Highlights d Flow cytometry, RNA-seq, and protein and image analyses reveal brain TME complexity d Glioma IDH mutation status and brain metastasis primary tumors shape the brain TME d Microglia and monocyte-derived macrophages exhibit multifaceted activation d TME immune cells show disease-and cell-type-specific expression patterns

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year relative survival rate of 8% and is projected to be the second leading cause of cancer death by 2030, underscoring the urgency to develop new strategies to improve current therapeutic modalities for PDAC. Targeting pancreatic cancer stem cells (PCSCs), which are resistant to radiation and chemotherapy, is a promising strategy. A novel approach which can be readily clinically translated is to repurpose disulfiram (DSF), a drug for treating alcoholism, to target PCSCs. Chemoradiation or the combination of chemotherapy agents FOLFIRINOX, currently standard care for PDAC, can increase stemness in some established or primary PDAC cell lines. However, DSF in the presence of exogenously or endogenously supplied copper (Cu), when combined with chemotherapy or chemoradiation, targets both PCSCs and nonstem PDAC cells. Previously, we demonstrated that DSF/Cu effectively targets breast cancer stem cells in the context of fractionated radiation (FIR) by inhibiting the NF-κB-stemness gene pathway. Therefore, the hypothesis that PCSCs can be effectively targeted by incorporating DSF/Cu into the standard chemoradiation regimen consisting of 5-FU and FIR was investigated and found to be effective in vitro in targeting PCSCs, identified as either ALDH or CD24+/CD44+/ESA+ or sphere-forming cells, as well as nonstem PDAC cells. In vivo, the combination of IR+5-FU+DSF/Cu was more effective (72.46%) than either IR+5-FU (30.32%) or IR+FOLFIRINOX therapy (43.04%) in inhibiting growth of the mouse Panc02 tumor. These encouraging results provide a solid foundation for clinical trials to improve the outcomes of the current standard chemoradiation therapy regimen for PDAC.

Glioblastoma is the most dangerous brain cancer. One reason for glioblastoma's aggressiveness are glioblastoma stem‐like cells. To target them, a number of markers have been proposed (CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6). A comprehensive study of co‐expression patterns of them has, however, not been performed so far. Here, we mapped the multidimensional co‐expression profile of these stemness‐associated molecules. Gliomaspheres – an established model of glioblastoma stem‐like cells – were used. Seven different gliomasphere systems were subjected to multicolor flow cytometry measuring the nine markers CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6 all simultaneously based on a novel 9‐marker multicolor panel developed for this study. The viSNE dimensionality reduction algorithm was applied for analysis. All gliomaspheres were found to express at least five different glioblastoma stem‐like cell markers. Multi‐dimensional analysis showed that all studied gliomaspheres consistently harbored a cell population positive for the molecular signature CD44+/CD133+/ITGA6+/CD36+. Glioblastoma patients with an enrichment of this combination had a significantly worse survival outcome when analyzing the two largest available The Cancer Genome Atlas datasets (MIT/Harvard Affymetrix: P = 0.0015, University of North Carolina Agilent: P = 0.0322). In sum, we detected a previously unknown marker combination – demonstrating feasibility, usefulness, and importance of high‐dimensional gliomasphere marker combinatorics.