Yuzhen Du

The unfavorable prognosis of patients with bone metastasis of lung adenocarcinoma (LUAD) is largely due to both osteolytic destruction and tumor stem cell-mediated secondary metastasis. However, the mechanisms by which the bone microenvironment augments the stemness of LUAD cells remain inadequately understood. In this study, we developed a LUAD cell model with enhanced stemness, termed BM3, by subjecting the cells to three rounds of conditioning within the bone microenvironment. Our findings identified osteoclasts, rather than osteoblasts, as the principal stromal cells that facilitate the enhancement of stemness in BM3 cells. This facilitation was inhibited by the application of either a tumor necrosis factor-alpha (TNF-α) neutralizing antibody or an NF-κB inhibitor. Additionally, we established that the expression level of the secretory protein olfactomedin-like protein 3 (OLFML3) is positively associated with the stemness of BM3 cells and exerts a self-reinforcing effect on tumor cell stemness. Mechanistic analyses revealed that OLFML3 upregulates the pluripotency-associated factor developmental pluripotency-associated protein 2 (DPPA2), which subsequently promotes the expression of core stemness factors, including CD44, OCT4, and SOX2, thereby augmenting the stemness and metastatic potential of tumor cells. Clinical data suggest that serum OLFML3 may serve as a candidate biomarker for identifying LUAD patients with bone metastasis who are at increased risk of subsequent metastatic spread. Our study uncovers a novel mechanism in which osteoclasts within the bone microenvironment augment the stemness of LUAD cells via the TNF-α-NF-κB-OLFML3-DPPA2 signaling axis. This finding provides an essential theoretical foundation for developing strategies aimed at suppressing secondary dissemination following bone metastasis in LUAD.

Objective: To develop and validate an interpretable machine learning (ML) model for predicting malignant risk in patients with breast nodules using serum lipid biomarkers. Methods: This retrospective study included 899 patients with breast nodules (236 malignant) admitted between March 2022 and December 2024. Patients were randomly assigned to a training cohort ( n = 630) and an internal validation cohort ( n = 269) at a 7:3 ratio. Baseline clinical and laboratory data were collected upon admission. Following feature selection via LASSO regression, the predictive performance of 8 ML algorithms was evaluated and compared using receiver operating characteristic (ROC) curves. The optimal model’s performance was further corroborated using an independent temporal validation cohort of 190 patients (admitted Jan–Aug 2025). Model interpretability was addressed using SHapley Additive exPlanations (SHAP). Results: Nine key predictors were identified from 20 candidates by Lasso regression and clinical expertise. The random forest (RF) model outperformed other algorithms, achieving areas under the curve (AUC) values of 0.789, 0.782, and 0.825 for the training, internal validation, and temporal validation cohorts, respectively. Hosmer–Lemeshow tests ( p > 0.05) indicated high calibration between the predicted and observed risks. SHAP importance analysis revealed Fer, age, and CEA to be the top three predictive factors. Conclusion: The RF model based on serum lipid biomarkers serves as a robust, noninvasive tool for assessing breast cancer risk, showing significant potential for clinical decision support in screening programs.

The early stages of tumor bone metastasis are closely associated with changes in the vascular niche of the bone microenvironment, and abnormal angiogenesis accelerates tumor metastasis and progression. However, the effects of lung adenocarcinoma (LUAD) cells reprogrammed by the bone microenvironment on the vascular niche within the bone microenvironment and the underlying mechanisms remain unclear. This study investigates the effects and mechanisms of LUAD cells reprogrammed by the bone microenvironment on endothelial cells and angiogenesis, providing insights into the influence of tumor cells on the vascular niche within the bone microenvironment. The culture media from bone-metastatic LUAD cell A549-GFP-LUC-BM3 (BM3-CM) and A549-GFP-LUC (A549-CM) were separately applied to human umbilical vein endothelial cell (HUVEC). A colony formation assay, scratch assay, and tube formation assay were conducted to evaluate the proliferation, migration, and angiogenesis of HUVEC. Gene set enrichment analysis (GSEA) was conducted to identify enriched pathways, while reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme linked immunosorbent assay (ELISA) were performed to quantify hepatocyte growth factor (HGF), a protein that plays a crucial role in angiogenesis. Furthermore, the pivotal function of HGF and its underlying molecular mechanisms have been substantiated through the utilisation of recombinant proteins, neutralising antibodies, pathway inhibitors, immunofluorescence staining, and Western blot. BM3-CM demonstrated a more pronounced impact on the proliferation, migration, and angiogenesis of HUVEC compared to A549-CM. Bioinformatics analysis, combined with in vitro experiment, demonstrated that the secretory protein HGF was significantly elevated in BM3 cells and BM3-CM (P<0.05). The addition of HGF neutralizing antibodies to BM3-CM inhibited the promoting effect of BM3-CM on HUVEC (P<0.05), while the addition of recombinant HGF to A549-CM reproduced that promoting effect of BM3-CM on HUVEC (P<0.05). HGF can enhance the activation of YAP (Yes-associated protein) in HUVEC, and this promotion effect may be achieved by activating Src and activating YAP into the nucleus (P<0.05), but this effect can be inhibited by HGF neutralizing antibodies (P<0.05). Furthermore, the addition of recombinant HGF to A549-CM can recapitulate the YAP activation effect of BM3-CM in HUVEC (P<0.05). Bone microenvironment reprogrammed bone-metastatic LUAD cells BM3 promote the proliferation, migration, and angiogenesis of HUVEC through the HGF/YAP axis, potentially playing a significant role in the modifications of the vascular niche.

