Pulmonary metastasis is a major cause of disease progression and mortality in patients with osteosarcoma. The survival impact of pulmonary metastasectomy and the prognostic relevance of surgery-related factors remain incompletely defined. This systematic review and meta-analysis aimed to evaluate the association between pulmonary metastasectomy and survival outcomes and to identify key prognostic determinants. PubMed, Embase, Web of Science, and the Cochrane Library were systematically searched from inception to December 2025. Studies enrolling patients with histologically confirmed osteosarcoma and lung metastases that examined associations between surgery-related factors and survival outcomes were eligible. Outcomes included post-relapse survival (PRS) and post-metastasectomy overall survival (PMOS). Hazard ratios (HRs) with 95

Osteosarcoma is a highly malignant tumor with poor prognosis. Current CAR-T cell therapies for osteosarcoma are predominantly designed with single targets, but their efficacy remains unsatisfactory. In this study, a novel bispecific CAR-T cell was developed to provide an experimental basis for improving the therapeutic outcome of osteosarcoma. Single-cell RNA sequencing (scRNA-seq) identified two antigens highly expressed in osteosarcoma cells, ANXA2 and CD147, whose expression was further validated at the tissue level by qRT-PCR, flow cytometry, and immunohistochemistry. Based on a second-generation CAR backbone, a bispecific ANXA2/CD147 CAR-T construct was generated using magnetic bead sorting, primary T-cell culture, and lentiviral transduction, achieving a transduction efficiency of 47.1%. LDH release assays demonstrated that bispecific CAR-T cells exhibited significantly greater cytotoxicity against tumor cells than single-target and control groups. ELISA confirmed that bispecific CAR-T cells released higher levels of effector molecules, including GZMB and TNFα. In a subcutaneous CDX model, bispecific CAR-T cells displayed superior antitumor activity and greater T-cell infiltration. In a paw pad xenograft model, mice treated with bispecific CAR-T cells exhibited the smallest tumor volumes, lowest tumor weights, and reduced rates of lymph node metastasis. Furthermore, PDX models confirmed that bispecific CAR-T cells effectively suppressed osteosarcoma growth. ScRNA-seq of tumors derived from CDX models and immunohistochemistry revealed markedly increased infiltration of M1 macrophages in the bispecific group. Collectively, this study successfully generated a bispecific ANXA2/CD147 CAR-T cell with robust antitumor activity, providing a promising new strategy for the immunotherapy of osteosarcoma.

BACKGROUND:The leading cause of death in osteosarcoma (OS) is metastasis, to which chemoresistant cell subpopulations contribute. A major obstacle is the limited understanding of the metastatic mechanisms of these cells and the identification of effective drugs. PURPOSE:This research aimed to identify a chemoresistant, metastasis-prone OS subpopulation, elucidate its pro-metastatic mechanism via endothelial dysfunction, and investigate the natural compound formononetin (FMN) as a potential inhibitor. STUDY DESIGN AND METHODS:We performed an integrated analysis of bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) data from OS patients to characterize tumor heterogeneity and identify key target. In silico molecular docking and molecular dynamics simulations were utilized to predict the binding interaction between FMN and the candidate target, which was further validated via CETSA assays. We performed in vitro functional verification including Transwell assays, transendothelial migration assays and conditional co-culture experiments in two OS cell lines. Finally, in vivo animal experiments were carried out to verify the lung metastatic capacity mediated by CD44 as well as the anti-metastatic effect of FMN against lung metastasis. RESULTS:ScRNA-seq analysis revealed a chemoresistant tumor cell cluster (TC1) and TC1 cells exhibited close spatial proximity to a specific endothelial cell subtype (C5) associated with metastasis. Cellphone DB analysis identified the CD44 as a critical target of TC1-induced endothelial dysfunction. Proprietary algorithm analysis confirmed predominant CD44 expression by TC1 cells. Computational modeling demonstrated a stable binding interaction between CD44 and FMN with high affinity (-7.0 kcal/mol), and CETSA assays yielded a ΔTm50 = 3.2 °C. In vitro, CD44 overexpression enhanced OS cell migration, invasion, and endothelial barrier disruption. FMN treatment significantly inhibited these phenotypes in a concentration-dependent manner. Crucially, rescue experiments confirmed that FMN specifically attenuated the enhanced metastatic capabilities induced by CD44 overexpression. In vivo experiments showed that CD44 overexpression promoted lung metastasis, whereas FMN reversed the lung metastasis induced by CD44 overexpression. CONCLUSION:Our research uncovers a novel mechanism whereby chemoresistant OS cells facilitate metastasis via CD44-mediated endothelial dysfunction. We demonstrated that FMN is a highly promising therapeutic agent, which likely acts by directly binding to the CD44 protein, thereby inhibiting OS cell transendothelial invasion. This research provides new insights into the treatment of metastatic chemoresistant OS.

