conditioned media ( cm ) from a549 - parental and a549 - bm5 cells were used to treat mc3t3 e1 sub14 cells for 14 days, and the expression levels of ccl3, bmp6, col1a1 and icam - 1 were detected using western blot analysis. ( rt - qpcr analysis of osteoblast differentiation markers ( alpl, runx2, and bglap ) in mc3t3 - e1 subclone 14 cells cultured for 14 days with conditioned medium ( cm ) from a549 - parental or a549 - bm5 cells. gene expression was normalized to β - actin and calculated using the 2 method ( n = 6 ). ( adhesion assay of gfp - labeled a549 - parental and a549 - bm5 cells to mc3t3 - e1 subclone 14 cells. quantification showed that a549 - bm5 cells exhibited significantly greater adhesion than parental cells ( n = 9 ). ( gsea analysis shows that the go: 0007155 gene set ( heterophilic cell – cell adhesion via plasma membrane cell adhesion molecules ) is enriched in a549 - bm5 cells compared with the a549 - parental group. the heatmap below displays the expression of leading - edge genes in the corresponding samples. data are represented as mean ± 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 ). p > 0. 05, ∗, p < 0. 05; ∗∗, p < 0. 01, ∗∗∗, p < 0. 001, ∗∗∗∗, p < 0. 0001. to investigate osteoblast - related interactions, we employed mc3t3 - e1 subclone 14 cells and primary bmscs. transwell assays showed that a549 - bm5 cells significantly enhanced recruitment of osteoblast progenitors ( fig. 3d ). under osteogenic induction with cm for 14 days, osteoblast differentiation was markedly increased in the a549 - bm5 group, as demonstrated by alp staining ( fig. 3e ). we next evaluated the expression of potential mediators in mc3t3 - e1 subclone 14 cells treated with conditioned medium ( cm ) for 14 days by western blot. a549 - bm5 cm induced a clear increase in ccl3, with bmp6, col1a1 and icam - 1 showing a tendency to be higher ( fig. 3f ). at the transcriptional level, rt - qpcr analysis of mc3t3 - e1 subclone 14 cells cultured for 14 days under cm treatment further confirmed upregulation of osteoblast differentiation markers alpl, runx2, and bglap in the a549 - bm5 group ( fig. 3g ). finally, adhesion assays showed that a549 - bm5 cells displayed superior attachment to mc3t3 - e1 cells compared with parental a549 ( fig. 3h ). consistently, gsea revealed enrichment of the go: 0007155 gene set ( heterophilic cell – cell adhesion via plasma membrane cell adhesion molecules ) in a549 - bm5 cells compared with the a549 - parental group, and the accompanying heatmap shows the expression of the leading - edge genes, including vcam1, icam1, nectin4, and crtam, in the corresponding samples ( fig. 3i ). to investigate the bone metastatic potential of a549 - bm5 cells in vivo, we established mouse models using both left ventricular and intra - iliac artery injections. following left ventricular injection, the progression of skeletal metastasis was dynamically monitored by bioluminescent imaging ( bli ). all mice injected with a549 - bm5 cells developed substantial bone metastatic lesions within 14 days, whereas parental a549 cells showed only limited colonization. at the endpoint, a549 - bm5 – bearing mice exhibited a significantly elevated bone metastatic burden, which was further confirmed by gross anatomy, x - ray imaging, micro - ct reconstruction, and histological examination ( fig. 4a – c ). quantitative micro - ct analysis revealed pronounced ost eol ytic bone destruction in a549 - bm5 mice, including reduced bone integrity, decreased bone volume fraction ( bv / tv ), trabecular thickness ( tb. , and trabecular number ( tb. , along with increased trabecular spacing ( tb. 4the in vivo metastatic capacity of a549 - bm5 ( a ) an intra - cardiac injection was used to establish bone metastasis cdx models of a549 - parental and a549 - bm5 cells. in