| Background and purposeLung cancer is still one of the tumors with the highest incidence of cancer worldwide,at the moment and the leading cause of cancer-related deaths,and about 85% of these are non-small-cell lung cancer(NSCLC).In the pathological process of NSCLC,about 30%of patients will undergo brain metastasis(Brain metastasis,Br M).Consequently,exploring the mechanism of brain metastasis in non-small cell lung cancer,especially lung adenocarcinoma,and finding the factors that may affect its brain metastasis are of great clinical significance for the prevention and treatment of lung cancer brain metastasis.Studies have shown that the interaction between tumor and microenvironment affects the process of tumor metastasis.In the brain microenvironment(TME),microglia cells mainly play the role of immune surveillance and protection against infection,so they play an important role in tumor immunity.As a resident innate immune cell of the Central Nervous System(CNS),microglial cells have different functional states: the physiological state is a branched resting state;the stimulus(infectious or traumatic),they quickly transforms into an "Amoeba"-like activated phenotype,and plays a role in removing pathogenic factors.The research results show that there is infiltration of microglia in malignant tumor tissues in the brain,and the activated types of microglia can be divided into M1 type with cancer suppressing effect and M2 type with cancer promoting effect.microglia are involved in brain tumor occurrence and progression.Exosomes(exosomes)are small vesicles secreted by cells that have a double-layered membrane structure and can carry biological giants(including proteins,DNA,m RNA,mi RNA,and lipids),and transmit biological information through fusion with cells.Current researches have confirmed that TDE regulates the specific metastasis of tumor target organs.They found that exosomes produced by breast cancer cells can be preferentially taken up by brain and lung tissue cells to regulate the formation of the microenvironment before specific target organ metastasis.Gon(?)alo Rodrigues and others reported that the CEMIP protein contained in exosomes secreted by breast cancer cells can upregulate the expression of cytokines and chemokines in the brain,induce the formation of a pro-inflammatory microenvironment,and then promote the occurrence of brain metastases.Pukrop demonstrated that microglia Wnt-dependent activation promotes colonization of breast cancer cells in the brain.The results of these studies indicate that tumor-derived exosomes remodel the functions of target organ tissue cells by transmitting biological information contained to distant target organs,and provides a suitable "soil" environment for tumor cell migration.The tissue and cell components in the brain are diverse and the precise transmission of information between cells is complicated.The association between brain microvascular cells,pericytes,astrocytes,microglia and neurons forms functional“neurovascular units”(NVU),which together maintained the homeostasis of normal brain function.Recent studies indicate that neurovascular units play an important role in brain metastasis.Among the constituent cells of NVU,cerebral microvascular cells are the earliest brain tissue cells that receive TDE information.Studies have shown that exosomes from MDA-MB-231 breast cancer cells can transfer mi R-105 to brain microvascular endothelial cells,down-regulate the expression of tight junction protein ZO-1,thereby destroying the integrity of the blood-brain barrier.Our previous study upshot that exosomes derived from brain microvascular endothelial cells can promote the survival of small cell lung cancer cells(SCLC)in the brain.These findings suggest that brain microvascular endothelial cells may obtain the transfer information of TDE in the circulation as early as possible,and pass this information to other types of tissue cells in the brain through NVU.At present,however,there is no report on how exosomes derived from lung adenocarcinoma cells transmit brain metastasis information through NVU and thus affect the formation of metastatic microenvironment in the brain.We incubated the purified lung cancer-derived exosomes with brain microvascular endothelial cells,and analyzed the cytokines secreted by brain microvascular endothelial cells by ELISA Array,and screened the