The Role And Mechanisms Of CCR7 In Lymphatic Vessel Invasion Of Breast Cancer

The chemokine receptor CCR7, belonging to CC chemokine receptor superfamily, is a G protein-coupled receptor (GPCR) with seven transmembrane domain. It plays an important role in mediating the homing of DC and T cells, and is also associated with lymph node metastasis of many human carcinomas. Recent studies suggested that the chemokine receptor CCR7 is a novel biomarker that can predict lymph node metastases in T1 breast cancer, whereas the CCR7 ligand, CCL21, is highly expressed in lymphatic endothelial cells (LECs). So we presumed that CCL21/CCR7 may mediate lymphatic vessel invasion (LVI) in early breast cancer. Combining with recent researches of cancer stem cells, we think that breast cancer stem cells or tumorigenic breast-cancer cells may be involved in LVI.Our present study aimed to investigate the role of CCR7 in LVI of breast cancer and its mechanisms. This study includes three parts: (1) The microlymphatic vessels (MLD) in 102 cases of breast cancer and 20 cases of normal breast tissues were evaluated by immunohistochemical staining, using monoclonal antibody against podoplanin. The characteristics of microlymphatic vessels and the association of MLD and LVI with clinicopathological parameters were evaluated. The expression of CCR7 in breast cancer tisses was also detected to further analyze the relationship between CCR7 and LVI and clinicopathological parameters. (2) According a previously described method by Ponti et al, we isolated tumorigenic breast-cancer cells (or MCF-7 stem cells, MCF-7S cells) from MCF-7 breast cancer cell line to explore the expression and function activity of CCR7 in tumorigenic breast-cancer cells. (3) The role of CCR7 in the growth and chemotaxis of MCF-7S were studied by RNAi.The main results and conclusions1. Lymphatic vessels in breast cancer had significant heterogeneity from normal breast tissues, including three aspects:①The microlymphatic density (MLD) in breast cancers was significantly higher than that in normal breast tissues. The mean MLD of breast cancer and normal breast tissues was 8.31±5.09 and 4.78±1.90, respectively (P<0.01).②The lymphatic vessels in breast cancer was indicated by a more irregular shape and a larger open lumen, and it was observed that cancer cells cluster entered the lymphatic vessels through the open area. Some microlymphatic vessels were compressed to change into fissture-like shape. MLD did not correlate with age, tumor size, pathologic type, hormone receptor (ER/PR) status, or nodal status. While LVI was found to be significantly related to lymph node metastasis (P<0.01).2. CCR7 was expressed mostly in cytoplasm and membrane of breast cancer cells, and positively expressed in 60.8% of breast cancer tissues. The CCR7 expression was significantly correlated with LVI and lymph node metastasis ( P <0.01 ), but not with age, tumor size, tumor type, or hormone receptor (ER/PR) status. The results suggested that CCR7 may play an important role in mediating LVI in breast cancer.3. Our research confirmed that MCF-7 breast cancer cells were organized by heterogeneous cell populations with different biological markers and colony morphology including holoclone,paraclone and meraclone etc. We obtained MCF-7H from holoclone which was enriched with CD44+CD24-/Low cells by cloning sylinder method.4. We obtained MCF-7S cells growing as mammospheres with stem/progenitor cells properties from MCF-7H by seeding with lower density and culturing with stem cells culture medium. The mammospheres with good refraction and outgrowth were regular. The majority of cells in culturing mammospheres were stained positively for CD44 and negatively or low for CD24. Under differentiating conditions, MCF-7S could generate ductal-like structures and expressed luminal/ductal cells marker (CK18) and myoepithelial cells marker(CK14).5. The mammospheres were stained with immunofluorescence of CCR7. MCF-7S cells in the spheres expressed CCR7 mostly in the cytoplasm and membrane. The activation of CCR7 in breast cancer stem cell with CCL21 could trigger the increase of intracellular [Ca2+] and F-actin polymerization by laser confocal microscopy, showing that CCR7 in breast cancer stem cells had functional activity.6. The mRNA and potein of CCR7 could be supppressed by RNAi, which did not affect the growing of MCF-7S, but inhibited the chemoxis. The results suggested that CCR7 and its ligand may play a crucial role in mediating breast cancer stem cells lymphatic invasion.