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.

scSNPdemux: a sensitive demultiplexing pipeline using single nucleotide polymorphisms for improved pooled single-cell RNA sequencing analysis.

BACKGROUND: Here we present scSNPdemux, a sample demultiplexing pipeline for single-cell RNA sequencing data using natural genetic variations in humans. The pipeline requires alignment files from Cell Ranger (10× Genomics), a population SNP database and genotyped single nucleotide polymorphisms (SNPs) per sample. The tool works on sparse genotyping data in VCF format for sample identification. RESULTS: The pipeline was tested on both single-cell and single-nuclei based RNA sequencing datasets and showed superior demultiplexing performance over the lipid-based CellPlex and Multi-seq sample multiplexing technique which incurs additional single cell library preparation steps. Specifically, our pipeline demonstrated superior sensitivity and specificity in cell-identity assignment over CellPlex, especially on immune cell types with low RNA content. CONCLUSIONS: We designed a streamlined pipeline for single-cell sample demultiplexing, aiming to overcome common problems in multiplexing samples using single cell libraries which might affect data quality and can be costly.