Lactate metabolism was increased in glioma and positively correlated with M2-type TAM infiltration

Due to the Warburg effect, most solid tumors exhibit an upregulation in lactate metabolism, which leads to the acidification of the tumor microenvironment (TME). In glioma, lactate production is primarily mediated by lactate dehydrogenase A (LDHA), while the efflux is mainly facilitated by monocarboxylate transporter 1 (MCT1) [21,22,23,24,25].

To evaluate the levels of lactate metabolism in glioma, we analyzed the mRNA levels of LDHA and MCT1 (referred as SLC16A1 at the mRNA level) using bulk RNA-seq data from the TCGA and CGGA datasets. Our analysis revealed that the expression of LDHA in glioma was significantly elevated compared to normal tissues (Figs. 1A & S1A). Furthermore, the expression levels increased in tandem with the higher WHO grade of glioma. Kaplan–Meier analysis results revealed that high LDHA expression correlated with poor overall survival (OS) among glioma patients in both TCGA and CGGA datasets (Figs. 1B & S1A). Similar results were observed for the analysis of SLC16A1 (Figs. 1A & S1A). Subsequently, we assessed the protein levels of LDHA and MCT1 using clinical samples through western blot analysis. Consistent with the bioinformatics analysis, the results indicated increased protein expressions of LDHA and MCT1 in gliomas compared to peri-tumorous normal tissues (Peri-NT), with notably higher levels in glioblastomas (GBM) compared to low-grade gliomas (LGG) (Fig. 1C).

Fig. 1

Lactate metabolism was increased in glioma and positively correlated with M2-type TAM infiltration. A Different mRNA expression levels of LDHA or SLC16A1, between GBM and Normal group, or among grade of gliomas in TCGA dataset. B Kaplan–Meier survival curves revealed the correlation between LDHA or SLC16A1 mRNA expression and survival of glioma patients in the TCGA dataset. C Western blot analysis of LDHA and MCT1 protein expression in clinical samples. D Correlation analysis between the expressions of LDHA or SLC16A1 and CD68 or CD163, respectively, using bulk RNA-seq data from the TCGA dataset. E Representative pictures for immunofluorescence staining of clinical tissues for LDHA, MCT1, CD68 and CD163. Cell nuclei were counterstained with DAPI. Data are presented as the Mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

To investigate the relationship between lactate levels and macrophage infiltration, we performed correlation analysis involving lactate metabolism markers LDHA (for lactate production) and SLC16A1 (for lactate efflux), along with macrophages markers CD68 (for total macrophage infiltration) and CD163 (for M2-macrophage infiltration) using bulk RNA-seq data from the TCGA and CGGA datasets. The results demonstrated a positive correlation between the expressions of LDHA or SLC16A1 and CD68 or CD163, respectively (Figs. 1D & S1B). The correlation analysis in GBM dataset was also shown in Figure S1. Additionally, we further assessed the histological expressions of LDHA, MCT1, CD68 and CD163 by tissue immunofluorescence staining. In GBM tissue slides, we observed elevated expressions of LDHA and MCT1, along with conspicuous infiltrations of CD68+SOX2− TAM and CD163+SOX2− M2-TAM. In contrast, very weak or even no signals were observed in Peri-NT and LGG slides (Figs. 1E & S1E).

In summary, lactate metabolism (including production and efflux) was heightened in glioma, particularly in GBM. High levels of lactate metabolism indicated unfavorable survival outcomes for glioma patients. Furthermore, the level of lactate metabolism was positively correlated with M2-subtype TAM infiltration.

Tumor cells-derived lactate induced tumor-associated macrophages towards M2-polarization in glioma

To investigate the relationship between lactate derived from tumor cells and tumor-associated macrophages, we collected the conditioned medium (CM) from glioma cells, and employed it to stimulate THP1-differentiated M0-macrophage. It has been documented that PMA treatment could induce differentiation of human monocyte line THP-1 into M0-macrophage [32, 33]. Firstly, we detected lactate concentrations in the cultural supernatants from various glioma cell lines U87, U251, and three cases primary GBM cells. After cultured in serum-free medium for 48 h, the lactate concentrations in the supernatants from glioma cells were detected as range from 6.70 mmol/L to 11.67 mmol/L, in stark contrast to the fresh medium (Fig. 2A). By utilizing the concentration gradient of tumor cells, we established lactate concentration gradient of CMs from U87 (2.68, 5.77 and 8.27 mmol/L), U251 (3.13, 6.23 and 10.17 mmol/L), and GBM3 cells (2.90, 6.033 and 10.53 mmol/L) which were subsequently used to treat M0-macrophage (Figs. 2B & S2A). As illustrated in Figs. 2C & S2B, the treatment with CMs from glioma cells resulted in elevated expressions of M2-macrophage markers CD206 and CD163, while showing no significant alterations in the M1-macrophage markers CD80 and CD86.

