| Background and purposeLung cancer is one of the high-incidence cancers in the world,and the tumor cells are markedly"encephalophilic"。The brain microenvironment is a relatively independent functional area,and the neurovascular units（NVU）composed of brain microvascular endothelial cells and nerve cells,microglia,and astrocytes maintain the steady state of the brain microenvironment.Interactions between tumors and the brain microenvironment contribute to the outcome of tumor colonization and survival in the brain.With the development of bioinformatics,researchers found that metastatic tumor cells are genetically reprogrammed to acquire cell characteristics adapted to growth in the new environment.Although primary tumors and brain metastases share a common ancestor,distinct evolutionary patterns have occurred at metastatic sites,demonstrating the plasticity of tumors.More recently,cancer hallmarks have included unlocking phenotypic plasticity.An in-depth understanding of the tumor plasticity exhibited by brain metastatic lung cancer cells under the influence of the brain microenvironment is critical for improving disease understanding and treatment.Several studies have demonstrated that brain metastases tumor cells,including lung cancer,breast cancer,and melanoma,undergo reprogramming under the induction of the brain microenvironment,and there are activation of axon guidance and synaptogenesis genes,enhanced neuronal properties,and developmental processes.Modeling Neurodevelopment.The field of tumor innervation has received increasing attention.It is dangerous for cancer cells to form synaptic connections with neurons.Studies have shown that cancer cells and neurons form excitatory synaptic connections between cells that promote tumor growth.The connection between cancer cells and neurons can be established through the release of neurotransmitters,which affects tumor colonization,cell infiltration,migration and proliferation in the brain.The establishment of tumor research models is very important to study the mechanism of tumor occurrence and development.Tumor formation has a complex microenvironment with abnormal cells and three-dimensional structures.To this end,this study will use animal experiments,tissue culture and cell levels to understand the growth of lung cancer after brain metastases.Through experiments,we found that lung adenocarcinoma cells acquired neuronal cell-like surfaces under the influence of the brain microenvironment.type.The GEO database was used to analyze the differentially expressed genes that were up-regulated in lung cancer cells after brain metastases compared with parental cells,and combined with the bioinformatic data obtained from the previous research of our group.The HOXB9 gene,a member of the HOX family of developmentally conserved genes that is more expressed in brain metastases than in situ lung cancers,was found.The HOX family,known as homeobox genes,encodes highly conserved homeobox transcription factors and is a regulator of cell differentiation and proliferation.During embryonic development,HOX genes play key roles in the axial configuration of a variety of animals and in regulating the regional properties of cells and tissues.In vertebrate-related studies,HOX genes are important in developing neural circuits in specific regions and in diversifying motor neurons.The functions of HOX genes during neuronal development include neuronal subtype determination,neuronal migration,axon guidance,axon branch formation,and neuronal circuit connections.It is also necessary for neuronal circuit formation,including synapse formation,maturation,and neural circuit adjustment.In addition,HOX genes also affect the survival and function of many types of nerve cells.HOXB9 is highly conserved.HOXB9 also plays an important role in a variety of human tumors,and its abnormal expression affects tumor formation.Studies have shown that high levels of HOXB9 are associated with poor prognosis in lung adenocarcinoma patients,low overall survival in colon cancer patients,high tumor grade and low overall survival in breast cancer patients,and advanced clinical staging in glioma patients.Studies have reported the role of HOXB9 in tumors,including promoting tumor cell EMT,affecting cell proliferation and cycle,or promoting angiogenesis.This study will explore how HOXB9,a conserved gene once involved in individual neurodevelopment,drives lung cancer cells to acquire the ability to survive in the brain.Explaining the transformation of malignant tumor phenotype from a developmental perspective.It provides new ideas for exploring the molecular mechanism of tumor cell plasticity,and provides potential risk predictors and intervention targets for clinical prevention and treatment of lung cancer brain metastases.Experimental methods1.To investigate the phenotype changes of lung cancer cells after brain