Differential response of patient-derived primary glioblastoma cells to metabolic and adhesion inhibitors

Abstract

This study aims to investigate the cellular response of Glioblastoma (GBM) to adhesion and metabolic inhibitors, focusing on cell migration and matrix adhesion properties. GBM is the most common incurable brain tumor. Despite decades of research into GBM's chemical and molecular classification, identifying mechanisms of drug resistance has been challenging. Studies on inhibitors targeting cancer cell migration and proliferation rarely consider the heterogeneous migration properties among cells, which may impact patient responses to treatment. In this work, tissue samples were obtained from spatially distinct locations with different 5-aminolevulinic acid (5-ALA) fluorescent intensities-including strongly fluorescent tumor cores, a weakly fluorescent tumor rim, and non-fluorescent tumor margins. These samples were previously shown to be associated with significantly different motility and adhesion properties. We tested the response of tumor cells to adhesion and metabolic inhibitors using metabolic MTT and Cell Titer Glo viability assays, respectively. We also monitored cell survival using time-lapse microscopy, while culturing them on low-modulus polydimethylsiloxane (representing the stiffness of brain tissue). Metabolic viability assays revealed substantial heterogeneity in drug potency across cells from different regions of the tumor. Highly fluorescent tumor core cells were significantly more resistant to an F0F1 ATP synthase inhibitor (Gboxin), and a FAK inhibitor (GSK2256098), while their proliferation ceased post-treatment in vitro. In contrast, cells derived from non-fluorescent tumor margins exhibited higher potency for the ATP synthase inhibitor (Gboxin), but their proliferation persisted post-treatment. Our study demonstrates a correlation between the adhesive and migration properties of cells and their sensitivity to therapeutics in different regions of the tumor within individual patients and between patients with GBM.

Keywords: Drug discovery; Glioblastoma; Mechanobiology.

Fig. 1

Differential treatment response of GBM cells to metabolic and adhesion inhibitors. A Collection of three strongly fluorescent tissue samples derived from the tumor mass (S from patient A, S1 and S2 from patient B, data shown in red on graphs), two nonfluorescent tumor margins (N1 and N2 from patient A, data in blue), and 1 weakly fluorescent tissue sample (W from patient B, data in green). The color code that represents different parts of the tumor is followed by the subsequent figures. B Cell viability (Cell Titer Glo assay) as a function of Gboxin concentration (in micromolar). Error bars represent mean ± SEM from three independent experiments. The response curves for each line is obtained from the mean of three biological replicates. The vertical lines mark the IC50 concentration of Gboxin for each cell line. C Cell viability (MTT assay) as a function of GSK concentration (in micromolar). Error bars represent mean ± SEM from three independent experiments. The response curves for each line is obtained from the mean of three biological replicates. The vertical lines mark the IC50 concentration of GSK for each cell line

Fig. 2

Effect of GSK and Gboxin treatments on GBM cell viability and cell division. Error bars, where appropriate, represent mean ± SEM at least three biological replicates. The number of asterisks indicate the magnitude of the p-value, P, obtained from Barnard’s test for statistical difference between control and treatment. (*P < 0.05, **P < 0.01, ***P < 0.0001, ****P < 0.000001). A Snapshots of cultured cells from different cell lines at before and after treatment with GSK (see Supplementary Fig. 2 for Gboxin). Scale bar: 100 µm. B Cell proliferation of the different cell lines following GSK treatment. C Cell proliferation of the different cell lines following Gboxin treatment

Fig. 3

Relationship between GBM cell–matrix adhesion strength (characterized by the shear forces required to detach the cells from their substrate), cell migration (characterized by the slope of cell mean squared displacement (MSD)) [18] and drug potency (IC50 in micromolar) for different cell lines. A Relationship between GSK drug potency (IC50) and cell adhesion. Error bars represent SEM from at least 2 independent experiments. B Relationship between GSK drug potency (IC50) and cell migration. C Relationship between Gboxin drug potency (IC50) and cell adhesion. Error bars represent SEM from at least 2 independent experiments. D. Relationship between Gboxin drug potency (IC50) and cell migration