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Infiltrative growth is a major cause of the high lethality of malignant brain tumors such as glioblastoma (GBM). The study of the contribution of biomechanical processes to GBM invasion is an emerging field. We show here that GBM cells upregulate the guidance receptor Plexin-B2 to gain invasiveness by modulating their biomechanical properties. Deletion of Plexin-B2 in GBM stem cells limited tumor spread and shifted invasion paths from axon fiber tracts to perivascular routes. On a cellular level, Plexin-B2 adjusts cell adhesiveness, migratory responses to different matrix stiffness, and actomyosin dynamics, thus empowering GBM cells to leave stiff tumor bulk and infiltrate softer brain parenchyma. Correspondingly, gene signatures affected by Plexin-B2 were associated with locomotor regulation, matrix interactions, and cellular biomechanics. On a molecular level, the intracellular Ras-GAP domain contributed to Plexin-B2 function, while the signaling relationship with downstream effectors Rap1/2 appeared variable between GBM stem cell lines, reflecting intertumoral heterogeneity. Our studies have established Plexin-B2 as a modulator of cell biomechanics that is usurped by GBM cells to gain invasiveness. Ongoing investigations focus on the regulation of the biomechanical properties of cell membrane and cell actomyosin cortex through plexins that provide GBM cells with the mechanical dynamics to penetrate to restricted space.

Tumor hypoxia is linked to poor outcome for glioblastoma (GBM), a highly malignant brain cancer, but the underlying mechanisms and the environmental factors that initiate tumor hypoxia are poorly understood. We tracked tumor hypoxia in GBM in immunocompetent mice with a hypoxia sensitive fluorescent reporter combined with single cell transcriptomics. We found that hypoxic GBM cells are quiescent, immunosuppressive and display a mesenchymal transition, all of which are linked to malignant potency. We also captured in vivo hypoxia gene signature, which is more represented in recurrent GBM and predicts worse outcome. Interestingly, hypoxic GBM cells is a diverse population, consisted of four subclusters, and enriched for immune pathways. Concordantly, our reporter highlighted a distinct geographic pattern of immune cells in hypoxic regions, with phagocytic tumor-associated macrophages (TAMs) and CD8+ cytotoxic T cells (CTLs) congregated in hypoxic cores confined by hypoxic GBM cells in pseudo-palisading patterns. Mechanistically, this is a dynamic temporospatial process, requiring cytokine CCL8. Remarkably, the sequestered TAMs also experience hypoxia, and they are reprogrammed to express immunotolerant markers by factors released from hypoxic GBM cells. Contrary to the conventional viewpoint that hypoxia arises from rapid tumor expansion outstripping vascular supply, we discovered anticancer immunity as an important driving force of tumor hypoxia; attenuating immune responses by implanting GBM in host mice with immunodeficiency or IL1β deletion greatly decreased GBM hypoxia. Analyses of human patient GBM samples highlighted a connection of mesenchymal subtype, immune response, and tumor hypoxia, all contributing to poor survival. Altogether, our study revealed a reciprocal influence of anti-tumor immunity and tumor hypoxia, which has significant ramifications for prognosis and immunotherapy for GBM.

Glioblastoma (GBM) is the most common malignant primary brain tumor. The nature of invasiveness of GBM makes complete surgical resection difficult. However, how GBM cells achieve wide infiltration in the brain is poorly understood. Microglia, the resident immune cells in the brain can support GBM growth and invasion, but the underlying mechanisms remain elusive. Here, we show that microglia are activated in a wide field away from tumor boundaries, ahead of tumor cell infiltration. Invading GBM cells are in close contact with microglia, progressively aligned with one another in the direction of tumor invasion. Moreover, ECM is also aligned with the infiltrating tumor and microglia, which may serve as invasion tracks in the brain. Mechanistically, we demonstrate that microglia direct cellular alignment and ECM remodeling in the invasion tracks through an axon guidance receptor Plexin-B2. Myeloid-specific ablation of Plexin-B2 perturbs microglia and tumor cell alignment, microglia migration, ECM organization, and GBM invasiveness. Together, our data reveal a hitherto under-appreciated role of microglia in providing directional cues for GBM invasion through physical interaction and alignment of ECM and tumor cells, thus providing new insights and novel molecular targets in curbing GBM invasion.

Longitudinal tracking of tumor growth using non-invasive bioluminescence imaging (BLI) is a key approach for in vivo cancer studies, but the current method of firefly luciferase (Fluc) BLI has quantitative limitations, as it is only suited for detection of tumors of considerable sizes at advanced stage, typically in the order of >105 cells. Recently, Akaluciferase (Akaluc) has been developed as an alternative BLI system that offers higher signal strength and better light penetration of tissue due to its red-shifted emission. Here, we established Akaluc BLI as a new sensitive method for in vivo tracking of glioblastoma (GBM) expansion in intracranial transplant models. In multiple GBM cell lines, including the frequently used U87MG and GL261, as well as patient-derived glioma stem cells (GSC), we demonstrate that Akaluc-expressing GBM cells produced more than 50-times brighter BLI signals in vitro and up to 100-fold higher signal intensities in vivo over Fluc-expressing counterparts. The higher sensitivity of Akaluc BLI permits early in vivo detection of intracranial GBM transplants starting as early as 4 hours after implantation and with as little as 5,000 transplanted GSC. We also reveal a prolonged engraftment period in intracranial GSC transplants before wide dissemination into host brain parenchyma. Akaluc BLI is also advantageous for longitudinal monitoring of therapeutic effects of chemoradiation for GBM and detection of early phase of tumor relapse. Thus, Akaluc BLI offers an important addition to the tool box for cancer research. SIGNIFICANCE: The high sensitivity of Akaluc bioluminescence is a significant improvement for the non-invasive tracking of tumors in preclinical cancer studies, including detection of small incipient tumors and micro-metastasis.

Glioblastoma (GBM) is the most common and lethal brain cancer that invariably recurs after therapy due to presence of resistant GBM cells within hypoxic and peri-necrotic regions. Eradicating such GBM cells, which constitute a major source of tumor recurrence, is important to curb disease relapse. An endogenously expressed, spatially sensitive hypoxia reporter would therefore be a valuable tool to evaluate hypoxic zones in GBM in detail, and to measure the efficacy of hypoxia-activated drugs. For this purpose, we engineered a lentiviral vector that carries a hypoxia reporter, consisting of HIF response elements (HRE) that drive expression of UnaG fluorescent protein, which fluoresces independent of oxidative maturation. We validated the sensitivity of our reporter in vitro using U87MG, GBM2, and patient-derived GBM stem cell lines, and we performed intracranial transplantations of GBM cells in SCID mice to identify cells undergoing hypoxic stress in in vivo microenvironment. In addition, GL261 murine GBM cells with hypoxia reporter were intracranially implanted in C57BL/6 mice as syngeneic model for studies on immune responses. Brains from our transplant studies were dissociated and single-cell RNA sequencing (Drop-Seq) was performed to investigate heterogeneity in response to hypoxia within GBM cells and the cellular composition of microenvironment. We will also apply a hypoxia-activated prodrug, Evofosfamide (Evo), in our ongoing studies that can potentially eradicate hypoxic tumor cells and increase T cell infiltration and reverse immune suppression. As hypoxic niches are thought to confer resistance to radiation therapy (XRT), combining XRT with Evo could thus improve therapy efficacy. Our hypoxia gene reporter, combined with single-cell transcriptomics, could therefore serve as an effective tool to enable fundamental investigation of GBM microenvironment and could be used to evaluate therapies targeting tumor microenvironment to enhance GBM patient survival.