Lactate dehydrogenase A regulates tumor-macrophage symbiosis to promote glioblastoma progression

Abundant macrophage infiltration and altered tumor metabolism are two key hallmarks of glioblastoma. By screening a cluster of metabolic small-molecule compounds, we show that inhibiting glioblastoma cell glycolysis impairs macrophage migration and lactate dehydrogenase inhibitor stiripentol emerges as the top hit. Combined profiling and functional studies demonstrate that lactate dehydrogenase A (LDHA)-directed extracellular signal-regulated kinase (ERK) pathway activates yes-associated protein 1 (YAP1)/ signal transducer and activator of transcription 3 (STAT3) transcriptional co-activators in glioblastoma cells to upregulate C-C motif chemokine ligand 2 (CCL2) and CCL7, which recruit macrophages into the tumor microenvironment. Reciprocally, infiltrating macrophages produce LDHA-containing extracellular vesicles to promote glioblastoma cell glycolysis, proliferation, and survival. Genetic and pharmacological inhibition of LDHA-mediated tumor-macrophage symbiosis markedly suppresses tumor progression and macrophage infiltration in glioblastoma mouse models. Analysis of tumor and plasma samples of glioblastoma patients confirms that LDHA and its downstream signals are potential biomarkers correlating positively with macrophage density. Thus, LDHA-mediated tumor-macrophage symbiosis provides therapeutic targets for glioblastoma.

(q) Immunoblots of P-ERK, ERK, YAP1, P-STAT3, STAT3, and actin in LDHA-depleted (shLdha) CT2A cells treated with or without lactate (1 mM).(r, s) RT-qPCR for Ccl2 (r) and Ccl7 (s) in CT2A cells expressing shC and shLdha) treated with or without lactate (1 mM).n = 4 independent samples.A representative example of three replicates is shown for (c-j and m-q).The experiments were independently repeated at least two to three times.Data from multiple replicates are presented as mean ± SEM.Statistical analyses were determined by Student's t-test (a, b, k, r, s) and Pearson's correlation test (l).Source data are provided as a Source Data file.
Inhibition of glioblastoma cell LDHA affects cell cycle, apoptosis, and proliferation.(a, b) Representative images (a) and quantification (b) of flow cytometry cell cycle analysis in CT2A cells expressing shRNA control (shC) and Ldha shRNAs (shLdha).n = 3 independent samples.(c, d) Representative images (c) and quantification (d) of flow cytometry cell cycle analysis in CT2A cells treated with or without isosafrole (10 mM).n = 3 independent samples.(e, f) Representative images (e) and quantification (f) of flow cytometry apoptosis analysis in CT2A cells expressing shC and shLdha.n = 3 independent samples.(g, h) Representative images (g) and quantification (h) of flow cytometry apoptosis analysis in CT2A cells treated with or without isosafrole (10 mM).n = 3 independent samples.(i, j) Representative images (i) and quantification (j) of colony formation in CT2A cells expressing shC and shLdha.n = 3 independent samples.(k, l) Representative images (k) and quantification (l) of colony formation in CT2A cells treated with or without FX11 (8 mM) or stiripentol (10 mM).n = 3 independent samples.The experiments were independently repeated at least two times.Data from multiple replicates are presented as mean ± SEM.Statistical analyses were determined by one-way ANOVA test (b, f, j, l) and Student's t-test (d, h).Source data are provided as a Source Data file.S9.LDHA is expressed in glioblastoma cells and macrophages, and inhibition of LDHA reduces glycolytic activity in macrophages.(a) Immunoblots of LDHAi cell lysates of Raw264.7 macrophages expressing shRNA control (shC) and Ldha shRNAs (shLdha).A representative example of three replicates.(b) UMAP dimensional reduction of single cells from tumor samples of a cohort of four glioblastoma patients 41 .