Fig 1.
Mammary carcinoma cells with forced expression of TFF3 increased angiogenic behavior of HUVEC.
(A) Monolayer proliferation of HUVEC after co-culture with MCF-7 cells with forced expression of TFF3 in 10% FBS conditions. (B) Monolayer proliferation of HUVEC after co-culture with MCF-7 cells with forced expression of TFF3 in 0.2% FBS conditions. (C) HUVEC cell cycle progression after 24 hours co-culture with MCF-7 cells with forced expression of TFF3 in serum-free (SF) and 10% FBS conditions. (D) HUVEC apoptotic cell death after 24 hours co-culture with MCF-7 cells with forced expression of TFF3 in serum-free and 10% FBS conditions. (E) HUVEC migration after 24 hours co-culture with MCF-7 cells with forced expression of TFF3 in serum-free conditions. (F) HUVEC invasion after 24 hours co-culture with MCF-7 cells with forced expression of TFF3 in serum-free conditions. (G) and (H) HUVEC tubule formation in vitro in the Matrigel after 12 hours co-culture with MCF-7 cells with forced expression of TFF3. Total tubule length (G) and total tubule number (H) were assessed. (I) Representative light photomicrographs of HUVEC tubule formation in vitro in the Matrigel after 12 hours co-culture with MCF-7 cells with forced expression of TFF3. MCF-7 cells with empty vector (MCF7-Vec) was used as control. β-ACTIN was used as input control in semi-quantitative RT-PCR and Western blot analyses. *, P < 0.05; **, P < 0.01; ***, P < 0.001; scale bar, 200 μm.
Fig 2.
Mammary carcinoma cells with forced expression of TFF3 promoted tumor angiogenesis in vivo.
(A) IHC analysis of CD31 and CD34 protein expressions in xenograft tumors formed by MCF-7 cells with forced expression of TFF3. (B) Microvessel density (CD31) was assessed by quantifying percentage area of CD31 labeled cells in xenografts formed by MCF-7 cells with forced expression of TFF3. (C) CD34 was assessed by quantifying percentage area of CD34 labeled cells in xenografts formed by MCF-7 cells with forced expression of TFF3. MCF-7 cells with empty vector (MCF7-Vec) used as control. **, P < 0.01; ***, P < 0.001; scale bar, 50 μm.
Fig 3.
Depletion of TFF3 by siRNA in mammary carcinoma cells decreased angiogenic behavior of HUVEC.
(A) Semi-quantitative RT-PCR analysis of TFF3 mRNA level in MCF-7 cells with depletion of TFF3 (MCF7-siTFF3) and control siRNA vector cells (MCF7-siVec) after 24 hours transient transfection. (B) Western blot analysis of TFF3 protein in MCF-7 cells with depletion of TFF3 and control siRNA vector cells after 48 and 72 hours transient transfection. (C) Monolayer proliferation of HUVEC after co-culture with MCF-7 cells with depletion of TFF3 in 10% FBS conditions. (D) Monolayer proliferation of HUVEC after co-culture with MCF-7 cells with depletion of TFF3 in 0.2% FBS conditions. (E) HUVEC cell cycle progression after 24 hours co-culture with MCF-7 cells with depletion of TFF3 in serum-free and 10% FBS conditions. (F) HUVEC apoptotic cell death after 24 hours co-culture with MCF-7 cells with depletion of TFF3 in serum-free and 10% FBS conditions. (G) HUVEC migration after 24 hours co-culture with MCF-7 cells with depletion of TFF3 in serum free conditions. (H) HUVEC invasion after 24 hours co-culture with MCF-7 cells with depletion of TFF3 in serum free conditions. (I) and (J) HUVEC tubule formation in vitro in the Matrigel after 12 hours co-culture with MCF-7 cells with depletion of TFF3 in serum-free conditions. Total tubule length (I) and tubule number (J) were assessed after 12 hours incubation. (K) Representative light photomicrographs of HUVEC tubule formation in vitro in the Matrigel after 12 hours co-culture with MCF-7 cells with depletion of TFF3. MCF-7 cells with control siRNA vector (MCF7-siVec) was used as control. β-ACTIN was used as input control in semi-quantitative RT-PCR and Western blot analyses. *, P < 0.05; **, P < 0.01; ***, P < 0.001; scale bar, 200 μm.
Fig 4.
TFF3 enhanced IL-8 expression in mammary carcinoma cells and HUVEC.
