Study Of Glioma Malignant Biological Behavior Mediated By TGF-β In Tumor Microenvironment

Part I Galunisertib inhibits glioma vasculogenic mimicry formation induced by astrocytesIntroductionGliomas are the most lethal intracranial tumors due to their high capacity of proliferation and invasion into healthy brain tissue, which preclude complete surgical resection 1. As glioma invasion and proliferation rely on angiogenesis2, the potential of anti-angiogenic therapy to inhibit glioma progression has been investigated3. However, recent studies showed that although anti-angiogenic therapy might delay tumor progression, it failed to prolong long-term surviva14,5. What is worse, some evidence suggests that anti-angiogenic therapy might elevate the risk of tumor adaptation and invasion in hypoxic and ischemic environments6,7. First introduced by Maniotis et al. in 19998, vasculogenic mimicry is characterized by tumor cells forming tubular structures that transport erythrocytes and plasma in order to nourish tumors, independent of endothelial blood vessels. These structures have also been found in other types of tumors including breast9, lung10, and ovarianll. As in gliomas, vasculogenic mimicry was also detected predominantly in high-grade medulloblastomas and there was a significant association between vasculogenic mimicry and medulloblastoma grade12. Researchers have suggested that vasculogenic mimicry enabled gliomas to survive in hypoxic and ischemic environments 13, and thus explain the limitations of anti-angiogenic therapy14. Apart from anti-angiogenic therapy, anti-vasculogenic mimicry therapy should be considered for treatment of gliomasl5.However, investigation of the mechanisms of vasculogenic mimicry stimulation and inhibition are required.In the brain, hypoxia, ischemia, and the presence of glioma cells cause chronic inflammation resulting in recruitment of cell types such as astrocytes and microglia; reactive astrocytes often in turn surround gliomas and brain metastases16,17. Although the physiological function of astrocytes is to protect neurons18, they also seem to enhance tumor cell survival signaling pathwaysl9 and increase their resistance to chemotherapy. In addition, reactive astrocytes express numerous genes that support tumor cell survival in a paracrine manner20, where hypertrophic astrocytes secrete chemokines that promote tumor survival and invasion21,22. Specifically, reactive astrocytes have been shown to secrete TGF-β,which increases tumor cell proliferation, as well as connective tissue growth factor and metalloproteases, facilitating glioma invasion23.Galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF- RI, is one of the few TGF-β pathway inhibitors currently under clinical investigation in glioma patients24. In recent clinical trials24,25, galunisertib improved glioma prognosis. However, in vitro experiments have not been able to explain its mechanism of action and the relationship between galunisertib and astrocytes has not yet been reported. Our research uncovers the effects of galunisertib on gliomas, particularly on vasculogenic mimicry. Our results also show the potential influence of galunisertib on autophagy, an important process responsible for tumor metabolism and invasion. These findings suggest a new strategy for discovery of novel vasculogenic mimicry therapeuticsObjectiveThe main aims of our study are to expands our understanding of the role of astrocytes in gliomas and demonstrates that galunisertib inhibits glioma vasculogenic mimicry induced by astrocytes, thus providing potential target for curing glioma.Methods and results1. Human astrocytes promote vasculogenic mimicry in glioma cell line A172 As shown in results, astrocytes stained with glial fibrillary acidic protein (GFAP) were more abundant in glioblastomas (GBM) than in normal brain tissue. The astrocytes in the GBM clustered, forming a border encompassing the tumors, which differs from the grid distribution of normal brain tissue. And in Brdu proliferation assay, significantly more Brdu+ cells were detected in the NHA/A172 co-culture system than in the A172 cells cultured alone. Cell migration was also increased in the co-culture system compared to A172 cells alone Next we studied the role of astrocytes in vasculogenic mimicry. The NHA/A172 co-culture group demonstrated more rapid tube formation.2. Astrocyte-induced vasculogenic mimicry required TGF-β1 secretion and was inhibited by galunisertibWe explored whether the anti-angiogenesis drug bevacizumab could inhibit astrocyte-induced vasculogenic mimicry. Not surprisingly, there was no significant difference in tube formation after bevacizumab treatment. To screen potential target cytokines, a suspension array technique was employed. We found that TGF-β1 was highly expressed in the supernatant of NHA/A172 co-culture system and the suspension array results were confirmed by qRT-PCR