The Mechanism Of Macrophages In Promoting Lung Metastasis Of Breast Cancer

Background and AimsBreast cancer is the most common type of cancer in women.Metastasis is the leading cause of breast cancer-related mortalities.Lung is one of the most common metastatic target organs in breast cancer,but the underlying mechanisms remain largely unclear.Currently,effective therapeutic treatment of lung metastases remains highly challenging.Once tumor metastasis occurs,the existing treatment methods are difficult to produce lasting clinical efficacy.Therefore,early detection and intervention of lung metastasis is of great significance for the prognosis of breat cancer.The theory of"seed and soil" is an important theoretical basis in the study of tumor metastasis in recent years.According to this theory,tumor cells themselves are not enough to lead to metastasis,but need the interaction and joint participation between tumor cells and target organ microenvironment.Tumor metastasis is composed of a series of complex and interrelated processes,which can be divided into two phases:pre-metastatic phase and metastatic phase.The pre-metastatic phase is the preparation process of tumor metastasis.Before the arrival of circulating tumor cells in the metastasis target organ,the primary tumor has changed the microenvironment of the metastasis target organ through a series of mechanisms to make it suitable for tumor cell colonization.This pre-modified microenvironment is called "pre-metastatic niche".In recent years,the cells and factors involved in the formation of pre-metastatic niche have gradually become the targets for the treatment of tumor metastasis.The stage of tumor metastasis is the process that tumor cells fall off from the primary tumor,migrate to the target organ changed in advance via the blood vessels,extravasate into the surrounding tissue and form metastatic nodules.Both of these two stages play an important role in tumor metastasis.Macrophage is one of the most abundant inflammatory cell in the tumor microenvironment.A large number of studies have shown that macrophages play an important role in all stages of tumor metastasis.In primary tumor,macrophages can promote the invasion and migration of tumor cells,which is conducive to the shedding of tumor cells from the primary tumor site and reaching distant organs along the blood and lymphatic circulation.In addition,macrophages at the metastatic site also play an important role in promoting tumor metastasis.They could be recruited to the metastatic site to create a suitable environment before the arrival of tumor cells.They also directly act on tumor cells to promote their adhesion,extravasation,and early colonization at the metastatic site.Therefore,the role of macrophages in tumor metastasis has attracted more and more attention,but the specific role and mechanism of macrophages is still unclear.The study is divided into two parts.The first one is to study the mechanism of macrophage recruitment and differentiation in lung metastasis and its role in promoting the remodeling of pre-metastatic lung tissue with 4T1 breast cancer model.In the second part of study,we investigated the mechanism of macrophages in promoting the malignant metastasis potential of breast cancer cells.Part 1 Mechanism of macrophages promoting ECM remodeling in premetastatic lungObjective1.To verify that primary tumor promote the recruitment and differentiation of macrophages in the pre-metastatic lung.2.To investigate the mechanism of macrophages involved in regulating ECM remodeling in the pre-metastatic lung.Methods1.Primary tumor promote the recruitment and differentiation of macrophages in the pre-metastatic lung.1.1 Establishment of 4T1 breast cancer model:Female BALB/c mice,aged 6-8 weeks(20-22 g)were used to establish the 4T1 breast cancer model.In the 4T1 group,1×1064T1 cells were subjected to suspending in 0.1 ml of serum-free RPMI 1640 and injection into each BALB/c mouse’s paired abdominal mammary glands.Similarly,PBS was administered as a control in isometric quantities.1.2 Histological studies:On day 0,7,14,21 and 28,five mice in the control and 4T1 groups were sacrificed.The left lung of the mouse was used for H&E staining.1.3 Immunofluorescence and western blot were used to detect the effect of primary breast cancer on the recruitment of macrophage in lung tissue.1.4 Transwell and adhesion assay were used to detect the recruitment and differentiation of THP-1 monocytes response to the condition mediam(CM)of tumor cell.1.5 CM of tumor cells induced RAW264.7 and THP-1 derived human macrophages to polarize toward tumor associated macrophages(TAMs).2.Macrophage-secreted S100A4 supports breast cancer metastasis by remodeling the extracellular matrix(ECM)in the pre-metastatic niche.2.1 Western blot and immunofluorescence were used to observe the expression of activated fibroblast marker protein α-smooth muscle actin(α-SMA)and fibronectin.2.2 Clodronate liposomes(CL,100 mg/kg)were delivered 24 hours intravenously after the 4T1 cells were implanted into the mammary fat pad of BALB/c mice,followed by recurrent injections of 50 mg/kg each fourth day.The control group was intravenously injected with isometric amounts of PBS liposomes(PL).On day 14,we measured the number of macrophages and activated fibroblasts in lung tissue.On day 28,the number and size of lung metastases were detected by HE staining.2.3 The TAMs were cocultured with normal lung fibroblasts in vitro to detect the effect on the activation and migration of fibroblasts.2.4 Double immunofluorescence staining was used to detect the expression and cell origin of S100A4 in lung tissue.2.5 The TAMs were transfected with control or S100A4 siRNA and then cocultured with lung fibroblasts in a transwell system.The expression of α-SMA and fibronectin in fibroblasts was measured by western blot.2.6 The effect of S100A4 on the activation and migration of lung fibroblasts was detected by western blot and Transwell assay.2.7 