The Study Of The Interaction And Mechanism Between Microglia And Tumor Cells In The Microenvironment Of Lung Cancer With Brain Metastases

Objective:Brain metastases,the most common tumors in the brain parenchyma,have been considered a terminal disease stage with a poor prognosis and short survival time.However,the treatment options for brain metastases are still limited.Lung cancer is the most common tumor that metastasizes to the brain.The brain metastases microenvironment consists of cellular and non-cellular components.As resident macrophages in the brain,microglia form a major component of the brain immune system.In recent years,many researches have reported the critical role of microglia in brain tumor progression.As essential mediators of intercellular communications,tumor-derived extracellular vesicles have been recognized as important factors in tumor metastases.However,the special effects of exosomes from lung cancer on the function of microglia compared with exosomes from other tumors remain unknown.In this study,we compared the effects of exosomes of lung cancer and nasopharyngeal carcinoma on the functions of microglia and attempted to explore the impact of lung cancer cells prone to brain metastases on the lipid metabolism and functions of microglia,and search for new therapeutic targets and strategies for brain metastases.Methods:In this study,we extracted the exosomes of tumor cells by differential centrifugation.The morphology and size of these exosomes were shown by transmission electron microscopy,and the diameter of exosomes was analyzed by nanoparticle tracking analysis technology.The expression of exosome markers was detected by western blot,and the exosomes phagocytosis of microglia cells was detected by PKH67.Mice brain metastases models in vivo and co-culture models of lung cancer cells and microglia in vitro were established,and lung cancer patient samples of brain metastases were collected.Immunohistochemical staining,immunofluorescence staining,western blot,transcriptomics,flow cytometry,RT-qPCR and gas chromatography were used to detect lipid metabolism and functions of microglia.The formation and sizes of brain tumors were observed by live imaging of small animals.Phosphoprotein antibody chips were used to screen cellular pathways.Moreover,sequencing and analysis of miRNAs in exosomes were performed.Results:Transcriptome sequencing showed that the two exosomes had significantly different effects on the expression of genes related to lipid metabolism in microglia cells.Especially,lung cancer-derived exosomes significantly promoted the expression of Stearoyl CoA desaturase 1(SCD1)in microglia.In co-cultured models,the expression of SCD1 and the number of lipid droplets in microglia were increased.In vivo,a large number of microglia clustered around brain metastases in both mice models and patients with brain metastases,furthermore,the expression of SCD1 and lipid droplets in microglia were increased.In vitro,A549-BrM cells could reduce the response of microglia to inflammatory stimuli,while Cay 10566 could restore the inflammatory response of microglia.In addition,microglia could promote the proliferation of tumor cells and the addition of SCD1 inhibitors inhibited the proliferation of tumor cells.Although no significant effect of SCD1 inhibitors on brain tumor growth was observed,SCD1 inhibitors combined with CSF1R inhibitors significantly reduced tumor burdens in brain metastases of mice.Furthermore,lung cancer cells prone to metastases could significantly activate the STAT3 signaling pathway in microglia to increase SCD1 expression.Sequencing of extracellular miRNAs derived from lung cancer exosomes showed that miR1246 was the most highly expressed miRNAin lung cancer,and the plasma level of miR1246 in lung cancer patients was higher than that in healthy controls.Overexpression of miR1246 in microglia could promote the activation of the STAT3 signaling pathway and the increase of SCD1 expression.Conclusions:Lung cancer cells in the brain metastases microenvironment can promote the expression of SCD1 and the accumulation of lipid droplets in microglia.Increased activity of SCD1 in microglia could reduce its response to inflammatory stimuli and promote tumor cell proliferation.Moreover,SCD1 inhibitors could ameliorate the effect of the tumor on microglial inflammatory reactivity,therefore reducing the proliferation of tumor cells.Furthermore,SCD1 inhibitors combined with CSF1R inhibitors reduced tumor burden in mice with brain metastases.Importantly,lung cancer cells can deliver miR1246 into microglia through exosomes,causing activation of the STAT3 signaling pathway and promoting increased expression of SCD1 in microglia.Part Ⅰ:Comparative study on the effects of exosomes derived from lung cancer and nasopharyngeal carcinoma on microglia functionsObjective:Brain metastases,the most common tumors in the brain parenchyma,have been considered a terminal disease stage with a poor prognosis and short survival time.However,the treatment options for brain metastases are still limited.Lung cancer is the most common tumor to metastasize to the brain.Interestingly,nasopharyngeal carcinoma(NPC)is a malignant head and neck tumor,and the incidence of intracranial invasion is not unusual but the brain is the least common metastatic site in NPC patients.As essential mediators of intercellular