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Breast cancer cells induce cancer-associated fibroblasts to secrete hepatocyte growth factor to enhance breast tumorigenesis.

Tyan SW, Kuo WH, Huang CK, Pan CC, Shew JY, Chang KJ, Lee EY, Lee WH - PLoS ONE (2011)

Bottom Line: Here, we show that the human cancer-associated fibroblasts (CAFs) had higher activity in enhancing breast tumorigenecity compared to the normal tissue-associated fibroblasts (NAFs) isolated from the same patients.Co-culture with human breast cancer MDA-MB-468 cells in a transwell system enhanced NAFs to secret HGF as well as promote tumorigenecity.These results suggested that breast cancer cells could alter the nature of its surrounding fibroblasts to secrete HGF to support its own progression through paracrine signaling.

View Article: PubMed Central - PubMed

Affiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan Authority.

ABSTRACT
It has been well documented that microenvironment consisting of stroma affects breast cancer progression. However, the mechanisms by which cancer cells and fibroblasts, the major cell type in stroma, interact with each other during tumor development remains to be elucidated. Here, we show that the human cancer-associated fibroblasts (CAFs) had higher activity in enhancing breast tumorigenecity compared to the normal tissue-associated fibroblasts (NAFs) isolated from the same patients. The expression level of hepatocyte growth factor (HGF) in these fibroblasts was positively correlated with their ability to enhance breast tumorigenesis in mice. Deprivation of HGF using a neutralizing antibody reduced CAF-mediated colony formation of human breast cancer cells, indicating that CAFs enhanced cancer cell colony formation mainly through HGF secretion. Co-culture with human breast cancer MDA-MB-468 cells in a transwell system enhanced NAFs to secret HGF as well as promote tumorigenecity. The newly gained ability of these "educated" NAFs became irreversible after continuing this process till fourth passage. These results suggested that breast cancer cells could alter the nature of its surrounding fibroblasts to secrete HGF to support its own progression through paracrine signaling.

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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.
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pone-0015313-g001: 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.

Mentions: To compare the differential effects of CAFs and NAFs on breast tumorigenesis, we isolated fibroblasts of human breast cancer tissue and adjacent normal breast tissue from the same patients. These primary fibroblasts were grown to 100% confluent in culture and then evaluated for their abilities to promote cancer cells to form colony in soft agar. Using this soft agar colony formation system, we compared the effects of five pairs of CAFs and NAFs on the MDA-MB-468 cell colony formation in nutrition restricted medium, in which MDA-MB-468 cells could not form colonies in the absence of fibroblasts. Although both CAFs and NAFs were able to support MDA-MB-468 cells to form colonies, significantly more colonies (about 30–50% more) were formed when cells were co-cultured with CAFs (with the average about 650 colonies) compared to NAFs co-culture (about 490 colonies) (Figure 1A and 1B). Similar results were observed using another breast cancer cell line, SK-BR-3 (Figure 1C and 1D). Taken together, these results indicated that CAFs, compared to NAFs, significantly enhanced colony formation of these breast cancer cells.


Breast cancer cells induce cancer-associated fibroblasts to secrete hepatocyte growth factor to enhance breast tumorigenesis.

Tyan SW, Kuo WH, Huang CK, Pan CC, Shew JY, Chang KJ, Lee EY, Lee WH - PLoS ONE (2011)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020942&req=5

pone-0015313-g001: 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.
Mentions: To compare the differential effects of CAFs and NAFs on breast tumorigenesis, we isolated fibroblasts of human breast cancer tissue and adjacent normal breast tissue from the same patients. These primary fibroblasts were grown to 100% confluent in culture and then evaluated for their abilities to promote cancer cells to form colony in soft agar. Using this soft agar colony formation system, we compared the effects of five pairs of CAFs and NAFs on the MDA-MB-468 cell colony formation in nutrition restricted medium, in which MDA-MB-468 cells could not form colonies in the absence of fibroblasts. Although both CAFs and NAFs were able to support MDA-MB-468 cells to form colonies, significantly more colonies (about 30–50% more) were formed when cells were co-cultured with CAFs (with the average about 650 colonies) compared to NAFs co-culture (about 490 colonies) (Figure 1A and 1B). Similar results were observed using another breast cancer cell line, SK-BR-3 (Figure 1C and 1D). Taken together, these results indicated that CAFs, compared to NAFs, significantly enhanced colony formation of these breast cancer cells.

Bottom Line: Here, we show that the human cancer-associated fibroblasts (CAFs) had higher activity in enhancing breast tumorigenecity compared to the normal tissue-associated fibroblasts (NAFs) isolated from the same patients.Co-culture with human breast cancer MDA-MB-468 cells in a transwell system enhanced NAFs to secret HGF as well as promote tumorigenecity.These results suggested that breast cancer cells could alter the nature of its surrounding fibroblasts to secrete HGF to support its own progression through paracrine signaling.

View Article: PubMed Central - PubMed

Affiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan Authority.

ABSTRACT
It has been well documented that microenvironment consisting of stroma affects breast cancer progression. However, the mechanisms by which cancer cells and fibroblasts, the major cell type in stroma, interact with each other during tumor development remains to be elucidated. Here, we show that the human cancer-associated fibroblasts (CAFs) had higher activity in enhancing breast tumorigenecity compared to the normal tissue-associated fibroblasts (NAFs) isolated from the same patients. The expression level of hepatocyte growth factor (HGF) in these fibroblasts was positively correlated with their ability to enhance breast tumorigenesis in mice. Deprivation of HGF using a neutralizing antibody reduced CAF-mediated colony formation of human breast cancer cells, indicating that CAFs enhanced cancer cell colony formation mainly through HGF secretion. Co-culture with human breast cancer MDA-MB-468 cells in a transwell system enhanced NAFs to secret HGF as well as promote tumorigenecity. The newly gained ability of these "educated" NAFs became irreversible after continuing this process till fourth passage. These results suggested that breast cancer cells could alter the nature of its surrounding fibroblasts to secrete HGF to support its own progression through paracrine signaling.

Show MeSH
Related in: MedlinePlus