Fig.1 The association of glycolysis with tumor clinical outcomes and RT. (A-B) Kaplan-Meier survival curve presenting overall survival of patients in SKCM (A) cohort (n = 229 pairs) and UVM (B) cohort (n = 39 pairs) based on glycolysis signature genes. (C-D) Kaplan-Meier survival curve presenting overall survival of patients in SKCM (C) cohort (n = 229 pairs) and UVM (D) cohort (n = 20 pairs) based on DDR signature genes. (E-F) Pearson’s correlation between glycolysis and DDR from patients with SKCM (E) and patients with UVM (F). (G) Uniform manifold approximation and projection (UMAP) plot of cells from control and RT-treated melanoma tissues, colored by cell cluster. (H) Pathway enrichment analysis of cells from control and RT-treated melanoma tissues in (G) using KEGG pathways analysis. (I) UMAP plot of cells from melanoma tissues, clustered into 2 subpopulations by glycolysis signature. (J-K) Violin plots showing the glycolysis scores (J) and the DDR scores (K) of the tumor samples in (I). (L) Evaluation of LDHA and SLC16A1 expression levels. (M) Quantification of lactate level in A375 cells after different treatments (n = 3). Data of A-F and L were obtained from the TCGA database. Data are shown as mean ± SD, ∗P < 0.05 and ∗∗∗P < 0.001.#

为了打破这种耐药屏障，研究团队设计并合成了核壳结构纳米增敏剂。如图2所示，该材料以掺杂石墨烯量子点的聚 dopamine 为核心，外层包裹金纳米颗粒壳层，形成了尺寸约100纳米的GQD@PDA/Au纳米复合颗粒。金纳米颗粒利用高原子序数效应产生俄歇电子直接破坏DNA，而石墨烯量子点则在X射线照射下高效产生活性氧（ROS）。在650纳米近红外激光照射下，该增敏剂表现出优异且稳定的光热转换能力，可在5分钟内将局部温度提升至43℃至45℃的温和热疗范围。

Fig.2 Synthesis and characterization of GQD@PDA/Au. (A) Synthetic route of GQD@PDA/Au. (B) Transmission electron microscopy (TEM) images of GQD@PDA (left) and GQD@PDA/Au (right). (C-D) DLS (C) and UV-vis spectra (D) of GQD, GQD@PDA and GQD@PDA/Au. Red arrows represent the characteristic peaks. (E-F) Quantification of ROS levels in different solutions after radiation (4Gy) via TMB (E) and DHR123 (F) indicator. (G) Temperature elevation of GQD@PDA/Au at different concentrations (0, 20, 40, 80, 160, 320 μg/mL) under 650 nm laser irradiation. (H) Temperature profiles of GQD@PDA/Au under 650 nm laser irradiation at different power densities (0.5, 1, 2 W/cm2). (I) Infrared photothermal images of GQD@PDA/Au at different concentrations under laser irradiation (λ = 650 nm, 1 W/cm2). Data are shown as mean ± SD (n = 3), ∗∗P < 0.01 and ∗∗∗P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)#

细胞层面的体外实验证实了温和光热与放疗协同杀伤肿瘤的效果。如图3所示，GQD@PDA/Au能被肿瘤细胞高效内吞，并在温和光热刺激与放射线联合作用下显著抑制肿瘤细胞增殖，诱导细胞发生大量凋亡。流式细胞术与克隆形成实验均表明，三者联合治疗展现出最强的抑癌活性。

Fig.3  Intracellular uptake analysis and effect of GQD@PDA/Au against tumor cells in vitro. (A) TEM images of OMM2.3 cells treated with PBS or 80 μg/mL GQD@PDA/Au for 4, 8, or 12 h. Red arrows indicate the subcellular internalization of nanoparticles. (B–C) Viability of A375 (B) and OMM2.3 (C) cells after different treatments. (D) A375 and OMM2.3 cells stained with Calcein-AM/PI after various treatments. Calcein-AM (green, live cells) and PI (red, dead cells). Scale bar = 100 μm. I. Ctrl; II. GQD@PDA/Au (80 μg/mL); III. Laser (λ = 650 nm, 1.0 W/cm2, 5 min); Ⅳ. GQD@PDA/Au + Laser; Ⅴ. RT (4Gy); Ⅵ. GQD@PDA/Au + RT; Ⅶ. GQD@PDA/Au + Laser + RT. (E-F) Corresponding live/dead cell percentage of A375 (E) and OMM2.3 (F) under various treatment modalities. (G) Colony formation assay to assess the growth of A375 and OMM2.3 cells following different treatments. Representative images are presented. Data are shown as mean ± SD (n = 3), ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)#

在机制探索层面，转录组测序与代谢分析揭示了光热刺激调控代谢的分子路径。如图4所示，海马体代谢分析表明联合治疗组的细胞外酸化率和糖酵解水平显著下降，肿瘤细胞上清液中的乳酸水平降低超过50%。乳酸浓度的骤降直接抑制了NBS1蛋白K388位点的乳酰化修饰，阻止了同源重组修复关键蛋白RAD51的表达。彗星实验与γ-H2AX荧光焦点分析均证实，DNA损伤修复通路的阻断导致肿瘤细胞内双链DNA断裂程度剧烈增加。

