Neurons and glial cells in the central nervous system exhibit significant mechanosensitivity and can sense mechanical stimuli from the surrounding environment and convert them into biochemical signals to regulate their function (Cox et al., 2016; Tortorella et al., 2022). Piezo1 is expressed in neuronal cells, microglia, astrocytes, oligodendrocytes, and neural stem cells, and is activated when neuronal cells are stimulated by external tensile forces, activated when neuronal cells are stimulated by external changes in stretch force, osmotic pressure, and matrix stiffness (Hong et al., 2023), and participates in cellular physiological activities in a variety of ways, such as increasing cytoplasmic Ca²⁺ signaling or initiating Ca²⁺ inward flow and affecting the levels of downstream Ca²⁺ signaling proteins involved in neuronal function, through the c-Jun N-terminal kinase (JNK1) and mammalian target of rapamycin (mTOR) signaling pathways (Liu et al., 2021).

It was demonstrated that all histological subtypes of gliomas, except oligodendrogliomas, showed Piezo1 overexpression compared to normal brain tissue and that the expression level of Piezo1 was positively correlated with World Health Organization (WHO) grade and histopathology (Chen et al., 2018). Piezo1 expression was more significantly upregulated in high-grade gliomas (WHO grade III and IV) than in low-grade gliomas (WHO grade II). Isocitrate dehydrogenase (IDH) mutation, CpG island methylation (G-CIMP) phenotype, and 1p/19q co-deletion are important molecular biomarkers in gliomas to guide prognosis and treatment. Piezo1 expression was significantly higher in the IDH1 wild-type group of gliomas than in the IDH1 mutant group and was elevated in the 1p/19q-non-co-deletion group. Gliomas with non-G-CIMP phenotypes had higher levels of Piezo1 expression compared to G-CIMP phenotypes. Grade II oligodendrogliomas with IDH1 mutation and 1p/19q co-deletion had the lowest level of Piezo1 protein expression. IDH mutation established a glioma hypermethylation phenotype, and the deoxyribonucleic acid (DNA) structural domains spanning the Piezo1 promoter and 8000 bp upstream of the Piezo1 transcriptional start site (TSS) were also hypermethylated in IDH mutant gliomas. The methylation status of these probes was negatively correlated with Piezo1 messenger ribonucleic acid (mRNA) expression. Thus more aggressive IDH wild-type gliomas are epigenetically more inclined to upregulate Piezo1 at the transcriptional level (Zhou et al., 2020).

Kaplan-Meier survival analysis of multiple human glioblastoma datasets showed that patients with elevated Piezo1 expression had significantly worse overall survival (Figure 2). Multifactorial Cox regression analysis showed that Piezo1 overexpression was an independent prognostic factor for overall survival (OS) in glioblastoma patients (Qu et al., 2020b). These results suggest that Piezo1 can be used as a prognostic biomarker for glioblastoma patients in conjunction with molecular markers such as IDH wild-type, 1p/19q non-coding, and non-G-CIMP phenotypes.

Altered matrix stiffness in glioblastomas promotes elevated expression of piezo1 in glioblastomas, and Piezo1 is physically localized to the focal adhesions of glioblastoma cells, catalyzing the maturation and growth of focal adhesions through a force-dependent calcium signaling pathway. Piezo1 is one of the major ion channels conferring mechanosensitivity to GBM stem cells, and RNA of Piezo1 knockdown glioblastoma cell lines sequencing and dual-assay TCGA database analysis of Piezo1-related genes showed that Piezo1 is associated with activation of extracellular matrix (ECM), actin cytoskeleton remodeling and integrin adhesion signaling. In addition, Piezo1 expression transduces mechanical stimuli between glioblastoma cells, promoting tumorigenesis and progression (Hong et al., 2023). This finding could explain why the self-stiffening changes in glioblastomas are mainly caused by pressure gradients generated by the tumor itself rather than collagen deposition or cross-linking.

Piezo1 knockdown inhibited clonal growth of GBM cell lines. Tumor ball formation was blocked in GBM stem cells after Piezo1 knockdown. In vitro experiments, GBM cells were able to express Piezo1 short hairpin RNA (shRNA) in a doxycycline-dependent manner. Knockdown of Piezo1 expression by doxycycline-induced Piezo1 shRNA could be observed after treatment of glioblastoma model mice with doxycycline, which significantly suppressed GBM tumor growth and prolonged the survival time of the mice, suggesting that Piezo1 is critical for the maintenance and progression of glioblastomas once tumors form (Chen et al., 2018).

Regulated volume reduction (RVD) is an evolutionarily conserved process used by animal cells to restore normal volume when osmotic shock-induced swelling occurs. RVD plays an important role in a number of physiological processes, including the prevention of necrotic cell death induced by persistent cellular swelling, and is mediated primarily by the concerted activity of ion channels that mediate the passage of CI⁻ and K⁺ ions (Miyazaki et al., 2007). Volume-regulated anion channel (VRAC), a heterodimeric protein that mediates swelling-activated CI⁻ currents (ICl), has been identified during hypotonic cell swelling as the common and major channel responsible for CI⁻ transport during RVD in almost all vertebrate cells. Tension-activated K⁺ channels, voltage-dependent K⁺ channels, and Ca²⁺-activated K⁺ (KCa) large-, medium-, and small-conductance channels (BK, IK, and SK, respectively) also play key roles in volume regulation in many cell types (Caramia et al., 2019).

In addition to CI⁻ and K⁺ channels, nonselective Ca²⁺-permeable mechanosensitive channels (MSC) are also involved in cell volume regulation. During hypotonic cell swelling, MSCs sense changes in cell membrane tension and activate as a result. Their activation then triggers intracellular Ca²⁺ signaling, which in turn regulates various effectors. (Arniges et al., 2004).