metastasis1.1 Inject mouse lung cancer LLC cells into the brain of C57BL/6 mice to establish iterative brain metastases lung cancer cell lines LLC-Br M1,LLC-Br M2,LLC-Br M3.The cell viability of brain metastatic lung cancer cells was detected by MTS,and the cell cycle was detected by flow cytometry.The apoptosis of brain metastatic lung cancer cells was induced by H2O2,and the cell death resistance was evaluated by flow cytometry detection of apoptosis rate.Transwell was used to evaluate the invasion and migration ability of brain metastatic lung cancer cells.The LLC-Br M3 cells were cultured with Matrigel to form cell spheres with three-dimensional structure.The morphology of the spheroid was observed,and the viability of the spheroid was assessed by detecting the content of cellular ATP.The growth of LLC-Br M3 cells was observed after culturing LLC-Br M3 cells in spherical microplates.1.2 To establish a co-culture model of mouse lung cancer cell spheroids and C57BL/6mouse brain tissue slices,and to observe the culture of lung cancer cell spheroids on brain tissue slices with a fluorescent stereo microscope.The spheroids were immunofluorescently labeled with neural markers PSD95 and MAP2,and the cells were observed by laser confocal microscopy.1.3 A co-culture model of A549 cell spheroids and nude mouse brain tissue slices was established.Immunofluorescence was used to label CD31,and the culture of lung cancer cell spheroids was observed by laser confocal microscope.Immunofluorescence was used to label the neural markers PSD95 and MAP2,and the cell morphology and the labeling of neural markers were observed.1.4 Western Blot was used to detect the expression levels of PSD95 and MAP2 in brain metastatic lung cancer cells and lung cancer cells co-cultured with mouse brain tissue slices.To evaluate the expression of neural markers in lung cancer cells under the influence of brain microenvironment.1.5 Use bioinformatics to analyze the transcriptome sequencing data of brain metastases lung adenocarcinoma PC9 cells and parental cells from the Gene Expression Omnibus（GEO）database.2.To study the mechanism of neuronal cell-like changes in lung cancer cells under the influence of the brain microenvironment2.1 Combine the bioinformatics data obtained in the previous research of our group to screen for possible effects on lung cancer Genes by which cells acquire a neuronal-like phenotype.Detect the expression level of HOXB9 in brain metastatic lung cancer cells and lung cancer cells co-cultured with mouse brain tissue slices by Real-time PCR and Western Blot.2.2 LLC-Br M3 cells with knockdown of HOXB9 or LLC cells with high expression of HOXB9 were cultured with Matrigel to form cell spheres with three-dimensional structures.Observe cell spheroid morphology and evaluate the effect of HOXB9 on cell spheroid formation.2.3 The LLC cells with high expression of HOXB9 were stained with crystal violet,and the cell morphology was observed by microscope.After labeling the neural marker MAP2,the cell morphology and neural marker labeling were observed by laser confocal microscope.LLC cells with high expression of HOXB9 were co-cultured with mouse brain tissue sections,and the neural marker MAP2 was labeled.Laser confocal microscopy was used to observe cell morphological changes.2.4 Real-time PCR and Western Blot were used to detect the expression level of MAP2 in LLC cells with high expression of HOXB9 and LLC-Br M3 cells with knockdown of HOXB9,and to evaluate the correlation between HOXB9 and MAP2 expression.2.5 The A549 cells with high expression of HOXB9 were labeled with MAP2 or PSD95,and the cell morphology and the expression of neural markers were observed by laser confocal microscopy.Nude mouse brain tissue sections were co-cultured with A549 cells highly expressing HOXB9,and the morphological changes were observed by fluorescence microscopy.The co-cultured cells were labeled with neural markers MAP2 or PSD95,and the cell morphology and the expression of neural markers were observed by laser confocal microscopy.2.6 RNA-seq sequencing was used to obtain transcriptome data related to HOXB9.GO and KEGG enrichment analysis of up-regulated differentially expressed genes was performed.The up-regulated differentially expressed genes associated with axons were subjected to GO enrichment analysis.2.7 The HOXB9 immunoprecipitation-enriched sequences in A549 cells with high expression of HOXB9 were obtained by Ch IP technology and sequenced to analyze the distribution of HOXB9 Ch IP peaks in the functional regions of the genome and chromosomes.To analyze the Motif bound by HOXB9 and obtain regulated downstream genes.The combined analysis of Ch IP-seq and RNA-seq sequencing data screened HOXB9 as a transcription factor-regulated downstream target gene related to protrusion formation.2.8 Real-time PCR and Western Blot were used to