Glioblastoma cell glycolysis promotes macrophage migration. The metabolism signature and immune score in The Cancer Genome Atlas (TCGA) glioblastoma tumors have been defined based on gene expression data to infer the levels of tumor metabolism 30 and immune cell populations 31, respectively. To identify the potential connection between tumor metabolism and immunity that might influence glioblastoma tumor biology, the correlation analyses among the two signatures and patient survival were performed. We found that high tumor metabolism signature was correlated with poor outcomes (Fig. 1a) and correlated positively with immune score (Fig. 1b). Moreover, the metabolism signature was enriched in mesenchymal and IDH1-WT glioblastoma, but not related to the status of tumor recurrence, gender and MGMT promotor methylation (Extended Data Fig. 1a-e). These findings aligned with the Gene Set Enrichment Analysis (GSEA) on hallmark pathways, Gene Ontology Enrichment Analysis (GOEA) on the sub-ontologies of Biological Process, and KEGG Enrichment Analysis showing prominent representations of cytokine and chemokine signatures, immune response networks, and leukocyte and myeloid cell signatures in metabolism-high TCGA glioblastoma patient tumors compared to metabolism-low patient tumors (Table S1). To identify specific immune cells linked to tumor metabolism in glioblastoma, we audited the TCGA glioblastoma patient tumors for 18 types of immune cells using validated gene set signatures 3, 12, 13, 32, 33. As a result, macrophage and monocyte were identified as the top immune cell types correlating positively with the metabolism signature (Fig. 1c). Conversely, CD8+ activated T cells showed a negative correlation with the metabolism signature (Fig. 1c). Together, these findings suggest a connection between tumor metabolism and macrophage/monocyte infiltration in glioblastoma patient tumors.
Given the importance of macrophages in glioblastoma progression 8, we hypothesized that pharmacological inhibition of tumor metabolism-induced macrophage infiltration is a promising therapeutic strategy 5. We selected a cluster of 55 brain-penetrant small-molecule compounds with metabolic reprogramming functions (Table S2) and performed a screen focusing on macrophage migration using conditioned media (CM) from CT2A cells treated with or without these compounds at 10 µM. This screen resulted in identification of 24 compounds that significantly inhibited CT2A CM-induced macrophage migration (Fig. 1d and Extended Data Fig. 1f). Next, we performed a second round of screen with these 24 compounds at a lower concentration (5 µM) and found that 7 (stiripentol, lopinavir, ofloxacin, vorasidenib, IDH889, progesterone, and IOX4) of them impaired CT2A CM-induced macrophage migration (Fig. 1e and Extended Data Fig. 1g). Consistently, the LDH inhibitor stiripentol showed the strongest effect in these two rounds of screens, which led us hypothesize that glioblastoma cell glycolysis is essential for macrophage infiltration. To confirm it, we analyzed the single-cell RNA sequencing (scRNA-seq) data from 44 fragments of tumor tissues of 18 glioma patients, including 2 low-grade gliomas (LGG), 11 newly diagnosed glioblastoma (ndGBM), and 5 recurrent glioblastoma (rGBM) 34. Specifically, glioblastoma tumors containing tumor cells (Fig. 1f) and tumor-infiltrating myeloid cells (Fig. 1g) were analyzed. The glycolysis hallmark signature 35 was highly expressed in glioblastoma cells (Fig. 1h), which correlated positively with the abundance of macrophages and monocytes (Fig. 1i) and negatively with the level of microglia, but did not show a significant correlation with dendritic cells (DCs) (Extended Data Fig. 2a). Similarly, bioinformatics analyses in TCGA glioblastoma tumors demonstrated that high glycolysis signature correlated with increased immune score (Extended Data Fig. 2b); prominent representations of immune response networks, cytokine and chemokine signatures, as well as leukocyte and myeloid cell migration signatures (Table S3); and increased macrophages, monocytes, and to a lesser extent, DCs, and decreased microglia (Extended Data Fig. 2c). Finally, glycolysis signature, but not other metabolism-related hallmark signatures, was enriched in glioblastoma patient tumors compared to normal brain tissues (Extended Data Fig. 2d, e), and was increased in glioma cells of glioblastoma compared to LGG (Extended Data Fig. 2f).
