Integrated 2D, 3D, and In Vivo Modeling to Define CRK and CRKL Functions in Glioblastoma Invasion and Therapeutic Targeting
Presenter Status
Post-Doctorial Research
Abstract Type
Translational Research
Primary Mentor or Principal Investigator
Taeju Park
Presentation Type
Poster
Start Date
19-5-2026 11:00 AM
End Date
19-5-2026 12:00 PM
Abstract Text
Glioblastoma is the most aggressive primary malignant brain tumor in both adults and children. Despite maximal surgical resection followed by chemoradiation, median survival remains only about 15 months, largely because diffusely infiltrative tumor cells escape the surgery and drive recurrence. Dysregulation of CT10 regulator of kinase protein (CRK) and CRK-like (CRKL) has been implicated in malignant motility and associated with poor prognosis in several cancers, including glioblastoma. We hypothesized that CRK and CRKL cooperatively promote glioblastoma invasion and that their inhibition limits tumor invasion.
To test this hypothesis, we performed transient and stable knockdown of CRK, CRKL, or both in cultured glioblastoma cell lines, U-118MG and LN-229 using siRNA transfection and shRNA lentiviral transduction. Western blot analyses confirmed robust reductions in the target proteins following knockdowns. Using the xCELLigence real-time cell analysis system, we quantified the effects of CRK and CRKL loss on cell adhesion, migration, and invasion. CRKL knockdown significantly impaired all three cellular processes, whereas dual CRK and CRKL knockdown completely abolished adhesion, migration, and invasion, and induced morphological alterations. These results were consistent in the two glioblastoma lines, underscoring essential and overlapping roles for CRK and CRKL in glioblastoma cell motility.
To extend these findings into a three-dimensional (3D) context, we generated LN-229 spheroids and evaluated invasion into a collagen matrix following knockdown. The invasion deficits mirrored those observed in two-dimensional (2D) assays, supporting a critical requirement for CRK and CRKL in 3D tumor cell invasion.
We next tested this dependency in vivo by transplanting luciferase- and GFP-expressing LN-229 cells into the brains of immunodeficient NSG mice using a stereotactic device. IVIS imaging and GFP immunohistochemistry demonstrated in vivo tumor growth and widespread infiltration. In contrast, xenografts of tumor cells with CRK and CRKL dual knockdown showed a statistically significant survival benefit. Ongoing studies will evaluate the effects of CRK and CRKL knockdown on in vivo invasiveness by histological analysis.
Together, our integrated platform combining 2D impedance-based real-time cell analyses, 3D spheroid assays, and intracranial xenografts identifies CRK and CRKL as convergent and targetable regulators of glioblastoma invasion and highlights their potential as therapeutic nodes to enhance the efficacy of standard-of-care treatments.
Integrated 2D, 3D, and In Vivo Modeling to Define CRK and CRKL Functions in Glioblastoma Invasion and Therapeutic Targeting
Glioblastoma is the most aggressive primary malignant brain tumor in both adults and children. Despite maximal surgical resection followed by chemoradiation, median survival remains only about 15 months, largely because diffusely infiltrative tumor cells escape the surgery and drive recurrence. Dysregulation of CT10 regulator of kinase protein (CRK) and CRK-like (CRKL) has been implicated in malignant motility and associated with poor prognosis in several cancers, including glioblastoma. We hypothesized that CRK and CRKL cooperatively promote glioblastoma invasion and that their inhibition limits tumor invasion.
To test this hypothesis, we performed transient and stable knockdown of CRK, CRKL, or both in cultured glioblastoma cell lines, U-118MG and LN-229 using siRNA transfection and shRNA lentiviral transduction. Western blot analyses confirmed robust reductions in the target proteins following knockdowns. Using the xCELLigence real-time cell analysis system, we quantified the effects of CRK and CRKL loss on cell adhesion, migration, and invasion. CRKL knockdown significantly impaired all three cellular processes, whereas dual CRK and CRKL knockdown completely abolished adhesion, migration, and invasion, and induced morphological alterations. These results were consistent in the two glioblastoma lines, underscoring essential and overlapping roles for CRK and CRKL in glioblastoma cell motility.
To extend these findings into a three-dimensional (3D) context, we generated LN-229 spheroids and evaluated invasion into a collagen matrix following knockdown. The invasion deficits mirrored those observed in two-dimensional (2D) assays, supporting a critical requirement for CRK and CRKL in 3D tumor cell invasion.
We next tested this dependency in vivo by transplanting luciferase- and GFP-expressing LN-229 cells into the brains of immunodeficient NSG mice using a stereotactic device. IVIS imaging and GFP immunohistochemistry demonstrated in vivo tumor growth and widespread infiltration. In contrast, xenografts of tumor cells with CRK and CRKL dual knockdown showed a statistically significant survival benefit. Ongoing studies will evaluate the effects of CRK and CRKL knockdown on in vivo invasiveness by histological analysis.
Together, our integrated platform combining 2D impedance-based real-time cell analyses, 3D spheroid assays, and intracranial xenografts identifies CRK and CRKL as convergent and targetable regulators of glioblastoma invasion and highlights their potential as therapeutic nodes to enhance the efficacy of standard-of-care treatments.
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Poster Board Number: 35