| IntroductionCancer is one of the major public health problems worldwide.The research on the pathogenesis of cancer,the related factors affecting the cancer progression,and effective therapies for cancer has always been the focus of the biomedical field.Breast cancer is one of the most common malignancies and one of the main causes of cancer death in women.As a result of earlier detection and improved treatment,the mortality rate of breast cancer patients has gradually decreased,but there are still a large number of patients progressing to distant organ metastasis,and metastasis is the main cause of death of breast cancer.Breast cancer is a genetic and phenotypic heterogeneous disease.Breast cancer microenvironment contains breast cancer cells and a large number of infiltrating immune cells.It is of great significance to study the interaction between infiltrating immune cells and breast cancer in the microenvironment of breast cancer,as well as the impact on the process of cancer,and to find new targets for breast cancer treatment for basic research and clinical treatment of breast cancer.As an important immune cell in the body,macrophages not only play an important role in the occurrence and recovery of inflammation,but also play an important role in the tumor immune microenvironment.Studies have shown that macrophages can stimulate tumor angiogenesis,enhance the invasive ability of tumor cells,resist the killing effect of NK cells and T cells on tumor cells,promote tumor metastasis,and reduce the effect of chemotherapeutic drugs.Therefore,macrophage depletion may become an effective new irmmunotherapy method.CD169+macrophages,a subtype of macrophages,play an important role in removing pathogenic microorganisms,supporting bone marrow hematopoiesis and maintaining bone structure.At present,there are different opinions on the effect of CD169+macrophages on tumorigenesis and development.The function and related mechanism of CD 169+ macrophages in breast cancer microenvironment remain elusive.Therefore,systematic study of the effect of CD169+ macrophages on the progression of breast cancer can help us better understand the mechanism of CD169 macrophages on the development of breast cancer,broaden our understanding of CD 169+ macrophage-related tumor immunotherapy,and extend the theory knowledge of tumor immune microenvironment.Our findings from this project will provide new targets for breast cancer treatment.ObjectiveTo investigate the effects of CD169+ macrophages depletion on tumor growth,metastasis and complications of breast cancer in tumor-bearing mice,and to explore the possible mechanisms by which CD169+ macrophages suppress CD8+T cells in breast cancer microenvironment.Methods1.CD169-DTR mice on a C57BL/6 background were backcrossed to BALB/c mice for at least 10 generations.DNA from live progeny of heterozygous matings was genotyped by Southern blot analysis.Homozygous CD169-DTR mice were generated by intercrossing F11 heterozygous mice.2.CD169-DTR mice on a BALB/c background were used to inoculate 4T1 cells in situ to generate a mouse triple-negative breast cancer model.After inoculation of tumor cells for 1 week,the experimental group was given diphtheria toxin(DT)continuously to deplete the CD169 macrophages in CD169-DTR transgenic mice,meanwhile the tumor size was measured,until 28 days after inoculation.Then mice were sacrificed,tumor tissues,peripheral blood,femurs and spleens were collected,tumor weight was determined and tumor volume was calculated.3.CD169-DTR mice on a BALB/c background were used to inoculate 4T07 cells in situ to generate a mouse triple-negative breast cancer model.After inoculation of tumor cells for 10 days,the experimental group was given diphtheria toxin(DT)continuously to deplete the CD 169 macrophages in CD169-DTR transgenic mice,meanwhile the tumor size was measured,until 28 days after inoculation.Then mice were sacrificed,tumor tissues,peripheral blood,femurs and spleens were collected,tumor weight was determined and tumor volume was calculated.4.Starch was injected into the abdominal cavity of CD169-DTR transgenic mice on a BALB/c background.After 3 days,peritoneal macrophages were obtained by washing the abdominal cavity with serum-free medium.After 48 hours of culture in vitro,DT was given to observe the death of macrophages.5.The nucleated cells in peripheral blood of each group of mice were obtained after RBC was lysed by erythrocyte lysate,and CD8 T cells in the peripheral blood were labeled with FITC Rat Anti-Mouse CD3 and APC Rat Anti-Mouse CD8,then the proportion of CD8 T cells in the spleen was analyzed by flow cytometer.6.After removing the spleens of each group of mice,a single cell suspension was prepared by grinding,and CD8+T cells in the spleen single cell suspension were labeled with FITC Rat Anti-Mouse CD3 and APC Rat Anti-Mouse CD8,then the proportion