after the cells adhered, 200, 000 cells in 1000 μl suspension were added to each well and incubated at 37 °c for 30 min. the supernatant was discarded, and the wells were washed with phosphate - buffered saline ( pbs ). cell images were captured using a fluorescence microscope at 100 × magnification. five random fields were selected per well. the number of cells was counted, and the average cell number for each sample was calculated. all experiments were performed in triplic ate. total rna was extracted using the trizol reagent ( invitrogen, ca, usa ) according to the manufacturer ’ s protocol. rna purity and quantification were evaluated using the nanodrop 2000 spectrophotometer ( thermo scientific, usa ). rna integrity was assessed using the agilent 2100 bioanalyzer ( agilent technologies, santa clara, ca, usa ). then the libraries were constructed using vahts universal v6 rna - seq library prep kit according to the manufacturer ’ s instructions. the transcriptome sequencing and analysis were conducted by oe biotech co., ltd. ( shanghai, china ). the libraries were sequenced on an llumina novaseq 6000 platform and 150 bp paired - end reads were generated. about 50 m raw reads for each sample were generated. raw reads of fastq format were firstly processed using fastp and the low quality reads were removed to obtain the clean reads. then about 48 m clean reads for each sample were retained for subsequent analyses. the clean reads were mapped to the reference genome using hisat2. fpkm of each gene was calculated and the read counts of each gene were obtained by htseq - count. pca analysis were performed using r ( v 3. 2. 0 ) to evaluate the biological duplication of samples. differential expression analysis was performed using the deseq2. q value < 0. 05 and foldchange > 2 or foldchange < 0. 5 was set as the threshold for significantly differential expression gene ( degs ). hierarchical cluster analysis of degs was performed using r ( v 3. 2. 0 ) to demonstrate the expression pattern of genes in different groups and samples. the radar map of top 30 genes was drew to show the expression of up - regulated or down-regulate d degs using r packet ggradar. based on the hypergeometric distribution, go, kegg pathway, reactome and wikipathways enrichment analysis of degs were performed to screen the significant enriched term using r ( v 3. 2. 0 ), respectively. r ( v 3. 2. 0 ) was used to draw the column diag ram, the chord diag ram and bubble diag ram of the significant enrichment term. gene set enrichment analysis ( gsea ) was performed using gsea software. the analysis was used a predefined gene set, and the genes were ranked according to the degree of differential expression in the two types of samples. then it is tested whether the predefined gene set was enriched at the top or bottom of the ranking list. nanoflow reversed-phase chromatography was performed on an easy - nlc 1200 system ( thermo fisher scientific ). peptides were separated in 90 min at a flow rate of 300 nl / min on a 25 cm × 75 μm column ( 1. 6 μm c18, ionopticks ). mobile phases a and b were 0. 1 vol % formic acid solution and 80: 20: 0. 1 vol % acn: water: formic acid, respectively. the total run was 90 min ( 0 ∼ 66 min, 3 – 27 % b ； 66 ∼ 73 min, 27 – 46 % b ； 73 ∼ 84 min, 46 – 100 % b ； 84 ∼ 90 min, 100 % b ) or 60 min ( 0 ∼ 45 min, 5 – 27 % b ； 45 ∼ 50 min, 27 – 46 % b ； 50 ∼ 55 min, 46 – 100 % b ； 55 ∼ 60 min, 100 % b ). liquid chromatography was coupled online to a hybrid tims quadrupole tof mass spectrometer ( bruker timstof pro ) via a captivespray nano - electrospray ion source. capillary voltage was 1. 5 kv, dry gas temperature was 180 °c, and dry gas flow rate was 3. 0 l / min. the dual tims analyzer was operated at a fixed duty cycle close to 100 % using equal accumulation and ramp times of 100 ms. we performed dda in pasef mode with 10 pasef scans per topn acquisition cycle. the full ms scan range was set from 100 to 1700 m / z. the ion mobility range was 0. 75 – 1. 4 vs / cm2, and the collision energy range was 20 – 59 ev. the lc - ms / ms raw data were imported in maxquant ( version1. 6. 17. 0 ) for labeling free quantification analysis and the search engine was andromeda. the database was offered by researchers. all experiments were performed at least in triplic ate unless otherwise specified. data are represented as mean ± standard deviation ( sd ). p values were calculated using the unpaired student ’ s t test or mann – whitney u test, depending on the normality and homoscedasticity of the data. statistical significance was defined as follows: n. s., p > 0. 05; ∗, p < 0. 05; ∗∗, p < 0. 01; ∗∗∗, p < 0. 001; ∗∗∗∗, p < 0. 0001. graphs were generated using graphpad prism 9, with bar charts displaying individual data points together with mean ± sd. to establish a highly bone - metastatic subline derived from the lung adenocarcinoma a549 cell line, we utilized an in vivo selection strategy based on left ventricle injection ( fig. 1a ). a549 cells labeled with gfp and luciferase were injected into the left ventricles of nod / scid mice to establish a lung cancer bone metastasis mouse model. in - vivo bioluminescent imaging ( bli ) was immediately used to confirm successful injections, and bli was then continuously utilized to monitor the progression of bone metastatic lesions. approximately one month after injection, detectable bone metastatic lesions had emerged. at this point, the mice were humanely euthanized, and the bone metastatic tissues were collected, minced, and subjected to primary cell culture for subsequent in - vivo passaging. this sequence of left ventricle injection, primary culture, and reinjection was repeated for five consecutive cycles. during these cycles, both the incidence and the onset time of bone metastases significantly increased, thus validating the efficacy of this selection strategy. the cells obtained after five rounds of in - vivo selection were designated as a549 - bm5. fluorescence microscopy verified that all cells expressed gfp, demonstrating the successful derivation of a highly purified population of a549 - derived cells from the xenografts ( fig. 1b ). bright - field microscopy showed that, in contrast to the typical adherent epithelial - like morphology of parental a549 cells, a549 - bm5 cells adopted a more elongated, spindle - shaped appearance ( fig. 1b ). fig. 1the in vivo selection of a highly bone - metastatic a549 subline. ( schematic diag ram illustrating the establishment and characterization of the a549 - bm5 subline. ( morphological characteristics of a549 - parental and a549 - bm5 cells observed under bright - field microscopy and fluorescence microscopy ( gfp channel ) at 100× and 200× magnifications. the in vivo selection of a highly bone - metastatic a549 subline. ( schematic diag ram illustrating the establishment and characterization of the a549 - bm5 subline. ( morphological characteristics of a549 - parental and a549 - bm5 cells observed under bright - field microscopy and fluorescence microscopy ( gfp channel ) at 100× and 200× magnifications. to comprehensively clarify the in vitro characteristics of the a549 - bm5 cell line, we conducted a detailed comparison between it and its parental cells with respect to proliferation, migration, and epithelial - mesenchymal status. cell counting kit - 8 ( cck - 8 ) assays indicated that the a549 - bm5 cell line did not show a proliferative advantage over the parental a549 cells in vitro ( fig. 2a ). additionally, the colony formation ability of the a549 - bm5 cells was comparable to that of the parental cells ( fig. 2b ). cell cycle analysis revealed a reduction in the proportion of cells in the g0 / g1 phase and an elevation in the s-phase population, while the proportion of cells in the g2 / m phase remained largely unchanged when compared to the parental cells ( fig. 2c ).