Glioblastoma multiforme (GBM) is the most aggressive and highly vascularized brain tumor with poor prognosis. Endothelial cell-dependent angiogenesis and tumor cell-dependent Vasculogenic mimicry (VM) synergistically contribute to glioma vascularization and progression. However, the mechanism underlying GBM vascularization remains unclear. In this study, GBM stem cells (GSCs) were divided into high and low β8 integrin (ITGB8) subpopulations. Co-culture assays followed by Cell Counting Kit-8 (CCK-8), migration, Matrigel tube formation, and sprouting assays were conducted to assess the proliferative, migratory and angiogenic capacity of GBM cells and human brain microvascular endothelial cells (hBMECs). An intracranial glioma model was constructed to assess the effect of ITGB8 on tumor vascularization in vivo. Our results indicated that ITGB8 expression was elevated in GSCs and positively associated with stem cell markers in glioma tissues, and could be induced by hypoxia and p38 activation. ITGB8 in GSCs inhibited the angiogenesis of hBMECs in vitro, while it promoted the ability of network formation and expression of VM-related proteins. The orthotopic GBM model showed that ITGB8 contributed to decreased angiogenesis, meanwhile enhanced invasiveness and VM formation. Mechanistic studies indicated that ITGB8-TGFβ1 axis modulates VM and epithelial-mesenchymal transition (EMT) process via Smad2/3-RhoA signaling. Together, our findings demonstrated a differential role for ITGB8 in the regulation of angiogenesis and VM formation in GBM, and suggest that pharmacological inhibition of ITGB8 may represent a promising therapeutic strategy for treatment of GBM.
Glioblastoma multiforme (GBM) is the most malignant brain tumor and is highly resistant to combination therapies . Moreover, anti-angiogenic therapy has become a promising way to fight cancer . However, in a phase II study of patients with newly diagnosed GBM, those administered with bevacizumab and temozolomide showed prolonged progression-free survival, while no improvement in overall survival [3, 4]. Therefore, further investigations of anti-angiogenic therapy in GBM are warranted.
Vasculogenic mimicry (VM) was firstly observed by Maniotis et al. in human melanoma cells and considered as a marker of aggressive tumor . Analysis of xenograft models and human specimens unveiled that VM formation in patients with glioma usually predicts an unfavorable prognosis [6, 7]. Moreover, network formation in Matrigel has been widely used to evaluate the VM ability of tumor cells in vitro . And various types of tumor cells are associated with tube formation in Matrigel under hypoxic condition [9,10,11]. Hence, VM has been considered as a compensation of angiogenesis, particularly in response to hypoxia. Moreover, VM acts as a novel paradigm for tumor perfusion, providing nutrition for tumor growth and progression.
Epithelial-mesenchymal transition (EMT), is also associated with tumor aggressiveness and metastasis . Moreover, both VM and EMT could promote tumor cell motility and invasiveness. In addition, VM formation related signaling pathways including TGFβ, Notch, and Wnt, have also been shown to induce EMT process . Genes involved in angiogenesis and vasculogenesis are upregulated in aggressive cancer cells, including cadherin-5 (CDH5), EPH receptor A2 (EPHA2) and laminin gamma2 (LAMC2) . Matrix metallopeptidase 2 (mmp2) is also necessary in VM as it mediated extracellular matrix (ECM) remodeling via interacting with laminin 5γ2 chain . During EMT process, epithelial markers (E-cadherin, cytokeratin) were downregulated while mesenchymal markers (N-cadherin, vimentin) were increased in tumor cells.
Recently cancer stem-like cells (CSCs) have been highlighted in malignant neoplasms for its role in chemotherapy resistance and recurrence [16, 17]. CSCs are a subpopulation within cancer cells that capable to self-renew and give rise to multiple cell lineages . Recent studies demonstrated that a subpopulation of GBM cells could give rise to endothelial cells [19, 20]. Additionally, Hallani et al. and Scully et al. showed that some GSCs were able to transdifferentiate into smooth muscle cells or mural cells [21, 22]. Furthermore, Mani et al. illustrated that the induction of EMT in epithelial cells could lead to the elevation of stem cell markers .