and ELISA. Both mRNA levels in two experimental NHA groups. Using the in vitro tube formation assay, we observed that A172 cells treated with TGF-β1 demonstrated elevated tube formation compared to untreated A172 cells.3. Galunisertib inhibited SMa and VE-cadherin expression in the NHA/A172 co-culture system via Akt and Flk pathwaysTo further examine the roles of astrocytes and galunisertib in vasculogenic mimicry and to explore the potential mechanism, we studied molecular markers previously identified by Hendrix and Seftor et al. Western blot analysis revealed that co-culture with astrocytes elevated SMa, VE-cadherin, matrix metalloproteinase-2 (MMP-2), and MMP-9 protein expression in A172 cells. Meanwhile, treating the NHA/A172 cells with galunisertib dramatically down-regulated expression of these markers in A172 cells. Immunofluorescence also showed elevated SMa and VE-cadherin expression in DMSO-treated NHA/A172 cells and not in galunisertib-treated NHA/A172 cells. We also found that Akt and Flk phosphorylation in the galunisertib group varied dramatically compared to the NHA/A172 group. In addition, LC3B expression level was also changed which would be further discussed in the following part. To identify the precise role of these signaling pathways, we treated A172 and NHA/A172 cells with LY294002 (a PI3K-Akt pathway inhibitor), ZD6474 (a Flk pathway inhibitor), and SP600125 (a JNK pathway inhibitor). Using Western Blot, we found decreased SMa and VE-cadherin expression after LY294002 or ZD6474 treatment with A172 alone and A172/NHA control groups.4. Galunisertib impaired glioma autophagy, which in turn inhibited vasculogenic mimicryWe investigated whether galunisertib (TGF-β inhibitor) inhibits vasculogenic mimicry by regulating glioma autophagy. Immunofluorescence revealed that galunisertib did suppress glioma autophagy. In addition, the number of punctate LC3B structures, a standard marker of autophagy, was significantly decreased after galunisertib treatment. Transmission electron microscopy (TEM), the gold standard technique for identification of double-membrane autophagosomes, was then conducted. We found that galunisertib-treated cells had fewer autophagosomes. We thus examined the effect of galunisertib on glioma mitochondrial metabolism using seahorse XFe24. Results showed that NHA/A172 co-cultured cells had an elevated oxygen consumption rate (OCR) indicating more active ATP production and that galunisertib significantly lowered OCR. Recognizing the role of autophagy in vasculogenic mimicry, we hypothesized that autophagy inhibitors, such as chloroquine (CQ), may inhibit vasculogenic mimicry. As a result, the tube formation assay revealed that CQ mirrored the effect of galunisertib.5. Galunisertib inhibited tumor growth and vasculogenic mimicry in a xenograft tumor modelTo verify the in vitro findings, we established a xenograft tumor model using the A172 cell line. Mice were randomly assigned to two groups. No major side effects was observed throughout the study, galunisertib significantly reduced average tumor weight and volume compared to controls treated with normal tumor cells (NTC) alone. Importantly, we observed a pronounced decrease in tumor vasculogenic mimicry in the galunisertib-treated xenografts noted by the percentage of PAS"CD34+/PAS+CD34+cells identified using immunohistochemistry. Consistent with the in vitro observations, these data showed that galunisertib inhibited vasculogenic mimicry tube formation and proliferation in a xenograft tumor model.Conclusion1. Human astrocytes promote vasculogenic mimicry in glioma cell line A1722. Astrocyte-induced vasculogenic mimicry required TGF-β1 secretion and was inhibited by galunisertib3. Galunisertib inhibited SMa and VE-cadherin expression in the NHA/A172 co-culture system via Akt and Flk pathways4. Galunisertib impaired glioma autophagy, which in turn inhibited vasculogenic mimicry5. Galunisertib inhibited tumor growth and vasculogenic mimicry in a xenograft tumor modelPart Ⅱ Effects of human astrocytes on migration and invasion property of glioma cellsIntroductionGlioblastoma multiforme (GBM) is characterized by lethal aggressiveness and patients with GBM are in urgent need for new therapeutic avenues to improve quality of life. Current studies on tumor invasion focused on roles of cytokines in tumor microenvironment and numerous evidence suggests that TGF-β2 is abundant in glioma microenvironment and vital for glioma invasion. Autopagy is also emerging as a critical factor in aggressive behaviors of cancer cells; however, the relationship between TGF-β2 and autophagy in glioma has been poorly understood. Here we demonstrated that TGF-β2 activated autophagy in human glioma cell lines and knockdown of Smad2 or inhibition of c-Jun NH2-terminal kinase, attenuated TGF-β2-induced autophagy. TGF-β2-induced autophagy is important for glioma invasion due to the