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. 2023 Aug 8;11(8):e006457. doi: 10.1136/jitc-2022-006457

Figure 4.

Figure 4

In vivo modulation of the adenosine pathway reduces tumor growth and metastasis and improves the efficacy of cytotoxic treatment. (A) Schedule of adenosine inhibition (Adoi) treatment. Treatment was started following 12–14 days from implantation and continued for 2 weeks. Antibody anti-CD73 (2c5mIgG1, murine IgG1) was dosed two times per week intraperitoneally at 10 mg/kg. Adora2a inhibitor (AZD4635) was given by oral gavage two times a day at 50 mg/kg. (B–C) Mass spectrometry imaging (MSI) representative images (B) and MSI analysis graph with relative abundance (a.u.) (C) showing adenosine expression and distribution in PDAC allografts treated with vehicle+isotype or Adoi, at day 14 from treatment start (six mice per treatment group). Classification was obtained based on metabolites expression and is represented as follows: viable tumor (green line), necrotic margins (yellow line). (D) Tumor growth ratio (left) and weight (right) of 6419c5 allografts in C57Bl/6 mice treated with anti-CD73+AZD4635 (N=16) or vehicle+isotype (N=15). (E) Representative image (left) and graph (right) showing % of area of the lung analyzed occupied by metastasis (met/lung areas×100) in vehicle+isotype (N=12) and anti-CD73+AZD4635 (N=13), evaluated for the presence of spontaneous occurrence of lung metastases. Every dot represents a single mouse. Tissues were stained with an anti-p53 antibody to highlight the presence of cancer cells. A group of more than five p53-positive cells was counted as metastasis. (F) Schedule of 6419c5 tumor allografts 14-day treatment as following (N=7 mice per group): vehicles+isotype, AZD6738+gemcitabine, anti-CD73+AZD4635, AZD6738+gemcitabine + anti-CD73+AZD4635. (G) Tumor growth ratio (14 days) of 6419c5 tumor allografts treated as above (H) percentage change in the long diameter length following 14 days of treatment per group. Number of mice with stable disease (SD, <20% increase and <30% decrease) are shown at the bottom. (I) Schedule of KPC mice treatment as following: vehicles+isotype (12 mice), AZD6738+gemcitabine (11 mice), anti-CD73+AZD4635 (7 mice), AZD6738+gemcitabine + anti-CD73+AZD4635 (combo, 12 mice). Adenosine inhibition was administered until endpoint, while AZD6738+gemcitabine was allowed up to 3 weeks. (J) Survival analysis of KPC mice treated as above, including median overall survival (mOS). (K) Percentage of tumor volume increased in vehicles versus combo group at day 7 and 14 of treatment. All data are presented as mean±SEM. Statistical analysis was performed using Mann-Whitney test (D–E,K) mixed-effect model (F) and one-way analysis of variance with post hoc test analysis for multiple comparisons (C). Log-rank Mantel-Cox test to evaluate difference in survivals (J) p values are shown in the graphs and considered significant when p<0.05. IOT, immuno-oncology therapy; IP, intraperitoneal.