Integrins belong to the family of a type I transmembrane heterodimeric glycoprotein receptors for ECM proteins . β8 integrin (ITGB8) is crucial for the development of neuro-epithelial cells and microvasculature [25, 26]. ITGB8 was detected in multiple cancer types including lung adenocarcinoma, high grade serous ovarian cancer, gastric cancer and glioma, and correlated with poor survival [27,28,29,30]. Reyes et al. indicated that ITGB8 was overexpressed in GBM cells and correlated with diminished patient survival . However, the effect of ITGB8 on VM and EMT remains unclear.
In the present study, we measured the dynamic changes of ITGB8 in GBM stem cells, and also investigated whether ITGB8 contribute to the VM formation in orthotopic GBM model. Furthermore, we explored the mechanisms whereby elevated ITGB8 triggers VM and EMT process through TGFβ1-Smad2/3-RhoA signaling pathway.
β8 integrin is enriched in GSCs
We first examined ITGB8 expression in five human GSCs (GSC#1-GSC#5) and differentiated glioma cells. Immunoblot analysis revealed a robust expression of ITGB8, CD133 and Nestin in most of GSCs (Fig. 1A, C). GSCs were induced to differentiation in response to serum (10%) treatment. We next investigated the ITGB8 expression in GSCs serum-differentiated cells (GSDCs) and found that ITGB8 decreased significantly in GSDCs compared to that in GSCs (Fig. 1B, D). Additionally, β8+ GSCs showed robust self-renewal and proliferation capacities, while β8- GSCs showed decreased capacity to form spheres (Fig. 1E). We further examined the mRNA expression of ITGB8 in mixed glioma dataset from The Cancer Genome Atlas (TCGA) database. ITGB8 expression was significantly elevated in GBM and LGG compared to normal brain tissue (Fig. 1F). Specifically, ITGB8 expression was upregulated in GBM (grade IV) tissue rather than that of grade II and III (Fig. 1G). Moreover, high levels of ITGB8 indicated poor prognosis in glioma dataset (Fig. 1H and S1b). CD133, Nestin and SOX2 expression were positively associated with ITGB8 expression in TCGA and Chinese Glioma Genome Atlas (CGGA) databases (Fig. 1I and S1c), and we further confirmed that ITGB8 was closely correlated with Nestin expression in GBM specimens (Fig. 1J). Together, our findings indicated that ITGB8 was abundant in GSCs.
ITGB8 expression is regulated by hypoxia and p38 activation
GSC#1 and GSC#2 were further selected for in vitro experiments due to the robust ITGB8 expression. To evaluate ITGB8 expression under hypoxic condition, GSCs and GSDCs were exposed to varying levels of O2 for 24 h. We found that hypoxia-induced Decreased O2 concentrations for 24 h led to the upregulation of β8 integrin in GSCs but not GSDCs (Fig. 2A, B). The protein level of ITGB8 increased accompanied with Hypoxia-inducible factor 1α (HIF1α) accumulation in GSCs, but remained unchanged in GSDCs (Fig. 2C). In line with this, expression of ITGB8 was significantly attenuated in GSCs treated with si-HIF1α (Fig. 2D). These results were further confirmed by immunofluorescence (Fig. 2E). Additionally, bioinformatics analysis revealed a positive association between ITGB8 and HIF1a expression in GBM datasets (Fig. 2F). Moreover, gene set enrichment analysis (GSEA) revealed that hypoxia-induced genes were significantly correlated to ITGB8 in GBM (Fig. 2G).
As integrins were widely regulated by MAPK pathways in both tumors and normal tissues [31, 32]. GSCs#2 was treated with inhibitors targeting ERK (PD98059), P38 (SB202190) and JNK (SP600125) to examine ITGB8 expression in relation to these pathways. mRNA and protein levels of ITGB8 were mostly affected by p38 signaling (Fig. 2H, I and S2c). Previous study has shown that ITGB8 promoter region was located in area from −1280 to 69 bp of gene sequence with multiple putative transcription factor binding site (Fig. S2a) . We performed a luciferase reporter assay with various ITGB8 reporter constructs. The region from −491 to 69 bp was critical for transcriptional regulation of ITGB8 in GBM stem cells (Fig. 2J and S2d). Furthermore, a significant correlation between ITGB8 mRNA abundance and p38 phosphorylation was observed in TCGA glioma dataset (Fig. 2K). Furthermore, ITGB8 expression significantly decreased in GSCs pretreated with P38 inhibitor (Fig. 2L), and this was further confirmed by immunohistochemistry (IHC) staining in GBM samples (Fig. 2M). In summary, ITGB8 expression was regulated in GBM in response to hypoxia and p38 activation.