To biologically validate the role of glycolysis in triggering macrophage infiltration, we treated mouse glioblastoma cells (e.g., CT2A and GL261) and glioblastoma patient-derived GSC272 with a glycolysis inhibitor 2-deoxy-D-glucose (2-DG) 21, which can inhibit glycolysis as shown by reduced extracellular acidification rate (ECAR) and lactate production (Extended Data Fig. 2g-j). As expected, CM from 2-DG-treated glioblastoma cells and GSCs reduced the migration of macrophages, including mouse Raw264.7 macrophages (Fig. 1j-l and Extended Data Fig. 2k), primary mouse bone marrow-derived macrophages (BMDMs) (Fig. 1m and Extended Data Fig. 2l), human THP-1 macrophages (Fig. 1n and Extended Data Fig. 2m), and primary human BMDMs (Fig. 1o and Extended Data Fig. 2n), relative to the CM from control cells. Together, these findings suggest a critical role of glioblastoma cell glycolysis in regulating macrophage infiltration.
Glioblastoma cell LDHA promotes macrophage infiltration. To determine the molecular basis of tumor glycolysis in support of macrophage infiltration, we examined the connection between the expression of key glycolysis and tricarboxylic acid (TCA) cycle enzymes (e.g., HK1, HK2, HK3, PGM1, PGM2, LDHA, LDHB, MDH1, MDH2, FH, SDHA, SUCLA2, OGDH, IDH3A, IDH3B, IDH3G, CS, and ACO1) with patient survival, immune score, and macrophage signature in TCGA glioblastoma patient tumors. Following these analyses, LDHA was identified as the only gene that correlated negatively with patient survival and positively with immune score and macrophage signature (Extended Data Fig. 3a). Analysis of the scRNA-seq data 34 also identified LDHA as the only gene that was increased in glioma cells of glioblastoma (including ndGBM and rGBM) compared to LGG (Fig. 2a) and correlated positively with the abundance of macrophages and monocytes in glioblastoma patient tumors (Fig. 2b). Next, GSEA on hallmark pathways with the RNA-Seq profiling data from CT2A cells treated with or without a LDHA specific inhibitor FX11 demonstrated that FX11-treated cells displayed an impaired representation of immune response networks including interferon alpha and gamma responses, and inflammatory response (Fig. 2c). Similarly, LDHA-high glioblastoma patient tumors showed prominent representations of leukocyte and myeloid cell migration signatures, immune response networks, and cytokine and chemokine signatures (Table S4). Finally, GSEA on distinct immune cell signatures confirmed that macrophage and monocyte were the top immune cell types enriched in LDHA-high TCGA glioblastoma patient tumors (Extended Data Fig. 3b).
To further confirm the relevance of LDHA-mediated tumor glycolysis in promoting macrophage infiltration, we conducted shRNA-mediated LDHA depletion (shLdha) in glioblastoma cells, such as CT2A and GL261 (Fig. 2d), or treated them and GSCs with LDHA inhibitors (e.g., stiripentol and FX11). As expected, these modifications and treatments reduced lactate levels (Extended Data Fig. 3c-g) and ECAR (Extended Data Fig. 3h, i). Furthermore, CM from LDHA-depleted CT2A and GL261 cells reduced macrophage migration relative to CM from shRNA control cells (Fig. 2e-g and Extended Data Fig. 4a). Similarly, CM from stiripentol-treated CT2A cells (Fig. 2h, i and Extended Data Fig. 4b, c), GL261 cells (Fig. 2j and Extended Data Fig. 4d), 005 GSCs, a GSC line isolated from tumors with lentiviral transduction of brains with H-Ras and AKT in Trp53+/− mice 36, 37 (Extended Data Fig. 4e, f), and GSC272 (Fig. 2k, l and Extended Data Fig. 4g, h), induced significantly less migration of macrophages (including Raw264.7 macrophages, primary mouse BMDMs, THP-1 macrophages, and primary human BMDMs) than CM from untreated cells. In addition, CM from FX11-treated CT2A cells (Fig. 2m and Extended Data Fig. 4i), GL261 cells (Fig. 2n and Extended Data Fig. 4j), and GSC272 (Fig. 2o, p and Extended Data Fig. 4k, l) showed similar macrophage migration inhibitory effect. Finally, this phenomenon was reinforced by a scratch assay showing that CT2A CM-induced macrophage migration was impaired when glioblastoma cell LDHA was inhibited genetically and pharmacologically (Extended Data Fig. 4m-p). Together, these findings support a pivotal role of glioblastoma cell/GSC LDHA in triggering macrophage infiltration into the glioblastoma TME.
