| Background:Invasion and metastasis are the most basic biology behavior of malignant tumor and the main reasons of the death of breast cancer patients. The cell line with high metastatic potency and animal metastasis models are necessary tools in screening of metastasis related genes and molecular markers, researching metastasis mechanism, and exploring new medicine to anti-metastasis therapy for experimental studies in vitro and in vivo. According to the theory about the heterogeneity of tumor, subclones with different metastasis potency can be isolated from their parent cell line, which might be useful to the research of metastasis mechanism.Metastasis and recurrence are the main death reasons of breast cancer patients, and are difficult problems for clinical treatment. The metastasis of breast cancer is a complicative, multi-step and sequenced progress, regulated by different kinds of molecules and factors. Osteoglycin (OGN) and Integrin beta-like1(ITGBL1) were found down-regulated in breast cancer tissues comparing with their paired nomal tissues, and down-regulated in lymph node metastasis tissues relative to paired primary breast cancer tissues in our previous microarray analysis.Objective:The major objective was to isolate subclones with different metastatic potential from the human breast cancer cell line MDA-MB-435S; to further determine their role in carcinogenesis, development and metastasis of breast cancer, evaluate their clinical significance in breast cancer.Methods:Cell subclones with different metastatic potential were isolated from their parent cell line MDA-MB-435S by limiting dilution. Then the proliferation, invasion and migration potential of theses subclones were identified with cell biology methods in vitro. Clonality, cell population doubling time, cell cycle, invasion, adhesion, and migration capacity were evaluated by cloning experiments, cell growth curve, flow cytometry, matrigel invasion assay, adhesion assay, and migration assay respectively. Finally, subclone with high metastasis capability was verified by injecting into fat pad of SCID mice to observe its spontaneous metastasis ability in vivo. The mRNA expression levels of OGN and ITGBL1in16normal breast tissues,108primary tumors, and30lymph node metastases were detected by real time RT-PCR. The correlation between the OGN and ITGBL1mRNA levels and several clinicopathologic parameters, including clinical stage, pathologic tumor size, axillary lymph node status, nuclear grade, hormone receptor status, and local relapse or distant metastasis were analyzed by statistical methods.Results:By analyzing the invision and migration capacity of subclones from MDA-MB-435S in vitro, a subclone,14-E5, with high metastatic capability was isolated.14-E5subclone was polygon, with more pseudopodiums and smaller than MDA-MB-435S which is fusiform shape, has long pseudopodium.14-E5’s clonality is stronger than parents’s cell line MDA-MB-435S. Motility, invision capability, spontaneous metastasis capacity of14-E5cell clone is also stronger than MDA-MB-435S. The proportion of cells in DNA synthesis and mitosis was lower in14-E5cell subclone than in MDA-MB-435S. Cell population doubling time, heterogeneity adhesion capacity, tumorigenicity in14-E5cell clone and MDA-MB-435S had no difference.Both OGN and ITGBL1mRNA were down-regulated in primary cancer tissues comparing with their paired normal tissues (P<0.05), and down-regulated in metastases relative to paired primary cancer (P<0.05). Expression levels of OGN and ITGBL1mRNA were both lower in primary breast cancer tissues than in normal tissues (P<0.05), and they were both lower in metastases than in primary breast cancers (P<0.05). The OGN mRNA expression in breast primary cancer was associated with the age of patients, while no relationship was found with other clinical parameters such as tumor size, clinical stage, nuclear grade and lymph node status. The ITGBL1mRNA expression in breast primary cancer was associated with estrogen receptor (ER) status while no relationship was found with other clinical parameters. Kaplan-Meier analysis suggested that no correlation was found with OGN and ITGBL1mRNA expression level and incidence of metastasis, recurrence, or death in all breast cancer patients. Furthermore lower expression of ITGBL1mRNA was associated with poor outcome of breast cancer patients with positive lymph nodes.Conclusion:14-E5subclone with high metastatic capability will be a valuable tool to investigate mechanisms of metastasis, to explore anti-metastasis medicine and to supervise therapeutic efficacy of anti-metastasis. Down-regulation of OGN and ITGBL1mRNA are involved in carcinogenesis and metastasis of breast cancer and it might be a molecular marker for the diagnosis and monitoring the progress of breast cancer. |
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