Fig. 2

Tumor cells-derived lactate induced tumor-associated macrophages towards M2-polarization in glioma. A Lactate concentrations in CMs from glioma cell lines U87, U251, and three cases primary GBM cells. B Lactate concentration gradient in CMs from U87 and U251 cells. C Quantification of CD206, CD163, CD80 and CD86 mRNA expression in THP1- differentiated macrophages treated with CMs from U87 and U251 cells for 48 h. D Representative pictures for immunofluorescence staining for CD68, CD206 and DAPI of macrophages following stimulation with CMs from U87 or U251 cells for 48 h. E Lactate concentrations in CMs from U87 or U251 cells treated with SO or CHC. F Quantification of CD206 and CD163 mRNA expression in macrophages stimulated with CMs from U87 and U251 cells treated with or without SO and CHC. G Representative pictures for immunofluorescence staining for CD68, CD206 and DAPI of macrophages following stimulation with CMs from U87 or U251 treated with or without SO and CHC. Data are presented as the Mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

Moreover, previous studies have demonstrated that alterations in cell morphology offer a valuable means for evaluating the polarized phenotype of macrophages. Typically, M2-type TAMs exhibit a stretched and elongated morphology [18, 34]. In line with these observations, we noted the stretched and elongated morphology of macrophages after exposure to CMs from glioma cells (Figs. 2D & S2C). Alongside these morphological shifts, CMs from glioma cells also enhanced the expression of surface marker CD206 (Fig. 2D), instead of CD86 (Fig. S2D), indicating a shift towards M2-like polarization, evidenced by immunofluorescent staining.

To underscore the pivotal role of lactate in mediating the polarized effects of macrophages, we treated glioma cells with an LDHA inhibitor, sodium oxamate (SO), to inhibit lactate production, and an MCT inhibitor, α-cyano-4-hydroxycinnamate (CHC), to reduce lactate efflux, respectively [15, 21, 24, 35]. These treatments significantly reduced lactate levels in the supernatants of glioma cells (Figs. 2E & S2E). Subsequently, we stimulated M0-macrophages with CMs from glioma cells that had been subjected to treatment with or without SO and CHC. The reduced lactate levels in CMs led to the absence of upregulated M2-macrophage markers CD206 and CD163 (Figs. 2F & S2F-G). Additionally, as a positive control, we treated M0-macrophages with lactate (10 mM) alone, which also substantially increased the expressions of CD206 and CD163, as determined by qPCR. Similarly, akin to the molecular markers, stimulation with lactate in isolation or CMs from glioma cells induced macrophages to adopt the elongated and stretched morphology, which was not observed when lactate levels in CMs were downregulated using SO or CHC (Figs. 2G & S2H).

Overall, these results demonstrated that the lactate derived from the glioma cells drives TAMs towards an M2-polarized phenotype, thereby remodeling the TME in gliomas.

Lactate-stimulated M2-TAMs promoted glioma cells proliferation, migration, invasion, and mesenchymal transition

The M2-subtype tumor-associated macrophages (M2-TAMs) have been reported for its role in driving the malignant progression of tumor cells, including proliferation, migration, and invasion. In this study, we sought to investigate the effects of lactate-stimulated M2-macrophages on the malignant behaviors of glioma cells.