Glioblastoma cell LDHA promotes macrophage infiltration via upregulating CCL2 and CCL7. GSEA on KEGG pathways of CT2A cells with FX11 treatment versus control exhibited a prominent reduction of signatures related to chemokine and cytokine-cytokine receptor interaction (Fig. 3a), suggesting that LDHA in glioblastoma cells may regulate the expression of chemokines and cytokines. To elucidate such chemokines and/or cytokines governing macrophage recruitment in LDHA-high glioblastoma cells, we examined putative factors exhibiting a ≥ 2.0-fold change in CT2A cells (FX11 treatment versus control) and TCGA glioblastoma patient tumors (LDHA-high versus -low) using a human secreted protein dataset 38. This analysis led to identification of eleven genes (e.g., CCL2, CCL7, IL1B, IL1RAP, IL1RN, MMP9, NPY, PLAU, PROS1, S100A8 and SLPI) encoding secreted proteins that were upregulated in LDHA-high patient tumors compared to LDHA-low tumors and downregulated by LDHA inhibitor FX11 treatment in CT2A cells (Fig. 3b, c). To reveal the importance of these genes in glioblastoma tumor biology, we conducted bioinformatics analyses in TCGA glioblastoma tumors showing that the expression of most of these genes (except for NPY) correlated positively with macrophage signature, but only CCL2, CCL7, IL1RAP, PLAU, and S100A8 correlated negatively with patient survival (Extended Data Fig. 5a). RT-qPCR demonstrated a decreased expression of Ccl2, Ccl7, Plau, and S100a8, but not Il1rap, in CT2A and GL261 cells upon the treatment with LDHA inhibitor FX11 (Fig. 3d and Extended Data Fig. 5b). Reduced expression of Ccl2, Ccl7, Plau, and S100a8, was further confirmed by additional pharmacological (using LDHA inhibitor stiripentol) and genetic (using shLdha) strategies in CT2A cells (Fig. 3e, f) and GL261 cells (Extended Data Fig. 5c, d). To validate the capacity of CCL2, CCL7, PLAU, and S100A8 functioning as macrophage chemoattractants, we performed transwell migration assay showing that recombinant CCL2 and CCL7, but not PLAU and S100A8, protein-supplemented media increased the migration of Raw264.7 macrophages (Fig. 3g, h). Similar experiments in human GSC272 demonstrated that LDHA inhibitor stiripentol treatment reduced CCL2 and CCL7 expression (Fig. 3i, j) and secretion (Fig. 3k, l). Conversely, recombinant LDHA protein treatment increased the expression of CCL2 and CCL7 in both mouse CT2A cells and human GSC272 and rescued the impaired CCL2 and CCL7 levels in shLdha CT2A cells (Extended Data Fig. 5e-h). Consistent with the data from mouse Raw264.7 macrophages, recombinant CCL2 and CCL7 protein-supplemented media increased the migration of human THP-1 macrophages (Fig. 3m and Extended Data Fig. 5i). More importantly, the impaired macrophage migration induced by CM from shLdha CT2A cells was prevented by the treatment with recombinant CCL2 and CCL7 proteins (Extended Data Fig. 5j-m). The GOEA on the sub-ontologies of Biological Process and Molecular Function, and KEGG Enrichment Analysis in TCGA glioblastoma patient tumors also demonstrated that the migration of leukocytes and/or myeloid cells and the activity of chemokines and cytokines were the top CCL2- and CCL7-regulated processes (Tables S5 and S6).