Existing virtual perturbation methods can often infer directional changes by comparing predicted post-perturbation expression profiles with control cells. However, workflows that directly return direction-specific downstream candidate genes together with confidence scores, evidence support and interpretable summaries remain limited. We developed scGPA, an LLM-assisted workflow system for directional single-cell virtual gene perturbation analysis. scGPA starts from raw single-cell RNA sequencing data and performs quality control, normalization, dimensionality reduction, clustering and cell-group selection. It then constructs cell-group-specific wild-type regulatory networks using repeated subsampling, principal component regression (PCR)/Ridge-based network inference and CP tensor denoising. Based on these networks, scGPA simulates dose-aware virtual knockdown of the target gene and applies signed perturbation propagation to estimate the magnitude and direction of downstream transcriptional responses. LLM assistance is used for marker-based cell-type annotation, evidence-guided candidate prioritization and user-facing biological summarization. We benchmarked scGPA across five public Perturb-seq datasets and compared its performance with GEARS, scGPT and a random baseline. The overall correct prediction rate of scGPA was 23.0

Table S3, S5-S7, S10-S18, S21

BACKGROUND:Osteosarcoma is a rare malignant tumor originating from bone tissue. Despite advancements in neoadjuvant chemotherapy, the 5-year survival rate for osteosarcoma patients has plateaued around 60 % for the past fifty years, primarily due to the development of chemo-insensitivity. Cisplatin, a cornerstone in current treatment regimens, still has a low response rate in osteosarcoma patients, highlighting the need for strategies to enhance cisplatin sensitivity. PURPOSE:The purpose of this study is to explore the effects of formononetin, a bioactive compound, in sensitizing osteosarcoma cells to cisplatin. STUDY DESIGN:We utilized PDX models of osteosarcoma to evaluate the combined therapeutic effect of formononetin and cisplatin. Single-cell RNA sequencing and single-cell ATAC sequencing were performed on tumor tissues from these models to provide a detailed molecular profile of the treatment effects. METHODS:PDX models of osteosarcoma were established, followed by treatment with formononetin and cisplatin. A total of 7216 human-derived osteosarcoma cells and 89,558 mouse-derived cells were analyzed to assess their role in cisplatin sensitivity and tumor immune microenvironment changes. RESULTS:Our findings demonstrated that cisplatin insensitivity in osteosarcoma is strongly linked to ferroptosis. Formononetin sensitized osteosarcoma cells to cisplatin by inhibiting MAZ/GPX4 axis and inducing ferroptosis. Additionally, formononetin increased NK cell infiltration and immune activity, while reducing the infiltration of exhausted Cd8+ T cells and tumor-associated neutrophils, thereby reprogramming the tumor immune microenvironment and further enhancing cisplatin sensitivity. CONCLUSION:This study is the first to demonstrate that formononetin can enhance cisplatin sensitivity in osteosarcoma. By using osteosarcoma PDX models and performing comprehensive single-cell sequencing analyses, we identified formononetin as a promising sensitizer for cisplatin treatment. Our findings offer new therapeutic insights and mechanistic understanding that could help overcome cisplatin insensitivity in osteosarcoma and potentially improve patient outcomes.