Cell proliferation was assessed through both the CCK8 assay and the clone formation assay. We collected conditioned media from various macrophage treatments as described above, to examine their effects on the glioma cells. In comparison with the CMs from M0-macrophages, CMs from the lactate-stimulated M2-macrophages significantly increased the proliferation of glioma cells (Figs. 3A-B & S3A-C). Conversely, cell proliferation declined when lactate levels were reduced by SO and CHC. For migration and invasion, we conducted wound-healing migration and Transwell assays without Matrigel to assess the migratory capacity of glioma cells, and Transwell assays with Matrigel to evaluate invasion. CMs from lactate-treated macrophages notably boosted the motility of glioma cells, evidenced by a reduced scratch area and an increased number of migrating cells (Figs. 3C-D & S3D-G). In contrast, the migration and invasion capabilities of tumor cells were attenuated when the macrophages were stimulated with decline lactate levels. The molecular characteristics of MES subtype consistently indicate a more aggressive tumor state. The PN-to MES transition is recognized as the hallmark of malignant tumor progression, characterized by the upregulation of mesenchymal markers N-cadherin and Vimentin, and the downregulation of epithelial marker E-cadherin [6, 36, 37]. In the final analysis, we detected the expressions of proteins reflecting the malignance at the molecular levels. The expressions of N-cadherin and Vimentin were significantly upregulated by the treatment with lactate-stimulated macrophages, accompanied by the downregulation of E-cadherin. These expressional changes were reversed while the lactate levels were decreased (Figs. 3E & S3H-I).

Fig. 3

Lactate-stimulated M2-TAMs promoted glioma cells proliferation, migration, invasion, and mesenchymal transition. A-B Cell proliferation of U87 and U251 cells stimulated with conditioned media (CMs) from various pre-treated macrophages was assessed using the CCK-8 assay (A) and the clone formation assay (B-C). C-D Cell migration and invasion of U87 and U251 cells stimulated with CMs from various pre-treated macrophages were evaluated through wound healing assays (C) and Transwell assays (D). E Western blot analysis was performed to determine the expression levels of Vimentin, N-cadherin, and E-cadherin proteins in U87 and U251 cells stimulated with CMs from various pre-treated macrophages. Data are presented as the Mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

Above all, these results collectively demonstrated that lactate-stimulated M2-TAMs promote the malignant progression of glioma cells, influencing their proliferation, migration, and invasion, as well as the molecular characteristics of the PN-to-MES transition.

GPR65 on TAMs serves as the key sensor of lactate-stimulation, fueling glioma cells malignant progression

In the intricate landscape of the tumor microenvironment, the signal transduction triggered by lactate stimulation mainly relays on the lactate receptor HCAR1 (GPR81), and proton-sensing G-protein coupled receptors (GPRs), including GPR4, TDAG8 (GPR65), OGR1 (GPR68), and G2A (GPR132) [38, 39]. Each of these receptors exhibits distinct cellular distribution patterns and can activate various signaling pathways contingent upon specific cell types.

To investigate which receptor is responsible for the tumor-promoting effects of lactate-stimulation on TAMs in glioma, we analyzed the differential expression and cell distribution of these receptors. Our analysis of glioma datasets downloaded from The Cancer Genome Atlas (TCGA) unveiled significant differences in expression patterns between normal and tumor tissues, specifically an upregulation of GPR65, and a downregulation of GPR68 (Fig. 4A). Conversely, GPR4, GPR81 and GPR132 did not exhibit statistically significant differences (Fig. S4A-D). Additionally, an analysis of cell distribution in glioma using single-cell RNA-seq datasets from UCSC Cell Browser (https://gbm.cells.ucsc.edu) [30, 31] and Single Cell Portal (https://singlecell.broadinstitute.org, GSE131928) [11], revealed that only GPR65 was predominantly expressed on macrophages, while the others showed low expression level or ambiguous cell localizations (Fig. 4D-E & S4E-N). Furthermore, Kaplan–Meier analysis based on TCGA-dataset indicated that high expression of GPR65correlates with poor overall survival among glioma patients (Fig. 4B). This finding was corroborated by the higher expression of GPR65 in GBM samples than in peri-tumorous normal tissues (Fig. 4C). To ascertain the cellular localization of GPR65 in clinical samples, we assessed the co-expression of GPR65, the pan-macrophage marker CD68, and the M2-macrophage marker CD206 in 15 GBM specimens. Immunofluorescence analysis revealed substantial co-localization of GPR65 signals (42%-88%) with the macrophage marker CD68 (Fig. 4F). Furthermore, it was observed that GPR65 was co-expressed in 26%-77% of CD68 + TAMs and 69%-87% of CD206 + TAMs across different samples (Fig. 4F). In summary, these results collectively underscore that among the receptors implicated in lactate signaling, GPR65 is prominently expressed in GBM, with its primary localization on tumor-associated macrophages, especially M2-TAMs.