(c) UMAP dimensional reduction of macrophage and microglia (as highlighted) on the basis of CD68 expression pattern.(d) UMAP dimensional reduction of microglia (as highlighted) on the basis of CX3CR1 expression pattern.(e) Gene expression pattern representing single-cell gene expression of LDHA in macrophages, microglia (as highlighted), and other glioblastoma cells.Intensity of the blue color indicates the expression of individual cells.(f)Glycolytic activity (lactate level) assay on Raw264.7 macrophages expressing shC and shLdha in the presence or absence of glucose (55 mM).n = 3 independent samples.(g) Glycolytic activity (lactate level) assay on Raw264.7 macrophages treated with or without LDHA inhibitor FX11 (8 mM) or stiripentol (10 mM) in the presence or absence of glucose (55 mM).n = 3 independent samples.(h) Extracellular acidification rate (ECAR) of Raw264.7 macrophages expressing shC and shLdha.ECAR was obtained from the Seahorse experiments and glucose was added at indicated time point.n = 6 independent samples.(i) ECAR of Raw264.7 macrophages treated with or without LDHA inhibitor FX11 (8 mM) or stiripentol (10 mM).n = 6 independent samples.ECAR was obtained from the Seahorse experiments and glucose was added at indicated time point.The experiments for (a, h, and i) were independently repeated at least three times.Data from multiple replicates are presented as mean ± SEM.Source data are provided as a Source Data file.
. TAM-derived EVs deliver LDHA from macrophages to glioblastoma cells.(a) Quantification of average diameter and concentration of extracellular vesicles (EVs) isolated from control Raw264.7 macrophages and CT2A/GL261 conditioned media-educated macrophages (EMφ) based on nanoparticle tracking analysis.n = 3 independent samples.(b, c) Representative (b) and quantification (c) of DiD positive cells out of total CT2A cells incubated with DiD-labeled EVs (500 ng) isolated from Raw264.7 Mφ and EMφ expressing shRNA control (shC) and Ldha shRNAs (shLdha) for 24 hrs.Scale bar, 200 mm.n = 3 independent samples.(d, e) Representative (d) and quantification (e) of DiD positive cells out of total GL261 cells incubated with DiD-labeled EVs (500 ng) isolated from Raw264.7 Mφ and EMφ expressing shC and shLdha for 24 hrs.Scale bar, 200 mm.n = 3 independent samples.(f, g) Representative images (f) and quantification (g) of immunofluorescence for LDHA in GL261 cells incubated with EVs (500 ng) isolated from control Mφ, GL261 EMφ expressing shC or shLdha for 24 hrs.Scale bar, 200 mm.n = 3 independent samples.The experiments were independently repeated at least two to three times.Data from multiple replicates are presented as mean ± SEM.Statistical analyses were determined by one-way ANOVA test (a, c, e, g).Source data are provided as a Source Data file.
and quantification (b) of flow cytometry cell cycle analysis of shC and shLdha CT2A cells treated with EVs (500 ng) isolated from Raw264.7 macrophages and CT2A EMφ.n = 3 independent samples.(c, d) Representative (c) and quantification (d) of flow cytometry apoptosis analysis in shC and shLdha CT2A cells treated with EVs (500 ng) isolated from Raw264.7 Mφ and CT2A EMφ.n = 3 independent samples.(e, f) Representative (e) and quantification (f) of flow cytometry cell cycle analysis of shC and shLdha GL261 cells treated with EVs (500 ng) isolated from Raw264.7 Mφ and GL261 EMφ.n = 3 independent samples.(g, h) Representative (g) and quantification (h) of flow cytometry apoptosis analysis in shC and shLdha GL261 cells treated with EVs (500 ng) isolated from Raw264.7 Mφ and GL261 EMφ.n = 3 independent samples.The experiments were independently repeated at least three times.Data presented as mean ± SEM and were analysed by one-way ANOVA test (b, d, f, h).Source data are provided as a Source Data file.r i p e n t o l S t i r i p e n t o l + M φ E V S t i r i p e n t o l + s h r i p e n t o l + s h L d h a r i p e n t o l + s h L d h a Depletion of LDHA in macrophages impairs the pro-tumor effect of TAM-derived EVs.