micro - ct analysis of excised femora and tibiae was performed. raw projection images were reconstructed into cross - sectional slices using nrecon ( bruker ). quantitative bone morphometric parameters, including bone integrity, bone volume fraction ( bv / tv ), trabecular thickness ( tb. , and trabecular spacing ( tb. , were calculated using ctan software ( bruker ). three - dimensional reconstructions of bone microarchitecture and ost eol ytic lesions were generated with ctvox software ( bruker ). all analyses followed asbmr guidelines. for the cell viability assay, 1 × 10 cells per well were seeded into 96 - well plates ( corning, 3599 ) with three replicate wells per group and incubated overnight to allow for cell attachment. the following day, cell viability was assessed using the cck8 reagent kit ( yeasen, 40203es80 ) according to the manufacturer ’ s instructions. for the colony formation assay, 6 × 10 cells per well were seeded into 6 - well plates ( corning, 3516 ) and cultured for 10 to 14 days to enable colony development. surviving colonies were fixed with methanol for 30 min and subsequently stained with 0. 1 % crystal violet solution ( beyotime, c0121 ) for another 30 min. after thorough washing and air - drying, the number of colonies was counted. cells were collected from 6 - cm cell culture dishes, washed twice with phosphate - buffered saline ( pbs ), and then fixed in 70 % ethanol at − 20 °c overnight. following centrifugation and removal of the supernatant, the cells were washed once with pbs and stained with 500 µl of propidium io dide ( pi ) staining solution ( bd biosciences, 550825 ) for 10 min. the dna content was analyzed using a flow cytometer ( cytoflex s; beckman coulter ). migration and invasion assays were performed using 8 μm pore chambers ( corning, 353097 ). for the migration assay, 5 × 10 cells in 200 µl of serum - free culture medium were seeded into the upper chamber. for the invasion assay, the upper chamber membranes were pre - coated with matrigel ( corning, 356234 ), and 1 × 10 cells in 200 µl of serum - free culture medium were seeded into the upper chamber. in both assays, culture medium containing 10 % fetal bovine serum ( fbs ) was added to the lower chamber. after incubation for 16 – 20 h, the cells that had migrated or invaded through the membrane were fixed with methanol for 30 min and stained with 0. 1 % crystal violet for 15 – 20 min. nine random fields were photographed under a microscope ( olympus, ix73 ) at a magnification of 100×. cells were washed with prechilled pbs triplic ately, then lysed in buffer ( thermo, 78510 ) supplemented with protease inhibitors ( yeasen, 20123es50 ) and phosphatase inhibitors ( yeasen, 20109es20 ). cells were scraped and thoroughly lysed on ice for 30 min. lysates were centrifuged at 12, 000 rpm for 15 – 20 min, and the supernatants were collected. protein concentrations were measured using a bca assay kit ( thermo fisher scientific, 23227 ), and samples were normalized to equivalent concentrations. following the addition of 5× protein loading buffer ( yeasen, cat. no. 20315es20 ), samples were denatured at 100 °c for 10 min and stored at −80 °c. proteins were then subjected to sodium dodecyl sulfate – polyacr ylamide gel electrophoresis ( sds – page ) and transferred onto polyvinylidene fluoride ( pvdf ) membranes ( bio - rad, 1620177 ). membranes were blocked at room temperature for 2 h prior to incubation with primary antibod ies. the primary antibod ies used in this study were as follows: anti-n-cadherin ( 13116, cst ), anti-e-cadherin ( 3195, cst ), anti - zo - 1 ( 8193, cst ), anti - zeb1 ( 3396, cst ), anti-vimentin ( 5741, cst ), anti - claudin - 1 ( 13255, cst ), anti - β - catenin ( 8480, cst ), anti - mmp2 ( rm8377, biodragon ), anti - mmp3 ( rm8188, biodragon ), anti - mmp9 ( rm3763, biodragon ), anti - mmp13 ( rm8226, biodragon ), anti - ccl3 ( rm2987, biodragon ), anti - bmp2 ( bd - pt5651, biodragon ), anti - bmp3 ( bd - pt0499, biodragon ), anti - bmp6 ( bd - pt5655, biodragon ), anti - bmp7 ( bd - pt0503, biodragon ), anti - opg ( ab183910, abcam ), anti - rankl ( bd - pt5404, biodragon ), anti - opn ( rm4342, biodragon ), anti - col1a1 ( rm0325, biodragon ), anti - icam - 1 ( bd - pt2269, biodragon ), anti - rank ( bd - pt5881, biodragon ), anti - sp7 ( bd - pn0332, biodragon ), anti - beta - actin ( hrp - 66009, proteintech ) and anti - vinculin ( 26520 - 1 - ap, proteintech ). bands were visualized by enhanced chemiluminescence ( share - bio, sb - wb011 ), and densitometry measurements of the bands were acquired with quantity one software ( bio - rad ). bone marrow mesenchymal stromal cells ( bmscs ) and bone marrow – derived monocytes / macrophages ( bmms ) were isolated from 6 to 8 - week - old c57bl / 6j mice. under sterile conditions, femurs and tibias were dissected, and the ends of the bones were cut. bone marrow cells were collected by centrifugation at 10, 000×g for 5 s, and the pellet was subjected to red blood cell lysis for 5 min on ice. cells were then centrifuged at 450 × g for 3 min, washed once with pbs, and resuspended in α - mem complete medium. the suspension was plated in 10 - cm dishes, and after 24 h, adherent cells were predominantly bmscs, while non - adherent cells in the supernatant contained mainly monocytes. bone microenvironment cell recruitment assays were performed using 8 μm pore transwell chambers ( corning, 353097 ). a549 - parental and a549 - bm5 cells were seeded at appropriate densities in 24 - well plates. bone microenvironment cells ( 5 × 10 cells in 200 µl of serum - free culture medium ) were seeded into the upper chamber. after incubation for 16 – 20 h, the cells that migrated through the membrane were fixed with methanol for 30 min and stained with 0. 1 % crystal violet for 15 – 20 min. nine random fields per chamber were photographed under a microscope ( olympus, ix73 ) at 100× magnification. statistical analysis was performed using graphpad prism 9. under sterile and light - protected conditions, osteogenic induction medium was prepared by mixing 45 ml α - mem medium, 5 ml fetal bovine serum, 500 µl penicillin – streptomycin solution, 50 µl of 50 mg / ml vitamin c solution, 5 µl of 1 mm dexamethasone solution, and 500 µl of 1 m β - glycerophosphate solution. the medium was stored at 4 °c for future use. bone marrow mesenchymal stromal cells ( bmscs ) or mc3t3 - e1 subclone 14 cells were cultured to sufficient quantities and digested with trypsin. cells were seeded at a density of 5 × 104 cells per well in 24 - well plates. after cell adhesion, the medium was replaced with osteogenic induction medium, with medium changes every other day under light - protected conditions. according to the experimental design, conditioned medium ( cm ) derived from a549 - parental or a549 - bm5 cells was mixed at a 1: 1 ratio with osteogenic induction medium ( final concentration 50 % cm ) during the culture period. after 14 days of induction, alkaline phosphatase ( alp ) staining was performed. osteoclast differentiation induction medium was prepared by adding cytokines rankl ( 50 ng / ml ) and m−csf ( 10 ng / ml ) to α - mem complete medium. bone marrow macrophages ( bmms ) were cultured to sufficient quantities. adherent bmms were digested with versene dissociation solution and seeded into 96 - well plates at a density of 1 × 10 cells per well. after cell adhesion, the medium was replaced with osteoclast differentiation induction medium to induce differentiation, with medium changes every other day. according to the experimental design, conditioned medium ( cm ) derived from a549 - parental or a549 - bm5 cells was mixed at a 1: 1 ratio with osteoclast differentiation medium ( final concentration 50 % cm ) during the culture period. mc3t3 - e1 subclone 14 cells were seeded at an appropriate density in 6 - well plates.