The Relationship Between CD137 And Bone Metastasis Of Breast Cancer

Objective:As a pair of costimulatory molecules,CD137 and CD137 L belong to the TNF receptor superfamily members and TNF superfamily members,respectively,which are mainly expressed on the membrane surface of immune cells.In addition,it has been found that CD137 L can also be universally expressed on various tumor cells.Studies have found that,CD137 and CD137 L can coexist in different parts of the tumor tissue,suggesting that CD137/CD137 L may affect tumor progression.In the tumor microenvironment,tumor cells and macrophages exert their interaction through the CD137/CD137 L bidirectional signaling pathway.On the one hand,tumor cells regulate the migration and differentiation of monocytes through the CD137L-CD137 forward signaling pathway;on the other hand,monocytes/macrophages promote the EMT and metastasis of tumor cells through the reverse signaling pathway.Previous studies have found that CD137 and RNAK pathway share common downstream signaling pathways,and CD137/CD137 L bidirectional signaling pathway was found to affect RANKL-mediated osteoclast differentiation.However,it remains unclear whether CD137 can affect bone metastasis of breast cancer.Here,we will explore whether CD137 can regulate the migration of monocytes to the tumor microenvironment and affect the differentiation of monocytes into osteoclasts to form a microenvironment suitable for the colonization of tumor cells,which ultimately affects the bone metastasis of breast cancer.At the same time,we are trying to find new targets for the treatment of breast cancer with bone metastases.Methods: 1.First,we used siRNA libraries to screen for cytokine receptors associated with macrophages migration to the tumor microenvironment in the U937 cell line.At the same time,bioinformatics methods were used to analyze the relationship between CD137 and distant metastasis of breast cancer.2.ELISA assay was used to detect the serum levels of CD68 in patients with normal breast,primary breast cancer and metastatic breast cancer.Immunohistochemistry was used to detect the expression of CD137 and CD137 L in breast cancer tissues.Semi-quantitative PCR was used to detect the expression of CD137 L in 4T1,4T07 and EO771 mouse breast cancer cells.Western Blotting assay was used to detect the serum levels of sCD137 L in normal breast,primary breast cancer and metastatic breast cancer patients.3.CD137 silenced and overexpressed stable cell lines were constructed in RAW264.7 cell line labeled by Renilla and Fra-1 expression was assayed in two stable cell lines.The RNAseq method was used to detect the changes of gene expression in RAW264.7 cells induced by over-expression of CD137.Transwell and scratch assay were used to detect the effect of CD137 on the migration of RAW264.7 cells into the tumor microenvironment.TRAP assay was used to detect the effect of CD137 on the differentiation of RAW264.7 cells into osteoclasts.The effect of CD137 overexpression on the expression of osteoclast differentiation-related genes in RAW264.7 cells was detected by Real-time PCR assay.4.The 4T1 cells and RAW264.7 cells were co-injected subcutaneously to construct the primary breast cancer model in BALB/c mouse,and a spontaneous bone metastasis model of mouse breast cancer was constructed by excision of the primary tumor.Bone metastasis in breast cancer in mice was detected by Bioluminescenceimaging(BLI)and H&E staining.TRAP assay was used to detectosteoclast infiltration in bone metastases.Immunohistochemistry was used to detect the infiltration of Renilla-positive RAW264.7 cells.5.The spontaneous bone metastasis model of mice breast cancer was treated with Clodrolip to clear mononuclear macrophages.F4/80 staining of spleen was performed to detect the clearance of mononuclear macrophages by Clodrolip in mice.BLI and H&E staining were used to detect the bone metastasis and lung metastasis of breast cancer in mice.TRAP assay was used to detect osteoclast infiltration in bone metastases.6.The spontaneous bone metastasis model of mouse breast cancer was treated with NPs-CD137 Ab-F4/80.The bone metastasis and lung metastasis of breast cancer were detected by BLI and H&E staining.TRAP assay was used to detectosteoclast infiltration in bone metastases.Results: 1.The siRNA library screening found that CD137 affected the migration of monocytes into the tumor microenvironment.Bioinformatics analysis found that CD137 is associated with distant metastasis of breast cancer.2.Immunohistochemistry