To further confirm the role of glioblastoma cell CCL2 and CCL7 in macrophage infiltration, we first employed shRNAs to deplete CCL2 and CCL7 in CT2A and GL261 cells. As expected, CM from CT2A and GL261 cells expressing shCcl2 (Fig. 3n, o and Extended Data Fig. 6a-d) and shCcl7 (Fig. 3p, q and Extended Data Fig. 6e-h) induced significantly less macrophage migration than CM from shRNA control (shC) cells. Next, we depleted CCL2 and CCL7 in GSC272 and confirmed that CM from GSC272 expressing shCCL2 (Fig. 3r, s and Extended Data Fig. 6i, j) and shCCL7 (Fig. 3t, u and Extended Data Fig. 6k, l) inhibited the migration of THP-1 macrophages and primary human BMDMs compared to CM from shC cells. In summary, these results reinforce that the expression of CCL2 and CCL7 in glioblastoma cells/GSCs is regulated by LDHA and that glioblastoma cell CCL2 and CCL7 function as potent macrophage chemoattractants.
YAP1 and STAT3 transcriptional co-activators regulate LDHA-induced CCL2 and CCL7 expression in glioblastoma cells. To explore how LDHA regulates CCL2 and CCL7 expression, GSEA was utilized to catalog oncogenic signaling pathways modulated by LDHA in glioblastoma cells (LDHA inhibitor FX11 versus control) and TCGA glioblastoma patient tumors (LDHA-high versus LDHA-low). As a result, 35 overlapping pathways were identified, which include transcription factors (e.g., YAP1, ATF2, HOXA9, LEF1, and NRL), signaling pathways (e.g., YAP1, JNK/STAT, AKT/mTOR, Raf/ERK, and STK33), epigenetic factors (e.g., EED and EZH2), tumor suppressor genes and oncogenes (e.g., KRAS, TP53, RB, and SNF5), and others (e.g., IL2, RPS14, VEGFA, and WNT1) (Fig. 4a). By analyzing RNA-Seq data from CT2A cells focusing on above identified factors, we found that the expression of Hoxa9, Yap1, Eed, Ezh2, and Trp53 was downregulated by LDHA inhibitor FX11 treatment (Fig. 4b), which was confirmed by RT-qPCR analysis in both CT2A and GL261 cells (Fig. 4c and Extended Data Fig. 7a). Further studies on CT2A and GL261 cells treated with stiripentol (Fig. 4d and Extended Data Fig. 7b) or expressing shLdha (Fig. 4e, f) demonstrated that LDHA inhibition downregulated the expression of Hoxa9 andYap1, but had no effect on Eed and Ezh2. Next, we aimed to confirm whether YAP1, JNK/STAT, AKT/mTOR, Raf/ERK, and STK33 pathways are regulated by LDHA in glioblastoma cells. Western blotting demonstrated that shRNA-mediated depletion of LDHA or LDHA inhibitor (e.g., FX11 and stiripentol) treatment in CT2A cells and GSC272 significantly inhibited Phospho-ERK (P-ERK), YAP1, and P-STAT3 (Fig. 4g-i), but did not affect STK33, P-AKT, and P-STAT6 (Extended Data Fig. 7c, d). Moreover, the decreased P-ERK, YAP1, and P-STAT3 was confirmed in GL261 cells expressing shLdha or treated with LDHA inhibitors FX11 and stiripentol (Extended Data Fig. 7e-g). Finally, we treated mouse CT2A and GL261 cells and human GSC272 with ERK inhibitor ravoxertinib and found that such treatments significantly reduced YAP1, P-STAT3, and HOXA9 (Extended Data Fig. 7h-k). Together, these findings suggest that LDHA-directed ERK pathway regulates HOXA9, YAP1, and STAT3 transcription factors and/or signaling pathways in glioblastoma cells and GSCs.
