while bone lesions were observed, notable metastatic spread also occurred in other organs, including the lungs and abdominal viscera, potentially complicating the interpretation of bone - specific metastatic mechanisms. tan et al. established a bone metastasis model via intra - tibial injection of a549 cells. while this approach efficiently induces bone lesions, it bypasses key early steps of the metastatic cascade, limiting its relevance for mechanistic studies. moreover, the invasive procedure introduces tissue injury and allows cancer cells to enter circulation, increasing the risk of systemic spread. recent findings also suggest that injury - induced bone remodeling involving ng2 stromal cells may artificially promote tumor colonization, further compromising model specificity. this limitation was partially addressed by cai et al., who established the a549l6 subline through in vivo selection, which preferentially metastasizes to the spine. while a549l6 partially mimics the clinical pattern of lung cancer bone metastasis — typically initiating in the axial skeleton — it is less amenable to in vivo monitoring and is suboptimal for studying metastases to appendicular bones. in contrast, our a549 - bm5 subline exhibits a broader and more consistent bone - tropic pattern, including frequent involvement of limb bones, thereby offering a more versatile and observable model for investigating skeletal colonization and therapeutic interventions. however, given the unique biological features of vertebral bone — such as its distinc t skeletal stem cell populations and specialized metastatic niche — this model may be more appropriate for investigating spine - specific metastasis rather than broadly representative skeletal dissemination. collectively, these limitations underscore the urgent need for a lung adenocarcinoma model that not only exhibits stable and reproducible bone tropism but also ac curate ly reflects the general biological characteristics of skeletal metastasis — criteria that a549 - bm5 is well positioned to fulfill. given the clinical characteristics of bone metastasis, identifying biomarkers and therapeutic targets associated with early biological events of skeletal colonization holds significant research value. in this study, we evaluated the performance of the a549 - bm5 subline in combination with intra - iliac artery injection to model this process. remarkably, a549 - bm5 exhibited a clear proliferative advantage as early as five days post - injection, suggesting that it effectively recapitulates several key niches involved in the early stages of bone metastasis, including the perivascular niche and the osteogenic niche. consistent with the notion that organ - specific microenvironments can drive phenotypic divergence even among genetically similar cancer cells, a549 - bm5 exhibited not only stable proliferation in vitro but also acquired distinc t metastatic traits — including reduced cell adhesion, elevated emt marker expression, and significantly enhanced migratory and invasive capacities. these features, coupled with its robust and early skeletal colonization in vivo, suggest that repeated exposure to the bone microenvironment exerted strong selective pressure, enriching for a tumor subpopulation with specialized bone - homing potential. mechanistically, several components of the bone niche may have contributed to this phenotypic evolution. hypoxic conditions, characteristic of specific niches within the bone marrow microenvironment, are known to activate hif - 1α signaling, which in turn promotes emt and enhances metastatic capacity. tgf - β, abundantly released during ost eol ytic bone remodeling, has also been shown to induce mesenchymal transition and support metastatic seeding. additionally, extracellular matrix proteins such as collagen and osteopontin, along with mechanical cues like increased matrix stiffness, can activate integrin and focal adhesion kinase ( fak ) signaling pathways to further promote tumor cell motility and survival. moreover, accumulating evidence indicates that even subtle environmental cues can induce long - lasting transcriptional and epigenetic reprogramming in tumor cells during in vivo selection. therefore, the stable acquisition of bone - adaptive traits in a549 - bm5 may reflect not only selective outgrowth of pre - existing subclones, but also microenvironment - driven plasticity that enhances tumor cell fitness in the skeletal niche. importantly, the clinical relevance of the a549 - bm5 model was further substantiate d by transcriptomic and proteomic analyses, which not only revealed classical molecular hallmarks of bone metastasis but also aligned closely with clinical observations. previous studies in prostate cancer have shown that once tumor cells colonize the bone marrow microenvironment, they acquire osteomimicry features — such as the expression of alkaline phosphatase, osteocalcin, osteopontin, and bone morphogenetic proteins ( bmps ) — to support their survival and progression within bone tissue. interestingly, a549 - bm5 displayed a similar trend, with a transcriptomic profile resembling that of osteogenic cells and robust activation of the wnt signaling pathway — a critical regulator of both osteogenesis and bone metastasis — further reinforcing its bone - adaptive phenotype. in addition, a549 - bm5 showed marked enrichment of neuron - associated gene sets, suggesting a possible contribution of neurobiological processes to its metastatic behavior. although the role of neural regulation in lung cancer bone metastasis has been relatively underexplored, emerging evidence is beginning to highlight its importance. for instance, one study revealed that sensory nerves expressing calcitonin gene – related peptide ( cgrp ) are enriched at bone metastatic sites and promote tumor progression via the cgrp / crlr / p38 / hsp27 signaling axis. these findings provide compelling support for leveraging a549 - bm5 as a preclinical model to explore mechanisms underlying bone metastasis. taken together, a549 - bm5 offers a stable, bone - tropic, and clinically relevant model for lung cancer bone metastasis, characterized by distinc t transcriptomic and proteomic alterations compared to the parental a549 line and enhanced interactions with key components of the bone microenvironment. these features make it well - suited for mechanistic studies and therapeutic exploration targeting the skeletal niche. the immune system plays an important role in the process of bone metastasis. due to the limitations of lewis lung cancer cells, we chose a549 cells to establish a highly bone - metastatic subline, which limits the possibilities for immune-related studies. moreover, the in vivo selection strategy based on intracardiac injection bypasses the early steps of the metastasis cascade, such as pre - metastatic alterations in bone, the rise of bone - tropic metastatic seeds in primary tumors, and the perivascular niche and metastasis dormancy. this study did not generate new unique reagents. there is no original code in this study. any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. yujian xu: writing – original draft, visualization, methodology, investigation, data curation, conceptualization. yahan qin: software, methodology, investigation. wenjun chai: resources, methodology, formal analysis. ke xue: software, data curation. hongyu pan: writing – review & editing, supervision, conceptualization. mingxia yan: writing – review & editing, supervision, methodology, funding acquisition, conceptualization. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.