Fig.4  Transcriptome analysis and mechanism of sensitizing RT in vitro. (A) The heatmap of DEGs between control and GQD@PDA/Au + Laser + RT. Orange indicates high expression and blue indicates low expression. (B) The KEGG pathways analysis of DEGs to discuss the biological pathways affected by GQD@PDA/Au + Laser + RT. (C) The GO pathways analysis of DEGs concentrated in some specific biological processes. (D) GSEA enrichment plot of the negatively enriched biological processes term of canonical glycolysis. (E-F) GSEA showing the mountain plot for cytosolic DNA-sensing pathway (E) and positive regulation of interferon-beta production (F). (G-H) Seahorse Glycolytic Rate Assay showing the dynamic curve of extracellular acidification rate (ECAR) (G) and compensatory glycolysis (H) in tumor cells upon different treatments. (I) Quantification of lactate level in the supernatants of tumor cells after different treatments. (J-K) Western blot assay of NBS1 lactylation (J) and LDHA expression (K) in vitro following different treatments. (L) Cell viability following different treatments. (M) Immunofluorescence images of γ-H2AX (red) in tumor cells under various treatments. Scale bar = 20 μm. (N) Comet assays of tumor cells after different treatments. Scale bar = 10 μm. Data are shown as mean ± SD (n = 3), ns: no statistical significance, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)#

DNA损伤的加剧进一步触发了强烈的免疫激活效应。如图5所示，大量累积的胞质双链DNA激活了cGAS-STING信号通路，促使p-STING、p-TBK1和p-IRF3蛋白表达上调，显著增加了干扰素-β（IFN-β） 和白介素-6（IL-6） 的释放。同时，温和光热与ROS的协同作用诱导了强烈的免疫原性细胞死亡，促使钙网织蛋白表面外露和热休克蛋白释放。共培养实验显示，成熟树突状细胞的比例由对照组的15%提升至40%以上。

Fig.5 Activation of the cGAS-STING pathway and ICD effects in vitro. (A) Intracellular dsDNA levels in B16F10 cells after treatment with various formulations. Scale bar = 100 μm. I. Ctrl; II. GQD@PDA/Au (80 μg/mL); III. Laser (λ = 650 nm, 1.0 W/cm2, 5 min); Ⅳ. GQD@PDA/Au + Laser; Ⅴ. RT (4Gy); Ⅵ. GQD@PDA/Au + RT; Ⅶ. GQD@PDA/Au + Laser + RT. (B) Western blot assay of cGAS-STING pathway associated protein expression in B16F10 cells upon different treatments. (C-D) IFN-β (C) and IL-6 (D) in the supernatants of B16F10 cells after different treatments. (E) Intracellular ROS levels observed by probe DCFH-DA. Scale bar = 200 μm. (F) Immunofluorescence images of CRT (red, scale bar = 100 μm) and HMGB1 (green, scale bar = 50 μm) after different treatments. (G) Western blot images of HSPs after different formulations. (H) Ratio of extracellular ATP in the supernatants. (I) Quantitative analysis of BMDCs maturation (CD80+ CD86+ in CD11c+ cells) after 24h culture with different tumor cell groups. (J) Schematic illustration of co-culture system of cancer cells and dendritic cells (DCs). Data are shown as mean ± SD (n = 3), ∗P < 0.05 and ∗∗∗P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)#

活体动物实验全面验证了该增敏剂的抗肿瘤与免疫激活疗效。如图6所示，在黑色素瘤皮下移植模型中，联合治疗组实现了肿瘤的近乎完全消退，且未引起小鼠体重的明显波动或器官毒性。组织学检测确证了肿瘤组织内LDHA表达及NBS1乳酰化的同步下调。如图7所示，在双侧远端肿瘤模型中，对原发灶的联合治疗不仅促使肿瘤引流淋巴结中成熟树突状细胞比例升至40%以上，还显著增加了远端未治疗肿瘤内CD8+ cytotoxic T细胞和CD4+ 辅助T细胞的浸润数量，展现出强大的远隔效应，有效抑制了远端转移灶的生长。

Fig.6  In vivo antitumor performance of GQD@PDA/Au-based treatment. (A) Schematic illustration of the treatment schedule in mice bearing subcutaneous melanoma. (B-D) Digital image (B), volume curves (C), and tumor weights (D) of primary subcutaneous melanoma from different groups (n = 4). Scale bar = 2 cm. I. PBS; II. GQD@PDA/Au; III. Laser; Ⅳ. GQD@PDA/Au + Laser; Ⅴ. RT; Ⅵ. GQD@PDA/Au + RT; Ⅶ. GQD@PDA/Au + Laser + RT. (E) Representative H&E, Ki-67 and TUNEL staining images of primary subcutaneous melanoma slices after different treatments. Black scale bar = 50 μm and white scale bar = 20 μm. (F-G) Western blot assay for NBS1 lactylation (F) and LDHA expression (G) in tumor tissues following different treatments. Data are shown as mean ± SD, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.#