In human glioblastoma cells, Piezo1 is the main component of MSCs activated by cell swelling in glioblastoma cells, and is also primarily responsible for mechanotransduction during glioblastoma cell volume regulation. Ca²⁺ signaling can be amplified by activation of the mechanism, which is activated by the influx of MSCs activated by hypertonic cell swelling to activate IK and BK channels and VRAC, which are essential for RVD development. The cascade mechanism of Piezo1 activation from Ca²⁺ influx to IK/BK channel activation, which ultimately regulates cell volume, is an important signaling pathway for Piezo1 regulation of glioblastoma cell spreading, migration, and death (Michelucci and Catacuzzeno, 2024).

In glioblastoma tissues, the infiltration of isolated tumor cells into the peritumoral parenchyma often culminates in tumor migration and invasion. This situation greatly limits the success of surgical resection. The invasion of GBM cells into the confined space of healthy brain parenchyma requires that they be able to sense the presence of external barriers and forces from the environment and adjust their size and shape accordingly to pass through. It has been shown that glioblastoma cells use piezo1 channels to sense mechanical stimuli from the tumor microenvironment and deform the cell membrane (stretching, indentation, invagination, etc.), inducing an increase in cytoplasmic Ca²⁺ through, for example, activation of KCa channels and modulation of actin cytoskeletal polymerization, which allows for a change in the volume and shape of the cells required to invade healthy brain parenchyma.

KCa3.1 channels (also known as KCNN4, IK1 or SK4 channels) are moderately conductive calcium-activated potassium channels that regulate membrane potential and maintain calcium homeostasis. It has been shown to be involved in regulating GBM migration in response to C-X-C motif chemokine ligand (CXCL), serum and bradykinin in vitro. Studies have shown that the volume of invasive glioma cells is reduced by a maximum of 30%–35% compared to the volume of normal glial cells, which requires glioma cells to release all free unbound cytoplasmic water as well as K⁺ channels. Water permeably follows the release of ions through ion channels and cotransporter proteins, with a consequent change in cell volume. Inhibition of KCa3.1 channels retards cell invasion and migration (Watkins and Sontheimer, 2011).

Volume changes are critical in cell death, such as in apoptotic cell death, which is preceded by a contraction of cell volume called apoptotic volume decrease (AVD). In GBM cells, KCa channels are directly involved in AVD induced by the addition of astrocystin or TNF-α-related apoptosis-inducing ligand (TRAIL), which activate intrinsic or extrinsic pathways of apoptosis, respectively.

Ca²⁺ regulates glioblastoma maintenance, proliferation, migration, and cell cycle (Leclerc et al., 2016; Satir and Christensen, 2007). Most cancer cells obtain higher glucose utilization under aerobic conditions by producing ATP using glycolysis, a phenomenon known as the Warburg effect (Vander Heiden et al., 2009). When intracellular glucose is deficient, free fatty acid oxidation replaces glucose for cellular energy and maintains intracellular reactive oxygen species (ROS) levels and calcium homeostasis, Ca²⁺ is closely related to lipid metabolism (Li et al., 2018; Wires et al., 2017).

Sonodynamic therapy (SDT) is a non-invasive technique based on the combination of acoustic sensitizers and acoustic activation that disrupts the mitochondrial respiratory chain, leading to an increase in intracellular ROS levels and calcium overload, as well as inhibition of proliferation, invasion, and promotion of apoptosis in the biologically more aggressive grade IV glioblastomas (Chen et al., 2022). On the one hand, the mechanical force introduced by ultrasound activated the opening of the transient calcium channel Piezo1 on the membrane, maintained the open state of the Piezo1 channel by increasing the level of intracellular oxidative stress, prolonged the opening time of Piezo1, and increased the Ca²⁺ inward flow, which caused the cell to swell and activated the calcium overload pathway of the death mechanism. On the other hand, ROS generated by ultrasound-activated intracellular ultrasound sensitizers prolonged the opening time of Piezo1 and promoted Ca²⁺ entry into the cell to bind to lipid droplets to form complexes, thereby affecting lipid metabolism and preventing it from providing energy to glioblastoma cells, leading to cell death (Chen et al., 2022).

ROS generated by SDT activates the mitochondrial apoptotic pathway (Chen et al., 2017), leading to mitochondrial dysfunction and DNA damage, which sustains mitochondrial calcium uniporter (MCU) channel activity and induces Ca²⁺ overload in the mitochondria, further aggravating the mitochondrial dysfunction, generating more ROS, and forming a vicious cycle that ends in death.

Peritumoral oedema is an independent risk factor for poor prognosis and recurrence of glioblastoma (Schoenegger et al., 2009). Peritumoral oedema can exacerbate neurological signs and clinical symptoms in glioblastoma patients, such as seizures, neurological deficits, and increased intracranial pressure, and can even lead to brain herniation. In addition, peritumoral oedema can affect tumor exposure during surgery and make surgical resection more difficult (Wang et al., 2011; Hou et al., 2013). A recent study reported a linear correlation between the expression level of Piezo1 and the severity of peritumoral oedema, with preoperative magnetic resonance imaging (MRI) and corresponding Piezo1 immunohistochemical staining results in patients with different degrees of oedema. The higher the degree of oedema, the higher the Piezo1 expression (Qu et al., 2020a).

Peritumoral brain oedema (PTBE) includes vasogenic and cytotoxic oedema (Qu et al., 2020a; Liang et al., 2007). Vasogenic oedema is usually due to the degradation of tight junctions between endothelial cells (e.g., E-cadherin, N-cadherin, and b-catenin), which increases vascular permeability. Intravascular fluid then leaks out into the tissue space, further causing tissue oedema. The Piezo1 protein acts as a calcium channel to promote calcium ion influx into vascular endothelial cells, which in turn activates calcium-dependent calpain. Calpain further promotes the degradation of tight junctions between vascular endothelial cells and increases vascular permeability (Friedrich et al., 2019). High vascular permeability allows the extravasation of protein-rich fluid, leading to cerebral oedema. Extracellular cations enter neurons and glial cells through cation channels and accumulate intracellularly, while cation in-flow drives anion inflow, leading to increased intracellular osmotic pressure and entry of extracellular interstitial water molecules into the cells, resulting in cellular swelling and the formation of cytotoxic oedema occurs. Thus, inhibition of Piezo1 reduces clinical symptoms in glioblastoma patients and may be a therapeutic target for diseases involving blood-brain barrier collapse (Qu et al., 2020a).

