2440 - Tumor Immune Microenvironment Remodeling after Photon or Carbon Ion Radiation Combined with CTLA-4 Blockade in an Orthotopic Murine Osteosarcoma Model

Purpose/Objective(s):

Osteosarcoma exhibits a relatively immunologically cold tumor microenvironment, limiting responsiveness to immune checkpoint inhibitors. Using an orthotopic murine model to better recapitulate native immune context, we hypothesized that fractionated radiation induces stronger immune activation than single dose treatment, and that combining radiation with CTLA-4 blockade (C4) would amplify antitumor immunity and promote abscopal control. We further compared photon (?-IR) and carbon ion irradiation (Cion-IR) at equivalent biologic effect to determine whether radiation quality differentially remodels the TME and modifies response to C4.

Materials/Methods:

LM8 cells were implanted into one tibia of female C3H mice (primary tumor) and subcutaneously into the contralateral hind limb (distant lesion). On day14, the tumor bearing tibia received 16 Gy ×1 or 8 Gy ×3 ?-IR. Subsequent experiments used 8 Gy ×3 photons or biologically equivalent 4 Gy ×3 Cion-IR. Contralateral tumors were not irradiated. C4 (100 µg i.p. q2d ×3) was administered starting day14. Tumors were harvested 8days after irradiation, for flow cytometric analysis of CD8+T cells, Tregs, macrophages, CD103+cells, and functional markers. Bulk RNA sequencing was performed, followed by pathway enrichment analysis and immune infiltration estimation using computational deconvolution algorithms. Survival, growth of unirradiated tumors and metastases were assessed.

Results:

In photon treated tumors, fractionated irradiation increased immune infiltration versus controls, further enhanced by C4. Although ?-IR elevated Tregs, C4 reduced Tregs and augmented CTL responses and antigen presentation. RNA sequencing showed upregulation of cytotoxic genes, chemokines, and type I interferon pathways, with higher immune scores in the combination group. Survival improved with monotherapy and further with photon plus C4. Delayed growth of unirradiated tumors and reduced visceral metastases suggested abscopal effects.

In carbon ion treated tumors, Cion-IR alone markedly increased immune infiltration and CTL activation and induced DNA damage signatures. Orthotopic tumor volumes were significantly reduced in both IR and combination groups compared with controls. Although C4 reduced Tregs, it provided minimal additional improvement in immune infiltration or survival compared with Cion-IR alone, suggesting limited incremental benefit despite trends toward improved distant control.

Conclusion:

Using an orthotopic murine osteosarcoma model, we demonstrate that fractionated ?-IR enhances antitumor immunity compared with single-dose irradiation, and addition of C4 further converts the “cold” TME into an immune-activated state, improving survival, reducing metastasis, and promoting abscopal tumor control. In contrast, Cion-IR induces strong DNA damage and immune remodeling, but C4 provides limited incremental benefit in early TME reprogramming, despite potential effects on distant tumor control.