the reaction was stopped with excess edta ( 4 μl 0. 5 m ) followed by 75°c for 10 min. the dna was further extracted using phenol – chloroform and precipitated with ethanol. glyco gen carrier ( 5 μg ) was used at this stage, and the pellet resuspended in 80 μl water. the blunt - ended dna was then ligated to the lp21 – 25 linker. a stock of linker was prepared by mixing 80 μl lp21 ( 100 μm: gaattcagatctcccgggtca ) with 80 μl lp25 ( 100 μm: gcggtgacccgggagatctg aattc ) and 240 μl water. the mix was then placed on a 95°c hot block and the power supply turned off. when the block had cooled to room temperature, the lp21 – 25 linker was aliquoted and frozen. the 80 μl dna sample was split into two samples for ligation ( 5 μl 10× buffer, 3 μl lp21 – 25 linker, 3 μl ligase ( ne biolabs 400 u / μl and 39 μl dna ). this was mixed well with a pipette and left at 16°c overnight. following ligation, the dna was precipitated with ethanol, and resuspended in 42 μl water. the sample was amplified using vent exo - polymerase and a biotinylated lp25 primer. the mix ( 5 μl thermopol 10× buffer, 0. 5 μl b - lp25 ( 200 μm ), 2 μl vent exo - ( ne biolabs 2 u / μl ), 1 μl dntp ( 25 mm ) and 41. 5 μl dna ) was amplified with the following thermal cycle: ( 95°c 3 min, 95°c 30 s, 61°c 30 s, 72°c 30 s ) × 35 cycles. following amplification, the biotinylated products were extracted using dynal streptavidin beads. for each sample, 30 μl of beads ( dyabeads m - 270; dynal biotech ) were washed twice in 2× binding buffer ( 10 mm tris hcl ph7. 5, 1 mm edta, 2 m nacl ) using a magnet, and then resuspended in 50 μl of 2× binding buffer / sample. each post amplification sample ( 50 μl ) was then mixed with 50 μl of resuspended beads and incubated at room temperature on a rotator for 1 h. after binding, the samples were washed twice with 2× binding buffer, then once with 1× te. after the last wash, each sample was resuspended in 30 μl 0. 1 te. the paired samples that were split before ligation were then pooled, making a 60 μl final aliquot. this was heated at 95°c for 10 min to free the dna from the beads, and then stored at 4°c. real - time pcr quantificationwe have used stratagene brilliant sybr green qpcr master mix as our standard kit for quantification of genomic controls and samples. we have used a range of primer sets. the hmbs primers are: human — forward; atgctgcctatttcaaggttgt, reverse; gaatt ggaacattgcgacagt, and mouse — forward; cggagtcatgtccggtaac, reverse; cgaccaa tagacgacgagaa. primers from the tal1 locus ( human and mouse ) are available on request. amplification conditions and pcr mixes were as recommended by stratagene. genomic standard curves were calculated for each primer using serial 5 - fold dilutions from 50 ng genomic dna / pcr reaction. for each sample pcr, a 45 μl stock was made using 1. 3 μl of sample dna + 43. 7 μl water. this stock was used as 5 μl per real - time pcr reaction, and following amplification, quantified relative to the genomic dna standard curve, for each primer set. the quantifications shown in figure 3 represent the absolute quantification of dna at each primer set for the 5 μl pcr sample. the mrna expression levels of tal1 and stil in figure 4a were normalised relative to β - actin and plotted relative to mrna expression from normal human donor cd34 + cells, as described previously ( 11 ). lung cancer remains a leading cause of cancer - related mortality worldwide, with bone metastases representing one of the most common and devastating complications in non - small cell lung cancer ( nsclc ). at the time of diag nosis, 20 – 30 % of nsclc patients already exhibit bone involvement, and during disease progression, 35 – 60 % will develop bone metastases [,, ]. these skeletal lesions frequently result in severe skeletal - related events ( sres ), including pain, pathological fractures, spinal cord compression, and hypercalcemia, all of which markedly diminish patients ' quality of life and worsen clinical outcomes. although bone - targeted therapies such as denosumab have shown benefits in reducing skeletal complications, their efficacy remains limited and is often accompanied by adverse events. more importantly, the molecular mechanisms that govern skeletal colonization in lung adenocarcinoma remain poorly understood. a major limiting factor is the lack of lung adenocarcinoma cell lines with intrinsic bone - tropic potential, which restricts both mechanistic insights and the development of clinically relevant preclinical models. disseminated tumor cells ( dtcs ) reaching bone tissue must undergo complex interactions with various bone microenvironment cells at different stages, including the perivascular niche, the osteogenic niche, and the formation of the vicious cycle. given the complexity of these interactions, selecting appropriate modeling strategies and suitable cell lines is a critical foundation for advancing research on bone metastasis. among current strategies, methods such as left ventricular injection, intra - tibial or femur injection, intra - iliac artery injection, and tail artery injection have provided powerful approaches for quantitatively investigating various stages of the bone metastasis cascade. however, these methods often suffer from high experimental variability — an issue particularly pronounced in lung cancer bone metastasis research due to the lack of cell lines with bone - tropic potential, leading to high rates of non - bone metastases and inconsistent tumor engraftment. these limitations underscore the continued need for lung cancer models that are not only stable and reproducible, but also faithfully recapitulate the biological features of bone metastasis. although a few bone - metastatic lung cancer cell lines have been developed, their number remains limited, and each exhibits specific shortcomings in stability, bone tropism, or biological relevance — issues that constrain their broader applicability. this study aims to address these challenges by establishing a more reliable model. in the present study, we established a highly bone - metastatic subline, a549 - bm5, derived from the lung adenocarcinoma a549 cell line through an in vivo selection strategy. we systematically characterized its biological behavior both in vitro and in vivo, demonstrating enhanced bone - homing ability and metastatic tropism. furthermore, integrative transcriptomic and proteomic analyses revealed key molecular alterations associated with skeletal colonization. this subline offers a robust and biologically relevant platform for dissecting the mechanisms of lung cancer bone metastasis and for evaluating targeted therapies in a preclinical setting. human lung adenocarcinoma cell line a549 was obtained from the american type culture collection ( atcc ). the a549 cells and their derived subline cells were cultured using ham ’ s f - 12 k medium ( basal media, l450kj ). all media were supplemented with 10 % fetal bovine serum ( fbs ) ( yeasen, 40130es76 ) and 1 % penicillin – streptomycin solution ( basal media, s110jv ). cells were incubated at 37 °c in a humidified atmosphere containing 5 % co2. female nod / scid or balb / c nude mice aged 6 to 8 weeks were housed in standard specific pathogen - free ( spf ) laboratory animal facilities. in the left ventricular injection experiments, 1 × 10 a549 - parental or a549 - bm5 cells were injected into the left ventricle under ultrasound guidance. in vivo bioluminescent imaging ( bli ) was used to monitor tumor burden. after 4 to 5 weeks, the mice were humanely euthanized, and bone tissues were collected for subsequent experiments. in the intra - iliac artery injection experiments, following previously reported methods, briefly, 0. 5 × 10 a549 - parental or a549 - bm5 cells were injected into the iliac artery of female balb / c nude mice aged 6 to 8 weeks. at the end of the eighth day, the mice were humanely euthanized, and bone tissues were collected for subsequent experiments.