Establishment of U-87MG Cellular Fibrosis as a Novel in Vitro Model to Analyze Glioblastoma Cells' Sensitivity to Temozolomide

Abstract

Glioblastoma (GBM), a highly malignant brain tumor, arises within a complex microenvironment that plays a critical role in facilitating tumor progression, ensuring survival, and enabling immune evasion, ultimately contributing to therapeutic resistance. Cancer-associated fibrosis is increasingly recognized as a key factor in the tumor pathophysiology, particularly in extracranial cancers, and reported therapeutic strategies in several cancers consist of the current use of the standard-of-care treatment combined with anti-fibrotic drugs. However, it remains unclear how the fibrotic changes associated with the GBM microenvironment contribute to the transformation of GBM from a chemosensitive state to a chemoresistant one. Here, we developed an in vitro model that mimics a fibrosis-like mechanism using the U-87MG GBM cell line. To achieve this, we identified the optimal experimental conditions (i.e., U-87MG cultured in serum-deprivation medium in the presence of recombinant TGF-B1 at 5 ng/mL for 72 h) that effectively induced fibrosis, as suggested by the counter-regulated expression of E- and N-cadherin and sustained levels of α-SMA and collagen I. As expected, U-87MG fibrotic cells were demonstrated to be more resistant to TMZ (predicted EC₅₀ = 35 µM) as compared to the non-fibrotic counterpart (EC₅₀ not achieved here; predicted EC₅₀ = 351 µM). Accordingly, the anti-fibrotic uPAcyclin-a new derivative cyclic compound inspired as a A6 decapeptide drug-showed a significant cytotoxic effect, sensitizing resistant U-87MG fibrotic cells to TMZ. This highlights that targeting fibrosis may help to overcome TMZ resistance in GBM.

Keywords: GBM; chemoresistance; fibrosis; tumor-associated fibrotic alterations.

Figure 1

Analysis of fibrotic alterations associated with the in vitro model of fibrosis in GBM. (A) Representative bright-field microscopy images of the U-87MG cell line in the absence (on the top) and in the presence (downward) of TGF-β1 (5 ng/mL) after 72 h of treatment. Histograms of the analysis of the expression of the fibrosis-related marker (B), the downstream signaling pathways (C) in control (CTR), and fibrotic-like (FIBRO) U-87MG cell lines analyzed by Western blotting. Representative immunoblots are also shown. Band densities of the proteins were normalized to the respective loading control GAPDH. Pairwise comparisons were reported (Welch’s t-test, * p < 0.05, ** p < 0.01, ns no significant p-value). The heatmap (D) represents the panel of the immunodetection of phosphorylated proteins in the control (CTR) and the fibrotic-like (FIBRO) U-87MG cell lines. White boxes correlate with a lower p-kinases concentration and dark-purple boxes with a higher p-kinases concentration (see scale; mean pixel density). The fold-change thresholds within the boxes are reported, calculated as the ratio between the median values of p-kinases from the U-87MG FIBRO cell line, compared to the median values of p-kinases from the U-87MG CTR cell line (posed as CTR at 1). Results are representative of two independent experiments.

Figure 2

Effects of TMZ and anti-fibrotic compounds in an in vitro model of fibrosis in GBM. (A) CTR and FIBRO U-87MG cell lines were treated with different concentrations of TMZ (from 0 to 200μM) for 72 h. On the left, the graph shows the effect of TMZ on cell viability, analyzed by the MTT assay. On the left, the graph shows the effect of TMZ on cell proliferation, analyzed by the BrdU ELISA assay. (B) CTR and FIBRO U-87MG cell lines were treated with TMZ (25 μM) for 72 h. The bar graph shows the effect of TMZ on necrosis and late and early apoptosis, analyzed by Annexin V–PI staining by flow cytometry. In (C), the effect of TMZ on senescence was analyzed by SA-β-gal staining by flow cytometry. (D) CTR and FIBRO U-87MG cell lines were pre-treated with PFN (100 nM) overnight and then treated or untreated with TMZ (25µM). The histogram graph shows the percentage of viable cells analyzed by MTT assay. (E) CTR and FIBRO U-87MG cell lines were treated with A6 and uPAcyclin at different concentrations (1 and 10µM) in the presence or absence of TMZ (25µM). The histogram graph shows the percentage of viable cells analyzed by MTT assay. Pairwise comparisons were reported (one-way or two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant). Results were representative of three independent experiments, expressed as mean ± SD.