vivo bioluminescent imaging ( bli ) was employed to detect the bone metastatic burden ( n = 6 ). ( b ) representative gross anatomy, x - ray imaging and h & e staining of bone lesions from a549 - parental and a549 - bm5 groups. extensive ost eol ytic destruction was observed in a549 - bm5 – bearing mice. ( representative three - dimensional micro - ct images from a549 - parental and a549 - bm5 groups. ( quantitative analysis of bone integrity, showing significantly reduced integrity in the a549 - bm5 group compared with a549 - parental ( n = 6 ). ( quantitative analysis of bone volume fraction ( bv / tv ), revealing markedly decreased bone mass in the a549 - bm5 group ( n = 6 ). ( quantitative analysis of trabecular thickness ( tb. , showing thinner trabeculae in a549 - bm5 mice ( n = 6 ). ( quantitative analysis of trabecular number ( tb. , indicating fewer trabeculae in the a549 - bm5 group ( n = 6 ). ( quantitative analysis of trabecular spacing ( tb. , showing significantly increased spacing in the a549 - bm5 group ( n = 6 ). ( intra - iliac artery injection model of hindlimb bone metastasis. representative longitudinal bli monitoring shows significantly higher tumor burden in the a549 - bm5 group compared to a549 - parental. quantification of bone metastasis burden over time ( left panel ) and ex vivo bli of hindlimb bone tissues at day 8 ( right panel ) are shown ( n = 10 ). data are represented as mean ± 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 ). p > 0. 05, ∗, p < 0. 05; ∗∗, p < 0. 01, ∗∗∗, p < 0. 001, ∗∗∗∗, p < 0. 0001. the in vivo metastatic capacity of a549 - bm5 ( a ) an intra - cardiac injection was used to establish bone metastasis cdx models of a549 - parental and a549 - bm5 cells. in vivo bioluminescent imaging ( bli ) was employed to detect the bone metastatic burden ( n = 6 ). ( b ) representative gross anatomy, x - ray imaging and h & e staining of bone lesions from a549 - parental and a549 - bm5 groups. extensive ost eol ytic destruction was observed in a549 - bm5 – bearing mice. ( representative three - dimensional micro - ct images from a549 - parental and a549 - bm5 groups. ( quantitative analysis of bone integrity, showing significantly reduced integrity in the a549 - bm5 group compared with a549 - parental ( n = 6 ). ( quantitative analysis of bone volume fraction ( bv / tv ), revealing markedly decreased bone mass in the a549 - bm5 group ( n = 6 ). ( quantitative analysis of trabecular thickness ( tb. , showing thinner trabeculae in a549 - bm5 mice ( n = 6 ). ( quantitative analysis of trabecular number ( tb. , indicating fewer trabeculae in the a549 - bm5 group ( n = 6 ). ( quantitative analysis of trabecular spacing ( tb. , showing significantly increased spacing in the a549 - bm5 group ( n = 6 ). ( intra - iliac artery injection model of hindlimb bone metastasis. representative longitudinal bli monitoring shows significantly higher tumor burden in the a549 - bm5 group compared to a549 - parental. quantification of bone metastasis burden over time ( left panel ) and ex vivo bli of hindlimb bone tissues at day 8 ( right panel ) are shown ( n = 10 ). data are represented as mean ± 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 ). p > 0. 05, ∗, p < 0. 05; ∗∗, p < 0. 01, ∗∗∗, p < 0. 001, ∗∗∗∗, p < 0. 0001. to further assess whether a549 - bm5 cells could effectively mimic early biological events of bone colonization, we established a unilateral hindlimb metastasis model via intra - iliac artery injection. notably, differences in metastatic burden became apparent as early as day 5 – 7, with bli and ex vivo analyses showing markedly higher tumor load in a549 - bm5 compared to parental a549 controls ( fig. 4i ). we conducted a comprehensive characterization of a549 - bm5 cells at the transcriptomic and proteomic levels.