The Observation Of Phenotype Of TAMs In Primary Tumor And Bone Metastases

Background and Purpose:Macrophages are populated in all kinds of tissues and organs in human.They are the main actors of innate immunity,defending the first line of our immune system,playing a key role in clearing invading microbes and tissue debris.It has been reported that their function is confined and regulated by the microenvironment[1-3].Inflammatory cytokines and bacterial productions can induce the differentiation of macrophages into M1 phenotype.M1 macrophages express high level of IL(interleukin)-12 and low level of IL-10,participating in the up-regulation of inflammatory reactions.IL-4,IL-10,IL-13 and steroids can induce the differentiation of macrophages towards M2 phenotype.M1 macrophages express high level of IL-10 and low level of IL-12,enhancing immune tolerance[4-6].Tumor associated macrophages(TAMs)reside within tumor tissues.They differentiated from monocytes that are recruited from peripheral blood.In recent years,researchers show TAMs can interact with tumor cells,secret many cytokines and regulate the microenvironment.TAMs participate in the progression of tumors,playing a role in the angiogenesis of the tumor,stimulating the invading,the metastasis and the immune tolerance of tumors[7-9].The macrophage marker CD68 expression on tumor samples is positively related with the bad prognosis of patients with breast cancer,prostate cancer and pancreatic cancer [10-12].Breast cancer is one of the most common malignant cancers in female,which has 8% chances of bone metastasis.In the case of highly malignant cancer patients,bone metastasis can be as high as 69%[13].The cases of spine metastasis are about 2/3 chances of all the bone metastasis,which leads to the bone melting,the decreasing of bone quality.Spine metastasis is related to the increasing chances of compressing fracture[14-17],which can result in the nerve damage and paraplegia.The combination of cancer pain,bad prognosis after bone metastasis and an expectation of long surviving time lead to great pain both physically and psychologically.The number of infiltrating macrophage is high in breast cancer,which can be as high as 50% of the tumor cells in some cases[18].The infiltrating of TAM is positively related to the metastasis of tumors and an independent factor of prognosis of breast cancer[19].The mechanisms of the start,the development and the metastasis of breast cancer are unclear in breast cancer patients and especially in the most common invasive ductal carcinoma.The differences of TAMs in the original site and the bone metastasis site haven't been published,as we know.The analysis of the phenotype change of TAMs,during the process from the start,the development to the metastasis of cancer,may provide a clue and reference for cancer diagnosis[20].Whether the change of the tumor microenvironment lead to the TAM phenotype change and further regulate TAMs function is important because of the complicate process of tumor development.The current study used a self-development breast cancer model,i.e.MMTV-PyMT mouse to mimic the start,and the development of breast cancer.We compared the expression of M1 and M2 phenotype markers of macrophages,analyzed the phenotype of TAMs and provided evidences of the phenotype change during the tumor progressing.1.Methods1.1 Phenotype analysis of TAM in MMTV-PyMT mouse modelWe genotyped the MMTV-PyMT mice after the isolation of the genome DNA.We then divided the MMTV-Py MT mice into two groups,namely early stage cancer and late stage cancer according to the weight of the tumor and the age of the mice(16 week old and tumor smaller than 100 mm3 are in the early stage group,20 week old and the tumor larger than 800 mm3 are in the late stage group).We isolated cancer cells and then TAMs.The total RNA were isolated from the TAMs and reverse-transcribed into c DNA.The markers related to M1 and M2 phenotype were then analyzed using RT-qPCR.1.2 The comparative analysis of TAMs in the original site and the metastasis site.We looked up the cases of "breast cancer with bone metastasis" from 2006 to now in the clinical records of The General Hospital of PLA.We selected 6 clinical cases which has paraffin-embedded tumor pathological samples from both original site and bone metastasis site.We selected another 6 cases which has paraffin-embedded tumor samples with no metastasis.The samples were sectioned as 5?m thick for 6 consecutive sections.They were stained for HE,immunohistochemistry,and toluidine blue.The number of TAMs,M1 phenotype macrophages and M2 phenotype macrophages was recorded and analyzed.2.Results2.1.1 The genome DNA were analyzed using PCR.Tthe mouse which has the same band with positive control is identified as MMTV-PyMT mouse,and it can be used for following-up experiments.2.1.2 We monitored the tumor size of 10 MMTV-Py MT female mice with the age difference less than 1 week.The mice were divided into two groups.Mice with tumor smaller than 100 mm3 are in the early stage group.Mice with tumor larger than 800 mm3 are in the late stage group.2.1.3 We gated CD45+ cells as leucocytes and then gated MHC?+CD11b+ as TAM subsets(Figure 4).Leucocytes group are 2.07% of the tumor cells.TAM are 31.3% of Leucocytes.2.1.4.After the analysis of samples from early stage and late stage tumors using markers related to M1 and M2 phenotype,RT-q PCR analysis showed TAMs has a phenotype shift from M1 in early stage tumor to M2 in late stage tumor.2.2.1 CD68 could be used as a macrophage marker and CD163 could be used as an M2 macrophage marker.CD163+ cell/CD68+ cell ratio increased in the bone metastasis site compared with the original site,P<0.05.CD163+ cell/CD68+ cell ratio in the original site of breast cancer patients who have bone metastasis are higher than the ratio in the tumor of the breast cancer patients who doesn't have metastasis,P<0.05.CD163+ cell/CD68+ cell ratio in the metastasis site of breast cancer patients who have bone metastasis are higher than the ratio in the tumor of the breast cancer patients who doesn't have metastasis,P<0.01.CD68+ cell ratio in the original site are more than CD68+ cell ratio in the bone metastasis site in the breast cancer patients who have bone metastasis,P<0.01.2.2.2 The lymph node metastasis and bone metastasis are related to the ratio of CD163+ cells/CD68+ cells.3.Conclusions:1.In the MMTV-Py MT mouse model of breast cancer,TAMs has a M1 to M2 phenotype change from the early stage tumor to the late stage tumor.2.We observed that the M2 phenotype TAMs in the bone metastasis site are more than the TAMs in the site of the original site.The proportion of M2 phenotype macrophage in the original site of breast cancer patients who have bone metastasis is higher than the proportion in the tumor of the breast cancer patients who doesn't have metastasis.The number of both the CD68+ and the CD163+ cells in the bone metastasis site are less than cells in the original site,but the ratio of CD163+ cells/CD 68+ cells in the metastasis site is higher than the ratio in original site,which suggested that the positive proportion of M2 macrophages may be one of the reasons for promoting the progression of malignant tumors and even the occurrence of bone metastasis.