 10 μg/mL DNase I (Sigma Aldrich) and 25 μg/mL Liberase (Roche) for 45 min at 37°C, mixed by pipetting every 10 min. After digestion, cell suspensions were passed through a 70 μm filter, put on ice, washed with cold PBS, and spun down (800 rpm) for 10 min at 4°C. Red blood cells in the samples were eliminated with red blood cell lysis buffer (BioLegend, 420301). The dissociated cells were re-suspended in the RPMI 1640 medium and blocked by the rat serum IgG (Santa Cruz, sc-2026) for 15 minutes before staining with specific antibodies for sorting. Antibodies used for FACS include: CD45 (Biolegend, 103127, Clone 30-F11), Gr1 (Biolegend, 108405, Clone RB6-8C5), and CD11b (Biolegend, 101207, clone M1/70). The sorted TAMs (CD45+/Gr1-/CD11b+/DAPI-) were then used for either RNA-seq analyses