STING-induced blood-brain barrier opening combined with radiotherapy potentiates antitumor response in a high-grade glioma model
Shashwat Tripathi, Hinda Najem, Lisa Hurley, Ruochen Du, Crismita Dmello, Heba Ali, Kathleen McCortney, Karl J. Habashy, Peng Zhang, Craig M. Horbinski, Lara Leoni, Ryan J. Avery, Rimas V. Lukas, Timothy L. Sita, David R. Raleigh, Sean Sachdev, Roger Stupp, Maciej S. Lesniak, David M. Ashley, Daniele Procissi, Michael A. Curran, Irina Balyasnikova, Amy B. Heimberger
Shashwat Tripathi, Hinda Najem, Lisa Hurley, Ruochen Du, Crismita Dmello, Heba Ali, Kathleen McCortney, Karl J. Habashy, Peng Zhang, Craig M. Horbinski, Lara Leoni, Ryan J. Avery, Rimas V. Lukas, Timothy L. Sita, David R. Raleigh, Sean Sachdev, Roger Stupp, Maciej S. Lesniak, David M. Ashley, Daniele Procissi, Michael A. Curran, Irina Balyasnikova, Amy B. Heimberger
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Research Article Immunology Oncology Vascular biology

STING-induced blood-brain barrier opening combined with radiotherapy potentiates antitumor response in a high-grade glioma model

Abstract

Radiation therapy (RT) is the standard of care for glioblastoma but is not curative. Triggering the cGAS/stimulator of interferon genes (STING) pathway with potent agonists, such as 8803, exerts activity across high-grade glioma preclinical models. To determine if the combination of 8803 with RT warrants consideration in the up-front treatment setting and to clarify the underlying mechanisms of therapeutic activity, C57BL/6J mice harboring intracerebral CT-2A or QPP8v gliomas were treated with RT, intratumoral 8803, or both. The treatment with the combination resulted in 80% long-term survival in the CT-2A model but not in the radiation-resistant QPP8v model. This therapeutic effect was maintained in Sting–/– CT-2A cells, highlighting the direct role of the immune system in mediating the survival benefit. Single-cell RNA-Seq identified increased nitric oxide synthase 2 (Nos2) in inflammatory tumor-associated macrophages; however, the therapeutic effect was maintained in Nos2–/– mice. Additionally, 8803 reprogrammed the blood-brain barrier (BBB) by altering the Pecam and Cd147 pathways in endothelial cells; intracranial injection of 8803 induced bihemispheric BBB opening for up to 24 hours. Sting activation was visualized longitudinally using 3’-deoxy-3’-[18F]-fluorothymidine ([18F]-FLT) PET, which peaked 72–96 hours after 8803 administration. In summary, 8803 combined with RT triggers distinctive antiglioma immune reactivity, facilitates BBB opening, and warrants consideration for up-front clinical trials in glioblastoma, where treatment effects can be monitored using [18F]-FLT PET imaging.

Authors

Shashwat Tripathi, Hinda Najem, Lisa Hurley, Ruochen Du, Crismita Dmello, Heba Ali, Kathleen McCortney, Karl J. Habashy, Peng Zhang, Craig M. Horbinski, Lara Leoni, Ryan J. Avery, Rimas V. Lukas, Timothy L. Sita, David R. Raleigh, Sean Sachdev, Roger Stupp, Maciej S. Lesniak, David M. Ashley, Daniele Procissi, Michael A. Curran, Irina Balyasnikova, Amy B. Heimberger

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Figure 2

Ex vivo correlative analysis of the TME using scRNA sequencing and multiplex immunofluorescence.

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Ex vivo correlative analysis of the TME using scRNA sequencing and multi...
(A) Schema demonstrates that the therapeutic window analysis was conducted on brain samples collected 48 hours after 8803 administration. Brain samples were either freshly processed for scRNA-Seq analysis or formalin-fixed and paraffin-embedded for spatial multiplex immunofluorescence staining. (B) Uniform manifold approximation and projection (UMAP) plot showing the cellular composition of the CT-2A TME based on gene signatures of the scRNA-Seq dataset. (C) UMAP plot of the detailed clustering of the various immune populations present within the TME based on the RNA signatures. BAM, border-associated macrophage; TAM, tumor-associated macrophage. (D–F) Strip plots showing the differential abundance of immune cell types in CT-2A–bearing C57BL/6 mice based on treatment compared with control, log2(FC). The dot is colored when the P value is less than 0.05. The box plot represents the mean of significant points for immune populations with more than 3 significant clusters. Cell types with P ≥ 0.05 are colored in gray. P value calculated using negative binomial generalized linear model with weighted false discovery rate multiple correction. (G) t-Distributed stochastic neighbor embedding (t-SNE) plots show the cluster of Inflam-TAMs(2) (left panel) and the Nos2 gene expression within this population as a function of treatment condition (right smaller panels). (H) Heatmap of differential expression of Nos2, Pparg, Prkdc, Ythdf1, and Hmox1 genes based on treatment conditions: control, RT, 8803, and RT + 8803.