detect the expression level of Slit2 in LLC-Br M3 and lung cancer cells with high expression of HOXB9.Real-time PCR was used to detect the expression of Robo family genes in lung cancer cells with high expression of HOXB9.2.9 A549 cells overexpressing HOXB9 were mediated by si RNA to down-regulate Slit2and co-cultured with nude mouse brain tissue sections.The morphological changes of cells and the expression of neural markers PSD95 and MAP2 were observed by confocal fluorescence microscopy,and the protein expression levels of PSD95 and MAP2 in cells were detected by Western Blot.2.10 In situ injection of LLC and LLC cells with high expression of HOXB9 into the brain of C57 mice,the tumor formation in the mouse brain was observed by fluorescence stereo microscope,and the tumor formation area was determined by HE staining after embedding in paraffin.A co-culture system of mouse brain tissue slices and lung cancer cells was established using Transwell cell chambers.LLC cells overexpressing HOXB9,A549 cells overexpressing HOXB9,and A549 cells overexpressing HOXB9 with Slit2 down-regulated were co-cultured with brain tissue sections,and cell viability was detected by CCK8.H2O2 was used to induce apoptosis,and the apoptosis rate of LLC-Br M3 cells with knockdown of HOXB9 was detected.MTS was used to detect the cell viability of LLC-Br M3 cells with knockdown of HOXB9,and the cell cycle was detected by flow cytometry.2.11 To analyze the expression of HOXB9 in clinical lung cancer samples.Immunohistochemistry was used to detect the expression level of HOXB9 in lung adenocarcinoma tissue chips,the GEO database was used to analyze the expression level of different tumors in situ carcinoma and brain metastases,and the expression level of HOXB9 was analyzed using the sample data of lung adenocarcinoma patients in the TCGA database.The correlation between HOXB9 and patient survival and clinicopathological stage was analyzed.Experimental results1.Lung cancer cells in the brain microenvironment have the potential to acquire a neural-like phenotype1.1 The malignant phenotype of brain metastatic lung cancer cells was more pronounced after survival in the brain.The cell viability was enhanced and the G2/M phase of the cell cycle increased.As the accumulation of survival time in the brain shows the performance of adapting to the brain environment,the cell viability gradually increases.In addition,the ability of cell invasion and migration was also significantly improved.The three-dimensionally cultured mouse brain metastatic lung cancer cell spheres formed obvious protrusion structures.The cell viability of the spheroids was enhanced.Distant spread of mouse brain metastatic lung cancer cell spheres formed in spherical microplates.1.2 Brain metastases lung cancer cell spheroids were co-cultured with mouse brain tissue slices,and the cells extending outward from the spheroids had obvious morphological changes,which could mark neural markers.1.3 After co-culture of human lung adenocarcinoma A549 cell spheroids with nude mouse brain tissue slices,the spheroids formed protrusions.The cells exhibited neuron-like morphology and were labeled with neural markers PSD95 and MAP2.PSD95 accumulates at the ends of cell protrusions.1.4 The expression of neural markers PSD95 and MAP2 increased in lung cancer cells under the influence of brain microenvironment.1.5 Bioinformatics analysis proved that the gene expression profile of brain metastases lung cancer cells was changed,and the characteristics of nerve cells were enhanced.2.HOXB9 regulates Slit2 to induce the transformation of brain metastatic lung cancer cells to neural-like cells and affects their malignant phenotype2.1 The differentially expressed gene HOXB9 was screened to be up-regulated in brain metastases lung cancer compared with in situ carcinoma.The expression level of HOXB9in brain metastatic lung cancer cells was higher than that in orthotopic lung adenocarcinoma cells.2.2 The expression of HOXB9 affected the formation of protrusions in Matrigel-3D-cultured lung adenocarcinoma cell spheroids and brain metastatic lung cancer cell spheroids.Brain metastases lung cancer cell spheroids with knockdown of HOXB9 were inhibited from growing,and high expression of HOXB9 promoted lung adenocarcinoma cell spheroids to form protruding structures.2.3 The high expression of HOXB9 promotes LLC cells to undergo morphological changes,form protruding structures,and transform into neuron-like morphologies,and the protruding structures can be labeled with MAP2 by immunofluorescence.After the LLC cell spheroids expressing high HOXB9 were co-cultured with brain tissue slices,the cells spread out from the spheroids.And under the high magnification microscope,the cell morphology was obviously changed,and it turned into a neuron-like morphology.2.4 Western Blot and Real-time PCR demonstrated that high