To investigate the potential functional relevance of HOXA9, YAP1, and STAT3 in regulating CCL2 and CCL7 expression and macrophage infiltration, bioinformatics analyses in TCGA glioblastoma patient tumors were performed. As a result, we found that the expression of YAP1 and STAT3, but not HOXA9 and TP53, correlated positively with CCL2, CCL7, and macrophage signature (Extended Data Fig. 7l). Then, CT2A cells, GL261 cells, and GSC272 were treated with YAP1-TEAD interaction inhibitor verteporfin 39 and STAT3 inhibitor WP1066. The results of these experiments demonstrated that verteporfin treatment reduced P-STAT3, and, reciprocally, WP1066 treatment impaired YAP1 expression at both mRNA and protein levels (Fig. 4j, k). Moreover, the nuclear localization of STAT3 was reduced when CT2A cells harboring shLdha or treated with stiripentol and verteporfin (Extended Data Fig. 7m, n). Similarly, depletion of LDHA or treatment with stiripentol and WP1066 in CT2A cells reduced the nuclear localization of YAP1 (Extended Data Fig. 7o, p). These findings suggest that LDHA-regulated YAP1 and STAT3 are transcriptional co-activators 40, prompting us to investigate the role of YAP1 and STAT3 in transcriptional regulation of CCL2 and CCL7 in glioblastoma cells. Correspondingly, we observed specific YAP1 and STAT3 binding to the Ccl2 and Ccl7 promoters in CT2A cells, which was reduced upon LDHA depletion (Fig. 4l, m). Moreover, pharmacological treatment with verteporfin and WP1066 in CT2A and GL261 cells repressed Ccl2 and Ccl7 expression (Fig. 4n, o). To further investigate whether LDHA-regulated lactate contributes to this process, we treated LDHA-depleted glioblastoma cells with lactate and found that this treatment rescued the impaired signaling of P-ERK, YAP1, P-STAT3, CCL2, and CCL7 in shLdha CT2A cells (Extended Data Fig. 7q-s). Together, these findings suggest that YAP1 and STAT3 transcriptional co-activators contribute to LDHA/lactate–ERK axis-dependent CCL2 and CCL7 expression in glioblastoma cells.
Macrophage-derived LDHA-containing EVs promote tumor growth and activate the ERK-YAP1/STAT3-CCL2/CCL7 axis in glioblastoma cells. Once infiltrating into the glioblastoma TME, macrophages are educated to promote glioblastoma progression by secreting distinct factors and EVs 8. To mimic this process, we first utilized glioblastoma cell CM to educate macrophages (hereafter such educated macrophages are referred to as EMφ), and then examined the role of CM from EMφ on glioblastoma cells. As a result, we found that EMφ CM promoted LDHA expression in CT2A and GL261 cells (Fig. 5a, b), prompting a speculation that TAMs may support glioblastoma cell growth and survival via upregulating LDHA. To confirm the role of LDHA in glioblastoma cell biology, we performed cell cycle, apoptosis, and proliferation analyses in glioblastoma cells with or without LDHA inhibition. We found that CT2A cells expressing shLdha or treated with LDHA inhibitors (e.g., isosafrole, FX11 or stiripentol) displayed decreased G1 and upregulated G2–M fractions (Extended Data Fig. 8a-d), upregulated apoptosis (Extended Data Fig. 8e-h), and reduced proliferation (Extended Data Fig. 8i-l).