differential protein DKK-1.DKK-1 can competitively bind to LRP5 / 6 and inhibit Wnt signal activation.Studies have shown that DKK-1 is involved in the development of many tumors.DKK-1 is highly expressed in cholangiocarcinoma and is associated with lymph node metastasis;inhibiting the expression of DKK-1 in cancer cells can significantly inhibit the proliferation and migration of cancer cells.In breast cancer,the expression of DKK-1 shows different roles in bone metastasis and lung metastasis in breast cancer.DKK-1 antagonizes the non-classical WN signaling pathway to inhibit PTGS2-induced macrophages and neutrophils in lung metastasis Recruitment;and by regulating the activation of classic Wnt signaling in osteoblasts to promote bone metastasis,this suggests that DKK-1 has a diversity of molecular functions in tumor metastasis.There are reports in the literature that DKK-1 is highly expressed in non-small cell lung cancer,and serum DKK-1 content can be used as an indicator of disease prognosis.However,there is no relevant literature on the mechanism of DKK-1 in non-small cell lung cancer metastasis,especially brain metastasis.We intend to investigate whether DKK-1 produced by brain microvascular endothelial cells which lung cancer cell-derived exosomes induces to regulate the function of NVU constituent cells and thus affect the occurrence of lung cancer brain metastasis.Materials and Methods1 Acquisition and identification of lung adenocarcinoma-derived exosomes1.1 Ultrahigh-speed centrifugation was used to isolate and purify lung cancer cell-derived exosomes.Western blot and flow cytometry were used to identify the expression of exosome-related marker molecules.Transmission electron microscopy and nanoparticle measuring instrument were used to identify the structure and particle diameter of exosomes.1.2 A recombinant plasmid expressing CD63-m Cherry was constructed,transfected in lung cancer cell A549 cells,and co-cultured with HBMEC transfected with p EGFP-N1 plasmid.Laser confocal microscopy was used to observe whether exosomes derived from A549 cells were uptaked by HBMEC cells.2 Effect of lung adenocarcinoma-derived exosomes on the activity of microglia in the brain.2.1 Exosomes derived from mouse lung adenocarcinoma cells(Lewis Lung Cell,LLC)were inoculated into six-week-old C57 BL / 6 mice via the lateral ventricle or retrobulbar venous plexus,injected once every other day,after five consecutive times,using polyformaldehyde to perfuse the brain tissue and then preparaing the tissue oscillation brain slices.Immunofluorescence staining was used to observe the morphology and activation of microglia in the brain tissue.2.2 Lung adenocarcinoma-derived exosomes(human A549 cells and mouse LLC cells)were incubated with HBMEC/mouse brain microvascular endothelial cells(b.End.3 cells),collected the cell supernatant and incubated THP-1/BV2 cells,real-time Fluorescence quantitative PCR was used to detect the expression of M1(IL-1β,TNFα,TLR-4,)and M2(CD206,Arginase 1)type glial cell marker molecules in THP-1 / BV2 cells.3 The Transwell chamber was used to establish a monolayer culture model of brain microvascular endothelial cells,looking for differential extracellular factors secreted by brain microvascular endothelial cells which induced by lung cancer cell-derived exosomesUse Transwell chamber to culture HBEMCs on Transwell membrane,add lung cancer-derived exosomes in the upper chamber,incubate for 24 hours to collect upper chamber and lower chamber culture,and use ELISA Array to analyze the cytokines contained in the upper and lower chambers to screen for differential protein DKK-1.4 Lung cancer cell-derived exosomes induce brain microvascular endothelial cells to express DKK-14.1 Lung cancer-derived exosomes were incubated with brain microvascular endothelial cells,and the expression of DKK-1 in brain microvascular endothelial cells was detected by ELISA.4.2 The serum exosome isolation kit was used to collect exosomes derived from human serum of lung cancer patients,incubated with HBMEC cells,and detected the expression of DKK-1 by ELISA.5 Effects of DKK-1 on the biological function of lung cancer cells5.1 MTS detected the effect of DKK-1 on the proliferation ability of lung cancer cells.5.2 Scratch test detected the effect of DKK-1 on the migration ability of lung cancer cells.5.3 Apoptosis kit and flow cytometry analysis