Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2⁺, immunologically “cold”) melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50–150 mm³) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 µg daily, treatment days 6–10), and anti-CTLA-4 (100 µg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8⁺ and CD4⁺) and monocytes/macrophages (F4/80⁺) but no changes in FOXP3⁺ regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNγ, TNFα and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 (⁸⁶Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (∼12:1 brain tumor/brain and ∼8:1 flank tumor/muscle). When NM600 was chelated to therapeutic β-particle-emitting ⁹⁰Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies.

超过 60% 的晚期黑色素瘤患者会发生脑转移，并对生活质量和预后产生负面影响。在小鼠黑色素瘤模型中，我们之前展示了原位疫苗接种 (ISV) 方案，结合放射治疗和肿瘤内 (IT) 注射免疫细胞因子（IC：与 IL2 融合的抗 GD2 抗体），以及免​​疫检查点抑制剂抗CTLA-4 可强力消除外周侧腹肿瘤，但对同时发生的颅内肿瘤仅具有适度影响。在这项研究中，我们研究了低剂量大脑辐射与 ISV + 抗 CTLA-4 结合时增强针对脑肿瘤的抗肿瘤免疫的能力。B78 (GD2 ⁺，免疫学上“冷”）黑色素瘤肿瘤细胞被植入 C57BL/6 小鼠的侧翼和大脑右侧纹状体。胁腹肿瘤（50-150毫米³）处理以下先前优化ISV方案[辐射（12 Gy的×1，治疗第1天），IT-IC（50微克每日，治疗6-10天），以及抗CTLA- 4（100 µg，治疗第 3、6、9 天）]。与单独接受 ISV + 抗 CTLA-4、单独接受 WBRT 或不接受治疗（对照）的动物相比，在第 15 天额外接受全脑放射治疗（WBRT，4 Gy × 1）的小鼠的存活率显着增加（P< 0.001，对数秩检验）。WBRT 的时机至关重要，因为与 ISV + anti-CTLA-4 相比，第 1 天的 WBRT 给药并没有显着提高存活率，这表明 WBRT 对存活率的影响可能是通过免疫调节介导的，而不仅仅是直接的肿瘤细胞细胞毒性。T 细胞（CD8 ⁺和 CD4 ⁺）和单核细胞/巨噬细胞（F4/80 ⁺）适度增加，但 FOXP3 ⁺没有变化在将 WBRT（在第 15 天）添加到 ISV + 抗 CTLA-4 的情况下，在脑黑色素瘤肿瘤中观察到调节性 T 细胞（Treg）。细胞因子多重免疫分析显示，在 ISV + 抗 CTLA-4 中加入 WBRT（第 15 天）后，颅内黑色素瘤和对侧正常大脑均发生明显变化，促炎（如 IFNγ、TNFα 和 LIX/CXCL5）发生显着变化和抑制性（例如，IL10、IL13）细胞因子以及趋化因子（例如，IP-10/CXCL10 和 MIG/CXCL9）。我们测试了烷基磷酸胆碱类似物 NM600 作为靶向放射性核素疗法 (TRT) 向黑色素瘤脑肿瘤提供免疫调节辐射的能力。钇86 ( ⁸⁶Y) 与 NM600 螯合后通过尾静脉向患有侧腹和脑黑色素瘤的小鼠进行静脉注射，PET 成像显示在每个肿瘤部位（~12:1 脑肿瘤/脑和~8:1 侧腹肿瘤）的特异性积累长达 72 小时/肌肉）。当 NM600 与治疗性 β 粒子发射⁹⁰螯合时Y 并在治疗第 13 天给药，黑色素瘤脑肿瘤中的 T 细胞浸润和细胞因子谱发生了改变，就像在 WBRT 中观察到的那样。总体而言，我们的结果表明，添加低剂量辐射，与 ISV 对脑外肿瘤的给药时机适当，显着增加了患有黑色素瘤脑肿瘤的动物的存活率。这一观察结果具有潜在的重要转化意义，可作为一种治疗策略，用于增加大脑中肿瘤对全身免疫疗法的反应。