expression of HOXB9increased the expression of MAP2 in lung adenocarcinoma cells,while knockdown of HOXB9 decreased the expression of MAP2.2.5 The morphology of a few cells in A549 cells with high expression of HOXB9 was changed.The formed protrusions could be marked by MAP2 and PSD95,but the morphology of most cells had no obvious change.HOXB9 promotes human lung adenocarcinoma A549 cells to form neurite-like structures in the brain environment.After co-culture with nude mouse brain tissue slices,A549 cells with high expression of HOXB9showed obvious neuron-like morphological changes.Compared with the cells in the control group after co-culture,the number of cells with altered morphology was more and the protrusions were longer and branched,and the protrusions could mark the neural markers PSD95 and MAP2.2.6 Use RNA-seq technology to obtain transcriptome data related to HOXB9,and perform GO and KEGG enrichment analysis on up-regulated differentially expressed genes.KEGG was found to be enriched in the axon guidance signaling pathway,and multiple functional subsets related to nerves existed in the GO-enriched subset.GO enrichment analysis of up-regulated differentially expressed genes related to axons revealed that HOXB9 could affect axonogenesis,axon guidance,axon elongation and axon transport.2.7 A gene profile enriched for HOXB9 as a transcription factor was obtained using Ch IP-seq sequencing data analysis.The distribution of HOXB9 Ch IP Peak in the functional regions of the genome showed that the promoter region bound by HOXB9 as a transcription factor was 7.22%.The analysis obtained the distribution of peaks on chromosomes and predicted HOXB9 as a transcription factor binding motif.Combined analysis of Ch IP-seq and RNA-seq sequencing data,and GO enrichment analysis of genes up-regulated by HOXB9 as a transcription factor,it was found that there are multiple gene sets regulating nerves and axons.HOXB9 was found to regulate Slit2/Robo as a transcription factor in genes related to axon formation.2.8 High expression of HOXB9 can regulate the increase of Slit2 expression in lung cancer cells,and the expression level of Slit2 receptor Robo family genes is also significantly increased.2.9 A549 cells overexpressing HOXB9 down-regulated Slit2 by si RNA and co-cultured with nude mouse brain tissue sections.Laser confocal fluorescence microscopy showed that the cells could not form protrusion-like structures,PSD95 protein accumulated in the cells,and the expression of MAP2 in the cells was significantly reduced.Western Blot detection also verified that the protein expression levels of PSD95 and MAP2 were significantly reduced.2.10 HOXB9 affects the intracerebral viability of lung cancer cells.In situ injection into the brain of C57 mice,it was proved that LLC cells with high expression of HOXB9 had more tumor formation than control cells.CCK8 detection showed that the cell survival rate of LLC cells and A549 cells overexpressing HOXB9 in the brain microenvironment was significantly improved,and the cell survival rate of A549 cells overexpressing HOXB9was significantly reduced in the brain microenvironment after down-regulation of Slit2 by si RNA.In addition,mouse brain metastatic lung cancer cells had reduced cell viability after knockdown of HOXB9.2.11 To analyze the expression level of HOXB9 in samples from clinical lung adenocarcinoma patients.It was found that the expression level of HOXB9 in lung adenocarcinoma tissues was higher than that in adjacent tissues,and the expression level of HOXB9 in brain metastatic lung adenocarcinoma tissues was slightly higher than that in non-metastatic lung adenocarcinoma tissues,and there was no significant difference between negative and positive lymph node metastases in lung adenocarcinoma samples.The expression levels of brain metastases in lung adenocarcinoma or breast cancer were higher than in carcinoma in situ.Lung adenocarcinoma patients with high expression of HOXB9 had a shorter survival time,but the expression level of HOXB9 had no significant correlation with the clinical stage of lung adenocarcinoma.Conclusions1.Compared with in situ lung cancer cells,lung cancer cells with brain metastases have a more significant malignant phenotype,and the cell morphology undergoes neuron-like changes,and the expression levels of neural markers are increased,resulting in a neuron-like differentiation phenotype.2.Lung cancer cells with high expression of HOXB9 are more suitable for survival in the brain.The expression of HOXB9 promotes the neuronal cell-like transformation of lung cancer cells and helps lung cancer cells better adapt to the brain environment.3.HOXB9 mediates the neuronal cell-like phenotype of lung cancer cells to promote their brain survival by regulating the axon-forming protein Slit2/Robo pathway. |
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