To reveal how TAMs upregulate LDHA in glioblastoma cells, we depleted LDHA using shRNAs (Extended Data Fig. 9a) and inhibited LDHA using FX-11 in EMφ. Surprisingly, we noticed that LDHA inhibition in macrophages abolished EMφ CM-induced LDHA upregulation in glioblastoma cells (Fig. 5a, b), suggesting a potential for LDHA delivery from EMφ to glioblastoma cells. scRNA-seq data analysis on tumors from a cohort of four glioblastoma patients 41 demonstrated that LDHA was highly expressed in both glioblastoma cells and CD68+CX3CR1− macrophages, but not in CD68+CX3CR1+ microglia (Extended Data Fig. 9b-e). As expected, genetic and pharmacological inhibition of LDHA in macrophages reduced glycolysis as shown by the impaired lactate production (Extended Data Fig. 9f, g) and ECAR (Extended Data Fig. 9h, i). To investigate whether LDHA could be delivered from macrophages into glioblastoma cells via EVs, we treated EMφ with GW4869 (an EV biogenesis and release inhibitor) and found that this treatment abolished EMφ CM-induced LDHA upregulation in both CT2A and GL261 cells (Fig. 5c, d). Next, we purified EVs from CM of EMφ using ultracentrifugation and confirmed their identity nanoparticle tracking analysis (Extended Data Fig. 10a) and Western blotting for EV markers (e.g., CD63 and ALIX) and calnexin that is absent from EVs (Fig. 5e). Notably, CT2A and GL261 CM treatment did not affect the size and distribution of macrophage-derived EVs (Extended Data Fig. 10a), but increased LDHA levels in EMφ EVs, an effect that was abolished by shRNA-mediated Ldha depletion in macrophages (Fig. 5e). Moreover, EMφ EVs were labeled with the fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiD) and then incubated with glioblastoma cells. Recipient glioblastoma cells exhibited equal uptake efficiency for EVs from control macrophages, as well as CT2A EMφ and GL261 EMφ expressing shC and shLdha (Extended Data Fig. 10b-e). However, LDHA in glioblastoma cells was upregulated upon uptake of EVs from shC EMφ, but not from LDHA-depleted EMφ (Fig. 5f, g and Extended Data Fig. 10f, g). These findings reinforce the role of EVs in delivery of LDHA from EMφ to glioblastoma cells.
Next, we aimed to investigate the role of EMφ-derived EVs in regulating glioblastoma cell growth and survival. Among a series of cellular analyses in EMφ EV-treated glioblastoma cells, we found that EMφ EV treatment abolished Ldha knockdown-induced cell cycle transition from G1 to G2/M and apoptosis in CT2A (Extended Data Fig. 11a-d) and GL261 (Extended Data Fig. 11e-h) cells. Similarly, LDHA inhibitor stiripentol treatment-induced cell cycle transition from G1 to G2/M and apoptosis in CT2A and GL261 cells were rescued by the treatment with EVs from EMφ (Fig. 5h, i and Extended Data Fig. 12a-f). However, such effects were abolished or inhibited by depletion of LDHA in macrophages (Fig. 5h, i and Extended Data Fig. 12a-f).
Given the importance of glycolysis in tumor-macrophage symbiosis in glioblastoma, we further investigated the potential role of EMφ EVs in this process. Notably, we found that the impaired glycolytic activity as shown by reduced ECAR in LDHA-depleted glioblastoma cells was negated by the treatment with EVs from EMφ, but not LDHA-depleted EMφ (Fig. 5j). Moreover, treatment with EMφ EVs upregulated the levels of P-ERK, YAP1, P-STAT3, CCL2, and CCL7 in glioblastoma cells (Extended Data Fig. 12g, h). The decreased P-ERK, YAP1, P-STAT3, CCL2, and CCL7 in LDHA-depleted glioblastoma cells was rescued by the treatment with EMφ EVs, but not LDHA-depleted EMφ EVs (Fig. 5k-m). Together, these results demonstrate that TAM-derived LDHA-containing EVs can promote tumor growth by triggering a positive feedback loop between glioblastoma cell glycolysis and macrophage infiltration.