Exosomes are small extracellular vesicles (EVs) with a diameter of 40–160 nm (average ∼100 nm) that mediate intercellular communication (Kalluri and LeBleu, 2020). Exosomes contain components such as proteins, lipids, DNA, mRNA, and non-coding RNAs, which play important roles in tumor angiogenesis, invasion, metastasis, and drug resistance. Cyclic RNAs are a novel class of RNA transcripts produced by head-to-tail splicing of exons, which are produced and structured differently from mRNAs and therefore have unique cellular functions and potential biomedical applications (Memczak et al., 2013). Studies have shown that circular RNAs play an important regulatory role in glioblastoma. circZNF800 is a recently identified circular RNA that is highly expressed in glioblastoma stem cells. Studies have shown that glioblastoma stem cells secrete exosomes to enhance GBM cell proliferation and migration and inhibit apoptosis in glioblastoma. highly expressed circZNF800 regulates the expression of tumor-associated genes through the circRNA/PI3K/AKT axis and promotes glioblastoma progression (Zhang et al., 2024).

Micro RNAs (miRNAs) are small, highly conserved, small non-coding RNA molecules that control gene expression at the post-transcriptional level. It inhibits translation or promotes degradation of mRNAs by binding to complementary binding sites within the 3‘ untranslated region (3’UTR) of target messenger RNAs (mRNAs)(Bartel, 2004). miRNA-139 is located at 11q13.4 and exhibits anti-tumor and anti-metastatic activities in humans. It was shown that miR-139-5p was significantly reduced and negatively correlated with circZNF800 expression in GBM tissues compared with normal brain tissues. miR-139-5p inhibitor significantly promoted the expression of p-AKT protein, which further promoted the invasion of U251 and U87 cells and inhibited apoptosis of GBM cells, whereas the downregulation of circZNF800 eliminated this effect. Piezo1, a direct target of miR - 1395p, was regulated by circZNF800, and correlation analysis showed a positive correlation between the expression levels of circZNF800 and Piezo1 mRNA. In GBM cells, miR-139-5p significantly reduced the expression of Piezo1 mRNA. Whereas overexpression of circZNF800 significantly increased Piezo1 mRNA expression. CircZNF800 activated the Piezo1/AKT signaling pathway through phagocytosis of miR-139-5p and regulated proliferation, migration, and apoptosis of GBM cells (Zhang et al., 2024).

The extracellular matrix is the general scaffold that maintains the homeostasis of tissues and organs in vivo. It is also a key component of the cancer microenvironment that supports tumorigenesis. During tumor development and progression, a complex ECM network is established by fibrous or nonfibrous collagen, elastin, proteoglycans, glycoproteins, laminin, fibronectin, and other matrix proteins. The extracellular matrix not only provides nests for cancer cells and stromal cells, but also serves as a reservoir for growth factors and cytokines. In addition, the ECM interacts with stromal, parenchymal, precancerous or cancer cells to initiate different cell signaling cascades that stimulate cellular differentiation metastasis, autophagy, epithelial-mesenchymal transition (EMT), cell migration, invasion and metabolic reprogramming to promote cancer cell proliferation, migration, invasion, angiogenesis, and immune evasion. mechanical stimuli induced by both the ECM per se and by ECM sclerosis can activate cell membrane receptors and mechanosensors such as integrins, piezo1 and transient receptor potential ion channel subfamily V 4 (TRPV4), thereby modulating the malignant phenotype of tumor and stromal cells. In the glioblastoma tumor microenvironment, Piezo1 plays a particularly strong role in the ECM signaling pathway.

As a therapeutic target for glioblastomas, Piezo1 senses microenvironmental stiffness and converts mechanical stimuli into electrical and chemical signals that promote calcium influx. Elevated intracellular Ca²⁺ concentration catalyzes the assembly and maturation of focal adhesions and directly activates the integrin-focal adhesion pathway, which stimulates cell proliferation and regulates extracellular matrix remodeling, further increasing glioblastoma tissue stiffness in positive feedback. Enhanced stiffness between glioblastoma cells and the ECM microenvironment in turn regulates the activation of Piezo1.

Tumor cells and stromal cells can respond to mechanical signals induced by matrix sclerosis. ECM sclerosis typically induces mechanical perturbation of the lipid bilayer and activation of transient receptor potential (TRP) family channels and piezo1 channels, evolutionarily conserved ion channels that link ECM sclerosis-associated mechanical forces to cellular signaling pathways, particularly Ca²⁺ signaling in tumor and stromal cells. The cell surface receptor integrin is a mechanosignal transducer that can be activated by Piezo1 to promote cancer stem cells and drug resistance. Physical interactions between the extracellular structural domains of integrins and ECM proteins induce the assembly of cytoplasmic complexes consisting of scaffolding proteins (neuregulin, talin, stumpins, etc.), adhesion plaque kinase (FAK), Src, and PI3K/Akt, which orchestrate adhesion plaque and cytoskeletal assembly with stromal mechanical cues. The Rap1 GTPase also responds to the cytoplasmic signaling of integrins by stabilizing integrins and recruiting neuregulin to the adhesion plaque. Rap1 GTPase also responds to matrix sclerosis by stabilizing integrins and recruiting connexins to adhesion patches. In addition, Rho-associated coiled-coil kinases (ROCK) activation may be induced by ECM sclerosis, which then promotes integrin signaling, mitogen-activated protein kinase (MAPK) activation, and SNAIL stabilization. Integrins, integrin-linked kinase (ILK), SNAIL, and Src upregulate YAP expression and activation, thereby upregulating piezo1 expression.