(a) Quantification of flow cytometry cell cycle analysis of CT2A cells treated with extracellular vesicles (EVs, 500 ng) isolated from Raw264.7 macrophages (Mφ) and CT2A conditioned media-educated macrophages (EMφ), or treated with stiripentol (10 mM) in the presence or absence of EVs isolated from CT2A EMφ expressing shRNA control (shC) and Ldha shRNAs (shLdha).n = 3 independent samples.(b, c) Representative images (b) and quantification (c) of flow cytometry cell cycle analysis of GL261 cells treated with EVs (500 ng) isolated from Raw264.7 Mφ and GL261EMφ, or treated with stiripentol (10 mM) in the presence or absence of EVs isolated from GL261 EMφ expressing shC and shLdha.n = 3 independent samples.(d) Representative images of flow cytometry apoptosis analysis in CT2A cells treated with EVs (500 ng) isolated from Raw264.7 Mφ and CT2A EMφ, or treated with stiripentol (10 mM) in the presence or absence of EVs isolated from CT2A EMφ expressing shC and shLdha.(e, f) Representative images (e) and quantification (f) of flow cytometry apoptosis analysis in GL261 cells treated with EVs (500 ng) isolated from Raw264.7 Mφ and GL261 EMφ, or treated with stiripentol (10 mM) in the presence or absence of EVs isolated from GL261 EMφ expressing shC and shLdha.n = 3 independent samples.(g) Immunoblots of P-ERK, ERK, YAP1, P-STAT3, STAT3, and actin in CT2A cells treated with or without EVs (500 ng) isolated from CT2A Emφ.(h) RT-qPCR for Ccl2 and Ccl7 in CT2A cells treated with or without EVs (500 ng) isolated from CT2A EMφ.n = 6 independent samples.A representative example of five and three replicates is shown for (d) and (g), respectively.The experiments for (a-g) were independently repeated at least three times.Data presented as mean ± SEM.Statistical analyses were determined by one-way ANOVA test (a, c, f, h).Source data are provided as a Source Data file.inhibition reduces glioblastoma growth in vivo.(a, b) Representative images (a) and quantification (b) of H&E staining for CT2A tumors expressing shRNA control (shC) and Ldha shRNAs (shLdha).Tumors were harvested when control mice showing neurologic deficits or moribund.n = 3 independent samples.(c, d) Representative images (c) and quantification (d) of H&E staining for CT2A tumors treated with or without stiripentol (150 mg/kg, i.p., every other day for 6 doses).Tumors were harvested when control mice showing neurologic deficits or moribund.n = 3 independent samples.(e) Survival curves of C57BL/6 mice implanted with CT2A (2×10 4 cells).Mice were treated with isosafarol (150 mg/kg, i.p., every other day, 6 doses) beginning at day 8 postorthotopic injection.(n = 5 mice per group, sharing the control group with Fig. 6c).(f) Survival curves of C57BL/6 mice implanted with GL261 cells (2×10 4 cells).Mice were treated with stiripentol (150 mg/kg, i.p., every other day, 6 doses) and BLZ945 (200 mg/kg, oral gavage, every other day, 5 doses) beginning at day 8 post-orthotopic injection.n = 5, 7, 5 and 8 mice for control, BLZ945, stiripentol, and stiripentol + BLZ945 group, respectively.Data presented as mean ± SEM.Statistical analyses were determined by one-way ANOVA test (b), Student's t-test (d), and log-rank test (e, f).Source data are provided as a Source Data file.Inhibition of the LDHA-STAT2-CCL2/CCL7 axis reduces glioblastoma cell proliferation and macrophage infiltration in glioblastoma mouse models.(a, b) Immunofluorescence (a) and quantification (b) of Ki67 positive cells in CT2A tumors expressing shRNA control (shC) and Ldha shRNAs (shLdha).Scale bar, 50 mm.n = 3 independent samples.