Inhibition of LDHA-regulated tumor-macrophage symbiosis extends survival in glioblastoma mouse models. To further investigate the role of LDHA-mediated tumor-macrophage interplay in glioblastoma tumor biology, we utilized shRNA knockdown system to deplete LDHA in CT2A and GL261 tumors implanted into C57BL/6 mice. We found that LDHA depletion significantly inhibited tumor growth and extended survival in both glioblastoma mouse models (Fig. 6a, b and Extended Data Fig. 13a, b). Given the brain-penetrating ability of LDHA inhibitor stiripentol and isosafrole 42, we developed preclinical trials evaluating the anti-tumor effect of pharmacological inhibition of LDHA in glioblastoma mouse models. We found that stiripentol and isosafrole treatment impaired tumor growth and extended the survival of C57BL/6 mice implanted with CT2A cells, GL261 cells, and 005 GSCs (Fig. 6c-e and Extended Data Fig. 13c-e). Moreover, we developed a patient-derived xenograft (PDX) model in nude mice by intracranial implantation of GSC272 and found that stiripentol treatment also extended survival (Fig. 6f). To confirm that macrophages were the critical target of stiripentol in impairing tumor growth and progression, we compared the anti-tumor effect of stiripentol and BLZ945 (an CSF-1R inhibitor that can impair macrophage role in mice) in GL261-bearing mice. Each agent extended survival; however, their combination treatment did not exhibit additional anti-tumor effects (Extended Data Fig. 13f). On the histological level, immunofluorescence for Ki67 and cleaved caspase 3 (CC3) demonstrated that glioblastoma cell proliferation was dramatically reduced, whereas apoptosis was increased upon Ldha depletion (Extended Data Fig. 14a-d) and treatment with stiripentol and isosafrole (Extended Data Fig. 14e-h). Flow cytometry demonstrated that macrophages were profoundly reduced in LDHA-depleted CT2A tumors (Extended Data Fig. 14i-k) and LDHA inhibitor-treated GL261 (Fig. 6g, h), CT2A (Fig. 6i and Extended Data Fig. 14l-n) and 005 GSC tumors (Extended Data Fig. 14o, p). Similarly, immunofluorescence for F4/80 confirmed that infiltrating macrophages were profoundly reduced in CT2A tumors by inhibition of LDHA genetically (Extended Data Fig. 14q, r) and pharmacologically (Extended Data Fig. 14s, t). However, LDHA inhibition with stiripentol did not change macrophage apoptosis in CT2A tumors (Extended Data Fig. 14u, v).
To confirm the role of LDHA in regulation of the YAP1/STAT3–CCL2/CCL7 signaling axis in vivo, we performed immunofluorescence for YAP1 and STAT3 in tumors and ELISA for CCL2 and CCL7 in plasma from control and LDHA-inhibited glioblastoma tumor-bearing mice. We found that the nuclear level of YAP1 and STAT3 in CT2A tumors (Fig. 6j-m) and plasma level of CCL2 and CCL7 from glioblastoma tumor-bearing mice (Fig. 6n, o) were significantly reduced upon stiripentol treatment. Similarly, blockade of the YAP1/STAT3 signaling using STAT3 inhibitor WP1066 reduced plasma level of CCL2 and CCL7 and intratumoral macrophages in CT2A-bearing mice (Fig. 6p-r and Extended Data Fig. 14w). Next, we investigated the in vivo role of CCL2 and CCL7 by implantation of shC, shCcl2, and shCcl7 CT2A cells into the brains of C57BL/6 mice and found that depletion of CCL2 and CCL7 significantly extended survival (Fig. 6s) and reduced intratumoral macrophages (Fig. 6t and Extended Data Fig. 14x).
TAMs consist of pro-tumor and anti-tumor phenotypes and are usually biased toward a pro-tumor phenotype in glioblastoma 5, 6, 8, 43. We found that LDHA expression correlated positively with pro-tumor macrophage signature 32 in TCGA glioblastoma patient tumors (Extended Data Fig. 15a). CM from LDHA-inhibited (genetically and pharmacologically) CT2A and GL261 cells impaired the expression of a pro-tumor macrophage marker arginase 1 (Arg1) and the percentage of pro-tumor CD68+CD206+ cells in Raw264.7 macrophages (Extended Data Fig. 15b-g). Moreover, depletion of LDHA in glioblastoma cells or treatment with LDHA inhibitor stiripentol reduced pro-tumor CD45highCD11b+CD68+CD206+ macrophages in tumors from CT2A-bearing mice (Extended Data Fig. 15h-k). Similarly, pharmacologic inhibition of STAT3 or genetic depletion of CCL2 and CCL7 reduced pro-tumor CD45highCD11b+CD68+CD206+ macrophages in CT2A tumors (Extended Data Fig. 15l-o).