Piezo1 senses the stiffness of the microenvironment and converts mechanical stimuli into electronic and chemical signals that promote calcium influx. Elevated intracellular Ca²⁺ concentrations catalyze the assembly and maturation of focal adhesions, and piezo1 activates the integrin-FAK pathway when the extracellular structural domains of integrins interact with ECM proteins such as collagen, laminin, and tendon proteins, whose cytoplasmic structural domains are complexed with scaffolding proteins, including ankyrin and stumpyrin, as well as kinases, such as adhesion patch kinase (FAK) and Src. Through integrin-taIin, talin-FAK interactions, multiple FAK molecules accumulate at the focal adhesion site, resulting in the activation of FAK by phosphorylation and its association with other signaling molecules. Upon activation of FAK, its phosphorylated Tyr397 binds to the Src family of kinases, causing the PAK/Src complex by forming the FAK/Src complex. Src complex and causes phosphorylation of Paxillin and clustered regularly interspaced shortpalindromic repeats (CRISPR)-associated systems (Cas), which activate mitogen-activated protein kinase (MAPK), a kinase downstream of the rat sarcoma (Ras) pathway, via the junction proteins cysteine-rich receptor-like protein kinases (Crk) and growth factor receptor-bound protein 2 (Grb2) to control lesion adhesion and cytoskeleton assembly, regulate extracellular matrix remodeling, and increase the stiffness of glioblastoma tissues. and activate integrin-dependent intracellular kinase signaling to regulate cell adhesion, motility, proliferation, survival and differentiation (Chen et al., 2018).

It was shown that GBM stem cells were cultured in polyacrylamide hydrogels at different stiffness levels (100-5,000 Pa) in the normal human brain, low-grade glioblastomas, and high-grade glioblastomas. The percentage of proliferatively active GBM stem cells and the total number of cells increased with increasing stiffness, while Piezo1 knockdown eliminated this stiffness-dependent tumor cell growth (Chen et al., 2018).

Furthermore, many signaling pathways such as matrix metalloproteinase (MMP) family, tissue inhibitor of metalloproteinases (TIMP) family, mitogen-activated protein kinase (MAPK) family, phosphoinositide 3 kinase (PI3K) family, and others are positively correlated with high expression of Piezo1 during the pathology of glioblastoma development. Various pathways such as the mitotic cell cycle, focal adhesion, ECM, and tumor cell response to mechanical stimuli were altered after the knockdown of Piezo1. In the CGGA and TCGA databases, Piezo1 expression was positively correlated with the genes TIMP1, MMP2, MMP9, MAPK13, MAPKAPK2, PIK3R6, PIK3CD, PLOD1, and AKT2, and was also positively correlated with the genes TIMP1, MMP2, MMP9, MAPK13, MAPKAPK2, PIK3R6, PIK3CD, PLOD1, and AKT2, as opposed to low-grade glioblastomas (WHO classification II and WHO classification III) in glioblastomas (WHO grade IV) had significantly higher expression of all ECM-related genes positively associated with Piezo1. Among these genes, TIMP1, MAPK13, MAPKAPK2, and MAPKAPK3 regulate cell proliferation, PLOD1, MMP14, ADAM9, and PLAU control ECM remodeling, MMP2, and MMP9 control tumor-associated tissue remodeling; PIK3CD is involved in immune response, AKT2 regulates tumorigenesis, FHL3 controls actin cytoskeleton, and TAZ regulates the mechanosensitive HIPPO signaling pathway (Figure 4). Taken together, these data suggest that Piezo1 not only delivers mechanical inputs to promote glioblastoma growth but also positively regulates the ECM and other mechanotransduction mechanisms in tumors (Zhou et al., 2020).

In healthy brain tissue, glioblastoma-associated microglia (GAM) are highly branched, and branched microglia usually exhibit properties of M1-type macrophages that secrete pro-inflammatory factors to destroy pathogens. When inflammation, infection, trauma, or other neurologic diseases occur in the brain, microglia transform to the M2-type macrophage form. The transformed microglia have enlarged cell bodies, shortened protrusions, amoeboid morphology and activated phagocytosis. (Gieryng et al., 2017). GAM is the predominant inflammatory cell in the tumor micro-environment (TME), which not only exerts an immunosuppressive function and induces immune tolerance, but also promotes tumor growth and metastasis (Lin et al., 2024). Additionally, studies have shown that the immunosuppressive phenotype of GAMs correlates with GBM grade, with higher grades showing greater prevalence of tolerant or protumor GAMs (De et al., 2016). Neovascularisation in malignant tumors is one of the features that distinguish them from benign tumors, and the results of both clinical and experimental studies support the production of angiogenic factors [e.g., VEGF] by GAM to stimulate angiogenesis in glioblastomas. Studies have shown that IL-6 released from GAM keeps the blood-brain barrier in glioblastoma patients highly permeable by activating the Janus kinase 2/signal transducer and activator of transcription 3 (JAK/STAT3) pathway in endothelial cells and down-regulating the levels of intercellular junction proteins, which is responsible for the formation of vasogenic brain oedema (Couto et al., 2019). GAM-released IL-1β can also be used to stimulate the formation of vasculogenic brain oedema in glioblastoma cells by promoting glycerol-3-phosphate dehydrogenase 2 (GPD2) phosphorylation and glycolysis, thereby accelerating tumor proliferation and growth (Lu et al., 2020). In addition, microglia induce platelet-derived growth factor receptor (PDGFR) expression in mouse glioblastoma models and tumor cells from human patients, and the expression of this receptor stimulates glioblastoma cell migration, thereby accelerating tumor progression (Sherafat et al., 2021; Wallmann et al., 2018). Piezo1-mediated increases in cytoplasmic Ca²⁺ are involved in the inhibition of microglia proliferation. Piezo1 opening promotes extracellular Ca²⁺ endocytosis and releases Ca²⁺ stored in the endoplasmic reticulum into the cytoplasm, creating a cytoplasmic Ca²⁺ peak that interferes with the homeostasis of endoplasmic reticulum-mitochondrial Ca²⁺ communication and induces microglial dysfunction. Piezo1 is recruited to the endoplasmic reticulum via the R-Ras (which is most likely in an activated state), and then stimulates calcineurin signaling to maintain integrin activation, thereby enhancing cell adhesion. In vitro, microglia cultured on hardness-gradient hydrogels migrate to harder regions and are more responsive in these regions, suggesting that microglia may migrate towards and function in harder tumor tissue in vivo. Inhibition of Piezo1 expression modulates the affinity for integrins within microglia, thereby increasing the rate of cell migration (Hu et al., 2023). Bone marrow-derived macrophages (BMDM) migrate to the tumor site and accumulate in the central region of the GBM, exerting a predominantly immunosuppressive effect, whereas resident microglia have little or no immunosuppressive function. In grade II and III glioblastomas, microglia accounted for the majority of macrophages and lacked significant immunosuppressive activity (Miller and Hjelmeland, 2023). Activation of Piezo1 channel proteins by mechanical stimulation regulates macrophage functional differentiation, modulates dendritic cells (DC) responses triggers inflammation, and participates in T-cell activation, among many other ways, in immune cell infiltration in the tumor microenvironment (Pinton et al., 2019).