Fig. 4

GPR65 was the principal receptor mediating lactate stimulation in TAMs in glioma. A Differential mRNA expression levels of GPR65 among GBM, LGG, and Normal groups in TCGA dataset. B Kaplan–Meier survival curves for correlation between GPR65 mRNA expression and survival of glioma patients in the TCGA dataset. C Western blot analysis and quantification of GPR65 protein expression in clinical samples. D-E Cellular distribution of GPR65 in glioma using single-cell RNA-seq datasets from UCSC Cell Browser (https://gbm.cells.ucsc.edu) and Single Cell Portal (https://singlecell.broadinstitute.org, GSE131928). F Representative pictures and quantification for immunofluorescence staining of clinical tissues for GPR65, and / CD68 or CD206. Data are presented as the Mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

To validate whether GPR65 on TAMs indeed functions as the critical sensor for lactate signaling in the TME to drive tumor progression, we transfected TAMs with either shNC (negative control shRNA) or shGPR65 (GPR65-silencing shRNA). The efficacy of transfection was confirmed through qPCR and Western blot analysis (Fig. 5A-B). Subsequently, we collected conditioned media from these transfected TAMs, with or without lactate-stimulation, to treat glioma cells. The results revealed that the proliferative effects of lactate-stimulated shNC-TAMs on glioma cells were attenuated by GPR65 knockdown (Figs. 5C & S5A-C). Furthermore, GPR65 silencing alleviated the enhanced cell migration and invasion induced by lactate-stimulated shNC-TAMs in glioma cells (Figs. 5E-F & S5D-G). Consistent with the cellular behaviors, molecular changes related to mesenchymal transition were also reversed, characterized by decreased expression of Vimentin and N-cadherin, and increased expression of E-cadherin in glioma cells when GPR65 was inhibited (Figs. 5G & S5H-I). Together, these data collectively demonstrated that the tumor-promoting effects of TAMs were counteracted by GPR65-silence on TAMs.

Fig. 5

GPR65 on TAMs is essential for lactate-stimulation to promote glioma cells malignant progression. A RT-PCR and (B) Western blot analysis of GPR65 expression in macrophages transfected with shNC or shGPR65. C-D CCK8 (C) and clone formation assays (D) were performed to assess cell proliferation of U87 and U251 cells upon stimulation with CMs from TAMs transfected with shNC or shGPR65 following lactate stimulation. E–F Wound healing (E) and transwell assays (F) were conducted to evaluate cell migration and invasion of U87 and U251 cells upon stimulation with CMs from TAMs transfected with shNC or shGPR65 following lactate stimulation. G Western blot analysis the protein expression levels of Vimentin, N-cadherin, and E-cadherin in U87 and U251 cells upon stimulation with CMs from TAMs transfected with shNC or shGPR65 following lactate stimulation. Data are presented as the Mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

In conclusion, these findings collectively demonstrate that GPR65 on TAMs serves as the key sensor for lactate signaling in the tumor microenvironment, driving the malignant progression and mesenchymal transition of glioma cells.

GPR65 on TAMs promoted glioma cells malignant progression via HMGB1 secretion

To gain deeper insights into the mechanism of lactate-induced signaling through GPR65 on TAMs and its role in enhancing the malignant progression of glioma, we analyzed six potential cytokines and chemokines (CCL2, CCL5, CCL17, CCL18, HMGB1, and TGFB1) derived from TAMs, which were previously reported to be associated with lactate levels or pro-tumorigenic activities [14, 15, 18, 33, 40]. We found that the levels of HMGB1 was obviously elevated under lactate stimulation, which were also dropped down when GPR65 on TAMs was silenced (Fig. 6A). To substantiate this observation, we further found that the expressions of HMGB1 were increased along with the lactate concentration gradient at both mRNA and protein levels (Fig. 6B-C). Furthermore, the concentrations of HMGB1 in macrophage supernatants increased in response to lactate stimulation (Fig. 6D). However, the elevation of HMGB1 was inhibited by GPR65-silencing, as confirmed by western blot and ELISA analysis (Fig. 6E-F). Based on these findings, HMGB1 emerged as a potential candidate for validating the pro-tumor effects of lactate-GPR65 signals on TAMs in glioma.