were used to detect the effect of DKK-1on the anti-apoptotic ability of lung cancer cells.6 Effect of DKK-1 on the biological function of pericytesThe effect of DKK-1 on the proliferation of pericytes was measured by MTS.7 The effect of DKK-1 on the biological function of endothelial cells7.1 MTS detection of the effect of DKK-1 on the proliferation ability of HBMECs.7.2 Cell cycle kit labeled endothelial cells cycle.Flow cytometer was used to analyze the effect of DKK-1 on endothelial cell cycle.7.3 Apoptosis kit and flow cytometry analysis were used to detect the effect of DKK-1on the anti-apoptosis ability of HBMECs.8 Effect of DKK-1 on the biological function of microglia.8.1 DKK-1 was injected into the lateral ventricle of C57 BL / 6 mice and injected once every other day for seven consecutive times.After perfusion,brain tissue was taken to prepare oscillating brain slices.Immunofluorescence was used to observe the number of microglia and the number and activation status of microglia in the brain slices of mice.8.2 In the co-culture of brain microvascular endothelial cells and microglia,Real-time PCR and Western blot were used to detect the expression of M1 and M2 marker molecules.LLC cell-derived exosomes were used to treat brain microvascular endothelial cells;RNAi technology was used to down-regulate the expression of DKK-1 in brain microvascular endothelial cells in the co-culture system,and lung adenocarcinoma cells derived exosomes treated brain microvascular endothelium cells and observed the expression of M1 and M2 type marker molecules in microglia co-cultured with them.8.3 DKK-1 treated LPS or IL-4 activated microglia BV2 cells or monocyte THP-1cells.Real-time PCR and Western blot were used to detect the expression of M1 and M2 type marker molecules.8.4 DKK-1 treatment of LPS and IL-4 with different time axis activated BV2 cells.Real-time PCR detected the expression of M1(IL-1β,TNFα and TLR-4)and M2(CD206 and Arginase 1)type marker molecules.8.5 LLC Br M1 was co-cultured with BV2,and then add neutralizing antibodies such as DKK-1,Real-time PCR was used to detect the expression of M1 and M2 marker molecules in microglia.8.6 LLC Br M3 was co-cultured with BV2,and then add DKK-1 recombinant protein,Real-time PCR was used to detect the expression of M1 and M2 marker molecules in microglia.9 Lung adenocarcinoma cells were implanted in situ in the brain of mice,and brain metastatic cells of lung adenocarcinoma with different colonization ability were screened in the brain to analyze the expression of DKK-1.9.1 Six-week-old C57 BL / 6 mice were implanted with LLC cells in situ in the brain to form transplanted tumors within 14 days.Immunofluorescence was used to detect the number and activation of microglia in brain tumor tissue;immunohistochemical analysis of the brain the expression of DKK-1 in tumor tissues.9.2 Six-week-old C57 BL / 6 mice were implanted with LLC cells in situ in the brain,transplanted tumors were formed in the brain within 14 days,and the first round of lung adenocarcinoma brain metastasis(LLC Br M)was isolated and cultured.The second round of LLC Br M2 cells and the third round of LLC Br M3 cells were obtained respectively.Western blot analysis of the expression of DKK-1 in lung adenocarcinoma brain metastasis cells.9.3 Western blot was used to detect the expression of DKK-1 in lung cancer tissues and adjacent tissues,as well as the expression of DKK-1 in unpaired lung cancer metastatic cancer tissues.Results1 Lung cancer cell-derived exosomes can be endocytosed into cells by brain microvascular endothelial cells.Identification of lung cancer cell-derived exosomes: Western blot results showed that the relevant marker molecule CD63 was expressed in the isolated and purified exosomes.Flow cytometric analysis revealed the presence of exosome membrane protein CD63.Exosomes were observed by transmission electron microscopy the double-layer membrane vesicle structure and nanoparticle tracking technology detected that the diameter of exosomes was 30-150 nm.In summary,we obtained lung cancer cell-derived exosomes by ultra-high speed centrifugation.1.2 After co-culture of A549 cells transfected with p CMV-CD63-m Cherry plasmid and HBMEC transfected with p-EGFP-N1 plasmid,laser confocal images shown that exosomes derived by A549 cells entered HBMECs.2 Lung adenocarcinoma-derived exosomes regulate