transwell assays clearly demonstrated that the a549 - bm5 cells exhibited significantly enhanced migration and invasion capabilities compared to their parental counterparts ( fig. 2d ). western blot analysis showed a clear reduction of e - cadherin in a549 - bm5 compared with parental a549 cells. minor changes were also observed in n - cadherin, β - catenin, and snail ( fig. 2e ). notably, we observed that e - cadherin is significantly downregulated, while n - cadherin, β - catenin, and snail are upregulated. this pattern suggests the cells are undergoing epithelial - mesenchymal transition ( emt ), becoming more migratory and invasive. fig. 2the in vitro functions and epithelial - mesenchymal status of a549 - bm5 cell subline ( a ) cell counting kit - 8 assay: in vitro proliferation analysis of a549 - parental and a549 - bm5 cells ( n = 3 ). ( colony formation ability of a549 - parental and a549 - bm5 cells ( n = 3 ). ( flow cytometry was used to detect changes in the dna content distribution in the a549 - parental and a549 - bm5 cells ( n = 3 ). ( transwell assay assessing the migration and invasion abilities of a549 - parental and a549 - bm5 ( n = 9 ). ( western blot analysis of e - cadherin, n - cadherin, β - catenin and snail in a549 - parental and a549 - bm5 cells 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 vitro functions and epithelial - mesenchymal status of a549 - bm5 cell subline ( a ) cell counting kit - 8 assay: in vitro proliferation analysis of a549 - parental and a549 - bm5 cells ( n = 3 ). ( colony formation ability of a549 - parental and a549 - bm5 cells ( n = 3 ). ( flow cytometry was used to detect changes in the dna content distribution in the a549 - parental and a549 - bm5 cells ( n = 3 ). ( transwell assay assessing the migration and invasion abilities of a549 - parental and a549 - bm5 ( n = 9 ). ( western blot analysis of e - cadherin, n - cadherin, β - catenin and snail in a549 - parental and a549 - bm5 cells 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. tumor progression during bone metastasis relies on intricate interactions with a variety of cells in the bone microenvironment. to investigate the interaction capacity of a549 - bm5 cells with these cellular constituents, we examined their impacts on osteoclasts, osteoblasts, and their progenitor cells. using transwell assays, we found that a549 - bm5 cells exhibited a significantly enhanced ability to recruit osteoclast precursors compared with parental cells. both raw264. 7 cells and bone marrow – derived macrophages ( bmms ) were used as osteoclast precursor models, and in both conditions a549 - bm5 cells demonstrated stronger recruitment ( fig. 3a ). for osteoclast differentiation experiments, we specifically used bmms isolated from c57bl / 6j mice. after 14 days of culture under osteoclastogenic induction, a549 - bm5 cm significantly promoted osteoclast formation, as confirmed by trap staining and per - well quantification ( fig. 3b ). at the transcriptional level, rt - qpcr further revealed significant upregulation of osteoclast differentiation markers including tnfrsf11a, ctsk, car2, calcr, tm7sf4, acp5, and nfatc1 in the a549 - bm5 group ( fig. 3c ). fig. 3the capacity of a549 - bm5 cells to interact with bone microenvironment cells. ( transwell assays showing the recruitment capacity of a549 - parental and a549 - bm5 cells toward osteoclast precursors. both raw264. 7 cells and bone marrow – derived macrophages ( bmms ) were used as osteoclast precursor models. quantification indicates significantly stronger recruitment in the a549 - bm5 group ( n = 6 ). ( osteoclast differentiation assay using bmms. after 14 days of induction with conditioned medium ( cm ) from a549 - parental or a549 - bm5 cells, trap staining and per - well quantification confirmed markedly enhanced osteoclast formation in the a549 - bm5 group ( n = 3 ). ( rt - qpcr analysis of osteoclast differentiation markers ( tnfrsf11a, ctsk, car2, calcr, tm7sf4, acp5, and nfatc1 ) in bmms 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 ). ( transwell assays showing recruitment capacity of a549 - parental and a549 - bm5 cells toward osteoblast progenitors ( mc3t3 - e1 subclone 14 and bmscs ). quantification demonstrates enhanced recruitment by a549 - bm5 cells ( n = 6 ). ( mc3t3 - e1 subclone 14 cells and bmscs were induced for 14 days with osteogenic medium containing cm from a549 - parental or a549 - bm5 cells. alp staining demonstrated stronger osteogenic differentiation in the a549 - bm5 group ( n = 3 ). ( 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. the capacity of a549 - bm5 cells to interact with bone microenvironment cells. ( transwell assays showing the recruitment capacity of a549 - parental and a549 - bm5 cells toward osteoclast precursors. both raw264. 7 cells and bone marrow – derived macrophages ( bmms ) were used as osteoclast precursor models. quantification indicates significantly stronger recruitment in the a549 - bm5 group ( n = 6 ). ( osteoclast differentiation assay using bmms. after 14 days of induction with conditioned medium ( cm ) from a549 - parental or a549 - bm5 cells, trap staining and per - well quantification confirmed markedly enhanced osteoclast formation in the a549 - bm5 group ( n = 3 ). ( rt - qpcr analysis of osteoclast differentiation markers ( tnfrsf11a, ctsk, car2, calcr, tm7sf4, acp5, and nfatc1 ) in bmms 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 ). ( transwell assays showing recruitment capacity of a549 - parental and a549 - bm5 cells toward osteoblast progenitors ( mc3t3 - e1 subclone 14 and bmscs ). quantification demonstrates enhanced recruitment by a549 - bm5 cells ( n = 6 ). ( mc3t3 - e1 subclone 14 cells and bmscs were induced for 14 days with osteogenic medium containing cm from a549 - parental or a549 - bm5 cells. alp staining demonstrated stronger osteogenic differentiation in the a549 - bm5 group ( n = 3 ). (