| BackgroundLung cancer has been a big threat to human health due to its high morbidity and mortality in China.Metastasis is the leading cause of death in patients with lung cancer while brain metastasis(BM)is the most common distant metastasis site of lung cancer.BM refers to the spread of cancer cells from other organs to the brain,which consists of a series of pathophysiological processes,such as proliferation of primary cancer cells,migration of cancer cells,and metastatic tumor growth in the brain.A better understanding of the pathological process and related mechanisms underlying BM of lung cancer is critical for exploring new diagnostic and therapeutic approaches.However,the current approaches for lung cancer BM research have great limitations.The existing in vitro research platforms are difficult to reproduce the complicated pathological process and metastasis microenvironment of lung cancer BM,while in vivo animal models face difficulties in visual monitoring,low throughput and ethical restrictions.Therefore,there is an urgent need to develop a platform which can not only simulate the microenvironment in vivo,reproduce the complex metastasis process,but also realize real-time high-throughput monitoring for studies of BM.Microfluidic chip technology,which is recognized as one of the most important cutting-edge science and technology in the 21 st century,is expected to provide an ideal new technology platform for lung cancer BM research due to its advantages such as strong maneuverability and miniature high-throughput.Herein,this project intends to build a bionic chip for lung cancer brain metastasis based on microfluidic technology,realizing the visual reproduction and real-time monitor of BM pathological process,and then apply it to the study of molecular mechanisms of BM,which will further clarify the molecular mechanisms related to BM,and explore the application value of microfluidic chip in the BM researches.This study is divided into the following three parts.Methods1.Construction of a microfluidic bionic chip model for lung cancer brain metastasisThe chip consisted of two bionic organ units – an upstream ‘‘lung” and a downstream ‘‘brain” characterized by a functional “blood–brain barrier(BBB)” structure.The upstream "lung" simulates respiratory movement and air-blood exchange,and reproduces the development of lung tumor in a physiological environment close to the body.The downstream "brain" achieves multi-cell co-culture by connecting the vascular channels with the brain parenchymal chamber through micro-gaps.The chip is exposed to continuous vascular shear forces controlled by a micropump to build functional BBB.Based on this,the metastatic trajectory of lung cancer cells is monitored in real time,providing a new technical platform for the follow-up study of the molecular mechanism of lung cancer brain metastasis.2.Establishment of highly brain metastatic lung cancer cell line model and proteomics databaseMice were inoculated intracardially with the human lung cancer cell line PC9 and the brain metastatic subpopulation was extracted to isolate PC9 clones with more metastatic activity.After two rounds of in vivo selection and in vitro expansion,the PC9-Br M3 cells were selected as the highly brain metastatic cell populations.TMT labeling,high-performance liquid chromatography grading technology,and mass spectrometry-based quantitative proteomics technology is used to identidy the differential proteins in lung cancer BM compared to the parental cell lines.Functional enrichment and cluster analysis are conducted to further screen for key molecules,providing evidence for mechanism research of BM.3.Molecular mechanism of AKR1B10 in promoting lung cancer brain metastasisUnder the combined application of the new microfluidic bionic chip platform and traditional in vivo and in vitro research platforms,the role and molecular mechanism of AKR1B10(Aldo-keto reductase family 1 B10),a key molecule screened from the proteomics database,in brain metastasis of lung cancer is clarified,providing strong evidence support for that AKR1B10 can act as a new diagnosis biomarker and target for BM.Results1.Construction of a microfluidic bionic chip model for lung cancer brain metastasisThe microfluidic bionic chip model of lung cancer brain metastasis was successfully constructed,which realized the simulation of the primary pulmonary physiological functions including the breathing rhythm and air-blood exchange in the upstream "lung" and the physiological barrier function of blood-brain barrier in the downstream "brain".On the basis of this,the chip successfully realized real-time visual monitor of the entire BM process,from the growth of primary tumor to its breaking through the BBB,and finally reaching the brain parenchyma,providing a new methodology platform for further basic research.2.Establishment of highly brain metastatic lung cancer cell line model and proteomics databaseThe highly brain metastatic lung cancer cell line was successfully constructed,providing a reliable cell line model for the subsequent establishment proteomics databases and molecular studies.By applying TMT labeling,high-performance liquid chromatography fractionation technology,and mass spectrometry-based quantitative proteomics technology,the changes in protein expression profiles in lung cancer BM were clarified.The difference in fold values exceeded 1.3 times as a significant increase,and less than 1 / 1.3 as Significantly down-regulated the standard of change.It was found that there were 200 proteins significantly up-regulated and 203 proteins down-regulated in highly brain metastatic lung cancer cell line compared with parental cells.Key molecules such as AKR1B10,ALDH1A1,S100A4,METTL7 A,MCAM,GSTM1,GPX4,FBLN1,and CTSF were screened out by bioinformatics analysis such as protein interaction network analysis,KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway and GO(Gene Ontology)enrichment analysis,providing important clues for further mechanism research.3.Molecular mechanism of AKR1B10 in promoting lung cancer brain metastasisAKR1B10,which is one of the key differential proteins in proteomics database,was selected for multi-platform verification.It was found that the expression of AKR1B10 was significantly increased in BM group,whether in cell lines,biomimetic chip model or clinical serum samples.It can be used as a new biomarker for the diagnosis of BM from lung cancer.By combining with bionic lung cancer BM model and traditional in vivo and in vitro research methods for intervention studies,it was further confirmed that AKR1B10 promoted BM of lung cancer cells by regulating the expression of matrix metalloproteinase(MMP2,MMP9)via MAPK signal pathway which lead to the destruction of the blood-brain barrier structure.Conclusions1.This study successfully constructed a microfluidic biomimetic chip model of lung cancer BM,realizing the visualization and dynamic observation of complex pathological processes and simulating the microenvironment in vivo to the maximum degree.It provides a new technology platform for the follow-up study of the molecular mechanism of lung cancer BM.2.The lung cancer high brain metastasis cell line was successfully constructed,and key molecules that may play an important role in the pathological process of BM were screened using proteomics technology,providing an omics basis for subsequent molecular mechanism verification studies.3.By using the chip and traditional models synergistically,we first defined the diagnostic value of AKR1B10 and its role and mechanism in promoting lung cancer BM,which is of great significance for clinical diagnosis and treatment of lung cancer BM.4.The successful application of microfluidic bionic chip in the molecular mechanism research of brain metastasis shows the great potential of organoid chip to assist the basic research of oncology. |
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