BNCT Triggers Immunogenic Cell Death in B16 Melanoma Without Accompanying T-Cell Infiltration--Implications for Combination Immunotherapy

Objective: Boron neutron capture therapy (BNCT) is a targeted radiotherapy with highly selective tumor killing; its potential to induce immunogenic cell death (ICD) and modulate the tumor immune microenvironment has attracted attention. However, whether BNCT-induced immune activation can overcome the T-cell exclusion barrier of immunologically "cold" tumors and translate into intratumoral effector T-cell infiltration remains unclear. Methods: A B16 melanoma model was established in C57BL/6 mice. Mice were assigned to CK, N10/N20 (neutron irradiation alone for 10 or 20 min), BPA, and BNCTN10/BNCTN20 groups (BPA 500 mg/kg plus 10 or 20 min irradiation) to evaluate tumor suppression and survival. In a separate cohort (CK, N20, and BNCTN20), the most efficacious regimen (BNCTN20) was compared with CK on days 3 and 7: H&E staining; immunohistochemistry/immunofluorescence for gamma-H2AX, HMGB1, cleaved caspase-3, PD-L1, CD8+, and granzyme B; ELISA for cytokines and chemokines; and ICP-OES for 10B distribution. Results: BNCT significantly suppressed tumor growth. When CK tumors reached the humane endpoint (~2000 mm3), N20 tumor volume was half that of CK (TGI ~ 49.8%), whereas BNCTN20 reached only one tenth (TGI ~ 88.9%, p < 0.001), with prolonged survival (p < 0.001). BNCT induced gamma-H2AX-associated DNA damage, HMGB1 release, and cleaved caspase-3-mediated apoptosis; upregulated PD-L1; induced a Th1-type response (elevated IFN-gamma/TNF-alpha, reduced IL-10); and enhanced CXCL9/10 signaling. However, intratumoral CD8+ T-cell infiltration and granzyme B expression did not increase, indicating that this activation was not translated into a local effector T-cell response. Conclusions: BNCT inhibited tumor growth and prolonged survival while inducing ICD and a systemic Th1 response; nevertheless, systemic activation failed to translate into local CD8+ T-cell infiltration, while PD-L1 was upregulated, a "strong direct killing but blocked immune translation" profile in the B16 cold tumor. PD-L1-associated adaptive immune resistance may limit this conversion, providing rationale for combining BNCT with immune checkpoint blockade.

Quan, R., Huang, H., Zhang, L., Zhang, C., Chang, S., Wu, M., Zhang, S., Zhang, L., Dai, Z., Gu, L.

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