of CD8+ T cells in the spleen was analyzed by flow cytometer.7.Digest the tumor tissue with type IV collagenase and DNase to make a single cell suspension of tumor tissue.The cells were stained with FITC Rat Anti-Mouse CD3 and APC Rat Anti-Mouse CD8.Stained cells were washed with PBS and analyzed by flow cytometry.8.The thymi of 4T1 tumor-bearing mice were collected,photographed and weighed.9.The spleens of mice were harvested,photographed and weighed.The compressive strength of spleen was measured using a mechanical property testing machine.10.The spleens of mice were fixed with formaldehyde,paraffin-embedded and stained with HE.The thickness of splenic capsule and the number of trabeculae in spleen of mice in each group were observed and recorded.11.After the femurs of each group were obtained,the soft tissue on the surface of the femur was removed.The femurs were dried at 100 C for 48 hours to constant weight and weighted.12.After the femur was dried to constant weight,the femur bone density of each group was measured by dual energy X-ray bone density detector.13.Bone marrow cells were flushed out of femurs.After dilution,the cells were counted under a microscope to calculate the total number of cells in the bone marrow.14.For erythroid cells analysis,the bone marrow cells were incubated with APC Rat Anti-Mouse Ter119 and PE Rat Anti-Mouse CD71 antibodies.After staining,flow cytometry was performed for the quantification of CD71+Ter119+ cells.The reticulocytes and erythroblasts of bone marrow were analyzed by flow cytometric analysis through thiazole orange and Terl 19-APC double staining.15.After co-culture of primary macrophages with 4T1 cells,the cells were labeled with APC Rat Anti-Mouse CD169 and PE Rat Anti-Mouse PD-L1.The expression of PD-L1 on macrophage was detected by flow cytometry.16.Primary macrophages and 4T1 tumor cells were co-cultured on cell slide,fixed and perforated,and stained with APC Rat Anti-Mouse CD169,PE Rat Anti-Mouse PD-L1 and DAPI.Laser confocal analysis was used to analyze the expression level of PD-L1 on macrophages and 4T1 cells.17.The lungs of 4T1 metastatic mice were taken out,fixed with Bouin's stain,photographed,and then counted the number of pulmonary nodules in each group.18.Lung of 4T1 metastasis model mice was taken out,fixed with formaldehyde,paraffin-embedded,stained with HE,and then the number of lung metastases was counted under the microscope.19.The peripheral blood,spleen,and lung tumor were obtained from the 4T1 metastasis mice,and the single cell suspension was prepared,and CD8+T cells in the spleen single cell suspension were labeled with FITC Rat Anti-Mouse CD3 and APC Rat Anti-Mouse CD8,then the proportion of CD8+T cells in the mice was analyzed by flow cytometer.20.Western blot was used to detect changes in the expression levels of Cyclin-D1,MCL-1,BCL-XL,E-cadherin and Vimentin in tumor tissues,and the phosphorylation levels of ERK1/2.Results1.CD169-DTR homozygous mice on a BALB/c background were obtained after 11 generations of continuous hybridization identification.2.In the 4T1 triple-negative breast cancer mouse model,the growth rate of tumors was significantly slower and the tumor weight was significantly lighter at the end of the experiment in CD169-DTR tumor-bearing mice given diphtheria toxin,compared with the wild-type tumor-bearing mice,wild-type tumor-bearing mice given diphtheria toxin and CD169-DTR tumor-bearing mice given the saline.The results indicated that the 4T1 tumor was significantly inhibited after the CD169+macrophage was depleted in the 4T1 triple-negative breast cancer mice model.3.In the 4T07 breast cancer model,the growth rate of tumors was significantly slowed down after the depletion of CD 169+macrophages,and the weight of tumors was significantly reduced at the end of the experiment.This result indicates that CD169+macrophages can promote the growth of 4T07 tumors in the 4T07 breast cancer model.4.Peritoneal macrophages of BALB/c background mice cultured in vitro were given diphtheria toxin,and it was found that CD169-DTR transgenic mouse-derived peritoneal macrophages showed cell death under light microscope after administration of diphtheria toxin,such as obvious rounding and increased transmittance.And wild-type mouse-derived peritoneal macrophages showed no cell death after administration of the same concentration of diphtheria toxin,indicating that CD 169-DTR of BALB/c background mice are similar to CD 169-DTR of C57BL/6 background mice which are able to efficiently deplete CD 169+macrophages after administration of DT.5.In the 4T1 and 4T07 breast cancer mice model,the proportion of CD8+T cells in tumor,spleen and blood was significantly higher in CD169-DTR tumor-bearing mice group given diphtheria toxin,compared with the wild-type tumor-bearing mice group,wild-type tumor-bearing mice group given diphtheria toxin and CD169-DTR