Background
Breast cancer is a major malignancy affecting females worldwide. It is the most common cause of death from cancer in women [
1‐
3]. Cell lines are widely used in laboratory research and particularly as
in vitro models in cancer research. Apart from MCF-7, the most commonly used breast cancer cell line in the world derived from a pleural effusion in the Michigan Cancer Foundation [
4], a number of other cell lines are routinely used as breast cancer models, such as BT20, MDA-MB-231, MDA-MB-435s, and T-47D [
5]. Moreover, most
in vitro studies using breast cancer cells are based on a few well-characterized cell lines, such as MCF-7, ZR-75-30, T-47D and MDA-MB-231, which have been established in culture for over 30 years[
4,
6,
7]. Furthermore, most of these long established breast cancer cell lines are not derived from primary breast tumors, but from tumor metastases, especially aspirates or pleural effusions.
We also found that these routinely used breast cancer cell lines were mostly derived from Caucasians or African-Americans, such as MCF-7, ZR-75-30, T-47D and MDA-MB-468, but very rarely cell lines were derived from xanthoderm, particularly from Asians. In American Type Culture Collection (ATCC), there were more than 70 breast cancer cell lines, but only one of them was derived from Asian(Hs 739.T, ATCC NO.: CRL-7477) and one from East Indian (HCC1954, ATCC NO.: CRL-2338). And there were few standard models to study the pathogenic mechanism at molecular level and cell signaling pathway of breast cancer for Asian patients. Even in some studies on tumor pathologic features in Chinese patients, only the cell lines derived from Caucasians or African-Americans were used, so there were few experimental systems to study the pathogenic mechanism of breast cancer for Asian patients [
8]. In our previous work about the breast cancer associated gene BRCA1, we also used the cell lines ZR-75-30 and MDA-MB-435S as investigate objects[
9]. In China, the incidence rate of breast cancer has been seen increasing significantly both in urban and rural areas in the last three decades[
10,
11]. So it is quite necessary to establish new breast cancer cell lines from xanthoderm to study the pathogenic mechanism and therapeutic methods.
In this report, we established and characterized three new breast cancer cell lines BC-019, BC-020 and BC-021 from three Chinese patients. These cell lines which are positive for estrogen receptor (ER), progesterone receptor (PR) and C-erbB-2, could provide us with new experimental materials to study the pathogenic mechanism and to screen for new therapeutic reagents against breast cancer.
Discussion
Well-characterized human cancer cell lines are important resources for studying cancer cell biology, as well as for developing new strategies against cancer cell growth and progression[
28]. In this study, we reported the establishment and characterization of three new breast cancer cell lines, BC-019, BC-020 and BC-021, which were derived from breast invasive ductal carcinoma tissues of Chinese female patients.
The newly established cells grew as adherent monolayer with characteristic epithelial morphology. The cultured cells maintained consistent morphology from the primary culture to the subsequent subculture passages. These cells have grown continuously for over 6 months and have undergone more than 45 passages. They appeared to be permanent cell lines since growth continued after recovery from cryopreservation. The population-doubling time of BC-019, BC-020 and BC-021 were 36 hours, 35 hours and 46 hours, respectively.
We did not add any insulin to the medium in primary culture to maintain the cell lines, while it was necessary for most of routinely used breast cancer cell lines, such as MCF-7, T-47D, MDA-MB-435S and so on[
4,
7,
29]. We found that the absence of insulin in the culture medium did not affect the characteristics of these three cell lines. Therefore, it is more convenient to culture these cell lines.
The contaminations would do great harm to cell cultures. In our contamination tests, no bacteria and fungi or mycoplasma could be found in the three cell lines. Isoenzyme analysis was used to verify the origin of species. We analyzed the lactate dehydrogenase and malate dehydrogenase of the new cell lines. The results indicated that the origin of the three cell lines was identical to HeLa cell line. So the three breast cancer cell lines were derived from human tissues and there were no other species cross-contamination during the proliferation. Meanwhile, all the three cell lines were positive for the expression of estrogen receptor, progesterone receptor and C-erbB-2. This result demonstrated clearly that the three cell lines were derived from human breast cancer tissues.
The karyotypes of the three breast cancer cell lines showed chromosomes with hyperdiploidy and complex rearrangements. The pronounced heterogeneity suggested that these three cell lines consisted of karyotypically different clones. The main heterogeneity of the three cell lines was chromosome fracture at the centromeres and most of the fractured chromosomes could not be identified. The fractured chromosomes were in larger number in BC-021 than in the other two cell lines. The hyperdiploidy (> 50 chromosomes) may arise from fracture of the chromosomes.
The three breast cancer cell lines had been proved to have severe tumorigenicity when injected into nude mice, and their tumorigenicity was higher than that of MCF-7 cell line. Metastasis could be occasionally observed in nude mice injected with BC-019 cells and BC-021 cells, but not in mice injected with BC-020 cells and MCF-7 cells. It was well known that breast cancer cell lines tend to be less tumorigenic and less metastatic than other cell lines derived from lung, renal and colon carcinoma when injected subcutaneously in nude mice[
30]. Interestingly, these three cell lines could form tumors at 100% rate when injected in nude mice within two weeks, and the maximum dimension of these tumors reached 2.0 cm within 5 weeks post-injection. The size of the tumor was bigger than that formed by MCF-7 under same conditions. It seems that these newly established breast cancer cell lines could be new
in vivo models for studying the biology of breast cancer.
Most of the previously established breast cancer cell lines used in research did not derive from primary breast tumors, but from tumor metastases, especially ascites or pleural effusions. While primary cultures are indispensable for direct comparison to the tissue of origin, immortalized cell lines are necessary for long-term studies [
31,
32]. Therefore, these new cell lines will be valuable tools in breast cancer research. Not only are cells directly isolated from the tumor site, but also detailed pathology is available to allow the characteristics of the culture to be compared with those of the original tumor.
Moreover, most of the published breast cancer cell lines were derived from Caucasians, which limited the study of pathogenic mechanism of breast cancer among Asian patients. To resolve this problem, we established three breast cancer cell lines which were derived from Chinese and provided a research resource and tool to compare the difference of cells and tumors between the two races.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CS, MG, DL, LM and LH carried out all experimental work. CS and CZ conceived ideas, analyzed data and wrote manuscript. JC contributed in collecting tissues of the patients and pathology assay. CS, LH and CZ analyzed results and evaluated manuscript. All authors read and approved the final manuscript.