| Part 1Objective:Tumor microenvironment play a significant role in tumor growth, which composed of tumor cells and tumor stromal cells.The purpose of study is to investigate the impact and significance of the tumor microenvironment in the culture and identified of the breast cancer stem cells. Thereby explore the lymphatic invasion, lymph node metastasis of malignant high biological behavior of invasive micropapillary carcinoma, then lay the basis for future research.Methods:Cells isolated from primary beast cancer tissues were cultured in vitro with serum-free medium PCM-2 and the supernatant of cultured fibroblasts respectively. MCF-7 breast cancer cell line as a control group. Through observing the status of microspheres to explore the formation mechanism and clinical significance in invasive micropapillary carcinoma of breast cancer.Results:1. With the extension of the incubation time, the diameter of microshperes(P=0.002) increased gradually in PCM-2. The cells in the supernatant of cultured fibroblasts were presented mostly spindle cell growth, the number of cells increased day by day. The morphological characteristics of MCF-7 have a similar result compared to primary breast cancer cells in different culture solutions.2. The growth rate of primary breast cancer cells in the supernatant of cultured fibroblasts was faster than in PCM-2. The growth rate of MCF-7 with similar result compared to primary breast cancer cells (P=0.004).3. The expression of ALDH1 in 5d was reduced compared to Id in PCM-2. Nevertheless, primary breast cancer cells which cultured in 1d was negative expression of E-cadherin and in 5d exhibited weak of it. The expression of Vimentin in 5d was slightly higher than 1d. In addition, the expression of ALDH1 was higher in PCM-2 than the supernatant of cultured fibroblasts comparatively4. In comparison with the supernatant of cultured fibroblasts, ALDH1 and Oct-4 were up-regulated in the MCF-7 from the serum-free medium PCM-2. E-cadherin was down-regulated. All the same time, ALDH1 was up-regulated in the primary breast cancer cells from the serum-free medium PCM-2. E-cadherin was down-regulated.Conclusion:Serum-free culture can be one best methods to enrich breast cancer stem cells. Including tumor cells themselves, tumor stromal cells, tumor-associated macrophages, carcinoma-associated fibroblasts, microvascular and a varitay of cytokines are the important parts of tumor microenvironment, which palys a crucial role in the growth and development of tumor cells and the evolution of breast cancer.Part 2Objective:Invasive micropapillary carcinoma of the breast cancer is a new type in 2003 WHO breast and female reproductive system pathology and genetics classification, which has high malignant biological behavior such as high lymphatic invasion, high lymph node metastasis. In order to explore new methods of treating breast cancer, more and more researchers pay much attention to the tumor microenvironment. TAMs play a very important role in the tumor microenvironment. This study aim to investigate the expression and clinical significance of CD68 and CD 163 in IMPC.Methods:Immunohistochemistry was used to detect the expression and clinical significance of CD68 and CD 163 in 68 cases IMPC(72 cases invasive ductal carcinoma, not otherwise specified, IDC-NOS as control group), which archives in breast cancer pathology in Tianjin Medical University Cancer Hospital between January 2006 to December 2007.Results:1. The expression of CD68 and CD 163 in the cancer nest, intratumoral and peritumoral stromal both IMPC and IDC-NOS. But only stromal tumor of CD68+ macrophages expression in IMPC higher than IDC-NOS and the difference between the two groups was statistically significant (P=0.022)2. Stromal tumor of CD68+ macrophages expression in IMPC (47/68,69.1%) higher than IDC-NOS group (37/72,51.5%), CD68+ macrophages in IMPC stroma positively correlated with pathological stage, histological grade, lymph node metastasis, vascular invasion, and Ki-67 expression (P<0.05), and inversely correlated with the expression of ER (P=0.037).3. The rate of CD68 and CD163 expression in peritumoral stromal(63/68,92.6%; 65/68,95.6%) of IMPC was higher than intratumoral stromal(47/68,69.1%; 29/68,42.6%), and the difference was statistically significant(rs=0.508, P＜0.001; rs=0.462, P<0.001).The expression of CD68 was higher than CD 163 in both intratumoral stromal and the cancer nest of IMPC, the difference was statistically significant(rs=0.529, P＜0.001; rs=0.908, P＜0.001).4. Kaplan-Meier survival curve analysis showed that CD68+ macrophages in the stroma of IMPC were significantly associated with worse progression-free survival (P=0.027), and no significant correlation with overall survival (P=0.188).Conclusion:1. The expression of stromal tumor of CD68+ macrophages in IMPC higher than IDC-NOS group, CD68+ macrophages in IMPC stroma positively correlated with histological grade, and inversely correlated with the expression of ER.2. Stromal tumor of CD68+ macrophages expression in IMPC positively correlated with lymph node metastasis, vascular invasion. In our previous studies, the expression of microvessel density in stromal of IMPC was higher than IDC-NOS, and positively correlated with lymph node metastasis. Maybe TAMs produced vascular endothelial growth factor, MMP-9 and immunomodulatory factors(such as IL-10) promote the formation of new blood vessels in stromal of IMPC and lymph node metastasis, vascular invasion.3. CD68+ macrophages in IMPC stroma positively correlated with Ki-67 expression (P<0.05), which speculates the tumor cells of IMPC secreted various factors (such as epidermal growth factors) can recruit TAMs, thus contributing to the progress of IMPC.4. CD68+ macrophages in the stroma of IMPC were negatively associated with progression-free survival, may contribute to the development of the immune potential therapeutic target for breast cancer. |
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