Kevin A. Rosa González, B.S
L. Kucheryavykh, PhD

Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico

Introduction

Glioblastoma (GBM) is a highly aggressive brain cancer with a median survival of less than a year. Our studies have shown a correlation between activation of proline-rich non-receptor tyrosine kinase (Pyk2) in GBM cells and cytokine expression by tumor-associated myeloid cells (TAMs). This activation increases levels of monocyte chemoattractant protein 1 (CCL2), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL10, IL12, and vascular endothelial growth factor (VEGF) while elevating CD86+/CD206+ TAM populations. We hypothesize that Pyk2 signaling in GBM regulates cytokine release, suppressing immune responses in the tumor microenvironment.

Methods

Primary human GBM cell lines and the GL261/C57Bl/6 mouse glioma implantation model were used. CRISPR/Cas9 was employed to knock out Pyk2 (Pyk2KO) in both GL261 and primary human GBM cells. Cytokine levels were quantified using a cytokine antibody array combined with RT-PCR analysis to assess cytokine gene expression and release. Flow cytometric analysis was conducted to quantify the expression of programmed death-ligand 1 (PD-L1) on cell surfaces and to analyze TAMs in Pyk2KO and wild-type (WT) tumors.

Results

Analysis of cytokines in the cell culture conditioned media from Pyk2KO and Pyk2WT cells showed reduced CCL2 and CCL5 release in Pyk2KO cells compared to Pyk2WT cells. RT-PCR analysis of myeloid cells isolated from Pyk2WT and Pyk2KO tumors in the GL261/C57Bl/6 GBM model revealed significant downregulation of key pro-tumorigenic factors, including CCL2, CCL12, CCL5, tumor necrosis factor (TNF), VEGF, and epidermal growth factor (EGF) in Pyk2KO tumors compared to Pyk2WT tumors. Flow cytometry showed fewer myeloid-derived suppressor cells (MDSCs), more dendritic cells (DCs), and increased TNF/IFNγ-expressing CD8+ T cells in Pyk2KO tumors. Pyk2KO tumors also had decreased pro-tumorigenic Ly6C+/CD206+ cells and an upregulation of inflammatory Ly6C+/CD86+ myeloid cells.

Conclusion

These findings suggest that Pyk2KO tumors exhibit an immunocompetent microenvironment with enhanced phagocytic and cytotoxic function compared to Pyk2WT tumors.

Funding

This study was supported by NIH Grants 1R15CA287203 and 1R16GM153522.

IACUC Approval Number

035-2023-09-01