Visualizing cancer-originating acetate uptake through monocarboxylate transporter 1 in reactive astrocytes in the glioblastoma tumor microenvironment

Abstract

Background: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explore 11C-acetate PET as an imaging technique for detecting reactive astrogliosis.

Methods: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and monocarboxylate transporter 1 (MCT1) expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis.

Results: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with a worse prognosis.

Conclusions: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas.

Keywords: PET imaging; acetate; glioblastoma; monocarboxylate transporter 1; reactive astrogliosis.

Graphical Abstract

Figure 1.

Regions of ¹¹C-acetate uptake at the tumor boundary in patients with glioblastomas. (A) Representative images of ¹¹C-acetate PET and MRI in patients with glioblastoma and IDH1-mt astrocytoma. (B) SUVR of ¹¹C-acetate uptake correlated with the WHO 2021 classification. (C) The median tumor volume in ¹¹C-acetate PET images was significantly larger than that seen on MRI in glioblastoma (n = 38) (20.80 (IQR 8.99–59.39) versus 7.72 (IQR 1.80–26.14)). (D–E) Western blot for MCT1 expression and ¹⁴C-acetate uptake in mouse primary astrocytes and various human glioma cell lines (U87MG, U373MG, T98G, and U87mt). (F–G) Immunofluorescence images of GFAP, Ki-67, MAO-B, and MCT1 in human glioma. C, Cortical; S, Star-shape astrocyte; L, Linear-shape astrocyte; T, Tumor. (H–I) Immunofluorescence images showing GFAP, Ki-67, MAO-B, and MCT1 in human glioma. (J) Immunofluorescence images of CD133, MAO-B in human glioma. (K) Pearson’s correlation coefficients for MAO-B and CD133 colocalization assays. (L) The proportion of MAO-B+ or CD133+ in Ki-67+ cells in IDH1-wildtype glioma (n = 5). (M) Representative image of Sholl analysis of an astrocyte according to S, L, or T region in human glioma. (N–Q) Quantification of GFAP area, the number of Ki-67+ cells, MAO-B, and MCT1 immunoreactivity of GFAP + astrocytes in glioblastoma versus IDH1-mt gliomas (n = 3). Scale bars, 50 μm (F), (G); 20 μm (H and I). Data are presented as mean ± SEM. ^*P < .05, ^**P < .01, ^***P < .001, ^****P < .0001 by 1-way ANOVA with Tukey’s test (C), unpaired 2-tailed t-test (O and Q), Kruskal–Wallis test (B), (N) left, (P) left, or Mann–Whitney U-test (N) right, (P) right.

Figure 2.

Reactive astrogliosis and ¹¹C-acetate uptake in patient-derived xenograft models. (A–B) Immunofluorescence images showing GFAP, MAO-B, and MCT1 in glioblastoma-TS models. (C) Immunostaining images showing MCT1 in glioblastoma-TS models. (D–E) Quantification of MAO-B immunoreactivity of GFAP + astrocyte and MCT1 positive area on the peritumoral (Peri-T.) and contralateral regions (Ctr.) in glioblastoma-TS models (n = 3). Scale bars, 500 μm (A) left; 20 μm (A) middle; 10 μm (A) right, (B), and (C). Data are presented as mean ± SEM. ^***P < .001, ^****P < .0001 by unpaired 2-tailed t-test (D and E).

Figure 3.

Molecular factors of glioblastoma that induce reactive astrogliosis. (A) Heatmap of Z-scores calculated from count per million values of GFAP, MAO-B, STAT3, SOX9, SLC16A1, SLC16A3, CD44, ALDH1A1, ACSS1, and ACSS2 related to astrocytic reactivity or acetate metabolism in DMEM or U87MG-CM-treated primary mouse AST. (B) Z-scores and raw data of GFAP, STAT3, SOX9, MAO-B, and SLC16A1. (C) Schematic diagram illustrating preparation of U87MG or U87-IDH1-CM. (D) Western blot for GFAP, MAO-B, and MCT1 expression. (E) MCT1 mRNA expression. (F) ¹⁴C-Acetate uptake in U87MG and U87mt-CM-treated AST. (G) MCT1 expression in shMCT1-transfected AST. (H) MCT1 expression (mRNA) in shMCT1-transfected AST. (I) ¹⁴C-Acetated uptake after shMCT1-transfection in AST. (J) The concentration of acetate in U87MG-CM. (K) J-scaling HSQC spectra of the methyl group on ¹³C₂-acetate on the CM of U87MG treated with 20 mmol/L U-¹³C₆-glucose. (L) Peak volume for the doublet of the methyl group from the spectra normalized to trimethylsilyl propionate (TSP) and protein. (M) Western blot for GFAP, MAO-B, and MCT1 expression in acetate-treated AST. Data are presented as mean ± SEM. ^*P < .05, ^**P < .01, ^***P < .001, ^****P < .0001 by 1-way ANOVA with Tukey’s test (E, F, and J) or unpaired 2-tailed t-test (H, I, and L).

Figure 4.

Inhibition of MAO-B and MCT1 decreases reactive astrogliosis and ¹¹C-acetate uptake in the PDX model. (A) Representative images of MRI, ¹¹C-acetate PET, PET, and MRI fusion with or without KDS2010 administration in the PDX mouse model. (B) SUVr of ¹¹C-acetate in the peritumoral and contralateral regions (n = 4). (C) Immunofluorescence images of GFAP expression in mouse models. (D and G) Immunofluorescence images of S100β, MCT1, GFAP, and MAO-B expression. (E, F, H, and I) Quantification of GFAP, S100β, MCT1, and MAO-B expression ± KDS2010 administration in PDX mouse models (n = 4). (J) Representative images of MRI, ¹¹C-acetate PET, PET, and MRI fusion with or without astrocyte-specific MCT1 gene-silencing around PDX glioblastoma cells. (K) SUVr of ¹¹C-acetate in the peritumoral and contralateral regions (n = 4). (L) Immunofluorescence images showing GFAP expression in mouse models. (M and P) Immunofluorescence images showing S100β, MCT1, GFAP, and MAO-B expression. (N, O, Q, and R) Quantification of GFAP, S100β, MCT1, and MAO-B expression ± astrocyte-specific MCT1 gene-silencing of PDX glioblastoma cells (n = 4). Scale bars, 500 μm (C), (L); 10 μm (D), (G), (M), (P). Data are presented as mean ± SEM. ^*P < .05, ^**P < .01, ^***P < .001, ^****P < .0001 by unpaired 2-tailed t-test (B) or Mann–Whitney U-test (E–I, N–R).

Figure 5.

Relationship between volume of reactive astrogliosis and prognosis in patients with glioblastoma and cancer stem cells. (A) Immunofluorescence images showing GFAP, Ki-67, and CD133 in the peritumoral region of glioblastomas. (B) The proportion of GFAP+ and/or CD133+ in Ki-67+ cells in glioblastomas (n = 3). (C) The ¹¹C-acetate uptake area beyond the contrast-enhanced MRI was defined as Volume_ACE–MRI. (D) Kaplan–Meier survival analysis showed significant differences in PFS between the 2 groups with a Volume_ACE–MRI cutoff value of >5.06 cc (P = .0134). (E) Kaplan–Meier Survival analysis also showed significant differences in overall survival (OS) between the 2 groups with a Volume_ACE–MRI cutoff value of >5.06 cc (P = .0206). Scale bars, 10 μm (A). Data are presented as mean ± SEM. ^****P < .0001 by 1-way ANOVA with Tukey’s test (B).