Multiomic screening of invasive GBM cells reveals targetable transsulfuration pathway alterations
Joseph H. Garcia, … , Sanjay Kumar, Manish K. Aghi
Joseph H. Garcia, … , Sanjay Kumar, Manish K. Aghi
Published November 16, 2023
Citation Information: J Clin Invest. 2024;134(3):e170397. https://doi.org/10.1172/JCI170397.
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Research Article Metabolism Oncology

Multiomic screening of invasive GBM cells reveals targetable transsulfuration pathway alterations

Abstract

While the poor prognosis of glioblastoma arises from the invasion of a subset of tumor cells, little is known of the metabolic alterations within these cells that fuel invasion. We integrated spatially addressable hydrogel biomaterial platforms, patient site–directed biopsies, and multiomics analyses to define metabolic drivers of invasive glioblastoma cells. Metabolomics and lipidomics revealed elevations in the redox buffers cystathionine, hexosylceramides, and glucosyl ceramides in the invasive front of both hydrogel-cultured tumors and patient site–directed biopsies, with immunofluorescence indicating elevated reactive oxygen species (ROS) markers in invasive cells. Transcriptomics confirmed upregulation of ROS-producing and response genes at the invasive front in both hydrogel models and patient tumors. Among oncologic ROS, H2O2 specifically promoted glioblastoma invasion in 3D hydrogel spheroid cultures. A CRISPR metabolic gene screen revealed cystathionine γ-lyase (CTH), which converts cystathionine to the nonessential amino acid cysteine in the transsulfuration pathway, to be essential for glioblastoma invasion. Correspondingly, supplementing CTH knockdown cells with exogenous cysteine rescued invasion. Pharmacologic CTH inhibition suppressed glioblastoma invasion, while CTH knockdown slowed glioblastoma invasion in vivo. Our studies highlight the importance of ROS metabolism in invasive glioblastoma cells and support further exploration of the transsulfuration pathway as a mechanistic and therapeutic target.

Authors

Joseph H. Garcia, Erin A. Akins, Saket Jain, Kayla J. Wolf, Jason Zhang, Nikita Choudhary, Meeki Lad, Poojan Shukla, Jennifer Rios, Kyounghee Seo, Sabraj A. Gill, William H. Carson, Luis R. Carette, Allison C. Zheng, David R. Raleigh, Sanjay Kumar, Manish K. Aghi

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Figure 6

Targeting CTH inhibits GBM invasion.

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Targeting CTH inhibits GBM invasion. Analyses used t test (A and G), ANO...
Analyses used t test (A and G), ANOVA with post hoc Tukey’s test (B), Pearson’s correlation (E), or Kaplan-Meier test (H). (A) GBM43 cells with knockdown were less invasive in 3D hydrogels based on bulk invasive area (left; P < 0.05; n = 6 regions of interest across 3 devices) and number of detached invasive cells (right; P < 0.001), with invasive cell morphology unaffected by CTHkd (right; P > 0.05; n = 16 regions of interest across 3 devices). (B) Spheroid invasion assays revealed that increasing cysteine from 200 to 250 μM reversed the slowed invasion caused by CTHkd (n = 24 spheres across 3 independent experiments). (C–E) GBM43 cells with CTH knockdown of CTH were seeded into invasion devices, after which cells from core and invasive fractions were assessed using the NanoString 770 metabolic gene platform. (C) GSEA: 6/13 upregulated pathways were shared with control cells invading hydrogels (green). (D) Heatmap depicting normalized gene expression (NGE) of cells in the invasive versus core hydrogel fractions for CTHkd (red bars) and control GBM43 cells (black bars) (n = 3/group), with uniform gene-expression changes across control GBM43 versus CTHkd cells suggesting similar transcriptional profiles among invasive GBM cells regardless of CTH expression. (E) Scatter plot depicting gene expression fold change for individual genes in invasive versus core fractions for GBM43 control (x axis) and GBM43 CTHkd (y axis). The high correlation between fold change in invasive GBM43 control versus CTHkd cells (P < 0.001) means that metabolic transcriptional patterns change during invasion similarly regardless of CTH expression. Purple dots indicate genes with discordant expression changes in control GBM43 versus CTHkd cells, which are scant (20/322 total genes = 6.2%). (F–H) Intracranial GBM43 PDXs expressing mCherry along with dCas9 or dCas9 with sgRNA targeting CTH (F and G) were less invasive with CTHkd (median ± 95% CI shown; P = 0.002; n = 9/group) based on fractal analysis of images of tumors and their surrounding brain, which yields fractal dimension, a measure of invasive tumor growth as a continuous number between 1 and 2, with higher numbers representing greater invasiveness and (H) exhibited unchanged survival with CTHkd (P = 0.1; n = 9–10/group). Original magnification, ×10 (left); ×20 (right). Scale bars: 25 mm (left); 1,000 mm (right). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.