Fig. 6

Lactate-stimulation GPR65 on TAMs promoted glioma cells malignant progression via secreting HMGB1. A RT-PCR analysis of various potential TAM-derived chemokines and cytokines in PMA-treated THP1 cells transfected with shNC or shGPR65, with or without lactate stimulation. B RT-PCR and (C) Western blot analysis of HMGB1 expression in macrophages under lactate concentration gradient stimulations. D HMGB1 concentrations in CMs from macrophages under lactate concentration gradient stimulations. E Western blot analysis of HMGB1 expression in macrophages transfected with shNC or shGPR65, with or without lactate stimulation. F HMGB1 concentrations in CMs from macrophages transfected with shNC or shGPR65, with or without lactate stimulation. G-H CCK8 (G) and clone formation assays (H) were performed to assess cell proliferation of U87 and U251 cells stimulated with CMs from various pretreated macrophages with HMGB1 inhibitor or rHMGB1. I-J Wound healing (I) and transwell assays (J) were conducted to evaluate cell migration and invasion of U87 and U251 cells stimulated with CMs from various pretreated macrophages with HMGB1 inhibitor or rHMGB1. K Western blot analysis of Vimentin, N-cadherin, and E-cadherin protein expression levels of U87 and U251 cells stimulated with CMs from various pretreated macrophages with HMGB1 inhibitor or rHMGB1. Data is presented as the Mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. shNC group. nsp > 0.05, and ###p < 0.001 vs. shGPR65 group

To elucidate the pivotal role of HMGB1 in CMs derived from lactate-stimulated TAMs, we introduced recombinant HMGB1 (rHMGB1) as a supplement into CMs from shGPR65 TAMs to stimulate tumor cells. Concurrently, we employed a selective inhibitor, glycyrrhizin, to reduce HMGB1 levels in CMs from shNC-TAMs. Consequently, the inhibition of HMGB1 levels in CM from shNC TAMs using glycyrrhizin led to a notable reduction of tumor cell proliferation, while the exogenous addition of rHMGB1 rescued the enhanced growth of glioma cells in GPR65-silenced TAMs (Figs. 6G-H & S6A-C). Similarly, rHMGB1 promoted the migration and invasion of glioma cells, while treatment with glycyrrhizin alleviated these processes (Figs. 6H-I & S6D-G). The tendencies of mesenchymal transition induced by rHMGB1 was also mitigated by glycyrrhizin (Figs. 6K & S6H-I). Summarize all, the tumor-promoting effects of lactate stimulated TAMs were mediated by GPR65 through the secretion of HMGB1.

GPR65 on TAMs activated by lactate stimulates HMGB1 secretion via the cAMP/PKA/CREB signaling pathway

GPR65, as a proton-sensing G-protein coupled receptors, typically triggers an increase in cAMP upon stimulation, subsequently activating the downstream PKA-CREB (PKA: protein kinase A; CREB: cAMP-response element binding protein) signaling pathway [41, 42]. To investigate the signaling molecules responsible for lactate-mediated HMGB1 secretion, we examined changes in this pathway.

We observed an increase in cAMP production with rising lactate levels (Fig. 7A). Furthermore, downstream PKA expression increased, enhancing the phosphorylation of CREB (pCREB), a recognized indicator of cAMP-PKA activation (Fig. 7B). However, GPR65 silencing reduced cAMP production and concurrently inhibited PKA and pCREB expression (Fig. 7C-D). The generation of cAMP is mediated by adenylate cyclase (AC), which catalyzes ATP to release a pyrophosphate [43]. To elucidate the roles of cAMP and PKA involving in HMGB1 secretion regulated by GPR65, we employed SQ22536 to reduce cAMP levels by inhibiting AC activity and H89 to inhibit PKA activation (Fig. 7E). Treatment with SQ22536 and H89 respectively resulted in a downregulation of HMGB1 expression, with both PKA and pCREB expressions being suppressed, while cAMP levels were only decreased by SQ22536, not H89 (Fig. 7F-G). This illustrated that PKA functions as a downstream molecule of cAMP. Additionally, an AC activator, Forskolin, was used to re-activate AC in GPR65-silenced cells for further validation. When AC was re-activated in GPR65-silenced TAMs by Forskolin, cAMP levels increased, along with the phosphorylation of CREB increased, and HMGB1 production was upregulated (Fig. 7H-I).

