Spatial fractionated radiotherapy (SCART) remodeling of the tumor immune microenvironment and changes to ablative effects in murine models: Spatial transcriptomic and histopathologic evidence.

e15100 Background: Spatially fractionated radiotherapy (SFRT) delivers heterogeneous dose distributions to improve the therapeutic ratio. SCART (Spatially Compressed Ablative Radiotherapy), an advanced SFRT modality, offers physical advantages, but its immunobiological mechanisms remain poorly defined. This study evaluated SCART's effects on intratumoral immune architecture and ablative efficacy in a murine hepatocellular carcinoma model. Methods: Subcutaneous H22 tumors were established in BALB/c mice and treated when the short axis ≥10 mm and volume ≥700 mm³. Groups: SCART (21 Gy × 3 fractions; heterogeneous plan with central peak 21 Gy and peripheral valley 5 Gy), SBRT (uniform 21 Gy × 3), and single-fraction (21 Gy × 1). Tumors were collected 72 hours after treatment initiation. Histology (H&E) assessed necrosis and gross immune infiltration. High-definition spatial transcriptomics (0.5 μm resolution) profiled gene expression and cellular spatial organization. Analyses comprised differential expression, KEGG/GO enrichment, and spatial multimodal integration. CD45⁺/PTPRC⁺ immune populations were extracted, clustered by unsupervised machine learning, and annotated using SingleR plus AI-assisted marker- and literature-guided refinement to map immune cell distributions. Results: Baseline immune barrier: Spatial transcriptomics identified a native immune barrier at the tumor margin in untreated tumors, with fewer CD45⁺ cells at the periphery versus core—an effect not evident on H&E. Post-irradiation recruitment: SCART substantially increased peripheral immune-cell infiltration, especially at dose-gradient interfaces, exceeding the effect observed with SBRT. SCART versus SBRT: SCART elicited broader immune-activation gene programs and higher immune-cell infiltration density while reducing dose to adjacent normal tissues. The central high-dose SCART region produced effects comparable to SBRT; the intermediate- and low-dose regions of SCART represent critical intratumoral zones for immune responses. Fractionation benefit: The 21 Gy × 3 SCART regimen yielded superior 3D conformality relative to single-fraction irradiation. Conclusions: SCART remodels the spatial immune microenvironment by breaching peripheral immune barriers and promoting immune-cell recruitment, demonstrating superior immunomodulatory and ablative effects versus conventional SBRT. Fractionated SCART (21 Gy × 3) enhances conformality and central necrosis, indicating potential for reduced radiation energy requirements. These findings furnish combined immunological and dosimetric evidence to support the clinical translation of SCART.