ITGB8 correlates with reduced angiogenesis
To explore the potential interaction between GSCs and human brain microvascular endothelial cells (hBMECs), GSCs were co-cultured with hBMECs under different conditions (Fig. S3a). hBMECs co-cultured with β8+GSCs showed diminished proliferative capacity compared to those with β8− GSCs (Fig. 3A, B). Migration assay showed that the number of hBMECs crossed the Matrigel layer toward β8+ GSCs was significantly lower than that toward β8− GSCs (Fig. 3C). Next, hBMECs were seeded on Matrigel-coated lower plates to investigate the tube formation (Fig. S3b). The hBMECs co-cultured with β8+ GSCs showed attenuated network formation compared to those with β8− GSCs (Fig. 3D). Moreover, we established an in vitro angiogenesis model, in which hBMEC spheroids were located in the lower chamber with collagen I solution (Fig. S3c). An inhibited capacity of hBMECs spheroid-based angiogenesis was observed when co-cultured with β8+GSCs compared to that with β8−GSCs (Fig. 3E). The proliferation of hBMECs significantly decreased when cultured in conditioned medium from β8+GSCs (Fig. S4a-c), furthermore, tube formation (Fig. S4d) and sprouting angiogenesis (Fig. S4e) were also attenuated when compared to that of hBMECs cultured with β8−GSCs.
As ITGB8-TGFβ signaling contributes greatly to endothelial cells development of retinal , we co-cultured hBMECs with β8+ or β8− GSC#1. The protein levels of phosphorylated Smad, and p21 were elevated in brain endothelial cells cultured with β8+ GSC#1 while c-myc expression was decreased accordingly (Fig. 3F). We also established orthotopic xenografts in nude mice with β8+ or β8− GSC#2, and the vessel quantification revealed an increased angiogenesis in β8− GSC#2 intracranial tumors comparing to that of β8+ GSC#2 (Fig. 3G). Together, these findings suggested that ITGB8 alleviated angiogenesis in GBM.
ITGB8 induces VM and EMT process
Primary GBM cells and A172 cells showed enhanced migration when co-cultivated with β8+ GSCs rather than β8− GSCs and neutralizing antibody treated-GSCs (Fig. S3d, Fig. 4A, B). GSC#2 was added into the upper insert and G4 was seeded on Matrigel in the lower plate, and the tube formation ability of GBM cells was evaluated (Fig. S3e). Tubule formation of G4 increased significantly when co-cultured with β8+ GSC#2, which was alleviated by β8-neutralizing antibody pretreatment (Fig. 4C, D). Tumor cells undergo EMT would display a remodeling of actin . G4 and G10 cells co-cultured with β8+ GSC#2 showed increased filopodia formation, which was attenuated when GSCs were pretreated with β8-neutralizing antibody (Fig. 4E, F). Immunoblot analysis revealed augmented CDH5, MMP2, N-Cadherin and vimentin expression in GBM cells when co-cultivated with β8+ GSCs. And forced expression of ITGB8 in β8− GSCs led to the upregulation of these molecules in co-cultured GBM cells. Meanwhile, downregulating ITGB8 in β8+ GSC#2 caused declined expression of CDH5, MMP2, N-Cadherin and vimentin (Fig. 4G). These results were further confirmed by ICC and IHC staining analysis (Fig. 4H–J). Additionally, we performed PAS/CD34 dual staining to detect VM in GBM tissues (Fig. 4K). A classic VM pattern was characterized as PAS-positive and CD34-negative . VM quantity was significantly associated with ITGB8 expression (Fig. 4L). In addition, survival analysis demonstrated a positive association between ITGB8 expression and adverse outcomes (Fig. 4M).