tumor-bearing mice group given the saline,The results indicated that depletion of CD169+macrophages can significantly increase CD8+T cells in tumor tissues to inhibit turner growth.6.In the 4T1 triple negative breast cancer model,the thymus of the tumor-bearing mice was significantly smaller than that of the control group,and the size and weight of the thymus of the tumor-bearing mice increased significantly after the depletion of CD169+ macrophages.7.In the breast cancer models of 4T1 and 4T07 mice,there were obvious splenomegaly paraneoplastic syndrome in the tumor-bearing mice,and the max stress of the spleen was significantly reduced.After the depletion of CD169+ macrophages,the splenomegaly of the tumor-bearing mice was significantly inhibited,and the limit stress of the spleen was significantly increased.8.In the 4T1 triple-negative breast cancer model,the splenic capsule of the tumor-bearing mice became thinner,the number of splenic trabeculae in the spleen decreased significantly.After the depletion of CD169+ macrophages,the splenic capsule of the tumor-bearing mice increased significantly,and the number of splenic trabeculae in the spleen increased significantly.9.In breast cancer models of 4T1 tumor bearing mice,femur dry weight and bone mineral density of mice were significantly reduced,indicating that there was serious bone destruction in breast cancer models of 4T1 mice,and the risk of bone fracture in tumor-bearing mice was significantly increased.After the depletion of CD169+macrophages,there was no significant change in femur dry weight and bone mineral density of tumor-bearing mice.10.In breast cancer models of 4T1 mice,the femur of mice became white obviously,and the total number of cells in bone marrow decreased significantly.This indicated that there was serious destruction of bone marrow hematopoietic system in breast cancer models of 4T1 mice.After the depletion of CD 169+ macrophages,the color of femur and the total number of cells in bone marrow of tumor-bearing mice did not change significantly.11.Flow cytometry analysis showed that PD-L1 was highly expressed in peritoneal CD 169+macrophages cultured in vitro.The expression of PD-L1 on the CD 169+macrophages co-cultured with 4T1 tumor cells increased further.12.Laser confocal imaging showed high expression of PD-L1 in peritoneal CD169+macrophages cultured in vitro.The co-culture with 4T1 tumor cells showed that PD-L1 expression in CD169 macrophages was increased,and the expression level of PD-L1 on the CD169+macrophages was significantly higher than that of 4T1 tumor cells.13.In the 4T1 triple-negative breast cancer mice model with lung metastasis,the total number of metastatic nodules was significantly less after staining with Bouin's dye and the pathological examination of lung paraffin sections after HE staining showed significantly less lung metastases in CD169-DTR tumor-bearing mice group given diphtheria toxin,compared with the wild-type tumor-bearing mice group,wild-type tumor-bearing mice group given diphtheria toxin and CD169-DTR tumor-bearing mice group given the saline.The results indicated that depletion of CD169+macrophages can significantly inhibit lung metastasis of 4T1 triple-negative breast cancer in the 4T1 triple-negative breast cancer model with lung metastasis.14.In the lung metastasis model of 4T1 triple-negative breast cancer,the proportion of CD8+T cells in peripheral blood,spleen and lung tumor nodules was significantly higher than that in other tumor-bearing mice after the depletion of CD 169+macrophages,indicating that the anti-tumor immunity of tumor-bearing mice was enhanced after the depletion of CD169+macrophages,and then the metastasis of tumor was inhibited.15.In 4T1 tumor tissues,the expression levels of Cyclin-Dl,MCL-l,BCL-XL and Vimentin were significantly decreased after CD169+macrophages were depleted,and the expression of E-cadherin was significantly increased.The level of cytochemistry decreased significantly,indicating that the depletion of CD169+ macrophages can reduce the expression levels of Cyclin D1 in tumor tissues and decrease the level of anti-apoptotic proteins,thereby inhibiting the proliferation of tumor cells,promoting apoptosis of tumor cells,and inhibiting tumor cells.Signal transduction pathway,inhibits the viability of tumor cells,inhibits the activation of EMT signaling pathway,and reduces the metastatic potential of tumorsConclusionCD169+ macrophages can inhibit CD8+T cells by expressing PD-L1 in breast cancer models,and exert immunosuppressive effects to promote tumor progression.Depleting CD169+ macrophages can increase the number of CD8+T cells in breast cancer models which inhibit malignant behaviors such as growth and metastasis of tumors,at the same time,it has certain inhibitory effect on paraneoplastic syndrome. |
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