Pulsed Low-dose-rate Radiotherapy Remodels the Tumor Immune Microenvironment and is Associated With Crosstalk Between SPP1⁺ tumor-associated macrophages and GZMK⁺ CD8⁺ T cells

Pulsed low-dose-rate radiotherapy (PLDR) is a promising reirradiation strategy for refractory solid tumors because of its potential to reduce normal tissue toxicity; however, its immunomodulatory mechanisms remain poorly understood. In this study, we retrospectively analyzed clinical outcomes and treatment-related adverse events in 16 patients with refractory solid tumors treated with PLDR. Using MC38 tumor-bearing mice, we compared PLDR and conventional radiotherapy (cRT) under biologically equivalent dose conditions and performed single-cell RNA sequencing to characterize treatment-associated remodeling of the tumor immune microenvironment (TIM). Multiplex immunofluorescence staining and spatial analysis were further conducted in an independent cohort of 114 patients with rectal cancer to evaluate the clinical relevance of identified immune features. PLDR achieved comparable local tumor control to cRT while reducing treatment-related toxicity and improving overall survival (OS) in mouse models. In the combination-treatment setting, single-cell analyses showed that PLDR + immunotherapy with an anti-PD-1 antibody combined with the targeted agent anlotinib (ITT), compared with cRT + ITT, was associated with enrichment of Gzmk⁺ CD8⁺ T cells, reduced Spp1⁺ tumor-associated macrophages (TAMs) abundance, and altered cell–cell communication between these populations. In our independent rectal cancer cohort, the spatial distance between SPP1⁺ TAMs and GZMK⁺ CD8⁺ T cells was independently associated with OS. These findings suggest that the interaction between SPP1⁺ TAMs and GZMK⁺ CD8⁺ T cells may be closely associated with PLDR-related TIM remodeling and may also serve as a potential prognostic biomarker in rectal cancer.