ITGB8 modulates VM formation and invasion of glioblastoma cells through TGFβ-RhoA signaling
Previous studies revealed ITGB8/ TGFβ axis as a prominent angiogenesis regulator during CNS development [36, 37]. β8+ GSCs produced more abundant TGFβ1 in culture medium comparing to that of β8− GSCs (Fig. 5A). We transfected glioma cells with shTGFβ to eliminate the autocrine effect of TGFβ. G4-shTGFβand A172-shTGFβ, pretreated with neutralizing antibody targeting TGFβ1 or LY2109761 (an inhibitor of the TβRI), were co-cultured with β8+ GSCs and subsequently subjected to migration and tube formation assay analysis. TGFβ1 blockage or TGFβ receptor inactivation led to attenuated migratory and tube formation capacities (Fig. 5B–D). It also significantly decreased the expression of CDH5, N-Cadherin and vimentin (Fig. 5E). TGFβ is released in a latent complex containing TGFβ, latency associated protein (LAP), and a latent TGFβ binding protein (LTBP) . Previous experiments have shown that ITGB8 is expressed on the surface of astrocyte and bind to the LAP of TGFβ1 in perivascular astrocyte and T lymphocyte [39, 40]. We conducted an immunoprecipitation assay in GSCs transfected with control vector or sh-β8 integrin. GSC#2 and GSC#4 expressing ITGB8 were detected with co-immunoprecipitated latent TGFβ1 (Fig. 5F). Confocal imaging analysis revealed a co-localization of ITGB8 and latent TGFβ1 on the cell membrane of GSC#2 (Fig. 5G). The cellular level of p-Smad3 as an indicator of the canonical TGFβ-smad2/3 pathways, and p-Akt, active RhoA, p-ERK, p-JNK as indicators of non-canonical TGFβ pathways in GBM cells treated with exogenous TGFβ were evaluated. Interestingly, elevated expression of p-Smad3, and activated RhoA was observed in response to TGFβ stimulation (Fig. 5H). P-Smad3 and Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) expression in G4-shTGFβ1 cells co-cultured with β8− or β8+ GSC#2 positively correlated with β8 integrin expression (Fig. 5I). To further illustrate the role of Smad2/3 and RhoA signaling in regulating ITGB8-induced invasive phenotype and VM formation, we pretreated TGFβ1 knock-out G4 and A172 cells with SIS3 (a specific inhibitor of Smad3 phosphorylation) or Y27632 (a RhoA kinase inhibitor), followed by co-cultivation with β8+ GSCs. The migration and tube formation capacities in G4-shTGFβ1 cells were significantly decreased (Fig. 5J, K). Inhibition of Smad3 phosphorylation or RhoA kinase also decreased the expression of CDH5, N-Cadherin and vimentin (Fig. 5L). F-actin staining revealed a similar effect of SIS3 and Y27632 on cytoskeleton alteration. Filopodia formation in G4-shTGFβ1 cells increased when co-cultured with β8+ GSC#2 than that with β8− GSC#2, however, it was attenuated when treated with SIS3 or Y27632 (Fig. 5M, N). Taken together, these findings suggested that GSCs-derived ITGB8 regulated GBM cells by mediating TGFβ/Smad/RhoA signaling pathway.
ITGB8 is associated with VM in an intracranial xenograft model
To explore ITGB8-mediated angiogenesis and VM in vivo, we established a xenograft model by orthotopically implanting mCherry-labeled β8− or β8+ GSC#2 into nude mice. Mice with β8+ GSCs exhibited a decreased tumor growth rate when compared to mice implanted with β8− GSC#2 (Fig. 6A, B). The endothelium-based vascular channel presented positive lectin and CD34 staining, while VM channel was characterized by positive lectin and mCherry staining (Fig. 6C). These results indicated that the VM formation in β8+ GSCs-xenograft GBM was significantly enhanced (Fig. 6D, E). Furthermore, the β8+ GSCs-xenograft was positively correlated with augmented expression of β8 integrin, p-Smad3 and ROCK1, as well as indicators for VM and EMT (Fig. 6F). In summary, these findings indicated that ITGB8 plays a critical role in VM formation of GBM in vivo.