communications,tumor-derived exosomes have been recognized as important factors in tumor metastases.Microglia as primary immune cells in the brain play important roles in the tumor environment.In this study,we compared the effects of exosomes of lung cancer and NPC on the functions of microglia and explored whether tumor cells with different metastatic tendencies of the brain have different effects on the remodeling of microglia.Methods:In this study,we extracted the exosomes of tumor cells by differential centrifugation,and their morphology and size were shown by transmission electron microscopy,the diameter of exosomes was analyzed by nanoparticle tracking analysis technology(NTA),the expression of exosomes markers was detected by western blot,and the exosomes were labeled with PKH67 to show being phagocytized by microglia.Exosomes were added into the microglial medium,and the effects of exosomes on inflammatory marker proteins,proliferation,migration,phagocytosis and secretion of microglia were detected by western blot,CCK8 proliferation assay,wound healing assay,immunofluorescence staining,profiler human cytokine array and Elisa kits.Transcriptome sequencing was used to analyze the differential gene expression.Results:Exosomes derived from lung cancer and NPC cells were successfully extracted by differential centrifugation and were identified with typical exosome diameter and marker expression.Moreover,microglia could take up these exosomes.The exosomes from nasopharyngeal carcinoma and lung cancer could affect the morphology of microglia and increase the proliferation,phagocytosis and release of inflammatory cytokines IL-6,IL-8 and CXCL1,but there were no significant effects on the expression of inflammatory markers and migration ability of microglia.Transcriptome sequencing showed that the two exosomes had significantly different effects on the expression of genes related to lipid metabolism in microglia.Conclusions:Lung cancer-derived and NPC-derived exosomes can change microglial morphology and promote the phagocytosis and secretion of cytokines,however there is no significant difference between them.The effects of two exosomes on lipid metabolismrelated genes in microglia were significantly different.These results provide the direction for further study on the mechanism of lung cancer brain metastasis.Part Ⅱ:Lipid metabolism of microglia in lung cancer brain metastases microenvironmentObjective:The brain metastases microenvironment consists of cellular and non-cellular components.As resident macrophages in the brain,microglia form a major component of the brain immune system.In recent years,many researches have reported the critical role of microglia in brain tumor progression.In our previous studies,it was observed that the effects of exosomes from lung cancer cells and nasopharyngeal carcinoma cells on microglia lipid metabolism were different,and lung cancer cells could significantly increase the expression of stearoyl-CoA desaturase 1(SCD1)in microglia.Some studies have shown that changes in cellular lipid composition and intracellular lipid droplet accumulation would affect the functions of immune cells including tumor-associated macrophages.However,there is no study observing the changes in microglial lipid metabolism in the brain metastases microenvironment.In this study,the changes in lipid metabolism of microglia cells were further observed in vivo and in vitro based on the results of previous studies.Methods:In this part,lung cancer cells with different brain metastases tendencies were cocultured with microglia,and western blot and RT-qPCR techniques were used to detect the expression of SCD1 and other genes;Intracellular fatty acid concentrations were detected by gas chromatography;The numbers of lipid droplets were detected by flow cytometry and immunofluorescence staining.Moreover,luciferase-labeled A549-BrM cells were injected into the left cardiac ventricle of mice.To confirm successful brain metastases,mice were intraperitoneally injected with D-luciferin and the photon flux from the whole body was observed and imaged.Next,hematoxylin staining and immunofluorescence staining were used to observe brain tumors and the expression of SCD1 and lipid droplet numbers in frozen slices of mice brains.Flow cytometry was used to detect the numbers of lipid droplets in microglia and separate mice brain microglia,then microglial RNA was extracted to detect SCD1 expression levels.In patient samples,HE and immunohistochemical staining were used to detect the number and morphology of microglia and the expression of SCD1 protein in the brain metastases area.Plasma lipid detection results were collected to analyze the characteristics of common plasma lipid metabolism parameters in lung cancer patients with brain metastases.Results:In vitro,microglia co-cultured with A549-BrM cells upregulated the expression of SCD1 and the 18:1 n-9/18:0,18:2/18:0 ratio,the numbers of lipid droplets in microglia were increased.In vivo,a large number of microglia clustered around brain metastases in both mouse models and patients with brain metastases.Furthermore,the expression of SCD1 and lipid droplets in microglia were increased.In patients,it was observed that the SCD1 staining of the metastases area was significantly darker than that of the normal area,and the analysis of clinical parameters showed that the total cholesterol