The hypoxic niche in glioblastoma is maintained by myeloid produced creatine

Glioblastoma (GBM) is the most common brain tumor malignancy in adults that is characterized by unique niches termed pseudo-palisading necrosis. These hypoxic regions are a defining feature of the disease which promote chemoresistance, radioresistance, and ultimately drive disease progression. Understanding the generation and sustainment of these regions is critical to understanding how effectively treat the disease. Multi-omics analysis of human GBM patients reveals that the creatine transporter, Slc6a8 is specifically expressed by tumor cells in hypoxic and necrotic regions. Surrounding these regions are tumor-associated myeloid cells (TAMCs), which are the most abundant infiltrating immune cell in GBM. Surprisingly, the TAMCs that surround the hypoxic pseudo-palisading regions express the enzymes necessary to produce creatine. Therefore, we hypothesize that TAMC-derived creatine promotes the generation of the pseudo-palisading necrotic niche in GBM, and that this metabolic crosstalk promotes both GBM fitness and therapy resistance. Previous work has identified that the hypoxic pseudo-palisading niche contains glioma stem cells (GSC), which are generated by the hypoxic stress of these regions. Furthermore, Slc6a8 is directly regulated by hypoxia, suggesting creatine uptake exerts a role on GSC phenotypes. Thus, the first aim of this proposal is to examine how creatine transport influences GSC phenotypes in both human and mouse models of GBM. In this aim, we will also utilize inducible models of GBM that recapitulate the genetic and pathologic features of human GBM to determine if Slc6a8 is necessary for the formation of the hypoxic pseudo-palisading niche in tumors. Our preliminary data indicate that TAMCs isolated from both mice and humans with GBM are proficient producers of creatine. Furthermore, we found that this metabolic phenotype is specific to the tumor microenvironment (TME) and is induced by extracellular lactate. Thus, the second aim of this proposal will examine how the ablation of creatine biosynthesis by TAMCs controls tumor growth and progression in mouse models of GBM. This aim will also determine how lactate induces the creatine biosynthetic phenotype of TAMCs in GBM. The third aim of this proposal is to examine if a clinically relevant inhibitor of creatine transport influences GBM growth. We will test how this inhibitor works in the context of chemo and radiotherapy to determine how creatine uptake influences GBM recurrence. To establish translatable value from this work, we will generate tumor samples and patient-derived xenografts from patients, screen them for expression of creatine metabolic genes, then assess sensitivity to creatine metabolic inhibitory therapy. The results of this aim will identify if blocking TAMC-to-tumor metabolic communication is a feasible strategy for GBM therapy.