Targeting Radiation-Induced Glioblastoma Invasion - PROJECT SUMMARY Recurrent Glioblastoma (rGBM) displays aggressive tumor invasion throughout the brain and spinal cord. The aggressive invasive behavior of rGBM emerges following initial fractionated radiotherapy, which induces mesenchymal transition and subsequent extracellular matrix (ECM) remodeling. However, the role of this ECM remodeling in establishing the immunosuppressive tumor microenvironment of invasive rGBM remains unclear. We found that Collagen type I and III (COL I & III) showed minimal expression in the normal brain, whereas it was highly upregulated in rGBM. Through genetic knockout and pharmacological inhibition, we demonstrated that COL I & III play crucial roles in establishing the immunosuppressive tumor microenvironment (TME). Mechanistically, COL-activated PI3K signaling enhances TGFβ1 expression, subsequently promoting the induction of regulatory T cells and exhausted T cells in the TME. Based on our findings, we developed two therapeutic approaches targeting invasive rGBM. First, we used blood-brain barrier (BBB)-permeable PI3K inhibitors to suppress COL-mediated signals. We confirmed that a PI3K inhibitor improved the immunosuppressive TME and extended survival in mouse models of invasive rGBM. Second, we engineered CBD-IL-12, a fusion protein combining a collagen-binding domain with the immunostimulatory cytokine IL-12. CBD-IL-12 demonstrated selective accumulation to tumor and enhanced therapeutic efficacy in rGBM mouse model, particularly when combined with PI3K inhibitors. For clinical translation, we engineered a humanized version of CBD-IL-12 and confirmed its safety in canines. Also, we analyze the therapeutic efficacy of human CBD-IL-12 against human invasive rGBM model in humanized mouse reconstituted human immune environment. The humanized CBD-IL-12 demonstrated remarkable therapeutic effects, resulting in a significant reduction of both tumor burden and spinal dissemination. Based on these findings, we hypothesize that radiation- induced COL-PI3K-TGFβ signaling contributes to the immunosuppression of invasive rGBM, and these cells can be targeted using BBB-permeable COL signaling inhibitors and CBD- IL-12 immunotherapy. We will investigate this through three specific aims: (1) Determine how COL-PI3K-TGFβ signaling induces immunosuppressive TME in invasive rGBM using genetic knockout approaches and signal inhibitors; (2) Examine how CBD-IL-12 modulates the immunosuppressive TME by analyzing chemotactic factors and dendritic cells induction; and (3) Evaluate the therapeutic synergy between CBD-IL-12 and BBB-permeable COL signaling inhibitors in clinically relevant syngeneic rGBM mouse model and humanized rGBM mouse models. Upon successful completion of these studies, we will gain crucial insights into immune evasion mechanisms of invasive rGBM and establish a strong foundation for clinical translation. This novel therapeutic strategy could be broadly applicable to other COL-expressing invasive malignancies, potentially transforming the treatment landscape.
