Fig. 8: Systemic or intratumoral CCR8⁺ T_reg cell depletion combined with VEGF blockade restrains KP adenocarcinoma progression.

a, Schematic of the experimental design; s.c., subcutaneous. b, Tumor growth dynamics upon the indicated therapeutic interventions. The data represent mean values of tumor volume measurements (left). Adjusted P values for day 20 measurements: PBS-IgG versus DT-IgG P < 0.0001; PBS-IgG versus PBS-αVEGF P = 0.0004; PBS-IgG versus DT-αVEGF P < 0.0001; DT-IgG versus PBS-αVEGF P = 0.0328; DT-IgG versus DT-αVEGF P = 0.0109; PBS-αVEGF versus DT-αVEGF; P = 0.0005. Representative image of tumor volumes at day 20 (center). Kaplan–Meyer survival curves followed by log rank (Mantel–Cox) of KP tumor-bearing mice (right). The ‘survival’ time reflects the end point of the experiment when tumor volume in individual mice reached 1 cm³; adjusted P values: PBS-IgG versus DT-IgG P = 0.0012; PBS-IgG versus PBS-αVEGF P > 0.05 (NS), PBS-IgG versus DT-αVEGF P = 0.0078; DT-IgG versus PBS-αVEGF P > 0.05 (NS); DT-IgG versus DT-αVEGF P = 0.05; PBS-αVEGF versus DT-αVEGF P = 0.0186. c,d, Quantification of the indicated immune cell subsets and frequencies of activated (CD44^hi CD62^lo), proliferating (Ki67⁺) and IFN-γ-producing TCRβ⁺ CD4⁺ and TCRβ⁺ CD8⁺ cells in tumor samples shown in Fig. 8b in the indicated experimental groups of mice analyzed on day 20. e, Representative HIF1α and TUNEL staining of KP tumor sections. f, Quantification of HIF1α expression and apoptosis (TUNEL staining) in KP tumor sections; staining areas and signal intensity normalized by the total area and mean background intensity, respectively. 3–5 tumors from each experimental group were analyzed. (PBS-IgG N = 5; DT-IgG N = 4; PBS αVegf N = 3; DT αVegf N = 3) with four sections per individual tumor sample. Data represent the mean ± s.e.m. g, Proportion of intratumoral T_reg cells on day 20 after KP tumor transplantation. Data represent the mean ± s.e.m. of one of two independent experiments; N = 8. h, Tumor growth dynamics upon the indicated therapeutic interventions. Gray arrows indicate days of neutralizing antibody administration. The data represent mean values of tumor volume measurements (left). Adjusted P values for day 20 measurements: IgG versus αCCR8 P < 0.0001; IgG versus αVEGF P < 0.0001; IgG versus αCCR8-αVEGF P < 0.0001; αCCR8 versus αVEGF P = 0.0434; αCCR8 versus αCCR8-αVEGF P = 0.0044; αVEGF versus αCCR8-αVEGF P < 0.0001. i, Quantification of proportion and absolute numbers of intratumoral and splenic T_reg cells following treatment (left) and the corresponding T_reg cell numbers in spleens in the treated animals (right). Data in h and i represent the mean ± s.e.m. of one of two independent experiments, IgG N = 10, CCR8 N = 10, αVegf N = 8, CCR8-αVegf N = 8. j, Tumor growth dynamics upon the indicated therapeutic interventions (left). Gray and black arrows indicate timing of neutralizing antibody and CCR2 inhibitor (CCR2i) administration, respectively. The data represent the mean ± s.e.m. values of tumor volume measurements. Adjusted P values of day 20 measurements: IgG versus αCCR8 P = 0.0009; IgG versus αVEGF P < 0.0001; IgG versus CCR2i P < 0.0001; IgG versus αCCR8-αVEGF P < 0.0001; IgG versus αCCR8-CCR2i P < 0.0001; αCCR8 versus αVEGF P = 0.9982; αCCR8 versus CCR2i P = 0.6138; αCCR8 versus αCCR8-αVEGF P < 0.0001; αCCR8 versus αCCR8-CCR2i P = 0.0041; αVEGF versus CCR2i P = 0.9551; αVEGF versus αCCR8-αVEGF P = 0.0003; αVEGF versus αCCR8-CCR2i P = 0.0363; CCR2i versus αCCR8-αVEGF P = 0.0018; CCR2i versus αCCR8-CCR2i P = 0.2271; αCCR8-αVEGF versus αCCR8-CCR2i P = 0.4530. Plots include data from two independent experiments combined with nine animals in each group in experiment 1 (IgG N = 9, αCCR8 N = 9, αVEGF N = 9, CCR2i N = 9, αCCR8 N = αVEGF-9, αCCR8-CCR2i N = 9) and 4–6 animals per group in experiment 2 (IgG N = 4; CCR2i N = 6; CCR8-CCR2i N = 6). k, Kaplan–Meyer survival curves followed by Log-rank (Mantel–Cox) of KP tumor-bearing mice. The ‘survival’ time reflects the end point of the experiment when tumor volume in individual mice reached 1 cm³. Adjusted P values: IgG versus αCCR8 ***P < 0.0001; IgG versus αVEGF ***P < 0.0001; IgG versus CCR2i ***P < 0.0001; IgG versus αCCR8-αVEGF ***P < 0.0001; IgG versus αCCR8-CCR2i ***P < 0.0001; αCCR8 versus αVEGF P = 0.5687 (NS); αCCR8 versus CCR2i P = 0.7411 (NS); αCCR8 versus αCCR8-αVEGF ***P = 0.0002; αCCR8 versus αCCR8-CCR2i P = 0.0342; αVEGF versus CCR2i P = 0.8054 (NS); αVEGF versus αCCR8-αVEGF ***P = 0.0006; αVEGF versus αCCR8-CCR2i P = 0.0666 (NS); CCR2i versus αCCR8-αVEGF ***P = 0.0003; CCR2i versus αCCR8-CCR2i P = 0.6749 (NS); αCCR8-αVEGF versus αCCR8-CCR2i *P = 0.0489. Plots include data from two independent experiments combined with 5–11 animals in each group in experiment 1 (IgG N = 9, αCCR8 N = 9; αVEGF N = 9; CCR2i N = 11; αCCR8-αVEGF N = 9; αCCR8-CCR2i N = 5) and 4–10 animals per group in experiment 2 (IgG N = 7; CCR2i N = 10; CCR8-CCR2i N = 4). In b–d, h and i, plots are representative of one of two experiments with 8–10 mice per group each, at day 20 after transplantation. Number of mice per group in b and c: PBS-IgG N = 10; DT-IgG N = 10; PBS-αVEGF N = 9; DT-αVEGF N = 9; number of mice per group in h and i: IgG N = 10; αCCR8 N = 10; αVEGF N = 8; αCCR8-αVEGF N = 8.

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