(A) IL-8 promoter reporter activity (full length, -4800 to + 104 bp) in MCF-7 cells with forced expression of TFF3 and control vector cells. MCF-7 cells with empty vector (MCF7-Vec) was used as control. (B) Semi-quantitative RT-PCR analysis of IL-8 mRNA level in MCF-7 with forced expression of TFF3 and control vector cells. (C) ELISA analysis of IL-8 protein secreted to the medium by MCF-7 cells with forced expression of TFF3 and control vector cells. (D) IHC analysis of IL-8 protein expression in xenograft tumors formed by MCF-7 cells with forced expression of TFF3. (E) Percentage of IL-8 labeled cells in xenograft tumors formed by MCF-7 cells with forced expression of TFF3 and control vector cells. (F) IL-8 promoter reporter activity (full length, -4800 to + 104 bp) in MCF-7 cells with depletion of TFF3 and control siRNA vector cells. MCF-7 cells with control siRNA vector (MCF7-siVec) was used as control. (G) Semi-quantitative RT-PCR analysis of IL-8 mRNA level in MCF-7 with depletion of TFF3 and control siRNA vector cells. (H) ELISA analysis of IL-8 protein secreted to the medium by MCF-7 cells with depletion of TFF3 and control siRNA vector cells. (I) Semi-quantitative RT-PCR analysis of IL-8 mRNA level in HUVEC co-cultured with MCF-7 cells with forced expression of TFF3 and control vector cells. (J) ELISA analysis of IL-8 protein secreted to the medium by HUVEC co-cultured MCF-7 with forced expression of TFF3 and control vector cells. β-ACTIN was used as input control in semi-quantitative RT-PCR and Western blot analyses. **, P < 0.01; ***, P < 0.001; scale bar, 50 μm.
Fig 5.
Depletion of IL-8 in mammary carcinoma cells with forced expression of TFF3 abrogated the stimulatory effect of TFF3 on HUVEC migration, invasion, and tubule formation in vitro.
(A) ELISA analysis of IL-8 protein secreted to the medium by MCF7-Vec and MCF7-TFF3 transiently transfected with control siRNA and IL-8 siRNA after 48 hours transfection. Scrambled control siRNA (siControl) was used as control. MCF7-Vec transiently transfected with scrambled control siRNA was a baseline. (B) HUVEC migration after 24 hours co-culture with MCF-7 cells with forced expression of TFF3 transiently transfected with control siRNA and IL-8 siRNA in serum-free conditions. (C) HUVEC invasion after 24 hours co-culture with MCF-7 cells with forced expression of TFF3 transiently transfected with control siRNA and IL-8 siRNA in serum-free conditions. (D) and (E) HUVEC tubule formation in vitro in the Matrigel after 12 hours co-culture with MCF-7 cells with forced expression of TFF3 transiently transfected with control siRNA and IL-8 siRNA in serum-free conditions. Total tubule length (D) and tubule number (E) was assessed using ImageJ analysis software. (F) Representative light photomicrographs of HUVEC tubule formation in vitro in the Matrigel after 12 hours co-culture of with MCF-7 cells with forced expression of TFF3 transiently transfected with control siRNA and IL-8 siRNA. **, P < 0.01 as compared with MCF7-Vec or MCF7-TFF3 transiently transfected with scrambled control siRNA, respectively. Scale bar, 200 μm.
Fig 6.
Monoclonal antibody inhibition of IL-8 and CXCR2 decreased TFF3 stimulated HUVEC tubule formation in vitro.
(A) and (B), HUVEC tubule formation in vitro in the Matrigel after 12 hours co-culture with MCF-7 cells with forced expression of TFF3 treated with IgG control or 50 μg/mL of anti-IL-8 monoclonal antibody in serum-free conditions. IgG was used as control. HUVEC co-cultured with MCF7-Vec treated with IgG control was a baseline. Total tubule length (A) and tubule number (B) was assessed using ImageJ analysis software. (C) Representative light photomicrographs of HUVEC tubule formation in vitro in the Matrigel, in which HUVEC co-cultured with MCF7-Vec and MCF7-TFF3 treated with IgG control or 50 μg/mL of anti-IL-8 monoclonal antibody. **, P < 0.01 as compared to HUVEC co-cultured with MCF7-Vec or MCF7-TFF3 treated with IgG control. (D) and (E) HUVEC tubule formation in vitro, in which MCF-7 cells with forced expression of TFF3 co-cultured with HUVEC treated with IgG control or 20 μg/mL of anti-CXCR2 monoclonal antibody. MCF7-Vec co-cultured with HUVEC treated with IgG control was as baseline. Total tubule length (D) and tubule number (E) was assessed. (F) Representative light photomicrographs of HUVEC tubule formation in vitro in the Matrigel, in which MCF7-Vec and MCF7-TFF3 co-cultured with HUVEC treated with IgG control or 20 μg/mL of anti-CXCR2 monoclonal antibody. **, P < 0.01 as compared to MCF7-Vec or MCF7-TFF3 co-cultured with HUVEC treated with IgG control. Scale bar, 200 μm.
