Neuro-Oncology | HK2 Gene Knockout Influences Immune and Angiogenesis-Related Pathways in Glioblastoma Microenvironment

This study reveals the non-canonical regulatory role of HK2 gene in the glioblastoma microenvironment, including modulation of immune response and angiogenesis-related pathways. It provides new theoretical basis for HK2-targeted anti-tumor therapies.

 

Literature Overview
This 'Rapid Literature Summary' reviews and synthesizes three studies on glioblastoma. The studies investigate HK2 gene's regulatory functions within the tumor microenvironment, prognostic value of peripheral blood immune cell subsets, and the impact of stromal cells on T cell dynamics using 3D organoid models. Technical approaches include RT-qPCR validation, flow cytometry analysis, and CODEX multiplex fluorescence imaging.

Background Knowledge
Glioblastoma (GBM) is the most aggressive primary brain tumor, where its microenvironment plays critical roles in disease progression and treatment resistance. Hexokinase 2 (HK2) serves as a key enzyme in glycolysis, but evidence suggests it participates in non-metabolic regulation in GBM. Peripheral blood immune subsets such as monocytes, CD8+ T cells, and myeloid-derived suppressor cells (MDSCs) correlate with survival duration and radiological features. Meanwhile, 3D organoid models offer a more physiologically relevant platform for studying GBM-immune cell interactions.

 

 

Research Methods and Experiments
The first study employed RNA-seq and RT-qPCR validation to analyze HK2 knockout (HK2KD) effects on glioblastoma cell lines (U87-MG and IN859), followed by Gene Ontology (GO) enrichment analysis to identify regulated biological processes. The second study conducted flow cytometry analysis on peripheral blood mononuclear cells (PBMCs) from 57 newly diagnosed GBM patients to investigate associations between immune cell subsets and radiological features (e.g., FLAIR volume, necrosis, midline shift) and overall survival (OS). The third study developed a 3D organoid model co-culturing glioma stem cells (GSCs) with T cells and stromal cells, analyzing T cell activation markers and immune checkpoint molecules (CD69, CD25, LAG3, TIM3, PD-1) through combined immunohistochemistry and spatial distribution analysis.

Key Conclusions and Perspectives

• HK2KD demonstrates significant enrichment in inflammation- and immune response-related biological processes without activating glycolysis-associated pathways.
• HK2KD upregulates pro-inflammatory cytokines (e.g., CSF2, IL1A/1B/6, TNF) and chemokines (e.g., CCL3, CXCL1/2/3/5/6/8), suggesting its role in remodeling the immune microenvironment.
• Downregulation of HIF2A, VEGF, and FGF2 in HK2KD models indicates potential anti-angiogenic mechanisms, offering novel strategies for anti-angiogenic therapies.
• Elevated monocyte and MDSC levels in GBM peripheral blood correlate with poor prognosis, whereas high immature NK cell counts associate with prolonged survival independent of steroid use.
• Immature NK cells show positive correlation with ECOG performance status (PS), suggesting their potential as prognostic biomarkers linked to radiological features including FLAIR volume, midline shift, and necrosis.
• In the 3D organoid model, stromal cell co-culture increases CD4+ T cell expression of CD69 and PD-1, indicating their regulatory influence on T cell activation.
• Stromal cells in 3D systems do not alter T cell spatial distribution but modify their activation phenotypes, supporting stromal cell targeting in immunotherapy.
• CODEX multiplex fluorescence imaging enables co-localization analysis of multiple biomarkers, advancing spatial characterization of brain metastasis microenvironments.

Research Significance and Prospects
Collectively, these studies elucidate the regulatory mechanisms of HK2, immune subsets, and stromal cells in glioblastoma. Future research should explore HK2 as a synergistic target for immunotherapy or anti-angiogenic treatment, investigate stromal cell functions in immune editing, and integrate multi-omics and spatial omics technologies to decipher tumor microenvironment dynamics. Peripheral blood immune profiling could provide non-invasive prognostic biomarkers for clinical applications.

 

 

Conclusion
These three studies focus on glioblastoma microenvironment regulation, where HK2 knockout affects immune and angiogenesis pathways, peripheral blood immune subsets correlate with survival outcomes, and stromal cells modulate T cell phenotypes in 3D models. Findings suggest that targeting HK2 or stromal cells could enhance immunotherapy and anti-angiogenic efficacy. Multiplex fluorescence imaging offers new spatial analysis capabilities for brain metastasis microenvironments, while integration with single-cell sequencing and spatial transcriptomics will further resolve tumor microenvironment heterogeneity. The studies highlight the importance of experimental models (e.g., 3D organoids, gene-edited cells) in glioma research, establishing a solid foundation for translational studies.

 

A Sagerer, D Westphal, M Meinhardt, G Schackert, and T A Juratli. P02.13.B IMPLEMENTATION OF A MULTIPLEXED TISSUE IMAGING PROTOCOL FOR CHARACTERIZATION OF THE TUMOR MICROENVIRONMENT IN MELANOMA BRAIN METASTASES. Neuro-Oncology.