In the present study, we provided clinical, in vitro and animal evidences demonstrating that GSCs-derived ITGB8 exhibits anti-angiogenic effect on brain microvascular endothelial cells, and contributes to VM formation and EMT in GBM to support tumor invasion. Mechanistic investigation indicated that ITGB8 promotes VM formation and invasive phenotype in GBM cells via activating the TGFβ1/p-Smad/RhoA signaling pathway (Fig. 7).
Our results revealed that cancer stem-like cells derived from primary GBM displayed abundant ITGB8 expression. Moreover, we detected the loss of ITGB8 in GSCs with serum-induced differentiation in vitro. In fact, ITGB8 has been reported to be expressed in neural stem and progenitor cells of adult mouse brain [26, 41]. Similar to stem or progenitor cells, cancer stem cells possess the ability of self-renewal and differentiation and are crucial for tumor progression and metastasis . ITGB8 was recently reported to be upregulated in various types of aggressive tumors. Mertens-Walker et al. reported that ITGB8 expression was positively associated with EphB4 receptor tyrosine kinase in prostate cancer cells and played an important role in tumor cell motility . Jin et al. revealed ITGB8 as a determinant of pancreatic ductal adenocarcinoma radiochemosensitivity . Additionally, overexpression of ITGB8 led to the growth and metastasis of colorectal cancer . Moreover, expression of ITGB8 contributed to unfavorable prognosis of high grade serous ovarian cancers . Our data showed that ITGB8 in GSCs, but not differentiated glioma cells, could be induced by hypoxia (Fig. 2A, B). Low oxygen tension facilitated the maintenance of undifferentiated states of embryonic and neural stem cell phenotypes . In fact, previous studies have shown that ITGB8 is partly co-expressed with other stem cell markers such as CD133 .
ITGB8 in neuro-epithelial cells and astrocytes is crucial for cerebral angiogenesis and development [26, 36]. For instance, Mobley and colleagues showed that β8-/- mice displayed compromised blood-brain barrier properties . However, the effects of ITGB8 on tumor neovascularization are much more complex. Takasaka et al. reported that β8 integrin expression was associated with increased angiogenesis and a specific ITGB8 blocking antibody substantially diminished vessel density in mouse xenografts derived from MC38 colon carcinoma cancer cell line . While Fang et al. suggested that reduced ITGB8 expression in glioma cells favored angiogenesis . Our data implied that enriched ITGB8 in GSCs accounts for reduced GBM angiogenesis. VM contributes to the failure of anti-VEGF therapy in solid cancers, including GBM . We presented evidence in current study supporting the importance of β8 integrin in GSCs-mediated VM formation.
Mechanical investigations demonstrated that activation of the TGFβ/Smad signaling pathway is involved in ITGB8-mediated VM formation. Latent TGFβ1 is expressed in diverse tumor cells and can be activated by binding to ITGB8 . Our work suggested that the expression of ITGB8 in GSCs was positively correlated with TGFβ1 levels. In addition to the Smad-dependent pathway, non-Smad pathways including MAPK, Rho-like GTPase and PI3K/AKT signaling pathways have been reported in carcinogenesis and tumor progression [51,52,53]. Our study showed that ITGB8-TGFβ1 affected downstream activation of Smad2/3 and RhoA in glioma cells. Attenuation of VM or invasive phenotype may result from inhibition of the Smad or RhoA pathway.
We further established orthotopic GBM models in nude mice with either integrin β8+ or β8− primary GSCs. Mice in β8+ group were associated with increased VM formation and EMT. Since ITGB8 caused opposite prognosis in patients with GBM, we speculated that endothelial-based angiogenesis mainly accounts for the outcome and tumor progression in GBM-bearing mice when no therapeutic intervention was received. While VM may play a greater role in GBM patients that clinically undergo surgery or chemotherapy.
In summary, our study depicted a complex role of β8 integrin in glioma vascularization. We provided evidence that β8 integrin expression was inversely correlated with angiogenesis, while promoted VM formation via inducing TGFβ expression, and subsequently activating the RhoA signaling pathway. Therefore, β8 integrin may be a potential therapeutic target for GBM VM and invasion. Pharmacological inhibition of β8 integrin together with anti-VEGF agent would effectively suppress tumor vascularization and prolong survival of patients with GBM.