Finally, we aimed to investigate the role of TAM-derived LDHA-containing EVs in glioblastoma progression and treated shLdha CT2A-bearing mice with stiripentol and EMφ EVs. As expected, stiripentol treatment did not exhibit additional anti-tumor effects in LDHA-depleted tumors (Fig. 6u), supporting our above in vivo findings that LDHA is the key target of stiripentol. However, EMφ EVs treatment rescued the impaired tumor growth in LDHA-depleted CT2A tumors (Fig. 6u). To confirm the role of macrophage LDHA in this process, we generated macrophage-specific LDHA null (LDHA-mKO) mice by crossing LDHA flox mice with Lysozyme-Cre (LyzCre) mice. Orthotopic transplantation of CT2A cells into the brains of LDHA-mKO and WT mice showed significant survival extension in LDHA-mKO mice compared to WT mice (Fig. 6v). However, stiripentol treatment showed similar anti-tumor effects in both WT and LDHA-mKO mice (Fig. 6v). Together, these results validate the importance of LDHA-regulated tumor-macrophage symbiosis in promoting glioblastoma progression and support a therapeutic potential of targeting this co-dependency in glioblastoma.
The LDHA–YAP1/STAT3–CCL2/CCL7 axis tracks with macrophages in glioblastoma patient tumors and is increased in glioblastoma patient plasma and EVs. The clinical relevance of above experimental findings was supported by bioinformatics using scRNA-seq data from 16 glioblastoma patients 34 showing that glioblastoma cell LDHA, YAP1, STAT3, and CCL2 correlated positively with macrophage abundance (Fig. 7a). Moreover, bioinformatics analyses in TCGA glioblastoma dataset confirmed that LDHA, YAP1, STAT3, CCL2, and CCL7 positively correlated with each other and with macrophage signature in patient tumors (Fig. 7b). Next, we performed immunofluorescence for LDHA and Mac-2 (a macrophage marker) in tumors from a cohort of 30 glioblastoma patients and found that LDHA signaling showed a positive correlation with the density of intratumoral macrophages (Fig. 7c, d). Since LDHA, CCL2 and CCL7 are genes encoding secreted proteins, we compared their protein levels in patient plasma showing that all of them were higher in glioblastoma patients than that in healthy controls, but such levels were not changed in meningioma patients (Fig. 7e-g). Moreover, plasma LDHA correlated positively with plasma CCL2 and CCL7 (Fig. 7h, i) and intratumoral macrophages in glioblastoma (Fig. 7j). Moreover, the median survival time of glioblastoma patients with high plasma LDHA (389 days) was lower than the patients with low plasma LDHA (675 days, Fig. 7k). However, it should be noted that this survival analysis did not reach statistical significance due to limited patient numbers (Fig. 7k). Moreover, plasma LDHA, CCL2, and CCL7 levels were not related to the status of recurrence, gender, age, and MGMT methylation in glioblastoma patients (Extended Data Fig. 16). Finally, we examined the levels of LDHA in plasma EVs from a cohort of healthy controls and glioblastoma patients and confirmed that LDHA in glioblastoma patient plasma EVs was significantly higher than that from healthy controls (Fig. 7l, m). Together, these correlative glioblastoma patient’s findings are consistent with the hypothesis that LDHA–YAP1/STAT3–CCL2/CCL7 axis drives macrophage infiltration, and suggest that LDHA, CCL2 and CCL7 might function as biomarkers for glioblastoma patients, although these data are still relatively preliminary.