(c, d) Immunofluorescence (c) and quantification (d) of cleaved caspase 3 (CC3) positive cells in CT2A tumors expressing shC and shLdha.Scale bar, 50 mm.n = 3 independent samples.(e, f)Immunofluorescence (e) and quantification (f) of Ki67 positive cells in CT2A tumors treated with or without stiripentol and isosafrole.Scale bar, 50 mm.n = 3 independent samples.(g, h) Immunofluorescence (g) and quantification (h) of CC3 positive cells in CT2A tumors treated with or without stiripentol and isosafrole.Scale bar, 50 mm.n = 3 independent samples.(i) Flow cytometry gating strategy for intratumoral CD45 high CD11b + CD68 + macrophages.(j, k) Representative (j) and quantification (k) of flow cytometry analysis for the percentage of CD68 + macrophages out of CD45 high CD11b + cells in CT2A tumors expressing shC and shLdha.n = 3 independent samples.(l) Representative of flow cytometry analysis for the percentage of CD68 + macrophages out of CD45 high CD11b + cells in CT2A tumors treated with or without stiripentol.(m,n) Representative (m) and quantification (n) of flow cytometry analysis for the percentage of CD68 + macrophages out of CD45 high CD11b + cells in CT2A tumors treated with or without isosafrole.n = 3 independent samples.(o, p) Representative (o) and quantification (p) of flow cytometry analysis for the percentage of CD68 + macrophages out of CD45 high CD11b + cells in 005 GSC tumors treated with or without stiripentol.n = 3 independent samples.(q, r) Immunofluorescence (q) and quantification (r) of F4/80 positive cells in CT2A tumors expressing shC and shLdha.Scale bar, 50 mm.n = 3 independent samples.(s, t) Immunofluorescence (s) and quantification (t) of F4/80 positive cells in CT2A tumors treated with or without stiripentol and isosafrole.Scale bar, 50 mm.n = 3 independent samples.(u, v) Immunofluorescence (u) and quantification (v) of Mac-2 (macrophage marker) and cleaved caspase 3 (CC3) positive cells in CT2A tumors treated with or without stiripentol.Scale bar, 20 mm.n = 3 independent samples.(w) Representative of flow cytometry analysis for the percentage of CD68 + macrophages out of CD45 high CD11b + cells in CT2A tumors treated with or without WP1066.(x) Representative of flow cytometry analysis for the percentage of CD68 + macrophages out of CD45 high CD11b + cells in shC, shCcl2, and shCcl7 CT2A tumors.A representative example of three replicates is shown for (l, w, and x).The immunofluorescence experiments were independently repeated at least two to three times.Data presented as mean ± SEM.Statistical analyses were determined by one-way ANOVA test (b, d, f, h, k, r, t) and Student's t-test(n, p, v).Source data are provided as a Source Data file.The correlation analysis between LDHA expression and pro-tumor macrophage signature in TCGA glioblastoma dataset.The pro-tumor macrophage signature was determined by a set of genes as reported previously 32 .R and P values are shown.(b) RT-qPCR for Arg1 in Raw264.7 macrophages following the treated with or without the conditional media (CM) from CT2A and GL261 cells expressing shRNA control (shC) and Ldha shRNAs (shLdha).n = 6 independent samples.(c, d) Representative (c) and quantification (d) of flow cytometry analysis for the percentage of CD68 + CD206 + cells in Raw264.7 macrophages treated with or without CM from CT2A and GL261 cells expressing shC and shLdha.n = 3 independent samples.(e) RT-qPCR for Arg1 in Raw264.7 macrophages following the treatment with or without the CM from FX11-treated CT2A and GL261 cells.n = 6 independent samples.(f, g) Representative (f) and quantification (g) of flow cytometry analysis for the percentage of CD68

Supplementary Fig. S5. CCL2 and CCL7 are the key chemokines responsible for LDHA- induced macrophage migration
. (a)The correlation analysis between cytokines (e.