Figure 6. Therapeutic Inhibition of IL-6 and mTOR Suppresses VEGFR TKI Withdrawal-Mediated Rebound Tumor Growth.

(A) Tumor volume comparison 23 days after orthotopic implantation of 4T1^P and 4T1^SuR cells (representing ST-W conditions) followed by treatment with α-mIL-6 antibody (BALB/c; n = 11–12).

(B) Combined analysis of differences in (presurgical) PT and (postsurgical) survival in an ortho-surgical model comparing α-mIL-6 (black dots) and α-hIL-6 (gray dots) treatment after LM2–4^P (SCID; n = 5) and LM2–4^SuR/ST-W (SCID; n = 10–18) cell implantation. Crossed lines represent the SD of α-mIL-6 (black cross) and α-hIL-6 (gray cross)-treated data derived from comparisons of PT burden data (vertical dotted line) and median survival data (horizontal dotted line). P values for PT burden (Student’s t test) and survival (Kaplan-Meier log-rank) are listed for comparisons between P and SuR cell lines, with significant values in bold.

(C and D) Quantification of phosphorylation levels of S6 and NF-κB-p65 proteins via western blot analysis for (C) LM2–4 SuR cell variants (n = 3) and (D) after 12.5 nM rapamycin treatment for 48 hr (n = 3).

(E) ELISA quantification of secreted IL-6 and IL-1α levels in conditioned media of LM2–4 SuR cell variants 48 hr after 12.5 nM rapamycin treatment (n = 4).

(F) Comparison of PT burden (left) and survival (right) in mice implanted orthotopically with LM2–4^SuR cells (representing ST-W conditions) and treated with rapamycin (SCID; n = 15).

(G) Comparison of 4T1^AxR/ST-W C₁₂FDG⁺ and 4T1^AxR/ST-W C₁₂FDG⁻ PT growth following orthotopic implantation in BALB/c mice (n = 6) and treated with rapamycin (starting at day 10).

(H and I) Comparison of C₁₂FDG⁺ and C₁₂FDG⁻ tumor responses to rapamycin treatment (compared to respective vehicle-treated control) based on (H) PT growth and (I) lung Met nodules at experiment termination (day 21).

Veh, vehicle; P, parental; SuR, sunitinib-resistant; AxR, axitinib-resistant; ST-W, short-term withdrawal; LT-W, long-term withdrawal; PT, primary tumor; Met, metastasis; Tx, treatment; Rap, rapamycin. One-sample Student’s t test was used for (D). Quantitative data are shown as mean ± SD. Treatment was stopped at day of surgery for (B), whereas it was continued until endpoint for (F). α-mIL-6 (100 μg/mouse/3 days); α-hIL-6 (400 μg/mouse/3 days). ST-W equaled 2 days in vitro; P cells treated with Su (5 μM) for 2 days. Overall Survival was based on Kaplan-Meier. Rapamycin (5 or 10 mg/kg/day). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared to untreated animals or parental cell lines, unless otherwise noted. Replicates and unadjusted uncropped images are shown in Data S1. See also Figure S5.