Compression force promotes glioblastoma progression through the Piezo1‑GDF15‑CTLA4 axis

Abstract

Glioma, a type of brain tumor, is influenced by mechanical forces in its microenvironment that affect cancer progression. However, our understanding of the contribution of compression and its associated mechanisms remains limited. The objective of the present study was to create an environment in which human brain glioma H4 cells experience pressure and thereby investigate the compressive mechanosensors and signaling pathways. Subsequent time‑lapse imaging and wound healing assays confirmed that 12 h of compression significantly increased cell migration, thereby linking compression with enhanced cell motility. Compression upregulated the expression of Piezo1, a mechanosensitive ion channel, and growth differentiation factor 15 (GDF15), a TGF‑β superfamily member. Knockdown experiments targeting PIEZO1 or GDF15 using small interfering RNA resulted in reduced cell motility, with Piezo1 regulating GDF15 expression. Compression also upregulated CTLA4, a critical immune checkpoint protein. The findings of the present study therefore suggest that compression enhances glioma progression by stimulating Piezo1, promoting GDF15 expression and increasing CTLA4 expression. Thus, these findings provide important insights into the influence of mechanical compression on glioma progression and highlight the involvement of the Piezo1‑GDF15 signaling pathway. Understanding tumor responses to mechanical forces in the brain microenvironment may guide the development of targeted therapeutic strategies to mitigate tumor progression and improve patient outcomes.

Keywords: CTLA4; Piezo1; compression force; glioma; growth differentiation factor 15; mechanosensor.

Figure 1.

Mechanical compressive stress increases the motility of human brain glioma cells. (A) A schematic of the mechanical pressure device. H4 glioma cells were cultured in a 6-well plate to form a monolayer, and compressive stress was applied with a predefined weight using glass coverslips. (B) A pressure of 0.75 mmHg was applied to the H4 cells for 12 h, followed by a 5-h observation under a time-lapse imaging microscope, where each cell lies at the origin (0,0) at t=0 h. The plots depict the motility of individual cells in 1 representative experiment. (C) Quantification of the migration speed of the individual cells in the control and pressure groups (n=3 independent experiments). (D) The effect of compression/compressive force on H4 cell migration was confirmed using a wound healing assay. Representative images of the wound healing assay for the control and pressure groups after 0, 12 and 24 h of pressure application. (E) Graph showing the percentage wound closure as quantified using ImageJ software (n=3). **P<0.01, ****P<0.0001, determined using unpaired t-test.

Figure 2.

Compressive solid stress increases the expression of the GDF15 and Piezo1 mechanosensors. (A) Comparisons of GDF15 and PIEZO1 gene expression between human GBM and normal brain tissues using data from The Cancer Genome Atlas and GTEx, analyzed with GEPIA. The red box plots represent GBM tissues (n=163) and the gray box plots represent non-GBM tissue (n=207). The figure was obtained from GEPIA. The expression levels of (B) GDF15 and (C) Piezo1 in human GBM tissue were compared with normal brain tissue by immunohistochemistry. The tissues were obtained from the Korea Brain Bank Network. Scale bars represent 50 µm and 10 µm. (D) H4 cells were subjected to compression for 12 h, and the gene expression levels of GDF15, PIEZO1 and MMP9 were evaluated using reverse transcription-quantitative PCR (n=3 independent experiments). (E) Piezo1 and GDF15 protein expression in H4 cells exposed to pressure for 12 and 24 h measured via immunoblotting analysis. *P<0.05, **P<0.01. GBM, glioblastoma multiforme; GDF15, growth differentiation factor 15; MMP9, matrix metalloproteinase 9; N, normal tissue; T, tumor tissue; TPM, transcripts per million.

Figure 3.

Knockdown of GDF15 and PIEZO1 using siRNA significantly reduces cell motility under pressure. (A) Motility plots of siCon or siGDF15 transfected cells subjected to pressure for 12 h and then observed for 5 h under a time-lapse imaging microscope. Plots depict the motility of individual cells in 1 representative experiment. (B) Quantification of the migration speed of individual cells (n=200). (C) Motility plots of siCon or siPiezo1 transfected cells subjected to pressure for 12 h and then observed for 5 h under a time-lapse imaging microscope. Plots depict the motility of individual cells in 1 representative experiment. (D) Quantification of the migration speed of individual cells (n=200). Cells with or without PIEZO1 knockdown were subjected to pressure for 12 h, and the gene expression levels of (E) PIEZO1 and (F) GDF15 were evaluated using RT-qPCR (n=3 independent experiments for each gene). Cells were subject to pressure with or without the addition of 10 µM BAPTA-AM for 12 h, and the gene expression levels of (G) PIEZO1 and (H) GDF15 were assessed using RT-qPCR (n=3 independent experiments for each gene). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, determined using one-way ANOVA followed by Tukey's test. Con, control; GDF15, growth differentiation factor 15; ns, not significant; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.

Figure 4.

Compressive stimuli regulate the expression of CTLA4 in neuroglioma cells. (A) H4 cells were subjected to 12 h of compressive stimuli and the expression of CTLA4 was analyzed using RT-qPCR. n=6 independent experiments; **P<0.01, determined by unpaired t-test. Cells transfected with (B) siCon or siGDF15 and (C) siCon or siPiezo1 were subjected to compressive stimulation for 12 h, and expression of CTLA4 was analyzed using RT-qPCR. n=3 independent experiments for each transfection procedure; *P<0.05, **P<0.01, ****P<0.0001 determined by one-way ANOVA followed by Tukey's test. (D) A proposed mechanism that promotes glioma progression through a mechanosensor that detects mechanical pressure in the brain tumor microenvironment. As GBM grows in a limited space, pressure builds between cells and surrounding tissues. The expression of Piezo1, a mechanosensor present in the cell membrane, increases, followed by the expression of GDF15. Subsequently, the immune checkpoint protein, CTLA4, is upregulated, enhancing the poor prognosis of glioma. Con, control; GBM, glioblastoma; GDF15, growth differentiation factor 15; ns, not significant; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.