Multiomic screening of invasive GBM cells reveals targetable transsulfuration pathway alterations
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
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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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 4

Invasive GBM cells exhibit increased ROS.

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Invasive GBM cells exhibit increased ROS. Analyses by paired (A–E) or un...
Analyses by paired (A–E) or unpaired (F) t tests. (A) Spheroid invasion assays in GBM43 cells incubated with JC-1 dye revealed increased mitochondrial membrane potential in invasive GBM43 cells (P < 0.01; n = 5 pairs). (B and C) MDA staining of (B) hydrogels and (C) patient specimens revealed increasing MDA in the edge versus the core of hydrogels (P < 0.001; n = 4 pairs) and patient specimens (P = 0.025; n = 3 pairs). (D and E) Nitrotyrosine staining of (D) hydrogels and (E) patient specimens revealed increased staining in the edge versus the core in the hydrogels (P < 0.05; n = 4 pairs), but not in the patient specimens (P = 0.5; n = 3 pairs). (F) While H2O2 increased invasion of GBM43 cells in HA hydrogels (P < 0.0001), ROS scavenger NAC did not affect invasion (P = NS) of GBM spheroids in HA hydrogel invasion assays (n = 24 spheres, collected across 3 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Original magnification, ×20 (B, C, D, E); ×10 (F). Scale bars: 200 μm (A); 100 μm (B, D); 50 μm (C, E); 200 μm (F).