Materials and methods
GBM specimen and cell culture
Brain tumor samples were obtained from consenting patients diagnosed as GBM. 73 paraffin-embedded GBM samples and corresponding clinicopathological data were collected from patients undergoing surgical operation in the department of neurosurgery at Zhujiang Hospital from 2013 to 2017.
Glioblastoma tissues were enzymatically digested and GBM stem cells were cultured in DMEM/F12 (Gibco, USA) medium supplemented with EGF (20 ng/ml, Peprotech, USA), bFGF (20 ng/ml, Peprotech) and B27 (1:50, Gibco).
Reagents, plasmids construction and siRNA
ITGB8 cDNA and plasmids construction, as well as siRNAs for target genes were designed and provided by Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, P.R. China). PCR primer sequences for IGFB8 and GAPDH were provided in supplementary table 2.
Sphere formation assay
Dissociated single GBM stem cells were seeded into 24-well plates and incubated in serum-free medium at 37 °C for 7 days. Diameters of 5 randomly selected tumor spheres were measured.
Cell proliferation and migration assay
Cell proliferation was measured via using CCK-8 and Edu assay. Migration assay was performed by using cell culture insert with 8-um pores in 24-well plates (Costar, USA).
Tube formation assay
Tube formation assay of glioma cells and hBMECs was carried out as previously described . Cells were cultured on Matrigel (BD Biosciences, USA) for 24 h and tubules were quantified.
Endothelium spheroid-based sprouting angiogenesis assay
Human TGF-beta1 ELISA kit (Proteintech) was used to measure the concentration of TGF-beta1 from GSCs-derived culture media according to manufacturer’s instruction.
Immunoblot Analysis and Immunoprecipitation assays
The immunoblot and immunoprecipitation assay were performed as described before . Lysates from certain cells were subjected to immunoblot analysis using antibodies which is listed in supplementary table 2.
Tissue immunohistochemical and immunofluorescence staining
Tissue IHC and IF staining were performed as previously reported. Briefly, specimens of surgical GBM tissues and xenograft samples were fixed, embedded and sectioned followed by immuno-staining. Antibodies used for targeted proteins were listed in supplementary table 2.
Luciferase reporter assay
Luciferase reporter assay was carried out according to the protocol described previously . Luciferase activity was measured using dual-luciferase reporter assay kit with Renilla luciferase activity as control (Promega, Mannheim, Germany).
In vivo xenograft assay
Five to eight-week-old Balb/c male mice were purchased from the Central Animal Facility of Southern Medical University. Fractionated β8+ or β8− GSCs cells (1 × 105 cells in 0.1 ml PBS) stably transfected with mCherry-LUC vector were orthotopically injected into the brain of Balb/c nude mice according to Ozawa’s instruction . Each group included 8 mice.
Vasculature quantification was measured according to the method previously reported . Three random specimens from each xenograft sample were subjected to CD34, mCherry and lectin (i.v.) staining. Lectin+/mCherry+ lumens stand for VM vessels and CD34+ lumens stand for regular endothelium-based vessels. ImageJ software was used for vessel density quantification.
All statistical analyses in this study were performed using Prism 8.0 (GraphPad Software Inc., USA) and R software. Data were expressed as mean ± SD. Sample size for each study was determined based on literature documentation of similar well-characterized experiments. Statistical significance was assessed by Student’s t-test or one-way ANOVA with Bonferroni correction for multiple comparisons. P value smaller than 0.05 was considered statistically significant. Statistical outlier analysis was calculated using the GraphPad Outlier calculator. Those significant outliers were excluded from data analysis.
All data generated during this study are included either in the main article or in the supplementary information files.
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This work was supported by the National Natural Science Foundation of China (No. 82002631 and No. 82072762).
The authors declare no competing interests.
Ethics approval and consent to participate
Animal study was performed with the permission of the Animal Care and Use Committee of Southern Medical University. And study protocol and informed consent were approved by the Ethical Committee of Zhujiang Hospital.
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Liu, Y., Xu, X., Zhang, Y. et al. Paradoxical role of β8 integrin on angiogenesis and vasculogenic mimicry in glioblastoma. Cell Death Dis 13, 536 (2022). https://doi.org/10.1038/s41419-022-04959-7