Separation, Culture， and Identification of Breast Cancer Stromal Fibroblasts_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

ZHANG Xiaoyao ¹ , KANG Hua ¹ , SUN Haichen ² , LIU Shuang ² , CUI Yeqing ² , ZHANG Lin ¹ , CAI Wei. ¹

1.Department of General Surgery， Xuanwu Hospital of Capital Medical University， Beijing 100053， China;;
2.Laboratory of General Surgery， Xuanwu Hospital of Capital Medical University， Beijing 100053， China;

Corresponding author：

Keywords：

Breast cancer; Stromal; Fibroblasts; Culture; Identification

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ObjectiveTo explore the methods of separation， culture， and identification of breast cancer stromal fibroblasts (BCSFs), which could build up a good basis for the further research of function. MethodsBreast cancer tissues were obtained during breast cancer operation， and were cut into pieces with size of 1 mm×1 mm×1 mm under aseptic conditions， then the pieces of the tissues were digested by collagenase Ⅰ and hyaluronidase. Finally the cells separated from the tissues incubated at 37 ℃ with 5% CO2 and 95% air humidified incubator. Morphological characteristics of the fibroblasts were observed under light microscope. The certain proteins were examined by immunohistochemistry （using CK， Vimentin， α-SMA, and TE-7 antibody） and flow cytometric analysis (CD34 and CD45). ResultsThe separated cells begin to attach to the wall of flask within 24 h and reached almost confluency in about 7 d to 10 d . According to identification, the successful rate of separation and culture of BCSFs was 90%（18/20）, and the characteristics of cells showed that morphological characteristics of the fibroblasts was flat spindle， rich cytoplasm, and a flat ovoid cystic nuclear. The fibroblasts in breast cancer tissues showed negative staining for cytokeratin， positive staining for vimentin， alpha-smooth muscle actin, and TE-7， and negative for CD34 and CD45 by flow cytometric analysis. ConclusionsThe fibroblasts in breast cancer tissues could be easily obtained by tissues cuting combined enzyme digestion and rocking technology in vitro. The present study provide an experimental foundation for further studies on fibroblasts in breast cancer.

Citation： ZHANG Xiaoyao,KANG Hua,SUN Haichen,LIU Shuang,CUI Yeqing,ZHANG Lin,CAI Wei.. Separation, Culture， and Identification of Breast Cancer Stromal Fibroblasts. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2011, 18(11): 1160-1164. doi: Copy

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14.	Farrow B, Albo D, Berger DH. The role of the tumor microenvironment in the progression of pancreatic cancer [J]. J Surg Res, 2008, 149(2): 319328.
15.	Witkiewicz AK, Casimiro MC, Dasgupta A, et al. Towards a new “stromalbased” classification system for human breast cancer prognosis and therapy [J] . Cell Cycle， 2009, 8(11): 16541658.
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1. 张晓耀，康骅．乳腺癌间质成纤维母细胞的生物学特性及研究现状 [J]．中国普外基础与临床杂志， 2011， 18（2）： 212215．.
2. Kojima Y, Acar A, Eaton EN, et al. Autocrine TGFβ and stromal cellderived factor1 (SDF1) signaling drives the evolution of tumorpromoting mammary stromal myofibroblasts [J]. Proc Natl Acad Sci USA, 2010, 107(46): 2000920014.
3. Joyce JA, Pollard JW. Microenvironmental regulation of metastasis [J]. Nat Rev Cancer, 2009, 9(4): 239252.
4. Taylor MA, Parvani JG, Schiemann WP. The pathophysiology of epithelialmesenchymal transition induced by transforming growth factorbeta in normal and malignant mammary epithelial cells [J]. J Mammary Gland Biol Neoplasia, 2010, 15(2): 169190.
5. Kang H, Watkins G, Parr C, et al. Stromal cell derived factor1: its influence on invasiveness and migration of breast cancer cells in vitro, and its association with prognosis and survival in human breast cancer [J]. Breast Cancer Res, 2005, 7(4): R402R410.
6. Surowiak P, Murawa D, Materna V, et al. Occurence of stromal myofibroblasts in the invasive ductal breast cancer tissue is an unfavourable prognostic factor [J]. Anticancer Res, 2007, 27(4C): 29172924.
7. Micke P, Ostman A. Tumourstroma interaction: cancerassociated fibroblasts as novel targets in anticancer therapy? [J]. Lung Cancer, 2004, 45 (Suppl 2): S163S175.
8. Barnas JL, SimpsonAbelson MR, Yokota SJ, et al. T cells and stromal fibroblasts in human tumor microenvironments represent potential therapeutic targets [J]. Cancer Microenviron, 2010, 3(1): 2947.
9. Anton K, Glod J. Targeting the tumor stroma in cancer therapy [J]. Curr Pharm Biotechnol, 2009, 10(2): 185191.
10. Sadlonova A, Mukherjee S, Bowe DB, et al. Human breast fibroblasts inhibit growth of the MCF10AT xenograft model of proliferative breast disease [J]. Am J Pathol, 2007, 170(3): 10641076.
11. Polyak K, Kalluri R. The role of the microenvironment in mammary gland development and cancer [J]. Cold Spring Harb Perspect Biol, 2010, 2(11): a003244.
12. Orimo A, Weinberg RA. Stromal fibroblasts in cancer: a novel tumorpromoting cell type [J]. Cell Cycle, 2006, 5(15): 15971601.
13. Surowiak P, Suchocki S, Gyrffy B, et al. Stromal myofibroblasts in breast cancer: relations between their occurrence, tumor grade and expression of some tumour markers [J]. Folia Histochem Cytobiol, 2006, 44(2): 111116.
14. Farrow B, Albo D, Berger DH. The role of the tumor microenvironment in the progression of pancreatic cancer [J]. J Surg Res, 2008, 149(2): 319328.
15. Witkiewicz AK, Casimiro MC, Dasgupta A, et al. Towards a new “stromalbased” classification system for human breast cancer prognosis and therapy [J] . Cell Cycle， 2009, 8(11): 16541658.
16. Hayashi S, Yamaguchi Y. Estrogen signaling in cancer microenvironment and prediction of response to hormonal therapy [J]. J Steroid Biochem Mol Biol, 2008, 109(35): 201206.
17. 崔娟，孙克勤，李霞，等．人牙龈成纤维细胞的原代培养及鉴定 [J]．中国医药导报， 2010， 1（3）： 2627．.
18. 马英智，张丽红，孙梅，等．人真皮成纤维细胞的分离培养及鉴定 [J]．中国老年学杂志， 2009， 29（16）： 20052006．.
19. 李锋，何晶，丁文龙．体外培养雪旺氏细胞纯化方法的探讨 [J]．解剖科学进展， 2006， 12（2）： 156159．.
20. Lazard D, Sastre X, Frid MG, et al. Expression of smooth musclespecific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue [J]. Proc Natl Acad Sci USA, 1993, 90(3): 9991003.
21. Goodpaster T, LegesseMiller A, Hameed MR, et al. An immunohistochemical method for identifying fibroblasts in formalinfixed, paraffinembedded tissue [J]. J Histochem Cytochem, 2008, 56(4): 347358.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Separation, Culture， and Identification of Breast Cancer Stromal Fibroblasts

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