showed that CD137 was mainly expressed in stromal cells of breast cancer tissues,whereas CD137 L was mainly expressed in tumor cells.Semi-quantitative PCR assay also found CD137 L expressed in 4T1,4T07 and EO771 cells.ELISA and Western Blotting assay found that the the content of CD68 and sCD137 in the serum of patients with metastatic breast cancer sCD137 L was significantly higher than the normal population and patients with primary breast cancer.3.CD137 silenced and over-expressed stable cell lines were successfully constructed in RAW264.7-RL cells and CD137 was found to regulate Fra-1 expression.RNAseq method was used to detect the expression of differential genes in RAW264.7-RL-CD137 cells.The results showed that there were 101 genes in RAW264.7-CD137 cells that were up-regulated or down-regulated.Transwell and Scratch experiments showed that CD137 regulated the migration of RAW264.7 cells into the tumor microenvironment.Anti-CD137 Ab and Fra-1 antagonists blocked the CD137 and Fra-1 pathways can significantly inhibit the migration of RAW264.7-CD137 cells to the tumor microenvironment.TRAP experiment found that CD137 regulated the differentiation of RAW264.7 cells into osteoclasts.Real-time PCR showed that the expression of osteoclast differentiation-related genes was significantly up-regulated in RAW264.7-CD137 cells after stimulation with M-CSF and RANKL.4?The 4T1-FL cells and RAW264.7-RL cells were co-injected subcutaneously to construct the spontaneous bone metastasis model of breast cancer in BALB/c mice.BLI and H&E staining showed that the incidence of bone metastases and tumor burden in 4T1-FL cells and RAW264.7-RL-CD137 cells co-injected group were significantly higher than those in the control group.TRAP experiments found that the number of TRAP positive osteoclasts in the bone metastases of RAW264.7-RL-CD137 cells co-injected group was significantly higher than that of the control group.IHC staining showed that the number of Renilla positive RAW264.7 cells in the bone metastases of RAW264.7-RL-CD137 cells co-injected group was significantly more than that in the control group.5.The spontaneous bone metastasis model of breast cancer in BALB/c mice was treated with Clodrolip to clear monocytes/macrophages.IHC staining of F4/80 in the spleens showed that Clodrolip significantly cleared monocytes/macrophages in mice.BLI and H&E staining showed that the clearance of monocytes/macrophages in mice significantly inhibited bone metastasis in breast cancer.TRAP experiment found that the number of TRAP positive osteoclasts in bone metastases of Clodrolip-treated group was significantly reduced.And we found that the depletion of monocytes/macrophages in mice can also significantly inhibit the occurrence of lung metastases in breast cancer.6.The spontaneous bone metastasis model of breast cancer was treated with NPs-CD137 Ab-F4/80 to block the CD137/CD137 L pathway in the monocytes/macrophages.BLI and H&E staining of bone specimens showed that blockade of the CD137/CD137 L pathway significantly inhibited bone metastasis in breast cancer.TRAP staining of bone metastases showed the infiltration of TRAP-positive osteoclasts in bone metastases treated with NPs-CD137 Ab-F4/80 was significantly lower than the control group.In addition,blockade of the CD137/CD137 L pathway also significantly suppressed the incidence of lung metastasis of breast cancer.Conclusion: 1.CD137 can regulate the migration of monocytes to the tumor microenvironment.Bioinformatics analysis found that CD137 is associated with distant metastasis of breast cancer.Taken together,we speculate that CD137 contribute to the migration of monocytes to distant organs and promote a favorable microenvironment for tumor metastasis.2.CD137/CD137 L signaling may be associated with distant metastasis of breast cancer.3.In vitro,CD137 can promote bone metastasis of tumor cells by regulating the migration of RAW264.7 cells to the tumor microenvironment and the differentiation into osteoclasts,forming a microenvironment suitable for the dissemination of tumor cells.4.It further confirms that CD137 promotes the migration and differentiation of monocytes into osteoclasts,thus forms a microenvironment suitable for the colonization of tumor cells in vivo.5.Monocytes/macrophages play an important role in the metastasis of breast cancer.Depletion of Monocytes/macrophages by clodrolip inhibits bone metastasis in breast cancer.6.Blockade of CD137/CD137 L signaling pathway inhibits bone metastasis in breast cancer...