Figure 1.
Breast cancer-associated fibroblasts enhanced breast tumorigenesis to a higher level than normal tissue-associated fibroblasts.
(A) CAF/NAF pairs from the same patients were isolated and subjected to soft agar colony formation assay using MDA-MB-468 cells. For each pair of fibroblasts examined, CAFs significantly enhanced colony forming ability of MDA-MB-468 cells to a higher level than NAFs. Data are mean ± SD of triplicate samples. (B) The average of colony number of MDA-MB-468 cells mediated by CAFs and NAFs from all samples was shown. Data are mean ± SD. (C) For each pair of fibroblasts tested, CAFs enhanced soft agar colony forming ability of SK-BR-3 cells more effectively than NAFs. Data are mean ± SD of triplicate samples. (D) The average of SK-BR-3 cell colony numbers mediated by CAFs and NAFs from all samples was shown. Data are mean ± SD. (E) CAF #199C significantly enhanced tumor growth in the NOD/SCID fat pads than its normal counterpart NAF #200N and the control (no fibroblasts). Tumor volume was determined every three days after injection. Data are mean ± SEM of tumors from 6 mice. Statistical significance between CAF #199C and NAF #200N was evaluated by Student's t-test. * P<0.05.
Figure 2.
Breast cancer-associated fibroblasts expressed higher HGF levels than normal tissue-associated fibroblasts.
(A) Regression of the cytokine/growth factor signal intensities in the conditional media from CAF #199C and NAF #200N was performed to evaluate the difference of the cytokines and growth factors secreted by CAFs and NAFs. The results revealed that HGF (4.57 fold) and TIMP-1 (1.41 fold) levels were significantly higher in the conditional medium from CAFs than those in the conditional medium from NAFs (array B). In contrast, lower levels of GCP-2 (0.17 fold) (array A), IGFBP-3 (0.48 fold), GRO family (0.44 fold), GRO-α (0.6 fold), ENA-78 (0.63 fold), GCSF (0.54 fold) (array B) and LAP (0.59 fold) (array C) were detected in the conditional media from CAFs compared to those in the conditional medium from NAFs. (B) Western blotting analysis revealed that the HGF protein levels in CAFs were higher than those in NAFs in all fibroblast pairs (upper panel). Whereas, there was no correlation observed between CAFs and IGFBP-3 protein expression (lower panel). (C) Enzyme-linked immunosorbent assay showed that the HGF amounts in the cultured media of CAFs were higher than those of NAFs in all fibroblast pairs. (D) Quantitative real-time RT-PCR analysis showed that the HGF mRNA levels in CAFs were higher than those in NAFs in all fibroblast pairs. (E) Real-time RT-PCR analysis revealed that the mRNA levels of SDF-1 α, SDF-1 β and SDF-1 γ varied in different samples. There was no association between the SDF-1 mRNA levels and CAFs. Data are mean ± SD of triplicate samples. * P<0.05. NS, no significant difference.
Figure 3.
Sequestration of the HGF activity reduced cancer-associated fibroblast-mediated soft agar colony formation of MDA-MB-468 cells.
(A) Neutralization of HGF activity by addition of 80 µg/ml anti-HGF antibody significantly reduced CAF #199C-mediated soft agar colony formation of the MDA-MB-468 cells. However, the anti-TIMP-1 antibody did not show any effect. (B) NAF #200N-mediated soft agar colony formation of MDA-MB-468 cells was not affected by addition of 80 µg/ml anti-IGFBP-3 antibody. Data are mean ± SD of three independent experiments. * P<0.05. NS, no significant difference.
Figure 4.
Pre-coculture with breast cancer cells enhanced the ability of normal tissue-associated fibroblasts to mediate breast tumorigenesis.
(A) The protocol of the co-culture system using MDA-MB-468 cells and NAFs was shown. NAF #200N co-cultured with MDA-MB-468 cells for four passages was indicated as 200N.E1-E4, respectively. Each fibroblast of 200N.E1-E4 was propagated in the absence of MDA-MB-468 cell co-culture and passaged from P.1 to P3. (B) The HGF protein expression in NAF #200N.E1-E4 was shown. The HGF protein levels in NAF #200N.E1 and E2 were reduced to the level in NAF #200N after three passages (P.3). However, the HGF protein levels in NAF #200N.E3 P.3 and E4 P.3 were significantly higher than those in NAF #200N. (C) Enzyme-linked immunosorbent assay showed that the HGF protein amounts in the cultured media of NAF #200N.E3 P.3 and E4 P.3, rather than E1 P.3 and E2 P.3, were higher than those of NAF #200N. (D) NAF #200N.E4 P.4 significantly enhanced soft agar colony formation of MDA-MB-468 cells to a higher level than NAF #200N. For (B), (C) and (D), data are mean ± SD of three independent experiments. (E) NAF #200N.E4 P.4 significantly increased the tumor growth in the NOD-SCID fat pads compared to NAF #200N. Tumor volume was determined every three days after injection. Data are mean ± SEM of tumors from 4 mice. Difference between NAF #200N.E4/CAF #199C and NAF #200N was evaluated by Student's t-test. * P<0.05.