Figure 3. GARP upregulation in murine mammary cancer cells promotes TGFβ activation, tumor growth, metastasis and immune tolerance.

(A) Concentration of active TGFβ in 3 days conditioned medium measured by ELISA, and immunoblot for GARP in 4T1 cells stably expressing GARP, sGARP or EV. (B) Treg differentiation assay. Naïve splenic CD4⁺CD25⁻ cells were stimulated with agonistic antibody against CD3 and CD28 for 3 days in the presence of 50% 72-hour conditioned media from 4T1-GARP, 4T1-sGARP, or 4T1-EV cells. Flow cytometry was then performed to define the percentage of Treg conversion from naïve non-Treg cells. (C) Female BALB/c mice were injected in the 4^th mammary fat pad with 5×10⁵ tumor cells. Tumor volume was measured every 3 days. (D) Three weeks after tumor injection, mice were sacrificed and the primary tumors were resected and weighted. (E) Lungs were isolated, paraffin embedded, and H&E stained for histological analysis. The number of micro-metastatic tumor nodules in the lungs were enumerated by a pathologist. (F) Portions of the primary tumors were isolated and embedded in OCT and snap frozen. The fresh frozen sections were stained for the presence of p-SMAD-2/3; representative images are shown. Scale bar: 20 μm. (G) Summary statistics for p-SMAD-2/3 staining intensity, defined independently by a pathologist (S.S). (H) Tumor-infiltrating lymphocytes were isolated and the percentages of CD4⁺CD25⁺Foxp3⁺ Tregs in CD4⁺ cells were determined by flow cytometry with representative flow plots. (I) Summary of the percentage of Tregs in the tumor microenvironment. *P<0.05; **P<0.01; ***P<0.001. Statistical significance was determined by two-tailed Student’s t-test or Two-way ANOVA, where appropriate. Data is representative of at least two independent experiments.