Defining the role of CRK and CRKL in glioblastoma invasiveness: from cell culture to mouse models

Glioblastoma is the most aggressive primary malignant brain tumor affecting both adults and children, with a median survival of only about 15 months despite the standard treatments of surgery, radiation, and chemotherapy. Complete surgical resection is hindered by the highly infiltrative nature of glioblastoma cells, making tumor invasion the most significant cause of recurrence. Dysregulation of CT10 regulator of kinase (CRK) and CRK-like (CRKL) proteins has been implicated in multiple cancers, including glioblastoma, in which their overexpression correlates with poor prognosis. We hypothesized that CRK and CRKL are essential in glioblastoma cell invasion and that their inhibition suppresses tumor infiltration and enhances treatment efficacy. To test this hypothesis, we performed transient and stable knockdown of CRK, CRKL, or both in glioblastoma cell lines (U118-MG and LN-229) using siRNA transfection and shRNA lentiviral transduction. Western blot analyses confirmed significant reductions in target protein levels. Using the xCELLigence real-time cell analysis system, we assessed the impact of CRK and CRKL knockdown on glioblastoma cell adhesion, migration, and invasion. CRKL knockdown substantially reduced all three processes, whereas combined CRK/CRKL knockdown completely abolished adhesion, migration, and invasion. These consistent results across three glioblastoma lines highlight essential, overlapping roles of CRK and CRKL in regulating glioblastoma cell motility. To examine the dependence of in vivo glioblastoma growth and invasion on CRK and CRKL, we transplanted LN-229 glioblastoma cells expressing luciferase and GFP into immunodeficient (NSG) mouse brains using stereotactic surgery. Luciferase and GFP enabled us to monitor in vivo tumor growth through IVIS imaging and identify infiltrating tumor cells by GFP immunohistochemistry. Histological analysis of the xenografted brains revealed diffuse invasion of glioblastoma cells into surrounding brain regions. Currently, we transplant glioblastoma cells with CRK/CRKL knockdown to determine the requirement of CRK/CRKL in glioblastoma invasion in vivo. Then, we plan to test the effects of CRK/CRKL-antagonist peptides we developed on glioblastoma invasion in the mouse brain. Our study highlights the promise of targeting CRK and CRKL as a therapeutic strategy to mitigate glioblastoma invasion and improve the efficacy of standard care for glioblastoma.