Study On The Role Of SDF-1 Played In The Microenvironment Of Bone Metastasis In Small Cell Lung Cancer

Background: Small cell lung cancer(SCLC)is a highly fatal cancer characterized by early and widespread metastases and the ability to rapidly develop resistance to chemotherapy.It is one of the pathological subtype of lung cancer,which is often accompanied by neuroendocrine characteristics.During the past decades,there is just little progress regarding the therapy on SCLC.For now,the surgery is still the main treatment for SCLC patients at limited stage,while chemotherapy and radiotherapy for SCLC patients at extensive stage.However,the emerging immunotherapy brings new prospect for SCLC.From the past to the present,we can see some changes in the tumor treatment strategy that we aimed to kill cancer cells in the past,while the current immunotherapy and anti-angiogenesis drugs are mainly targeted at the tumor microenvironment.The initiation and development of cancer are closely related to their surrounding microenvironment.Chemokines are important messengers transmiting signals between them.As a chemotactic factor constantly expressed by a series of organs,SDF-1 is involved in various tumors.Howere,it is still elusive about the role of SDF-1 played in SCLC,especially in its bone metastasises.So,this study aimed to explore the possible contribution of SDF-1 function to SCLC cells as well as its role in the bone metastasis of SCLC.Objective: To investigate the location of cancer cells in SCLC bone metastases and analyze the particularity of the microenvironment where the cancer cells are located.Then detect the expression of SDF-1 in the bone with metastatic lesions and explore the the effects of SDF-1 on the biological behaviors of SCLC cells in vitro,including proliferation,invasion,adhesion,apoptosis and cell cycle etc.At last,study the role of SDF-1-CXCR4 signal in SCLC bone metastasis by NOD/SCID mouse model,providing new therapeutic targets for the clinical treatment on SCLC.Methods: 1.Immunofluorescence staining was applied to detect the localization of cancer cells in SCLC bone metastases.2.The in vitro co-culture system was used to simulate the bone microenvironment of cancer cells in vivo for detecting the influence of osteoblasts on the invasion ability of SCLC cells and checking whether CXCR4 antagonist AMD3100 could interfere with this effect.3.ELISA was used to examine the secretion of SDF-1 by osteoblasts.4.Immunohistochemical staining was applied to detect the expression of SDF-1 in SCLC bone metastases lesions.5.MTT assay was used to test the effects of SDF-1on cell proliferation in SBC-3 cells and SBC-5 cells cultured in complete or serum-free medium.And figure out the effective concentration of SDF-1 that exerting its function.6.Transwell assay was used to determine the impact of SDF-1 on the invasive ability of SBC-3 cells and SBC-5 cells as well as whether AMD3100 could block this effect.7.Flow cytometry was used to explore the effects of SDF-1 on apoptosis and cell cycle in SBC-3 cells and SBC-5 cells under starvation.8.Adherence assay was performed to determine the effects of SDF-1 on the adhesion abilities of SBC-3 cells and SBC-5 cells with fibronectin and bone.9.Real time RT-PCR and Western blot was applied to detect the expression of CXCR4 and CXCR7 in SBC-3 cells and SBC-5 cells.10.Immunofluorescence staining was used to determine the localization and expression of CXCR4 and CXCR7 in SBC-3 cells and SBC-5 cells.11.Lentiviral transfection was applied to interfere the expression of SDF-1 in SBC-3 cells and enhance this expression in SBC-5 cells.Then the transfected cell lines was detected for their alibity on proliferation?migration and invasion.12.Bone matrix vectors coated with SDF-1 were implanted subcutaneously in NOD/SCID mice injected with SBC-5 cells to check if SDF-1 alone could cause SCLC cell metastasis.Meanwhile,the effect of AMD3100 on the formation of bone metastasis in mice was detected.Results: 1.In the bone metastasis lesions,SCLC cells are extremely close to HSCs(hematopoietic stem cells)and osteoblasts,and HSCs and osteoblasts are also closely adjacent.2.SCLC cells co-cultured with osteoblasts have stronger invasion ability,which could be impaired,to a certain extent,by CXCR4 antagonist AMD3100.3.Osteoblasts could secrete SDF-1.4.The expression of SDF-1in SCLC bone metastases was higher than that in normal bone tissue.5.SDF-1 has no effect on the proliferation of SBC-3 cells and SBC-5 cells cultured in complete medium,but can "protect" those cultured in serum-free medium.The effective concentration of SDF-1 was 100ng/ml.6.Transwell assay showed that SDF-1 could promote the invasion of SBC-3 cells and SBC-5 cells,which could be blocked by AMD3100.7.SDF-1 increased the apoptosis rate of SBC-3 cells and SBC-5 cells under starvation,and the cell cycle results further showed that SDF-1 increased the proportion of SBC-3 cells and SBC-5 cells in G1 phase.8.SDF-1 could enhance the adhesion ability of SBC-3 cells and SBC-5 cells to fibronectin and bone.9.At m RNA,CXCR4 and CXCR7 were shown a higer expression in SBC-3 cell than that in SBC-5 cells.As for protein,the expression of CXCR4 was consistent with the results of RT-PCR,but the expression of CXCR7 was shown a contrary result,with a higher expression in SBC-5 cells.Besides,both SBC-3 cells and SBC-5 cells could express SDF-1,and the expression level was higher in the former one.10.CXCR4 and CXCR7 were mainly located at the cytoplasm and membrane in SBC-3 cells and SBC-5 cells.And SBC-3 cells had a tronger epression of CXCR4,while SBC-5 cells had a tronger epression of CXCR7.11.Transfected cell lines were established and named as SBC-3-LV3-NC ?SBC-3-SDF-1-Ri ? SBC-5-LV5-NC and SBC-5-SDF-1.And down-expression of SDF-1 in SBC-3 cells could impair their proliferation and invasion ability;while over-expressing SDF-1 in SBC-5 cells could promote the cell to proliferate and invade.12.In NOD/SCID mice,no cancer cell was detected in the SDF-1-coated bone matrix vector,but the bone metastasis was inhibited by AMD3100.Conclusion: Cancer cells located in the HSC niche in SCLC bone metastasis lesions.This special microenvironment provided a protective microenvirment for cancer cells against immune attack and chemotherapy drugs.And SDF-1 played a crucial role in the formation of bone metastasis in SCLC.In vitro experiments,SDF-1 could improve the survival of SCLC cells under starvation by promoting them to enter G0 phase.In addition,SDF-1 could also enhance the cell invasion and adhesion.Consistently,CXCR4 antagonist AMD3100 could block the effect of SDF-1 exerted on the cell invasion in vitro and inhibit bone metastases in vivo.More importantly,we found that osteoblasts induced the invading of cancer cells expressing CXCR4 and/or CXCR7 to bone by secreting SDF-1,while SBC-5 reduced the concentration of SDF-1 in the primary tumor by expressing CXCR7 which had endocytosis on SDF-1.Then SBC-5 could escape from the primary location and migrate to organs expressing SDF-1.These findings suggested that SDF-1/CXCR4 signaling might be a new target for the treatment of bone metastasis in SCLC.