alteration of epithelial-mesenchymal transition and metabolism conversion, particularly influencing mitochondria trafficking and membrane potential (△Ψm). Autopaghy also initiated a feedback on TGF-β2 in glioma by keeping its autocrine loop and affecting Smad2/3/7 expression. A xenograft model provided additional confirmation on combination of TGF-p inhibitor (Galunisertib) and autophagy inhibitor (CQ) to better "turn off’tumor growth. Finally, our findings elucidated a potential mechanism of autophagy-associated glioma invasion and shed new light on the management of GBM by combining Galunisertib with CQObjectiveOur study was to explore the relationship of TGF-β and autophagy and their effect on glioma invasion and migration, thuw to provide potential target for glioma treatment and prolong the survival time of patients. Methods and results1. TGF-P2 and LC3B are highly expressed in glioma and correlated with each other indicating poor prognosis in glioma patients.By applying immunochemistry on samples with different pathologic grades obtained from patients in Qilu Hospital, we found that the percentage of LC3B and TGF-β2 positive cells was associated with the pathologic grade. Spearman correlation test also showed LC3B and TGF-β2 were both identified to be associated with the pathologic grades Then immunofluorescence images also indicated that these two molecules were co-localized in glioma tissues. We verified these conclusions in primary tumor samples and qRT-PCR analysis showed similar results in normal brain tissues and glioma patients We then added the survival time index and used the long-rank test to analyze. Data showed that high TGF-β2 expression indicated poor prognosis and short survival time in glioma patients.2. TGF-β2 induces autophagy in human glioma cell lines in a time and dose-dependent manner.To investigate the exact relationship between TGF-β2 and LC3B in glioma, we treated the tumor cells with a TGF-β2 dose gradient ranging from 0.1 ng/ml to 10 ng/ml, and after 24 h, Western blotting was used to detect autophagy levels. To determine the proper time required for TGF-P stimulation, we set a gradient time course and also tested the conversion ratio by western blot. Our results indicated that 24 h was appropriate to induce glioma autophagy. To confirm this phenomenon, we applied a lentiviral system and generated stably expressing GFP-LC3 cells to monitor autophagy (TGF-β2,10 ng/ml,24h). As transmission electron microscopy is the gold standard for identifying autophagosome double-membrane structure, we used transmission electron microscopy and found that glioma autophagosomes increased after TGF-β2 treatment Effects of 3-MA or Baf on autophagy flux mediated by TGF-β were also determined. Glioma cells were incubated with 3-MA (10 mmol/L) or Baf (10 nmol/L) during the treatment with TGF-β2 (Fig.2e) and results indicated that Baf could effectively inhibit TGF-β induced autophagy except for 3-MA. According to the results of a microarray that compared the control and TGF-β2 treatment groups, the mRNA levels of the autophagy related genes BECN1, ATG5, ATG7 were also up-regulated after TGF-P2 treatment.3. TGF-β2 induced autophagy is related to glioma invasion and migration.To explore the biological meaning of TGF-β2-induced autophagy in oncogenesis, we first introduced a stable siRNA expression system targeting the essential autophagy gene ATG5 and ATG7. Efficiency of siRNA was examined by Western blot. We used methods involving CCK-8 and BrdU proliferation assays to assess cell proliferation and observed no obvious effect after TGF-β2 treatment in normal tumor cells (NTC) or autophagy-deficient tumor cells. And we also carried out wound healing assays and Transwell assay in U251, T98, and U87 cell lines. In stable siATG5 and siATG7 tumor cells, autophagy impaired groups exhibited delayed wound closure compared to scramble group tumor cells when exposed to TGF-β2 stimulation.4. TGF-β2 induced EMT in glioma cells could be blocked by suppressing autophagyWe conducted qRT-PCR to determine the expression levels of MMPs in normal glioma cells and Baf-treated tumor cells with or without TGF-β2 stimulation. The Baf treatment down-regulated the mRNA expression of the mostly MMP family in cells exposed to TGF-β2, and in contrast control group with TGF-β2 showed increasing expression levels of most MMPs. We also used Western blotting to examine other EMT markers to assess the potential association between EMT, TGF-β2 and autophagy. N-cadherin, Vimentin, Slug2, and β-catenin increased in normal tumor cells (NTC) treated with TGF-β2 but decreased in autophagy-inhibited cells h. Besides immunofluorescence again demonstrated a direct change of Vimentin and N-cadherin between NTC and Baf-treated cells after treatment with TGF-β2.5. TGF-β2 and autophagy mediated energy reprogramming of glioma and mitochondria functionWe thus hypothesized that