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bioRxiv · 10.64898/2026.11.04.764456

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

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

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.

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Quan, R., Huang, H., Zhang, L., Zhang, C., Chang, S., Wu, M., Zhang, S., Dai, Z., Gu, L.. 2026-11-05. BNCT Triggers Immunogenic Cell Death in B16 Melanoma Without Accompanying T-Cell Infiltration--Implications for Combination Immunotherapy. https://doi.org/10.64898/2026.11.04.764456

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