Mechanical signaling has been identified as a potential trigger for inflammatory activation in microglia. Piezo1 has the ability to regulate microglial migration patterns and immune responses, and has been suggested to act as a mechanosensor for microglial cell lines. Piezo1 regulates the pro-inflammatory response of microglial cells upon activation by lipopolysaccharide (LPS), a commonly used surrogate for bacterial infections, inducing a transient changes. In vitro and in vivo, deletion of the Piezo1 gene in microglia reduced LPS-induced proinflammatory cytokine production, whereas Yoda1 increased LPS-stimulated proinflammatory cytokine production in microglia. Similar to microglia, macrophages lacking the Piezo1 gene exhibited reduced inflammation under both resting and endotoxin-stimulated conditions, accompanied by decreased proinflammatory cytokine production. These alterations may be related to Piezo1-mediated Ca2+influx and crosstalk between cytoskeletal and integrin proteins and piezo1 (Hu et al., 2023). While at the same time, some studies have shown that LPS can lead to microglia activation via Toll-like receptor 4 (TLR4), leading to activation of NF-κB signaling via the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways, which ultimately drives TNF-α and IL-6 expression and production. However Yoda1 inhibits LPS-induced NF-κB activation by activating piezo1-Ca2+ signaling. These results suggest that activation of piezo1 channels reduces LPS-induced microglia activation and proinflammatory cytokine production, and that NF-κB signaling may act as a major mediator of (Malko et al., 2023).

In addition to this, ROS generated by SDT stimulation interferes with mitochondrial oxidative respiration, allowing more Ca2+ to enter the mitochondria, leading to mitochondrial calcium overload and dysfunction. MCU inhibition due to mitochondrial dysfunction prevents mitochondrial calcium uptake and promotes M1 polarization (Tedesco et al., 2019). Early intervention of Ca2+ channels in combination with SDT increases the proportion of M1-type macrophages with pro-inflammatory and anti-tumor effects.

Piezo1, integrins, and YAP are also strongly associated with cancer immunity. High collagen density and piezo1 activation promote macrophage polarization and enhance their immunosuppressive phenotype, leading to a decrease in cytotoxic T cell numbers and proliferation and inducing the expansion of immunosuppressive myeloid cells. Studies have shown that deletion of piezo1 in T cells does not affect effector T cell function but increases immunosuppressive myeloid cell, suggesting that activation of piezo1 in T cells may enhance the immune response in autoimmune diseases. In addition, increased matrix stiffness impedes the ability of dendritic cells to elicit an immune response in vitro and inhibits dendritic cell migration, which is essential for activating T cells and eliciting an immune response. necessary for T cells and eliciting an immune response (Jiang et al., 2022).

Increased intracellular Ca²⁺ concentration plays a crucial role in angiogenesis and arterial remodeling (Moccia et al., 2014; Moccia et al., 2012). Growth factors and cytokines, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), angiopoietin, and stromal-derived factor-1α (SDF-1α), trigger potent Ca²⁺ signaling in vascular endothelial cells (Potenza et al., 2014; Moccia et al., 2003; Noren et al., 2016; Munaron and Fiorio Pla, 2000) recruiting large numbers of downstream Ca²⁺-dependent pro-angiogenic transcription factors. These transcription factors include but are not limited to the nuclear factor of activated t-cells (NFAT), nuclear factor κ b (NF-κB) and cyclic adenosine monophosphate (cAMP) -response element binding protein (CREB) (Noren et al., 2016; Zhu et al., 2008; Chen et al., 2016), myosin light chain kinase (MLCK) and myosin 2 (Tsai et al., 2014), endothelial-type nitric oxide synthase (eNOS)(Berra-Romani et al., 2013; Charoensin et al., 2017), extracellular signal-regulated kinase 12 (ERK 1/2) and AKT (Lyubchenko et al., 2011). Subsequent studies have demonstrated that endothelial Ca²⁺ signaling may also drive tumor angiogenesis, growth, and metastasis. Piezo1 regulates glioblastoma angiogenesis by engaging the Ca²⁺-dependent proteins eNOS and calpain, which promote the migration, alignment, and rearrangement of vascular endothelial cells in the direction of blood flow (Zhou et al., 2020; Barnes et al., 2017).

Piezo1 is highly expressed and co-localized in extensively angiogenic GBM endothelial cells and promotes aberrant angiogenesis in glioblastoma through the HIF-1α/VEGF pathway. Myosin 1b promotes angiogenesis through the VEGF/myc/myosin 1b/piezo1 axis. myosin 1b can block the degradation of hypoxia-inducible factor 1α (HIF-1α) (a marker of hypoxia) by inhibiting autophagosome-lysosome fusion to promote the expression of VEGF. myc is a transcription factor of myosin 1b, which is induced by VEGFA. Myc is a transcription factor of myosin 1b and plays a key role in VEGFA-induced myosin 1b expression. myosin 1b regulates the expression of Piezo1 and Ca²⁺ influx, and Piezo1 knockdown reverses VEGF-induced intracellular Ca²⁺ influx. Ca²⁺ influx induced by vascular endothelial growth factor (Lv et al., 2024).