TGF-β2-induced autophagy is enhanced to generate more ATP and materials for invasion. First we introduced the MitoTracker Red probe to observe the mitochondrial morphology and localization.n our experiment, tubular form was found to be a more favorable form in the cell invasion apex and underwent more trafficking. TGF-β2-treated NTCs favored the tubular mitochondria. Then TGF-β group also showed an increased percentage of the polarized form and Baf group showed more perinuclear form in mitochondria position. Apart from above two factors, mitochondrial membrane potential (△ Ψ m) is also a vital factor for ATP generation. We used a JC-1 probe to detect the changes of △ Ψm in mitochondria. We observed red fluorescence increasing after TGF-P2 treatment in NTCs and green fluorescence enhancing in Baf treatment tumor cells though also treated with TGF-β2. This phenomenon was confirmed by flow cytometry. Results also indicated that the phosphorylation of Drpl and the expression of Opa3 and Mitofusionl was responsible for the effect. Besides the mitochondria changing, tumor cells were also characterized for aerobic glycolysis (Warburg-like metabolism). We thus recurred to XFe24 analysis (Seahorse) and found that TGF-β2 also altered metabolism program towards to aerobic glycolysis which resulted in a higher consumption rate of oxygen (OCAR) and an increased extracellular acidification (ECAR) to sustain ATP providing and then inhibiting autophagy could block this process6. Autophagy induction by TGF-β2 is mediated by c-Jun NH2-terminal kinase and the Smad pathway and in turn affects TGF-β2 synthesis and secretionWe next investigated the exact mechanism of the process. We used Western blotting to determine whether either of these pathways functioned in this process. Inhibiting the AKT-PI3K-mTOR pathway by 3-MA did not attenuate TGF-β2-induced autophagy. Thus, we assessed the mTOR-independent pathway using the c-Jun NH2-terminal kinase (JNK) pathway inhibitor SP600125. This treatment efficiently down-regulated the LC3B-Ⅱ level in TGF-β2 treated glioma cells. TGF-β signals also went through downstream Smad and non-Smad pathways. Therefore we examined whether the Smad pathway also participated in autophagy induction. After transient siRNA with siSmad2, the addition of TGF-β2 did not stimulate autophagy in glioma. We next explored how autophagy influenced TGF-β2. The qRT-PCR results demonstrated that enhancing autophagy by rapamycin increased the R-Smad expression levels (Smad2, Smad3) and decreased the I-Smad expression level (Smad7). LY21457299 is a clinical applied TGF-β inhibitors, however Western blotting demonstrated that LY2157299 had little effect on the endogenous TGF-β2 levels. After blocking autophagy flux with CQ or siATG5, the TGF-β2 level decreased. And when we added exogenous lactate in cultured tumor cells, results confirmed that lactate could stimulate TGF-β2 and MMP2 expression and EMT-related protein changes, however CQ, except for LY2157299, could inhibit this process.7. In vivo xenograft models demonstrated that treatment with combined TGF-β2 and autophagy inhibition provides more benefits than galunisertib single application.We used tumor xenograft models to test this finding and explore the clinical significance. After planting U87 cells in severe combined immune deficiency (SCID) mice. We assigned the animals to the following treatment groups:PBS group, LY2157299 group, CQ group and LY2157299 combined with CQ group. LY2157299 combined with CQ was the most favorable treatment for the mice with tumor xenografts according to the survival time and tumor volumes results monitored by 3 Tesla MRI. Samples were obtained from the SCID mice and stained with HE, images showed that brains of groups with PBS was infiltrated with tumor cells among the normal brain and tumors borders, while combination treatment with LY2157299 and CQ showed sharp and clear borders between normal tissues and tumor. With IHC, we could also observed a decreased expression level of TGF-β in LY2157299 treated groups (P=0.0476) and combination drugs groups showed the least expression level (P=0.0056).Conclusion1. TGF-β2 and LC3B are highly expressed in glioma and correlated with each other indicating poor prognosis in glioma patients.2. TGF-β2 induces autophagy in human glioma cell lines in a time and dose-dependent manner.3. TGF-β2 induced autophagy is related to glioma invasion and migration.4. TGF-β2 induced EMT in glioma cells could be blocked by suppressing autophagy.5. TGF-β2 and autophagy mediated energy reprogramming of glioma and mitochondria function.6. Autophagy induction by TGF-β2 is mediated by c-Jun NH2-terminal kinase and the Smad pathway and in turn affects TGF-β2 synthesis and secretion.7. In vivo xenograft models demonstrated that treatment with combined TGF-β2 and autophagy inhibition provides more benefits than galunisertib single application.