| Background:Patients with triple-negative breast cancer(TNBC)do not benefit fromendocrinotherapy or targeted therapies,they have a higher rate of recurrence and a poorer prognosis than other subtypes of breast cancer.Therefore,exploring new therapeuticstrategies for TNBC patients is of great importance.With the development of cancer research,more attention have been paid to tumor microenvironme nt(TME).Tumor-associated macrophages(TAMs)are the most abundant immunocytes in the breast tumor microenvironme nt and hight density of TAMs are closely related to poor prognosis inTNBC.However,it remains unclear how TAMs support TNBC progression.In this study,a TNBC mouse model was established to to explore the mechanism underlying the pro-proliferative effect of TAMs.We also explored the therapeutic efficacy of targeting TAMs for the treatment of TNBC.Methods:1,A TNBC mouse model was established and flow cytometry was performed to analyze the features of TAMs isolated from 4T1 mammary tumors.Bone marrow-derive d macrophages were induced to a TAMs-like phenotype.2,Cell proliferation assay was conducted to evaluate the proliferation ability of 4T1 cells treated with TAMs supernatant or co-cultured with TAMs.We next investigated whether the exogenous supplement of macrophages could exert the pro-tumor effect.3,Co-culture system of TAMs and 4T1 cells was established in vitro.Transcriptiona l analysis was performed to identify pathways activated in TAM-stimulated 4T1 cells.Next,we validated the RNA expression of genes encoding six growth factors(EGF,HB-EGF,IGF-1,TGF-α,PDGFB,and TGFB3)that can lead to the pathway activation in the co-culture systems.We also verified the efficacy of targeted pathway inhibitor.4,Clodronate liposomes were applied to selectively deplete tumor-infiltra t in g macrophages in a 4T1 breast cancer mouse model.The targeted biomarkers within tumor tissue were analyzed by immunofluorescence staining and flow cytometry.5,Therapeutic efficacy of TAM-depletion in combination with a MAPK signaling inhibit or for the treatment of TNBC were analyzed.Results:1,TAMs in breast cancer were primarily a subpopulation with an M2 phenotype and we next isolated monocytes from the bone marrow of BALB/c mice and then treated them with M-CSF and IL-4 to induce the M2 phenotype.2,Incubation with TAMs or its supernatant significantly stimulated the proliferation of breast cancer cells compared to that with the vehicle control.TAMs and 4T1 cell co-injec tion resulted in increased tumor volumes and weights.3,Whole transcriptome analysis of 4T1 cells incubated with vehicle control or TAMs were performed.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses showed that MAPK signaling was significantly enriched in the TAMs-treated group.The relative expression of EGF,HB-EGF,IGF-1,and PDGFB were remarkably increased in the 4T1cell-treated macrophages.TAMs could significantly induce the expression of HB-EGF,TGFB3,and TGF-αin the 4T1 cell line.Blockade of MAPK signaling represses TNBC cell proliferation.4,Clodronate liposomes could reduce tumor volumes and weights.Immunofluoresce nc e staining of tumor tissues revealed a significant reduction of p-ERK1/2 levels with decreased macrophage infiltration after TAMs depletion.Flow cytometry demonstrated a decreased frequency of CD206~+TAMs and an increased percentage of CD8~+and CD4~+T cells in the clodronate liposome-treated groups.5,MEK inhibitors combined with TAMs depletion result in preferable therapeutic effects.Conclusions:1,TAMs in breast cancer are primarily a subpopulation with an M2 phenotype and TAMs enhance TNBC tumor growth.2,TAMs contribute to TNBC proliferation through MAPK pathway activation.Blockade of MAPK signaling by MEK inhibitor can significantly reduce the pro-proliferative effect mediated by TAMs.3,TAM depletion in mouse TNBC tumors decreases CD206~+TAMs,promotes CD4~+and CD8~+T cell infiltration,and reduces MAPK pathway activation.4,MEK inhibition combined with macrophage depletion result in preferable therapeutic effects. |
PDF Full Text Request