Biomaterial-Based In Situ CAR-M Engineering for NSCLC Treatment

Lung cancer remains the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for 85% of cases.1 While immune checkpoint inhibitors (ICIs) have improved treatment outcomes, many patients exhibit limited responses or develop resistance due to immunosuppressive tumor microenvironments (TME), restricting immune infiltration and dampening immune activation.1-2 Chimeric antigen receptor macrophage (CAR-M) therapy has shown potential in targeting solid tumors, but clinical translation remains limited due to poor macrophage persistence, inefficient tumor infiltration, and reliance on costly ex vivo cell modifications.3-4 To address these challenges, we propose a PEG-based biodegradable hydrogel for in situ CAR-M engineering, delivering 8–10 µg CAR-encoding mRNA per injection biweekly directly into the NSCLC TME.5 This approach leverages the natural abundance of tumor-associated macrophages (TAMs) to induce localized CAR expression, macrophage polarization, and enhanced anti-tumor activity.1 To evaluate this approach, we will use the K-rasLSL-G12D/+;p53fl/fl (KP) mouse model, which closely mimics human NSCLC by developing spontaneous tumors with a TAM-rich, immunosuppressive TME.6 This model is well-suited for testing in situ CAR-M reprogramming in a physiologically relevant system. To ensure immune system maturity and optimal tumor development, six-to-eight-week-old KP mice will be used.7 Luciferase-expressing NSCLC cells will enable real-time, non-invasive tumor tracking via bioluminescent imaging (BLI).8-9 Mice (n=40) will be divided into two groups: hydrogel-CAR-mRNA (treatment) and blank hydrogel (negative control), with each group consisting of 20 mice (10 male, 10 female) to account for sex-based differences in immune response.10 Key outcomes include CAR expression (flow cytometry), macrophage polarization (multiplex cytokine assay), and tumor regression (IVIS-BLI).11-15 We anticipate a significantly enhanced immune infiltration in the treatment group, increased M1 polarization and reduction in tumor burden. This biomaterial-based in situ CAR-M engineering strategy provides a scalable, cost-effective immunotherapy platform to overcome TME-induced resistance and improve treatment outcomes in NSCLC patients.