Fig. 7

GPR65 on TAMs activated by lactate stimulates HMGB1 secretion via the cAMP/PKA/CREB signaling pathway. A Levels of cAMP production in macrophages under lactate concentration gradient stimulations. B Western blot analysis of expressions HMGB1, PKA and pCREB in macrophages under lactate concentration gradient stimulations. C Levels of cAMP production in macrophages transfected with shNC or shGPR65, with or without lactate stimulation. D Western blot analysis of expressions HMGB1, PKA and pCREB in macrophages transfected with shNC or shGPR65, with or without lactate stimulation. E Levels of cAMP production in macrophages treated with SQ22536 and H89, with lactate stimulation. F Western blot analysis of expressions HMGB1, PKA and pCREB in macrophages treated with SQ22536 with lactate stimulation. G Western blot analysis of expressions HMGB1, PKA and pCREB in macrophages treated with H89. H Levels of cAMP production in macrophages treated with Forskolin, with lactate stimulation. I Western blot analysis of expressions HMGB1, PKA and pCREB in macrophages treated with Forskolin, with lactate stimulation. Data are presented as the Mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

In summary, GPR65 on tumor-associated macrophages responds to lactate stimulation, leading to HMGB1 secretion through the cAMP-PKA-CREB signaling pathway.

GPR65 knockdown or HMGB1 inhibition mitigates malignant progression of glioma in vivo

To assess the impact of inhibiting GPR65 or HMGB1 on tumor progression in an in vivo context, we subcutaneously injected U87 cells, with or without tumor-associated macrophages (TAMs), into nude mice to establish subcutaneous xenograft tumors. At 28 days post-injection, it became evident that the group with shNC-TAM (group II) exhibited larger tumor volumes compared to the group injected with tumor cells alone (group I). Conversely, GPR65 knockdown (group III) significantly attenuated tumor growth compared to the shNC-TAM group (group II) (Fig. 8A). Furthermore, at 42 days post-injection, the tumor volume was notably reduced in mice treated with the HMGB1 inhibitor glycyrrhizin (group IV, U87 + shNC + glycyrrhizin) compared to the untreated group (group II, U87 + shNC) (Fig. 8A-B). The corresponding tumor weights at 42 days post-injection followed a similar trend (Fig. 8C).

Fig. 8

GPR65 knockdown or HMGB1 inhibition mitigates malignant progression of glioma in vivo. A In a subcutaneous xenograft tumor model, the tumor volumes were measured and monitored every 7 days from each group of mice. (n = 4/group). B The pictures of corresponding subcutaneous xenograft tumors dissected from each group mice at 42 days post-injection. C Tumor weights from each group at 42 days post-injection. D Kaplan–Meier survival curve of each group mice of orthotopic glioma model (n = 6/group). E Representative images of IHC staining of HMGB1, Ki-67, Vimentin, N-cadherin, and E-cadherin in subcutaneous xenograft tumor. Data are presented as the Mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

Additionally, we conducted an orthotopic tumor model to assess the survival of tumor-bearing mice. The results demonstrated that the group with TAMs (group II) exhibited shorter survival compared to the group without TAMs. However, both GPR65 knockdown and HMGB1 inhibition via intraventricular injection of glycyrrhizin extended the survival of the mice (Fig. 8D).

To further elucidate these findings, we conducted immunohistochemistry staining to assess the expression of HMGB1, Ki-67, and mesenchymal transition markers, including Vimentin, N-cadherin, and E-cadherin. TAMs were found to enhance the expressions of HMGB1, Ki-67, Vimentin, and N-cadherin while reducing E-cadherin expression. Conversely, inhibiting GPR65 with shRNA or employing HMGB1 inhibition with glycyrrhizin effectively counteracted these tendencies (Fig. 8E).

In summary, our findings suggest that TAMs promote glioma progression and mesenchymal transition in an in vivo setting. However, the inhibition of GPR65 or HMGB1 alleviates these effects.