Figure 3.

The addition of CCR2/5i to RT and αPD-1 slowed the rate of tumor growth and prolonged survival in a PDAC orthotopic mouse model. (A) Treatment schema: On day 0, subcutaneous tumors formed by injecting the KPC tumor cells onto syngeneic wild-type C57Bl/6 mice ∼1–2 weeks before were dissected and divided into cubes of 2–3-mm diameter. One cube of tumor was immediately implanted orthotopically into the pancreas of each syngeneic wild-type C57Bl/6 mouse. After the surgery, mice were randomized into different treatment groups (six mice per group) as indicated. On day 5 (D5), pretreatment ultrasound was performed. Tumor-bearing mice were treated with RT (three fractions of 8 Gy daily on days 6–8), αPD-1, or IgG control (5 mg/kg i.p. twice weekly for 3 wk), and CCR2/5i (50 mg/kg by oral gavage twice a day continuously) on days indicated. Ultrasound was performed on days indicated. (B and C) Tumor size evaluated by ultrasound imaging until day 40 (B) and Kaplan–Meier survival curves in mice treated with different combinations of RT, αPD-1, and CCR2/5i (C). (D and E) The same experiment was repeated in the orthotopic mouse model with a different mouse PDAC cell line established from KPC mice. After tumor implantation, mice were randomized into four treatment groups (n = 5 per group) as indicated. Tumor size evaluated by ultrasound imaging until day 33 (D) and Kaplan–Meier survival curves in mice treated with different combinations of RT, αPD-1, and CCR2/5i (E). (F) Comparison of metastases between RT + aPD1 + CCR2/5i and RT + aPD1 groups combining the experiment in B and C and one repeated experiment (n = 5 per group), in total 11 mice per group. After the mice reached survival endpoint (day 140), at necropsy, numbers of mice with lung, liver, or peritoneal metastases were identified grossly and histologically. Surviving mice were free of tumors. χ² test was used to examine the correlation between treatment groups and metastasis rates. In the experiment in D and E, when the mice reached survival endpoint (day 63), all four surviving mice in the RT + aPD1 + CCR2/5i group were free of tumors; and the remaining one in the group did not have metastasis; all mice in the RT + aPD1 group had liver metastasis. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by log-rank test. All experiments were repeated at least twice.