Intratumor spatial heterogeneity facilitates therapeutic resistance in glioblastoma (GBM). Nonetheless, understanding of GBM heterogeneity is largely limited to the surgically resectable tumor core lesion while the seeds for recurrence reside in the unresectable tumor edge. In this study, stratification of GBM to core and edge demonstrates clinically relevant surgical sequelae. We establish regionally derived models of GBM edge and core that retain their spatial identity in a cell autonomous manner. Upon xenotransplantation, edge-derived cells show a higher capacity for infiltrative growth, while core cells demonstrate core lesions with greater therapy resistance. Investigation of intercellular signaling between these two tumor populations uncovers the paracrine crosstalk from tumor core that promotes malignancy and therapy resistance of edge cells. These phenotypic alterations are initiated by HDAC1 in GBM core cells which subsequently affect edge cells by secreting the soluble form of CD109 protein. Our data reveal the role of intracellular communication between regionally different populations of GBM cells in tumor recurrence.

Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the de novo lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and de novo lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated de novo lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma.

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1-AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors. These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. .

BackgroundCharacterization of the niches for stem-like tumor cells is important to understand and control the behavior of glioblastomas. Cell-cycle quiescence might be a common mechanism underlying the long-term maintenance of stem-cell function in normal and neoplastic stem cells, and our previous study demonstrated that quiescence induced by hypoxia-inducible factor (HIF)-1α is associated with a high long-term repopulation capacity of hematopoietic stem cells. Based on this, we examined human astrocytoma tissues for HIF-1α-regulated quiescent stem-like tumor cells as a candidate for long-term tumorigenic cells and characterized their niche histologically.MethodsMulti-color immunohistochemistry was used to visualize HIF-1α-expressing (HIF-1α+) quiescent stem-like tumor cells and their niche in astrocytoma (WHO grade II–IV) tissues. This niche was modeled using spheroids of cultured glioblastoma cells and its contribution to tumorigenicity was evaluated by sphere formation assay.ResultsA small subpopulation of HIF-1α+ quiescent stem-like tumor cells was found in glioblastomas but not in lower-grade astrocytomas. These cells were concentrated in the zone between large ischemic necroses and blood vessels and were closer to the necrotic tissues than to the blood vessels, which suggested that a moderately hypoxic microenvironment is their niche. We successfully modeled this niche containing cells of HIF-1α+ quiescent stem-like phenotype by incubating glioblastoma cell spheroids under an appropriately hypoxic condition, and the emergence of HIF-1α+ quiescent stem-like cells was shown to be associated with an enhanced sphere-forming activity.ConclusionsThese data suggest that the “peri-necrotic niche” harboring HIF-1α+ quiescent stem-like cells confers a higher tumorigenic potential on glioblastoma cells and therefore may be a therapeutic target to control the behavior of glioblastomas.