The amphiphilic peptide toxin GsMTx4 interferes with Piezo1 by modulating local membrane tension and does not act directly on Piezo1 itself (Suchyna, 2017). In unstressed membranes, GsMTx4 occupies a small region of the bilayer surface, stabilized by the positive charge of lysine residues. When pressure is applied to force the lipid to stretch, GsMTx4 sinks deeper into the membrane, forming a ‘regional reservoir’ to ‘clamp’ the tension applied to the outer layer. As a result, the efficiency of stimulus transport to the Piezo1 channel is disrupted, thereby inhibiting the Ca²⁺-mediated AKT/mTOR pathway, which plays a crucial role in tumor development (Gnanasambandam et al., 2017). Due to its specificity as a cationic mechanosensitive channel inhibitor, GsMTx4 has been widely used to study the physiological and pathological roles of Piezo1 channels. An increasing number of studies have begun to explore the potential of GsMTx4 as a new strategy for glioblastoma treatment (Chen et al., 2022).

Tetradecylamine-1-κ5n,2-κ5n,3-κ4n-di-μ-oxohexachlorotrisruthenium, also known as ruthenium red (RR), is a blocker of Piezo1-induced mechanosensitive currents, and has been widely used in biological studies as a non-selective inhibitor of calcium channels (Tapia and Velasco, 1997). RR inhibits the inward, but not the outward, mPiezo1 MA currents, suggesting the presence of a pore-blocking mechanism (Coste et al., 2012).

Gadolinium (Gd³⁺), a trivalent lanthanide, can inhibit Piezo1 by binding to intramembrane anionic phospholipids interfering with neighboring membrane lipids and does not block Piezo1 by directly blocking the channel pore (Ermakov Yu et al., 1998). Gd³⁺ efficiently binds to intramembrane anionic phospholipids such as phosphatidylserine, with a consequent increase in the membrane side pressure, which further stabilizes the Piezo1 channel towards the closed state (Ermakov et al., 2010).

Although ruthenium red and gadolinium exhibit inhibitory properties of piezo1, both are prone to off-targeting when acting as piezo1 inhibitors because they are non-specific blockers of many stretch-activated cation channels.

Amyloid (aβ) is a group of peptides that play a key role in the pathogenesis of Alzheimer’s disease. Similar to GsMTx4, the amphiphilic nature of Aβ peptides suggests that they can modulate membrane structure. Aβ peptides can affect cellular mechanotransduction by inhibiting Piezo1 activity. Accumulation of Aβ peptides is closely associated with upregulation of Piezo1 in reactive astrocytes (Velasco-Estevez et al., 2018) and microglial cells (Jäntti et al., 2022).

Saturated and polyunsaturated fatty acids Dietary fatty acids are important components of cell membranes and have a significant impact on their mechanical properties (e.g., bending stiffness and viscosity). Saturated margaric acid (n-heptadecanoic acid) stabilized the closed state of Piezo1 channels by increasing membrane order and binding stiffness in a concentration-dependent manner, inhibiting of Piezo1 channels (Romero et al., 2019). Several polyunsaturated fatty acids (PUFAs) are also negative modulators of Piezo1, e.g., arachidonic acid (AA) or eicosapentaenoic acid, either by mediating a metastable coupling between the inner pore helix (IH) and c-terminal extracellular structural domains (CED) or by disrupting the regulation of Piezo1 by other membrane proteins (Thien et al., 2024).

Tubeimoside I is a triterpenoid saponin extracted from the Chinese herb Bolbostemmatis Rhizoma, whose sugar chain is linked by 3-hydroxy-3-methylglutaric acid, forming a unique macrocyclic structure. Tubeimoside I had a significant inhibitory effect on Ca²⁺ responses in 293T cells overexpressing piezo1 by Yoda1. Tubeimoside I was also relatively selective for Pio 1 because it did not block Ca²⁺ currents induced by other mechanical actions. A negative correlation was shown between the concentration of Yoda1 and the inhibitory response of Tubeimoside I, and the two compounds may reversibly compete with each other for piezo1 in the Yoda1 binding site (Liu et al., 2020).

It was shown that TBMS1 inhibited viability and AKT phosphorylation in U251 cells. PI3K/AKT inhibitor lY294002 showed the same effect. Furthermore, the combination of TBMS1 and lY294002 enhanced these inhibitory effects (Cao et al., 2020).

The cell cycle control system is based on two major protein families, cell cycle proteins and cdKs. Normally, the expression levels of cell cycle proteins are dynamic, whereas cdKs act as catalytic subunits that stabilize the expression levels of cell cycle proteins. During the transition from G2 to M phase, cyclin B1 activates CDK1, leading to an increase in activated CDK1 (Arellano and Moreno, 1997). In glioblastoma cells, constitutive activation of the PI3K/AKT signaling cascade and reduced levels of p21 protein expression are common, leading to defective cell cycle progression (Yang et al., 2016). It has been shown that TBMS1 reduces cyclin B1, CDK1, and AKT phosphorylation levels and elevates the level of the cKd1 inhibitor p21, which inhibits DNA synthesis and induces G2/M phase block in glioblastoma cells by targeting the PI3K/AKT/p21 and p21/CDK1/cyclin B1 signaling cascades (Cao et al., 2020).

Apoptotic signaling pathways are divided into death receptor (exogenous) pathways and mitochondrial (intrinsic) pathways (Creagh, 2014). The Bcl-2 family, which includes anti-apoptotic components such as Bcl-2 and Bcl-xl, as well as pro-apoptotic components such as Bax, Bak, and Bad, is involved in the regulation of apoptosis through the mitochondrial pathway. Mitochondria-dependent signaling occurs through the cleavage of caspase-9, which subsequently activates downstream caspase-3, leading to the cleavage of various key cellular substrates, including ParP, thereby inducing apoptosis (Palmer et al., 2011). Previous studies have shown that TBMS1 prevents glioblastoma progression by triggering apoptosis through the PI3K/AKT-mediated Bcl-2 signaling pathway via TBMS1. In vitro experiments showed that after 24 h of TBMS1 action, U251 cells showed chromatin condensation, nuclear fragmentation, and formation of apoptotic vesicles.