and low-density lipoprotein in patients with brain metastases were higher than those in the healthy control group.Conclusions:A large number of microglia cells would aggregate in the brain metastases area.A549-BrM cells could promote the expression of SCD1 and the accumulation of intracellular lipid droplets.Increased expression of SCD1 and changes in plasma lipid metabolism were also confirmed in brain metastases samples from patients.In a word,the lipid metabolism of microglia in brain metastases microenvironment was altered.Part Ⅲ:The mechanism of microglial SCD1 on the cellular functions and progression of lung cancer brain metastasesObjective:Lipid metabolism is involved in microglia activation and a variety of cellular functions such as inflammatory signaling,migration,and phagocytosis.In peripheral tumors,some studies have shown that TAMs can promote tumor growth and metastases by inhibiting tumor immune monitoring.Moreover,fatty acid metabolism can control the immunosuppressive phenotype of TAMs,and lipid changes in TAMs can affect their inflammatory responses.However,the mechanism of microglia lipid metabolism and brain tumor progression in the brain tumor microenvironment remains unclear.Our results confirmed abnormal lipid metabolism in microglia cells and identified increased SCD1 expression and lipid droplet accumulation in microglia cells in the tumor microenvironment.Further experiments need to explore the effects of abnormal lipid metabolism in microglia on the functions of microglia and the growth of brain metastatic tumors.Methods:In this part,SCD1 inhibitor-Cay 10566 was used.Gas chromatography,western blot,immunofluorescence staining,and flow cytometry were used to observe the effect of the SCD1 inhibitor.At the same time,the changes in the inflammatory factors were detected by western blot and RT-qPCR in microglia co-cultured with tumor cells.EdU proliferation kit was used to detect the proliferation of tumor cells.To observe the treatment effect of SCD1 inhibitor,the method of intragastric administration in mice was used,and the formation and sizes of brain tumors were observed by live imaging of small animals.Results:Cay 10566 as an SCD1 inhibitor could significantly inhibit SCD1 activity and reduce lipid droplet formation in microglia.In vitro,A549-BrM cells could reduce the response of microglia to inflammatory stimuli,while Cay 10566 could restore the inflammatory response of microglia.In addition,microglia could promote the proliferation of tumor cells and the addition of SCD1 inhibitors to the co-culture system inhibited the proliferation of tumor cells,while SCD1 inhibitors had no direct effect on tumor cells.Although no significant effect of SCD1 inhibitors on brain tumor growth was observed,SCD1 inhibitors combined with CSF1R inhibitors significantly reduced tumor burdens in brain metastases of mice.Conclusions:Cay 10566 as an SCD1 inhibitor could reduce SCD1 activity and reduce lipid droplet accumulation in microglia.Increased SCD1 activity in microglia could reduce its response to inflammatory stimuli and promote tumor cell proliferation.Moreover,SCD1 inhibitors could ameliorate the effect of the tumor on microglial inflammatory reactivity,therefore reducing the proliferation of tumor cells in vitro.Furthermore,SCD1 inhibitors combined with CSF1R inhibitors reduced tumor burden in mice with brain metastases.Part Ⅳ:The molecular mechanism of lung cancer-mediated SCD1 upregulation in microglia in brain metastasesObjective:In the brain tumor microenvironment,many studies have confirmed that TAMs can interact with tumor cells and have an important role in the occurrence and development of tumors.In our previous experiments,lipid changes have been observed in microglia,especially the increased expression of SCD1.The reprogramming of lipid metabolism can cause alterations in their functions and promote the proliferation of tumor cells.However,the clear mechanism of lipid metabolism changes in microglia remains unknown.Here,we further investigate the detailed signaling pathways and mechanisms of brain metastases and microglia in lung cancer.Methods:In this study,phosphoprotein antibody chips were used to screen cellular pathways,and western blot was used to detect the activation and expression of proteins related to intracellular signaling pathways.Sequencing and analysis of miRNAs in exosomes were performed,and RT-qPCR was used to determine the expression levels of miRNAs.Plasma exosomes were extracted from lung cancer patients to detect miRNA expression.miRNA was transfected into microglia cells to observe the alterations of related genes.Results:Lung cancer cells prone to metastases could significantly activate the STAT3 signaling pathway in microglia,meanwhile,activation of STAT3 increased SCD1 expression in vitro.Sequencing of extracellular miRNAs derived from lung cancer cells showed that miR1246 was the most highly expressed miRNA in lung cancer,and the plasma level of miR1246 in lung cancer patients was higher than that in healthy controls.Overexpression of miR1246 in microglia could promote the activation of the STAT3 signaling pathway and the increase of SCD1 expression.Conclusions:Non-small cell lung cancer(NSCLC)cells can deliver miR1246 into microglia through exosomes,causing activation of the STAT3 signaling pathway and promoting increased expression of SCD1 in microglia.