Resistance to and associated toxic side effects of neoadjuvant chemotherapy remain major obstacles to improving the prognosis of osteosarcoma patients. Consequently, there is an urgent need to discover effective therapeutic agents with lower toxicity. In this study, the patient-derived xenograft (PDX) model was established and single-cell multi-omics sequencing was performed to comprehensively analyze changes in cellular heterogeneity and gene expression patterns of under formononetin treatment. We found that formononetin can significantly inhibit tumor growth in the osteosarcoma PDX model, on which the single-cell sequencing identified MYO1B as a key target mediating the anti-osteosarcoma effects of formononetin. In vitro experiments demonstrated that MYO1B overexpression enhanced the proliferation, invasion, and migration of osteosarcoma cells, while MYO1B silencing exhibited the opposite effects. Further investigation revealed that formononetin treatment markedly downregulated MYO1B expression, effectively suppressing the proliferative, invasive, and migratory phenotypes of osteosarcoma cells. Moreover, single-cell transcriptomic analysis of murine-derived cells showed that formononetin enhanced the cytotoxic activity of NK cells, promoted M1 macrophage polarization and inhibited M2 polarization, and reduced the proportion of senescent neutrophils, thereby alleviating the immunosuppressive state of the tumor microenvironment. Overall, our findings provide a comprehensive single-cell-level elucidation of the molecular mechanisms underlying the anti-osteosarcoma effects of formononetin, primarily involving downregulating the expression of MYO1B and remodeling the tumor immune microenvironment.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are crucial mediators of tumor-induced immunosuppression, while their heterogeneity and spatial dynamics across malignancies remain poorly understood. By integrating single-cell RNA sequencing data from 576 samples across 19 cancer types and spatial transcriptomics data from three distinct malignancies, we identified a PMN-MDSC population. This cell population demonstrated characteristic upregulation of immunosuppressive genes and was associated with poor prognosis across multiple cancer cohorts. Notably, TREM1 was highly expressed in PMN-MDSCs and may mediate immunosuppressive processes. Multiplex immunofluorescence demonstrated that TREM1+ PMN-MDSCs exhibited significantly higher distribution in tumor regions compared to non-tumor tissues. Spatial transcriptomics analysis revealed their co-localization with fibroblasts and exhausted T cells. Moreover, CellChat analysis showed that TREM1+ PMN-MDSCs remodeled the tumor microenvironment through interactions with diverse cellular components. Collectively, our study revealed the conserved immunosuppressive features and spatial interaction networks of TREM1+ PMN-MDSCs from a pan-cancer perspective, highlighting TREM1 as a pivotal therapeutic target to disrupt PMN-MDSC-mediated tumor immune evasion.

Osteosarcoma (OS) is highly malignant and easily prone to lung metastasis. The mechanisms of lung metastasis in OS remain unclear. The single-cell RNA sequencing (scRNA-seq) samples in this study included six primary osteosarcoma samples (published in-house data), two lung metastasis samples (GSE152048), and four normal bone tissue samples (GSE169396). To identify potential targets for metastasis, bulk RNA sequencing data from four primary tumors and four lung metastases (in-house data) were also analyzed. scRNA-seq identified five tumor cell subpopulations. CytoTRACE and lung metastasis scores indicated that the C1 subpopulation was most closely associated with lung metastasis. By intersecting lung metastasis-related genes identified via hdWGCNA analysis with differentially expressed genes from bulk RNA sequencing, SEC16B was identified as the key gene influencing lung metastasis. qRT-PCR results revealed that SEC16B expression was significantly downregulated in OS cell lines. Transwell assay demonstrated that overexpression of SEC16B significantly inhibited the invasion and migration capabilities of OS cells. Additionally, analyses using Scissor, CellphoneDB, and CSOmap suggested that fibroblasts, endothelial cells, and OS cells in the tumor microenvironment formed a pre-metastatic niche through mechanisms involving angiogenesis and extracellular matrix remodeling. Overall, this study identifies a new population that may promote lung metastasis by downregulating SEC16B in OS. Moreover, fibroblasts and endothelial cells in the tumor microenvironment play a critical role in OS lung metastasis.

