Targeting the Axl and mTOR Pathway Synergizes Immunotherapy and Chemotherapy to Butylidenephthalide in a Recurrent GBM

Figure 6

Clinical drug responses were evaluated in the patient-derived primary culture GBM cells. (a) Most of the primary culture cancer cells were CD133 (green) and SOX2 (red), highly expressed using flow cytometry analysis and immunofluorescence assay. Hoechst33342-labeled nuclei. Bar = 50 μm. MFI: mean fluorescent intensity. (b) Flow cytometry analysis and immunofluorescence assay in GBM primary cultured spheroid cells to detect the high expression of CD133 (green) and SOX2 (red) cultured with serum-free medium. Hoechst33342-labeled nuclei. Bar = 50 μm. MFI: mean fluorescent intensity. (c) Primary culture cancer cells, treated with (Z)-BP and temozolomide (active form MTIC was used), presented relatively low IC₅₀, compared to BCNU. Additionally, dosing (Z)-BP in IC₃₀ concentration could reduce the required amount of TMZ for determining the synergistic effect on eliminating primary culture gliomas. (d) Using (Z)-BP (200 or 400 μM) to expose the primary culture cells could reduce the level of MGMT, in comparison to vehicle control and BCNU (800 μM). Further, receptor tyrosine kinases including EGFR, phosphorylated EGFR, and Axl were also downregulated. Upstream mTOR activity was inactivated (p-mTOR) by (Z)-BP, and this could mutually affect Axl and EGFR signaling and then decrease the protein level of PD-L1. β-Actin was used as an internal control.

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