Overall, TBMS1 exhibited inhibition of cell growth, promotion of apoptosis, and induction of cell cycle arrest by inhibiting the PI3K/AKT-mediated signaling pathway in glioblastoma cells. This provides a theoretical basis for in vivo studies using TBMS1 as a potential cancer therapeutic compound (Cao et al., 2020). (Figure 5).

Although a variety of piezo1 inhibitors have been shown to inhibit tumor cell proliferation and promote apoptosis, most of the piezo1 inhibitors are non-specific inhibitors of piezo1 and have strong off-target properties. However, most of the piezo1 inhibitors are non-specific inhibitors of piezo1, with strong off-target properties, and most of the piezo1 inhibitors, except Tubeimoside I and GsMTx4, are unable to pass the blood-brain barrier, and they cannot act directly on glioblastoma tumor tissues, so their therapeutic application value is limited. At the same time, some inhibitors such as Dooku1 have poor solubility in body fluids, which seriously affects their activity in vivo.

Dendritic cells (DCs) are specialized antigen-presenting cells that play a crucial role in initiating immune responses to cancer immunotherapy through various activation pathways. Cell packets (BPs) are soft disk-shaped particles that modulate the phenotype of macrophages in vivo. The backpacks are prepared from biodegradable polymers using microcontact printing, and each backpack contains a cell adhesion layer, a poly (lactic-hydroxyglycolic acid) (PLGA) layer, a poly (vinyl alcohol) (PVA) layer and a second PLGA layer. Studies have shown that the “backpacks” can evade phagocytosis by macrophages, which enhances the stability of the drug in vivo (Shields et al., 2020). BPs represent an alternative approach for targeted delivery of immune activators and immune cells, providing prolonged immune modulation and enhanced therapeutic effects. Empty BPs act as modulators of DC immune function by activating cell-surface piezo1. Upon binding to DCs, BPs open piezo1 mechanoionic channels by binding to DCs, induce Ca²⁺ inward currents, activate local FAK and intracellular cytoskeletal remodeling to increase cellular stiffness, participate in the PI3K/Akt/mTOR cascade signaling to enhance IRF3 phosphorylation, and promote type I interferon production, and this activation leads to the initiation of the innate immunity-associated PI3K/Akt/mTOR/type I interferon signaling pathway. Radiation therapy combined with DC-BP (BPs that bind to DC cells) had a significant activating effect on the expression of mature dendritic cells, migrating dendritic cells, and lectin receptor-associated DCs, inducing a potent anti-tumor effect. Meanwhile, radiation therapy combined with DC-BP also significantly activated cytotoxic T lymphocyte (CTLs) in peripheral blood mononuclear cell (PBMCs), and activated T cells exhibited enhanced reactivity, leading to increased production of cytokines such as interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), which exhibited potent systemic antitumor effects. This approach holds great promise for synergistic radioimmunotherapy in cancer treatment, providing a multimodal therapeutic platform with great potential. The DC-BP vaccine combined with radiotherapy has been shown to significantly promote tumor shrinkage and to have the same killing effect on distant tumors in bladder cancer.

In the future, it could be tried to improve the local control rate and prolong the survival of glioblastoma patients who undergo surgical treatment with standard post-surgical adjuvant concurrent radiotherapy (total dose of 60Gy for more than 6 weeks with daily temozolomide, followed by 6 cycles of temozolomide maintenance therapy) with activation of piezo1 by the DC-BP vaccine, offering new possibilities for glioblastoma treatment (Yu et al., 2024).

The epidermal growth factor receptor (EGFR) gene is a tyrosine kinase receptor located on chromosome 7p11-p13, participating in key signaling pathways responsible for the cell cycle, growth, proliferation, migration, and survival of tumor cells. Mutations or overexpression are closely related to the development of more aggressive malignant phenotypes, leading to treatment resistance and poor clinical outcomes. After binding to endogenous ligands, EGFR forms homodimers or heterodimers, thereby activating the intracellular tyrosine kinase (TK) domain and causing phosphorylation of specific tyrosine residues. Under the operation of phosphorylated tyrosine, downstream PI3K/Akt, Ras/Raf/MAPK, JAK/STAT, and other pathways are activated. These pathways are closely related to the proliferation, invasion, and angiogenesis of gliomas, ultimately leading to the malignant biological behavior of gliomas. Currently, pan-targeted anti-angiogenic tyrosine kinase inhibitors (TKIs) targeting EGFR mainly act on the extracellular domain of EGFR, blocking intracellular signaling pathways such as MAPK and PI3K in tumor cells to control tumor growth, which is the direction of anti-EGFR treatment for gliomas. However, most of the current results are still in phase II clinical trials, and there is no reliable phase III clinical data to prove their effectiveness (Westphal et al., 2015; Lombardi et al., 2019).

