Mechanism Of CCL21/CCR7 Mediated Breast Cancer Stem Cells Invasion And Migration

Breast cancer is a malignant cancer with the highest morbidity and mortality in female patients worldwide. Tumor metastasis is the major factor leading to poor prognosis in breast cancer patients. Lymph node is the most common site of breast cancer metastasis, and the invasion and migration of breast cancer cells into lymphatic microvessels are the early events in breast cancer lymphatic metastasis. However, the mechanism of the process aforementioned is not fully understood yet.The key step of cancer metastasis into lymphatic microvessels includes the invasion and migration of breast cancer cells towards lymphatic microvessels and lymphangiogenesis. Recent studies have demonstrated that the interaction between CC chemokine receptor CCR7 and its ligand CCL21 played an important role in breast cancer lymph node metastasis. Cancer stem cells (CSCs) are considered as "seed" cells and the origin of recurrence and metastasis. Most of the metastasizing cells are CSCs. Our previous study found that CCR7 highly expressed in breast CSCs. Therefore, it's necessary to study the effect of CCL21/CCR7 in breast CSCs invasion and migration and its mechanism.Acquiring abundant high-purity CSCs is an important prerequisite for the above-mentioned research. At present, high-purity CSCs are sorted by flow cytometry and proliferated by short-term sphere culture. However, it is still unknown whether long-term sphere culture can maintain a high ratio of CSCs.In order to solve these problems, human breast cancer cell line MCF-7 was taken as the object of study and following researches were performed. Firstly, breast CSCs were sorted by flow cytometry, and identified and multiplied by sphere culture. Then the continuous variation of the CSCs ratio in long-term sphere culture was detected, and a mathematical model was established to explain the growth pattern of CSCs. Secondly, the mechanism that CCL21/CCR7 mediated breast CSCs invasion and migration was futher explored, as well as the possible role of CCL21/CCR7 and signaling pathway in lymphangiogenesis. The aim of this research was to investigate the molecular mechanism of breast cancer lymphatic metastasis and the biological characteristics of breast CSCs, so as to provide new perspectives in the treatment of breast cancer lymph node metastasis.Methods & Results1. Isolation and cultivation of breast cancer stem cellsMethods: CD44⁺CD24^-/low CSCs was sorted among MCF-7 cells by flow cytometry, and then cultured in serum-free medium to form mammospheres. The proportions of breast CSCs were detected continuously in long-term sphere culture. A mathematical model using ordinary differential equations was established and the model parameters were estimated to imitate the growth pattern of CSCs.Results: The CSCs ratio in MCF-7 breast cancer cells was 1.8%, and the CD44⁺CD24^-/low CSCs ratio was as high as 96.2% after sorting. CD44⁺CD24^-/low CSCs from MCF-7 cells could form compact spheres in serum-free medium. However, in long-term sphere culture, the observed CSCs ratios in mammospheres were gradually decreased and finally stabilized around 1.5%. The mathematical model could imitate the main growth pattern of CSCs and differentiated cancer cells in long-term sphere culture. Based on the model parameters, the gradual decrease of CSC ratio was mainly due to the asymmetric division of CSCs and the proliferation of differentiated cancer cells. Meanwhile, a small number of differentiated cells could spontaneously convert into CSCs in sphere culture.2. Effect of CCL21/CCR7 in breast CSCs invasion and migration and its mechanismMethods: Immunofluorescence staining and western blotting were performed to detect the CCR7 expression level in MCF-7 cells and MCF-7 CSCs. Two CCR7 siRNA interference lentiviral vectors were constructed and their effectivenesses were verified in MCF-7 CSCs. qRT-PCR, western blotting and ELISA were carried out to detect the MMP2/9 expression level, Erk1/2 expression and its phosphorylation level, as well as p38 expression and its phosphorylation level in MCF-7 CSCs and CCR7 siRNA interfering MCF-7 CSCs cultured with or without CCL21. The influences of Erk1/2 or p38 specific inhibition on MMP2/9 expression were also tested. In addition, the effects of CCL21 on the migration and invasion of MCF-7 CSCs in each group were assayed.Results: CCR7 expression level in MCF-7 CSCs was significantly higher than that in MCF-7 cells. Two CCR7 siRNA interference lentiviral vectors were constructed successfully and transfected into MCF-7 CSCs, which could inhibit CCR7 mRNA and protein expression in MCF-7 CSCs efficiently. CCL21 significantly increased MMP9 expression and Erk1/2 and p38 phosphorylation in MCF-7 CSCs, and CCR7 siRNA interference and Erk1/2 specific inhibition could reduce the upregulation of MMP9 mediated by CCL21. Meanwhile, CCL21 could induce the increased migration and invasion of MCF-7 CSCs with high CCR7 expression.3. Effect of CCL21/CCR7 on the VEGF-C expression in breast CSCs and its mechanismMethods: qRT-PCR, western blotting and ELISA were performed to detect VEGF-C mRNA and protein expression in MCF-7 CSCs and CCR7 siRNA interfering MCF-7 CSCs cultured with or without CCL21. Since the results in Section Two showed that CCL21 could significantly increase Erk1/2 and p38 phosphorylation, the influence of Erk1/2 and p38 specific inhibition on VEGF-C expression were also detected.Results: CCL21 significantly upregulated VEGF-C mRNA and protein expression in MCF-7 CSCs, as well as the protein secretion. However, p38 specific inhibition could reduce the upregulation of VEGF-C mediated by CCL21.Conclusions1. High-purity CD44⁺CD24^-/low breast CSCs can be obtained by flow cytometry sorting. However, long-term sphere culture cannot maintain a high ratio of CSCs, which is mainly due to the asymmetric division of CSCs and the proliferation of differentiated cancer cells.2. CCL21 secreted by lymphatic endothelial cells in lymphatic microvessels can interact with CCR7 expressed in breast CSCs, which can promote breast CSCs invasion and migration towards lymphatic microvessels in the following three aspects. Firstly, CCL21/CCR7 can induce breast CSCs migration. Secondly, CCL21/CCR7 can promote Erk1/2 phosphorylation, thus upregulate MMP9 expression, degradate extracellular matrix around the tumor cells and lymphatic microvessels, and further facilitate breast CSCs invasion. Thirdly, CCL21/CCR7 can promote p38 phosphorylation, upregulate VEGF-C expression in breast CSCs and induce the proliferation and migration of lymphatic endothelial cells, finally promote lymphangiogenesis.