Diffuse intrinsic pontine glioma (DIPG) is one of the most devastating pediatric brain tumors. It sits in the brainstem where it cannot be surgically removed, responds poorly to chemotherapy, and almost always is fatal within a year of diagnosis. It affects 400 children in the US per year, with a median age of 7.
Researchers have turned to chimeric antigen receptor (CAR) T cell therapies—an exciting frontier in cancer treatment—as an avenue for hope in treating DIPG. In this therapy, T cells are genetically engineered to bind to tumor cells, which triggers a coordinated chain of events that turns T cells into a highly active, targeted tumor cell killers.
CAR T cell therapies targeting B7-H3 (CD276), a marker expressed on DIPG tumors, have been tested in early phase clinical trials and demonstrated feasibility and tolerability. For the best chance of reaching the tumor, these cells are delivered intracranially into the cerebrospinal fluid, an approach that was pioneered by Drs. Nicholas Vitanza, Michael Jensen, and Julie Park at Seattle Children’s. However, many CAR T cells may struggle to travel into the tumor and exert their cancer-killing effects.
Why aren’t these CAR T cells reaching their intended destination? You can imagine the tumor as a school of fish somewhere deep in the ocean and CAR T cells as human divers searching for the fish. While the fish produce faint disturbances in the current, the divers would likely have trouble efficiently locating the fish without assistance.
Researchers Dr. Edward Song and colleagues in the Vitanza Lab at Seattle Children’s sought to equip these CAR T cells with better tumor-trafficking and communicating abilities. They previously detected increased levels of the chemokine CXCL10 in cerebrospinal fluid of pediatric brain tumor patients after injection of B7-H3 CAR T cells. Since CXCL10 attracts cells bearing its receptor, CXCR3, the team reasoned that increasing CXCR3 expression on the CAR T cells would enable them to home in on the tumor—much like swapping out the human divers with a pod of orcas, who have keen hunting skills and can communicate with each other to zero in on the hidden school of fish.
“Current CAR T cell technology is effective at treating blood cancers but faces many challenges in treating solid tumors, like brain tumors, and one major challenge is for CAR T cells to locate and travel to the tumors. Therefore, we improved our CAR T cells with an additional gene modification, the chemokine receptor isoform CXCR3-A, so that they could easily locate and travel to the tumors in the brain in a positive-feedback fashion.” first author Song shares. Their study was recently published in Nature Communications.
In the lab, when CAR T cells had to move through a porous membrane to reach DIPG cells, CAR T cells with CXCR3-A overexpression (“CAR-3A cells”) migrated more efficiently and caused more DIPG cell death than control CAR T cells. Through a serious of elegant control experiments, they showed that CXCR3-A doesn’t make CAR T cells intrinsically more lethal, it makes them better at navigating to tumor cells.
Upon engaging with their target tumor cells, the CAR T cells secreted the chemokine ligands of CXCR3, including CXCL10. A small number of CAR T cells initially reaching the tumor cells set off a positive feedback loop: they recognized B7-H3, released CXCL10 and inflammatory cytokines such as IFN-γ, DIPG cells responded by making even more CXCL10, and the CAR-3A cells swarmed in more efficiently than control CAR T cells.
To test whether this improved “homing” matters in vivo, the authors turned to orthotopic xenograft mouse models of DIPG. After brainstem implantation of luciferase-labeled DIPG cells, they treated animals with control CAR T cells or CAR-3A cells via intravenous (IV) or intracerebroventricular (ICV) delivery—routes already being used in pediatric trials. When given IV, both groups of CAR T cells were ineffective against the tumor. In contrast, when given ICV, CAR-3A cells accumulated more rapidly and extensively around the tumor than control cells and led to higher CXCL10 levels after treatment. “The Experimental Histopathology Shared Resource of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium provided essential support for our immunohistochemistry analyses that beautifully showed the enhanced migration of the CXCR3-A-modified CAR T cells into the DIPG tumor areas from our mouse models.” Song adds.
In mice bearing aggressive DIPG tumors, a single ICV dose of CAR-3A cells eliminated detectable bioluminescent tumor signal and most mice maintained complete regression over the study period. In contrast, control CAR T cells slowed growth, but tumors eventually rebounded in nearly all mice. A second, slower-growing DIPG model showed similar trends: improved tumor control and survival with CAR-3A. Importantly, the CAR-3A cells did not cause treatment-related toxicity. A resistance mechanism did interestingly emerge in one mouse—antigen escape with loss of B7-H3 and luciferase—lending importance to multi-antigen CAR strategies currently being tested in clinical trials at Seattle Children’s.
This work represents an exciting advancement by showing that tuning chemokine receptor expression can convert a modest endogenous chemokine signal into a powerful homing cue, creating a self-reinforcing loop of CAR T cell recruitment and tumor killing. “This concept of CXCR3-A-modified CAR T cells is readily applicable to CAR T cell therapies targeting other types of solid tumors.” Song proposes. “Now that we have addressed the issue of CAR T cell trafficking to the tumor, CAR T cell persistence in the tumor may raise another challenge to effectively treat solid tumors such as DIPG. Thus, we are underway to develop novel cell engineering strategies to further enhance CAR T cell efficacy by addressing several intrinsic and extrinsic factors that can affect CAR T cell persistence in treating DIPG.”
“We hope this new iteration of enhanced CAR T cell technology can ultimately bring cures to children with DIPG and save invaluable lives.”
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Drs. Myron Evans, Siobhan Pattwell, Vandana Kalia, Surojit Sarkar and Nicholas Vitanza contributed to this research.
The spotlighted research was funded by the Washington Research Foundation, The Invent at Seattle Children’s Postdoctoral Scholars Program, the We Love You Connie Foundation, the National Institutes of Health (NIH) National Cancer Institute R37CA289981 grant, the Yuvaan Tiwari Foundation, the McKenna Claire Foundation, the Team Cozzi Foundation, and the Pediatric Brain Tumor Research Fund Guild of Seattle Children’s.
Song EZ, Timpanaro A, Meechan M, Elena-Sanchez L, Li LZ, Jamet S, Lau DS, Winter LI, Dun MD, Foster JB et al. 2025. Engineered CXCR3-A expression enhances B7-H3-targeting CAR T cell migration and efficacy against diffuse intrinsic pontine glioma. Nature communications 16: 9914. https://doi.org/10.1038/s41467-025-64861-6.
Science Spotlight writer Kelly Mitchell is a postdoctoral fellow in the Paddison Lab at Fred Hutch Cancer Center. She utilizes live cell reporters and CRISPR screening to study how glioblastoma cancer cells resist chemotherapy and radiation treatment. She obtained her PhD in cellular biology from Albert Einstein College of Medicine.
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