Fig 7.
Depletion of STAT3 in mammary carcinoma cells by siRNA partially diminished the ability of TFF3 to stimulate mammary carcinoma cell IL-8 promoter activity and IL-8 protein expression as well as HUVEC migration, invasion, and tubule formation in vitro.
(A) IL-8 promoter reporter activity in MCF7-Vec and MCF7-TFF3 with depletion of STAT3 transiently transfected with an IL-8 promoter reporter vector (full length, -4800 to + 104 bp) and a pRL-CMV control reporter vector. Scrambled control siRNA was used as control. MCF7-Vec transiently transfected with scrambled control siRNA was as baseline. (B) ELISA analysis of IL-8 protein secreted to the medium by MCF7-Vec and MCF7-TFF3 transiently transfected with pcDNA vector containing STAT3 siRNA or control siRNA. *, P < 0.05; **, P < 0.01 as compared MCF7-Vec or MCF7-TFF3 transiently transfected with control siRNA. (C) HUVEC migration after 24 hours co-culture with MCF-7 cells with forced expression of TFF3 transiently transfected with control siRNA and STAT3 siRNA. (D) HUVEC invasion after 24 hours co-culture with MCF-7 cells with forced expression of TFF3 transiently transfected with control siRNA and STAT3 siRNA. (E) and (F) HUVEC tubule formation in vitro in the Matrigel after 12 hours co-culture with MCF-7 cells with forced expression of TFF3 transiently transfected with control siRNA and STAT3 siRNA in serum-free conditions. Total tubule length (E) and tubule number (F) was assessed. **, P < 0.01 as compared to MCF7-Vec or MCF7-TFF3 transiently transfected with control siRNA, respectively; Scrambled control siRNA was used as control; MCF7-Vec transiently transfected with scrambled control siRNA was a baseline.
Fig 8.
Exogenous recombinant hTFF3 increased HUVEC monolayer proliferation, migration, invasion, and tubule formation in vitro.
(A) HUVEC monolayer proliferation at different concentrations of recombinant hTFF3 in 10% FBS condition. (B) HUVEC monolayer proliferation at different concentrations of recombinant hTFF3 in 0.2% FBS conditions. HUVEC treated with different concentrations of recombinant hTFF3 (0.1, 1.0, 2.5, 5, 10 ng/mL of recombinant hTFF3) and BSA control (10 ng/mL). BSA was used as control. HUVEC treated with BSA control was a baseline. (C) HUVEC migration after 24 hours co-cultured with different concentration of recombinant hTFF3 or BSA control. (D) HUVEC invasion after 24 hours co-cultured with different concentration of recombinant hTFF3 or BSA control. (E) HUVEC tubule formation in vitro, in which HUVEC treated with different concentration of recombinant hTFF3 or BSA control were plated in the Matrigel. Total tubule length was assessed using ImageJ analysis software. (F) Representative light photomicrographs of HUVEC tubule formation in vitro, in which HUVEC treated with different concentrations of recombinant hTFF3 or BSA control. *, P < 0.05; **, P < 0.01; ***, P < 0.001 as compared with BSA control. Scale bar, 200 μm.
Fig 9.
TFF3 is a promoter of angiogenesis in mammary carcinoma.
TFF3 secreted from mammary carcinoma cells indirectly stimulated angiogenic behavior of endothelial cells to promote angiogenesis in mammary carcinoma via an IL-8/CXCR2 axis. STAT3 is one transcription factor responsible for the increased expression of IL-8 by TFF3. TFF3 also promotes angiogenesis by direct functional effects on endothelial cellular processes promoting angiogenesis. TFF3 stimulates angiogenesis to co-coordinate with the growth promoting and metastatic actions of TFF3 in mammary carcinoma to enhance tumor progression and dissemination.