Figure 2.

(A-D) G47Δ-mIL12 controls primary TNBC growth and inhibits metastasis in an orthotopic 4T1 mouse mammary carcinoma model. (A) Experimental schema. 1 × 10⁵ viable 4T1 cells in 100 μl PBS were injected into the right axillary mammary fat pad of female BALB/c mice. G47Δ-mIL12 (2 × 10⁶ PFU diluted in 25 μl PBS) or PBS injected intratumorally on day 6 (when tumors reached ~70 mm³), 9, 12, 15, and 18. Tumor volumes were measured every 2–3 days. (B) Tumor growth kinetics. Mean tumor volume of PBS injected tumors was compared to mean tumor volume of G47Δ-mIL12 injected tumors from day 10 to day 21. (C) Representative images of lungs. Mice from experiment 2B were euthanized on day 21 and lungs were fixed in Bouin's fixative and imaged at 24 hours post-fixation. Red asterisk indicates metastatic nodules at higher magnification in insets. (D) Total number of lung surface metastatic nodules counted with a stereo microscope. Mice from experiment 2B. Data presented by combining two independent experiments (n = 12 mice/group) and each experimental data are indicated by round- and square-shaped symbols. Mean ± SEM. Unpaired Student's t-test (two-tailed), ^**P ≤ 0.01, ^***P ≤ 0.001, ^****P ≤ 0.0001; d.p.i., days post-tumor implantation. (E-H) G47Δ-mIL12 treatment controls growth of treated and untreated contralateral tumors at late-stage of tumor development and extends survival. (E) Experimental schema. Orthotopic 4T1 tumors were established bilaterally in the right and left axillary mammary fat pads (1 × 10⁵ cells per mammary pad) on day 0. When tumors reached between 100 and 125 mm³ in volume, tumors located at the right axillary mammary fat pad intratumorally injected with PBS (n = 6) or G47Δ-mIL12 (10⁶ pfu/injection) (n = 8) on days 10, 13, 16, and 19, and the tumors at the left axillary mammary fat pad remained untreated and served as contralateral tumors. Tumor volumes periodically measured by caliper and mice were followed for survival until they become moribund or until tumors reached to their burden limit, i.e., 1.5 cm in size. (F, G) Tumor growth kinetics of treated and untreated contralateral tumors. Mean tumor volume of PBS treatment group was compared to mean tumor volume of G47Δ-mIL12 treatment group on day 16 and 19 by Unpaired Student's t-test (two-tailed). ^*P ≤ 0.05, ^**P ≤ 0.01. (H) Kaplan-Meier survival curve. Median survival of mice treated with G47Δ-mIL12 (27.5 days; 25% of mice surviving until the end of experiment on day 36) was compared to median survival of mice treated with PBS (23 days; P = 0.0001) by Log Rank test. ^****P ≤ 0.0001. d.p.i., days post-tumor implantation.