Fig 2: Flow cytometry assays for immune cells analysis of draining lymph nodes and spleens in the 4T1 model. (A–C) Representative flow cytometric images of T cells in draining lymph nodes. (A) CD8+ (CTL) and CD4+T cells populations. (B) IFN-γ expression in CTLs. (C) TOX expression in CTLs. (D) Quantification of T-cell subsets and effector markers in draining lymph nodes. (E–G) Representative flow cytometric images of T cells in spleens. (E) CD8+ (CTL) and CD4+T cell populations. (F) IFN-γ expression in CTLs. (G) TOX expression in CTLs. (H) Quantification of T-cell subsets and effector markers in spleens. (I–K) DC and macrophage subsets in spleens. (I) PD-L1 expression on cDC1 cells. (J) cDC2 cell population. (K) M1-like macrophage frequency. (L) Quantitative summary of DC and macrophage features in spleens. The two-tailed, unpaired Student’s t-test was used when two groups were compared. The symbol “*” represents differences compared with G1; the symbol “#” represents differences in pairwise comparisons. */#p<0.05, **/##p<0.01, ***p<0.001. cDC: conventional dendritic cells; CTLs, cytotoxic T lymphocytes; DC, dendritic cell; IFN, interferon; MHC, major histocompatibility complex; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; TOX:thymocyte selection associated high mobility group box protein.

Fig 3: D-alanine scanning identifies key amino acids of DPA. (A–B) PD-L1 expression in A375-PD-L1 and 4T1 cells and tumors was evaluated through western blot assay (A) and flow cytometry analysis (B). (C) PET/CT imaging of [68Ga]DPA in A375-PD-L1 tumor-bearing mice at 30 and 60 min after tail vein injection of 3.7 MBq radiopharmaceuticals. Tumors were indicated by white dashed circles (n=3). (D) Illustration of D-alanine scanning of DPA peptide. (E) PET/CT imaging of [68Ga]D1-12 in A375-PD-L1 tumor-bearing mice at 30 min after tail vein injection of 3.7 MBq radiopharmaceuticals. Tumors were indicated by white dashed circles (n=3). (F–G) Tumor-to-heart (F) and tumor-to-liver (G) ratios of [68Ga]DPA and [68Ga]D1-12 based on PET/CT imaging. All results are expressed as means±SEM, as indicated in at least three independent experiments. The two-tailed, unpaired Student’s t-test was used when two groups were compared. The symbol “*” represents differences compared with the [68Ga]DPA; *p<0.05, **p<0.01, ***p<0.001. PD-L1, programmed death-ligand 1; PET, positron emission tomography.

Fig 4: Flow cytometry assays for immune cells analysis of primary and distant tumors in the 4T1 model. (A–C) Representative flow cytometric images of T cells in primary tumors. (A) CD8+ (CTL) and CD4+T cells populations. (B) IFN-γ expression in CTLs. (C) TOX expression in CTLs. (D) Quantification of T-cell subsets and effector markers in primary tumors. (E–G) DC and macrophage subsets in primary tumors. (E) PD-L1 expression on cDC1 cells. (F) cDC2 cell population. (G) M1-like macrophage frequency. (H) Quantitative summary of DC and macrophage features in primary tumors. (I–K) Representative flow cytometric images of T cells in distant tumors. (I) CD8+ (CTL) and CD4+T cells populations. (J) IFN-γ expression in CTLs. (K) TOX expression in CTLs. (L) Quantification of T-cell subsets and effector markers in distant tumors. The two-tailed, unpaired Student’s t-test was used when two groups were compared. The symbol “*” represents differences compared with G1; the symbol “#” represents differences in pairwise comparisons. */#p<0.05, **/##p<0.01, ***/###p<0.001. cDC: conventional dendritic cells; CTLs, cytotoxic T lymphocytes; DC, dendritic cell; IFN, interferon; MHC, major histocompatibility complex; PD-1, programmed cell death protein-1; PD-L1, programmed death -ligand 1; TOX:thymocyte selection associated high mobility group box protein.

Fig 5: [177Lu]D3-6 combined with anti-PD-1 therapy achieved superior whole-body tumor inhibition. (A) Schematic illustration shows the design and mechanism of therapeutic evaluation. 4T1 tumor-bearing mice were randomized into six groups: saline (G1), [177Lu]D3 (G2), [177Lu]D3-6 (G3), anti-PD-1 (G4), [177Lu]D3 combined with anti-PD-1 (G5), [177Lu]D3-6 combined with anti-PD-1 (G6). (B) Tumor volume was measured every other day. (C–D) Individual tumor volume of each mouse in the six groups, shown for both primary (C) and distant tumors (D), to evaluate the antitumor efficacy of [177Lu]D3-6 and the combination therapy. (E) Tumor volume of primary and distant tumors on day 21. (F) Survival curve of six groups during the treatment. (G) The body weight of 4T1 tumor-bearing mice in each group was measured within 30 days. All results are expressed as means±SEM, as indicated in at least three independent experiments. The two-tailed, unpaired Student’s t-test was used when two groups were compared. The symbol “*” represents differences compared with the G1; the symbol “#” represents differences in pairwise comparisons. */#p<0.05. **/##p<0.01. ***/###p<0.001. i.t., intratumoral injection; i.p., intraperitoneal injection; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1.