Piezo1 activates EGFR through a typical epidermal growth factor (EGF) activation alternative pathway. During the steady-state cell turnover process, EGF activates EGFR signaling through tyrosine autophosphorylation, receptor internalization, and cytoplasmic extracellular regulated protein kinases (ERK) activation. Piezo1-dependent EGFR signaling (such as ERK activation and activator protein 1 (AP-1)induction) requires EGFR internalization through clathrin-mediated endocytosis (CME) rather than its kinase activity or Y1173 autophosphorylation, indicating that the internalization of the epidermal growth factor receptor is the key to piezo1 activation-induced AP-1. Internalized EGFR has a pro-survival function in kinase-independent cancer cells and is not affected by current tyrosine kinase-targeted therapy. The combination of Piezo1 inhibitors and TKI drugs provides a new direction for future targeted therapy of glioblastoma (Pardo-Pastor and Rosenblatt, 2023).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor-alpha family, induces apoptosis in cancer cells through death receptor activation (Lincoln et al., 2018; Fulda and Debatin, 2005). This ligand is expressed on the surface of activated natural killer (NK) cells, macrophages, DC, and T cells and plays a role in the innate immune response (Ralff and El-Deiry, 2018; Shepard and Badley, 2009). TRAIL has been found to trigger apoptosis in many tumor cell types by binding to death receptors 4 and 5 (DR4/5)(Lee et al., 2014). This binding leads to trimerization of the receptor, which then binds intracellularly to the Fas-associated death domain (FADD) and caspase-8 to form the death-inducing signaling complex (DISC). TRAIL is of particular interest in cancer therapy because of its ability to induce apoptosis in tumor cells without harming normal cells. However, xenograft animal studies have also shown that the use of TRAIL alone to treat the primary tumor itself is rarely successful. In addition, approximately 50% of tumor cell lines tested exhibit some degree of TRAIL resistance, and glioblastomas are particularly resistant to TRAIL. Therefore, for TRAIL to be a viable treatment, cancer cells must be sensitized to TRAIL (Lincoln et al., 2018; Fulda and Debatin, 2005).

One way in which TRAIL sensitization occurs is through the fluid shear stress (FSS) experienced by circulating tumor cells (CTCs) during circulation. Activation of the mechanosensitive ion channel Piezo1 via the agonist Yoda1 induces TRAIL sensitization under static conditions, and in vivo, Yoda1 significantly increased apoptosis in prostate, breast, and colon cancer cell lines compared to TRAIL alone (Hope et al., 2019). It was demonstrated that the combination of Yoda1 and TRAIL resulted in decreased GBM cell viability and increased late-stage apoptosis. The combination of FSS + TRAIL treatment resulted in a significant decrease in U87 and LN18 cell viability. These findings further demonstrated the efficacy of promoting TRAIL-mediated apoptosis through Piezo1 activation.

Combined treatment with Yoda1 and TRAIL increased mitochondrial depolarisation in GBM cells. Piezo1 has been shown to sensitize PC3 prostate cancer cells to TRAIL via the intrinsic apoptotic pathway, where Ca²⁺ activates calpain via Piezo1, subsequently amplifying death-inducing signals in the TRAIL-mediated apoptotic pathway (Knoblauch et al., 2023). These pro-apoptotic proteases induce pore formation in the mitochondrial membrane, which leads to mitochondrial outer membrane permeabilization (MOMP). Ultimately, MOMP leads to the release of apoptotic proteins through the mitochondrial pore, resulting in multiple cell death pathways (Hope et al., 2019; Chipuk et al., 2006).

Bax is a component of the intrinsic apoptotic pathway that triggers MOMP, and Bax channel blockers (BCBs) reduce mitochondrial depolarisation in GBM cells. Calpeptin, a calpain inhibitor, is an important component of the Piezo1 pathway. Yoda1 and TRAIL treatment inhibits calpain leading to a significant reduction in mitochondrial depolarisation.

Temozolomide (TMZ) is the standard chemotherapy used for the treatment of GBM.U87 cells are sensitive to TMZ, whereas LN18 cells are resistant to the drug. Studies have shown that LN18 cells are more sensitive to Yoda1 + TRAIL treatment than to TMZ treatment. U87 cells with low levels of Piezo1 expression responded comparably to both treatments. The sensitivity of these GBM cells to Yoda1 + TRAIL treatment suggests that the therapy may be successful in targeting tumors that are particularly resistant to current standard treatments. Thus Yoda1 + TRAIL combination therapy is expected to be a potential GBM treatment (Knoblauch et al., 2023).

Studies have shown that the abnormal activation of the PI3K/Akt/mTOR/NF-κB signaling pathway induced by EGFR promotes the proliferation of glioma cells and is related to MGMT promoter methylation or expression, subsequently enhancing glioma drug resistance, and the efficacy of chemotherapy drugs such as TMZ is weakened due to drug resistance. This is the main obstacle in the treatment of GBM. Studies have shown that TBMS1 can reduce the expression of EGFR in MGMT + cell lines by inhibiting the PI3K/Akt/mTOR/NF-κB signaling pathway. The combination of TBMS1 and TMZ strongly induces apoptosis in glioblastoma cells, accompanied by the formation of strong DSB, γH2AX foci, and an increase in PARP cleavage, and reduces the expression of MGMT in TMZ-resistant GBM cells, thereby reducing DNA damage repair and synthetic functions and restoring the sensitivity of MGMT + cells to TMZ. This study provides a theoretical basis for the combined application of TMZ and TBMS1 as a potential chemotherapy for MGMT + GBM patients (Tang et al., 2022).

Bevacizumab is a recombinant humanized immunoglobulin G1 (IgG1) monoclonal antibody that can bind to VEGF-A, inhibit its binding to VEGF receptor-2 (VEGFR-2), and subsequently inhibit the biological effects of VEGF, including affecting vascular permeability, proliferation, and the migration and survival of endothelial cells. The anti-angiogenic therapy represented by Bevacizumab normalizes tumor vessels, leading to excessive pruning of endothelial cells and perivascular cells, reduced vascular tortuosity, and decreased interstitial pressure, thereby improving oxygenation and delivering chemotherapy to tumor cells, amplifying the anti-tumor effect of chemotherapy. Meanwhile, Bevacizumab can bind to VEGF, preventing VEGF from binding to its receptors on the surface of endothelial cells, thereby pruning vessels, regulating vascular permeability, reducing brain edema caused by brain necrosis, and treating brain necrosis (Nayak et al., 2021). Piezo1 inhibitors can block VEGF activation caused by piezo1. In the future, they can be tried in combination with bevacizumab to reduce abnormal glioblastoma vessels and inhibit tumor progression.

In addition to this, the piezo1 agonist Yoda1 + TRAIL can successfully kill glioblastoma cells that are particularly resistant to current standard therapies by activating piezo1, providing a new idea for the treatment of glioblastoma resistant to temozolomide.