the function of microglia in the brain.2.1 LLC cells-derived exosomes were inoculated into C57 BL / 6 via the lateral ventricle or retrobulbar venous plexus.Immunofluorescence results showed that LLC-derived exosomes injected into the lateral ventricle can increase the number of activated microglia in the cerebral cortex.Intravenous injection through the retrobulbar venous the activation of microglia in the cerebral cortex.2.2 Establish a co-culture model of brain microvascular endothelial cells(HBMEC /b.End.3)and THP-1 cells / BV2 cells,treat brain microvascular endothelial cells with lung adenocarcinoma-derived exo,and analyze the activation of THP-1 cell / BV2 cell in the culture system by real time PCR.The results indicated that lung adenocarcinoma-derived exosomes can indirectly inhibit the activation of THP-1 cells /BV2 cells through brain microvascular endothelial cells.3 Lung adenocarcinoma-derived exosomes were co-cultured with HBMECs to obtain the differentially expressed protein—DKK-1.4.lung adenocarcinoma-derived exosomes induce brain microvascular endothelial cells to express DKK-1.4.1 Human and murine lung cancer cell-derived exosomes can induce brain microvascular endothelial cells to express DKK-1.4.2 LLC-derived exosomes injected by retrobulbar venous can induce mouse brain microvascular endothelial cells to express DKK-14.3 Exosomes from the serum of patients with lung adenocarcinoma can induce DKK-1 expression in brain microvascular endothelial cells.5.The effect of DKK-1 on the function of neurovascular unit constituent cells.5.1 DKK-1 has no effect on the proliferation and migration of lung cancer cells,and has a certain ability to resist apoptosis.5.2 DKK-1 had no obvious effect on the proliferation of pericytes.5.3 DKK-1 had no significant effect on the cell cycle,proliferation and anti-apoptosis ability of brain microvascular endothelial cells.6.DKK-1 inhibits the activation of resting microglia.6.1 Intraventricular injection of DKK-1 can reduce the number of activated microglia in the cerebral cortex and hippocampus of mice.6.2 Adding LLC-derived exosomes to the upper chamber of the HBMEC and BV2co-culture system can inhibit the expression of BV2 cell M1(IL-1β,TNFα,TLR-4)marker molecules and M2(Arginase 1,CD206)the expression of marker molecules;after using RNAi technology to down-regulate the expression of DKK-1 in brain microvascular endothelial cells in a co-culture system,the LLC-derived exo treatment of brain microvascular endothelium can inhibit the aforementioned effects.6.3 DKK-1 inhibits LPS-induced M1(IL-1β,TLR-4 and i NOS expression)microglial activation,and IL-4-induced M2(Arginase 1 and CD206)microglial activation.7.The expression of DKK-1 in lung cancer tissue in the brain is reduced.7.1 The expression of DKK-1 in lung cancer tissue in situ in clinical pathological tissues is higher than that in brain metastatic lung cancer tissues,and the expression level of DKK-1 in animal brain metastatic tumor tissues is reduced.7.2 In lung cancer brain metastasis cells(LLC Br Ms)with different colonization ability,the expression level of DKK-1 decreases with the increase of colonization ability in the brain.7.3 After antagonizing DKK-1 secreted by LLC Br M1 cells with neutralizing antibodies,it can promote the expression of M1 type marker molecules in BV2co-cultured with it and inhibit the expression of M2 type marker molecules.7.4 Co-culture with BV2 cells after treatment with DKK-1 recombinant protein in LLC Br M3 cells,inhibit the expression of M1 type marker molecules in co-cultured BV2,and promote the expression of M2 type marker molecules.Conclusion1.Lung adenocarcinoma-derived exosomes can indirectly regulate the activation of microglia in the brain by inducing DKK-1 expression in brain microvascular endothelial cells.2.DKK-1 from brain microvascular endothelial cells can inhibit the activation of microglia in the brain(M1 type and M2 type).3.When lung adenocarcinoma cells enter the brain,the expression of DKK-1 in the cells will decrease with the enhancement of lung adenocarcinoma cells’ colonization ability in the brain.The decrease in the expression of DKK-1 in lung adenocarcinoma cells weakens the inhibitory effect on microglial cells,so that microglial cells can be activated into M2-type microglia with cancer-promoting effects. |
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