The regulatory mechanisms underlying bone metastasis in lung adenocarcinoma (LUAD) are not yet fully understood despite the frequent occurrence of bone involvement. This study aimed to examine the involvement and mechanism of integrin subunit beta 3 (ITGB3) in the process of LUAD bone metastasis. Our findings indicate that ITGB3 facilitates the migration and invasion of LUAD cells in vitro and metastasis to the bone in vivo. Furthermore, ITGB3 stimulates osteoclast production and activation, thereby expediting osteolytic lesion progression. Extracellular vesicles (EVs) isolated from the conditioned medium (CM) of LUAD cells overexpressing ITGB3 determined that ITGB3 facilitates osteoclastogenesis and enhances osteoclast activity by utilizing EVs‐mediated transport to RAW264.7 cells. Our in vivo findings demonstrated that ITGB3‐EVs augmented the population of osteoclasts, thereby establishing an osteoclastic pre‐metastatic niche (PMN) conducive to the colonization and subsequent growth of LUAD cells in the bone. ITGB3 is enriched in serum EVs of patients diagnosed with LUAD bone metastasis, potentially facilitating osteoclast differentiation and activation in vitro. Our research illustrates that ITGB3‐EVs derived from LUAD cells facilitate osteoclast differentiation and activation by modulating the phosphorylation level of p38 MAPK. This process ultimately leads to the generation of osteolytic PMN and accelerates the progression of bone metastasis.

Bone is a common site for metastasis in lung adenocarcinoma, but the mechanism behind lung adenocarcinoma bone metastasis is still unclear. And currently, there is a lack of easily traceable and stable lung adenocarcinoma bone metastasis cell models, which limits the research on the mechanism of lung adenocarcinoma bone metastasis. The establishment of human lung adenocarcinoma cell line that are highly metastatic to bone, labeled with green fluorescent proteins (GFP) and fireflies luciferase (LUC), along with transcriptomic characterization, would be beneficial for research on lung adenocarcinoma bone metastasis and provide new experimental methods. The human lung adenocarcinoma cell line A549-GFP-LUC was injected into nude mice via the left ventricle to construct a bone metastasis model, and was domesticated in vivo for three consecutive times to obtain the human high bone metastasis lung adenocarcinoma cell line A549-GFP-LUC-BM3; cell counting kit-8 (CCK-8), colony formation assay, scratch wound assays, Transwell assay and Western blot were used to compare the proliferation and invasion abilities of A549-GFP-LUC-BM3 with the parental cells. A549-GFP-LUC-BM3 cells and parental cells were further analyzed by transcriptomic sequencing. Human high-bone metastatic lung adenocarcinoma cells A549-GFP-LUC-BM3 was successfully established. Compared to parental cells, this cells exhibited a significantly higher incidence of bone metastasis and enhanced in vitro proliferation, migration, and invasion abilities. Transcriptomic sequencing results revealed that the A549-GFP-LUC-BM3 cell line had 2954 differentially expressed genes compared to the parental cells, with 1021 genes up-regulated and 1933 genes down-regulated. Gene Ontology (GO) functional enrichment analysis indicated that the differentially expressed genes were primarily localized in cellular components such as the cell periphery. The molecular functions identified as significantly enriched included signaling receptor activity, calcium ion binding, and extracellular matrix structural constituent. Additionally, the biological processes found to be enriched were cell adhesion and biological adhesion. The enrichment analysis conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the differentially expressed genes were primarily involved in the metabolism of xenobiotics by cytochrome P450, retinol metabolism, drug metabolism-cytochrome P450, cell adhesion molecules, steroid hormone biosynthesis, and the nuclear factor kappa B (NF-κB) signaling pathway. The highly bone-metastatic human lung adenocarcinoma cell line with GFP and luciferase double labeling was successfully established. The biological behavior and transcriptome sequencing of the cell line suggest that it has a high bone-metastatic potential.

Due to its key roles in malignant tumor progression and reprograming of the tumor microenvironment, integrin β3 has attracted great attention as a new target for tumor therapy. However, the structure-function relationship of integrins β3 remains incompletely understood, leading to the shortage of specific and effective targeting probes. This work uses a purified extracellular domain of integrin β3 and integrin β3-positive cells to screen aptamers, specifically targeting integrin β3 in the native conformation on live cells through the SELEX approach. Following meticulous truncation and characterization of the initial aptamer candidates, the optimized aptamer S10yh2 was produced, exhibiting a low equilibrium dissociation constant (K_d) in the nanomolar range. S10yh2 displays specific recognition of cancer cells with varying levels of integrin β3 expression and demonstrates favorable stability in serum. Subsequent analysis of docking sites revealed that S10yh2 binds to the seven amino acid residues located in the core region of integrin β3. The S10yh2 aptamer can downregulate the level of integrin heterodimer αvβ3 on integrin β3 overexpressed cancer cells and partially inhibit cell migration behavior. In summary, S10yh2 is a promising probe with a small size, simple synthesis, good stability, high binding affinity, and selectivity. It therefore holds great potential for investigating the structure-function relationship of integrins.