Function Of Lgr5 Positive Cells In The Develpoment Of Mammary Glands And Breast Cancer Metastasis

The Wnt signaling pathway receptor Lgr5 has been regarded as a stem cell marker in multiple tissues.However,whether Lgr5 marks multipotent mammary gland stem cells remains controversial.To further explore this issue,we first established a 3D culture system based on Lgr5/R-spondin1 axis using Matrigel as matrix.Cultured in this system,single Lgr5 positive cells isolated from the mammary gland of Lgr5-lacZ transgenic mice could grow into mammary organoids at a ratio of 8±1%.The organoids contained basal cells around the periphery and luminal cells lining an interior cavity,which were similar to the structure of normal mammary ducts.Moreover,Lgr5 positive cells derived organoids were sustainable during long-term passaging.In contrast,Lgr5 negative cells could expand into primary organoids,but most of the cells in the organoids were luminal cells without ductal-like structure.And organoid-forming ability immediately dissipated upon passaging.The above experiments demonstrated that mammary Lgr5 positive cells had the ability to differentiate into different cell types and keep self-renewal during passaging,indicating that they may be multipotent mammary stem cells.Further,we have found that Lgr5 positive cells were mainly concentrated at the nipple area at different stages of the development of mammary gland,suggesting that Lgr5 positive cells may remain at the nipple area through asymmetric differentiation,while the differenciated cells distribute along the elongating gland ducts.Breast cancer is the leading cause of female death.Metastasis and recurrence are considered to be the main reasons.Recent study found that breast cancer stem cells might play an important role in the occurance and progression of the disease and they were found to be resistant to radiotherapy and chemotherapy.Based on that,our study investigated the role of Lgr5 positive tumor cells in breast cancer.Long-term in vitro culture of Lgr5 positive cells showed that some cells from tumor organoids could invade into the surrounding Matrigel and form new organoids there.Further,transwell assay revealed that the migrating efficiency of Lgr5 positive cells was 6 folds of that of Lgr5 negative tumor cells,while 4 folds more cells invaded into Matrigel in transwells than the negative cells.When cells were injected into mice by tail vein,Lgr5 positive tumor cells could form lung tumors at higher efficiency(67%)than Lgr5 negative cells(11%,P < 0.05).Moreover,Lgr5 positive cells leaded to more lung metastasis from primary tumor to the lung(100%)than other cells(33%,P < 0.05).At the same time,clinical data from Kaplan-Meier database indicated that breast cancer patients with higher expression of Lgr5 had an increased probability of metastasis than those with lower expression level(P < 0.05),and patients with higher expression of Lgr5 had shorter overall survival time compared with patients with lower expression of Lgr5.In summary,Lgr5 positive cells are prone to lead to breast cancer metastasis.Therefore,targeting and killing Lgr5 positive tumor cells are promissing to increase the cure rate of breast cancer.