BACKGROUND:Anterior pelvic ring injuries frequently involve obturator ring disruption. With advancements in surgical techniques and rising patient expectations, there is growing interest in repairing these injuries within anterior pelvic ring treatment strategies. While current approaches (e.g., transabdominal, ilioinguinal) are used, they present limitations. This study describes a Modified Lateral-Perineal Approach (MLPA) for treating obturator ring fractures. METHODS:We retrospectively reviewed 22 pelvic fracture patients with obturator ring injuries that had been treated via the MLPA. All patients underwent open reduction and internal fixation (ORIF) between June 2019-June 2024. According to the Tile classification, there were 16 type B fractures and 6 case of type C fractures. All patients had a reduction and fixation to the fracture of the superior pubic ramus and inferior ramus of pubis-ischium ramus. Incision length, operative time, blood loss, time to sitting/weight-bearing, union time, adductor strength (MRC 0-5), satisfaction and complications were recorded. The quality of fracture reduction was evaluated according to Matta radiographic criteria. The pelvic function was evaluated according to Majeed Pelvic Score at the last follow-up. RESULTS:Twenty-two patients (10 male, 12 female; mean age 49.7 ± 13.7 years) were included. Mean follow-up was 15 ± 3 months. Mean surgical parameters: incision length 8.5 ± 1.1 cm, operative time 106.4 ± 23.4 min, blood loss 89.1 ± 41.2 mL. All achieved "excellent" Matta scores. Functional recovery: sitting at 6.6 ± 3.4 days, partial weight-bearing at 15.7 ± 7.2 days, full weight-bearing at 8.9 ± 2.5 weeks. Radiographic union occurred at 9.5 ± 2.4 months. One-year Majeed score averaged 88.5 ± 4.5. Adductor strength was MRC grade IV in 3 patients and V in 19. All patients expressed satisfaction. There were no complications of nonunion, internal fixation failure, vascular injury, nerve palsy, or hernia. All of the patients were esthetically satisfied with the scar. CONCLUSION:The MLPA provides a safe and effective simultaneous exposure of the obturator ring's key structures: the superior pubic ramus, inferior ramus of pubis-ischium ramus. The advantage of this approache to repair the obturator ring include safety, simplicity, early weight-bearing, aesthetics, and satisfactory clinical efficacy with a low incidence of complications.

qPCR results of the expression of 4 deferent lncRNA in cytoplasm and nucleus. Three PCR by-wells were prepared for each sample.

INDEL was detected by whole-exome sequencing of 19 ccRCC samples.

Objective: Osteosarcoma (OS) is a rare and complex form of cancer that mostly affects children and adolescents. Pain is a common symptom for patients in OS which causes significant unhappiness and persistent aches. To date, there is minimal knowledge on the mechanisms underlying OS induced pain and few treatment options for patients. Previous genetic studies have demonstrated that the panel of four genes, artemin ( ARTN ), persephin ( PSPN ), glial cell line-derived neurotropic factor ( GDNF ), and neurturin ( NRTN ) are associated with the regulation of pain processing in OS and analgesic responses. Methods: In the present study, by utilising a scRNA-seq OS dataset, we aimed to measure the gene expression levels of four pain related genes, and compare them between the different cell types in human OS tissues and cell lines. Results: Within a complex and diverse range of cell types in OS tissues, including osteoblastic OS cells, carcinoma associated fibroblasts (CAFs), B cells, myeloid cells 1, myeloid cells 2, NK/T cells, plasmocytes, ARTN and NRTN genes had the highest expression in Osteoblastic OS cells, GDNF gene had a peak expression in carcinoma associated fibroblasts, and PSPN gene in endothelial cells. In addition, all four genes showed deferential pattern of expression in 16 OS cell lines. Conclusion: Future studies should investigate the potential to target deferentially expressed pain-related genes in specific cell types of OS for therapeutic benefit to improve the quality of life for patients living with pain caused by OS.

qPCR results after ASO (5717 and 5608) treatment of 2 lncRNAs in caki-2 and 786-O cell lines.Three or six PCR by-wells were prepared for each sample. NC, negative control.