Western blot was used to detect the changes of ERK,Wnt and FAK signaling pathways in fibroblasts treated with S100A4.Results1.Primary tumor promote the recruitment and differentiation of macrophages in the pre-metastatic lung.1.1 HE staining showed that,a large number of inflammatory cells were infiltrated in the lung tissues of 4T1 tumor-bearing mice compared with control mice.1.2 Primary tumor induced the increase of macrophages in the pre-metastatic lung.1.3 CM of tumor cells promoted RAW264.7 and THP-1 monocytes migration.1.4 THP-1 monocytes acquired the enhanced ability of adhesion in respond to CM produced by tumor cell.1.5 CM of breast cancer cells induced RAW264.7 and THP-1 monocytes to polarize toward tumor associated macrophages and they displayed a M2-like phenotype polarization.2.Macrophage-secreted S100A4 supports breast cancer metastasis by remodeling the extracellular matrix(ECM)in the pre-metastatic niche.2.1 The expression of α-SMA and fibronectin protein was increased in the premetastatic lung.2.2 In response to CL treatment,macrophage numbers were significantly reduced.Interestingly,the number of α-SMA+myofibroblasts in the pre-metastatic lungs of CLtreated mice was also decreased.In addition,the number and size of lung metastases were decreased.2.3 In vitro,TAMs promote the activation and migration of lung fibroblasts.2.4 The double immunofluorescent showed that there was a strong co-staining of F4/80 and S100A4,indicating that S100A4 is produced by macrophages present in the premetastatic lung.2.5 Reduction of S100A4 secretion by macrophages diminishes its pro-activation effect on lung fibroblasts.2.6 Extracellular S100A4 promotes the activation and migration of lung fibroblast.2.7 The ERK signaling pathway is involved in the S100A4-induced activation of lung fibroblasts.Conclusions1.Primary tumor promote the recruitment and differentiation of macrophages in the pre-metastatic lung.2.Macrophage-secreted S100A4 supports breast cancer metastasis by remodeling the ECM in the pre-metastatic niche.Part 2 Molecular mechanism of macrophages participating in regulating metastatic potential of breast cancer.ObjectiveTo explore the mechanisms of tumor associated macrophages(TAMs)in regulation of invasion,migration and EMT of breast cancer.Methods1.TAMs promote the invasion,migration and EMT of breast cancer.1.1 CM of breast cancer cells induced THP-1 monocytes to polarize toward tumor associated macrophages.1.2 In vitro,we co-culture of TAMs and breast cancer cells to detect the morphological changes of breast cancer cells.1.3 Cell migration and invasion capacity of breast cancer cells(MDA-MB-231 and MDA-MB-468)alone or co-cultured with TAMs was determined by the Transwell coculture system.1.4 The effect of TAMs on the EMT of breast cancer cells was analyzed by real-time PCR and western blot analysis.1.5 The influencre of macrophages on the growth of breast tumors and lung metastases in 4T1 mice were detected.The expression of EMT related protein in tumor tissue was detected by immunohistochemistry.2.TAMs secretion of IL6 promote EMT in breast cancer cells via upregulating the expression of TGM2.2.1 Real-time PCR and ELISA were used to detect the cytokines secreted by TAMs related to tumor EMT and metastasis.2.2 Breast cancer cells treated with IL-6 or control medium were evaluated their migratory、invasive and EMT abilities.2.3 The expression of TGM2 gene in breast cancer cells alone or treated with IL-6 was detected.2.4 To validate whether TGM2 was involved in the regulation of breast cell EMT,a small RNA interference(siRNA)or overexpressing plasmid targeting human TGM2 was transfected into breast cancer cells.Transwell assay and western blot were used to detect the effects of TGM2 gene on invasion,migration and EMT of breast cancer cells.3.IL6 activates TGM2 transcription via JAK2/STAT3 signaling.3.1 Western blot was used to detect the activation of signal pathway after IL6 induction.3.2 To validate the significance of the JAK/STAT3 pathway in mediating IL6-induced TGM2 activation,the inhibitor AG490 was added to the cell culture system.3.3 Double luciferase assay was used to detect the transcriptional activation of STAT3 on TGM2 gene.Results1.TAMs promote the invasion,migration and EMT of breast cancer.1.1 After co-culture with TAMs,the morphology of breast cancer cells has changed.1.2 TAMs induce EMT to promote migration and invasion of breast cancer cells.1.3 Real-time PCR and Western blot detection showed that TAMs could promote the expression of EMT related transcription factors and proteins in breast cancer cells.1.4 TAMs enhanced breast cancer tumorigenesis and lung metastasis in vivo.2.TAMs secretion of IL6 promote EMT in breast cancer cells via upregulating the expression of TGM2.2.1 The mRNA levels of IL6 emerged as the most prominently upregulated and abundant cytokine in TAMs than those in THP-1 macrophages.ELISA further showed that IL6 levels were significantly increased in the media from TAMs with compared t those from THP-1 macrophages or breast cancer cells.2.2 Transwell assay showed that IL-6 significantly enhanced the migrating and invasiv ability of breast cancer cells in vitro.2.3 Western blot showed the upregulation of TGM2 in breast cancer cells after cc culture with TAMs or induced by IL6.2.4 TGM2 upregulation promotes EMT in breast cancer cells.2.5 TGM2 knockdown inhibits the migrating,invasive and EMT ability of breas cancer cells promoted by TAMs in vitro.3.IL6 activates TGM2 transcription via JAK2/STAT3 signaling.3.1 Co-cultured with TAMs or stimulation of breast cancer cells with IL6 increased th expressions of p-JAK2 and p-STAT3.3.2 JAK2/STAT3 inhibitor AG490 treatment inhibited the activation of TGM2 in MDA-MB-468 induced by TAMs.3.2 STAT3 transcription factor can directly bind to the promoter region of TGM2 and promote the expression of TGM2.ConclusionTAMs promotes the malignant metastatic potential of breast cancer via IL6/STAT3/TGM2 activation.