OBJECTIVES:To investigate the inhibitory effect of polyphyllin VII (PP7) on osteosarcoma xenograft growth in mice and explore the underlying molecular mechanism. METHODS:Ultra‑performance liquid chromatography‑tandem mass spectrometry was used to analyze the main active components of Paris polyphylla. Six nude mice bearing patient‑derived xenograft (PDX) were randomized into two groups for treatment with 2 mg/kg PP7 gavage or saline every other day for 28 days, and the changes in tumor volume and mass were measured. In cultured 143B and HOS cells, the effect of PP7 treatment (0, 1.25, 2.5, 5, and 10 μmol/L) on cell proliferation was assessed with CCK‑8 assay, and Transwell assays were employed to examine the changes in cell migration and invasion. The target of PP7 was predicted by integrated analyses with single‑cell RNA sequencing (scRNA‑seq), bulk RNA sequencing (bulk RNA‑seq) and molecular docking and verified using Western blotting. In osteosarcoma cells transfected with SOHLH1 siRNAs or a negative control sequence, the effects of PP7 treatment (5 μmol/L) on cell migration, invasion, ferroptosis, reactive oxygen species (ROS) production and lipid peroxidation (LPO) were analyzed. RESULTS:PP7 was identified as one of the major active constituents of Paris polyphylla. In the tumor-bearing mice, PP7 treatment significantly lower the tumor volume and mass. In 143B and HOS cells, PP7 concentration‑dependently inhibited cell proliferation, and at 5 μmol/L, PP7 significantly inhibited cell proliferation, migration and invasion. Multi‑omics analysis identified SOHLH1 as a potential target of PP7, and Western blotting confirmed that PP7 upregulated SOHLH1 expressions at both the mRNA and protein levels. SOHLH1 silencing obviously attenuated the inhibitory effects of PP7 on cell migration and invasion and reduced PP7‑induced ferroptosis. CONCLUSIONS:PP7 suppresses osteosarcoma xenograft growth in mice by inducing ferroptosis via upregulating SOHLH1 expression.

As the most common primary malignant bone tumor, osteosarcoma (OS) is characterized by drug resistance and poor prognosis, highlighting the urgent need for promising therapeutic agents. Formononetin (FMN), a natural product derived from Spatholobi Caulis, has been reported to possess anti-tumor properties. However, its role in OS has not yet been elucidated. In the present study, we established an OS patient-derived xenograft model to investigate the effects of FMN and the underlying mechanisms of its effects on OS. When FMN treatment was completed, bulk transcriptome sequencing was conducted, and the analyses were combined with OS single-cell RNA sequencing (scRNA-seq) data. Results indicated that GTSF1 was up-regulated in OS but down-regulated after FMN intervention, which may regulate the apoptosis of OS cells. Furthermore, the qRT-PCR and IHC results demonstrated that GTSF1 expression was significantly up-regulated in OS cells, whereas FMN expression was down-regulated both in vitro and in vivo. Moreover, in vitro experiments revealed that FMN effectively promoted apoptosis and suppressed the proliferation, migration, and invasion of OS cells. Therefore, this study demonstrated that FMN exerts anti-OS effects by down-regulating GTSF1 expression, thus effectively promoting the apoptosis and inhibiting the proliferation of OS cells, making FMN a promising anti-OS drug.

SNV was detected by whole-exome sequencing of 19 ccRCC samples.