Forty novel diosgenin derivatives were designed and synthesized, and their anti-osteosarcoma activities were systematically evaluated in vitro and in vivo. Some of the compounds suppressed the proliferation of MG-63, U-2 OS, and 143B osteosarcoma cell lines in vitro. Among them, compound 5a displayed potent inhibitory activity against U-2 OS and 143B cells, with IC₅₀ values of 2.32 μM and 2.38 μM, respectively. Further in vitro studies revealed that compound 5a could inhibit cell migration, suppress colony formation, and induce apoptosis in U-2 OS and 143B cells. Mechanistic investigations demonstrated that compound 5a upregulated the expression of pro-apoptotic markers (cleaved PARP and Bax) and the epithelial–mesenchymal transition marker E-cadherin, while downregulating the anti-apoptotic marker Bcl-2 and epithelial–mesenchymal transition markers (Slug, Snail, N-cadherin, and Vimentin). Additional studies confirmed that the anti-osteosarcoma effects of compound 5a were mediated by activity of the HIPPO signaling pathway. Notably, compound 5a demonstrated favorable pharmacokinetic properties (T₁/₂ = 5.53 h) and exerted significant anti-osteosarcoma efficacy in murine models without apparent systemic toxicity. Importantly, it specifically suppressed pulmonary metastasis, a major clinical obstacle in osteosarcoma treatment. These findings underscore the potential of compound 5a as a promising candidate for further development against osteosarcoma.

This protocol aims to determine whether a patient-specific, multidimensional traction (MDT regimen), delivered alongside standard bracing and mirror-corrective exercises, can improve structural correction in moderate adolescent idiopathic scoliosis (AIS). The study is a pilot randomized clinical trial (RCT) enrolling skeletally immature adolescents (Cobb 20 - 45°) and allocating them 1:1 to (i) brace plus mirror-corrective exercises or (ii) the same program augmented by MDT. Traction vectors are individualized to the curve pattern and flexibility. Interventions are provided over a 3-month period. The MDT arm receives supervised sessions 2x a week, and all participants complete standardized daily exercises and wear braces according to guideline-based prescriptions. The primary outcome is the change in the major-curve Cobb angle from baseline to post-treatment, measured on standardized standing radiographs using consistent end vertebrae and blinded dual reading. Safety, adherence, and adverse events are monitored throughout. In results, adding MDT to bracing and exercises yielded a greater reduction in the major-curve Cobb angle. This protocol operationalizes a reproducible, clinic-ready workflow for parameterized multi-vector traction as an adjunct to conventional nonoperative care, offering a framework to maximize immediate in-brace correction and promote follow-up curve regression. This trial provides methodology and preliminary evidence to support larger, longer follow-up studies through skeletal maturity.

Aberrant long non-coding RNAs (lncRNAs) expression profoundly influences cellular proliferation of osteosarcoma cells, suggesting that they may serve as potential therapeutic targets. However, functional studies of lncRNAs remain largely theoretical and limited to a few validations. Colony formation assays, which specifically reflect the ultimate proliferative fate of single cells, are regarded as the gold standard for evaluating long-term proliferative potential. Here, this protocol established a reliable experimental protocol using colony formation assays in lncRNA-knockdown cells to evaluate the effect of lncRNA on osteosarcoma cell proliferative capacity. This experimental protocol provides a stable, cost-effective, and efficient assay to assess the regulatory effects of lncRNA on osteosarcoma cell proliferation. This approach is not only suitable for research on osteosarcoma cells but also serves as a feasible tool for lncRNA-regulated cell proliferative capacity in other tumors. This study included a detailed detection protocol of lncRNA-knockdown and colony formation assays, with an example of the small nucleolar RNA host gene 6 (SNHG6) knockdown in osteosarcoma cells (143B). Experimental results confirmed efficient suppression of SNHG6 expression, accompanied by a marked reduction in both the number and size of colonies. These findings suggest that SNHG6 is essential for sustaining the longterm proliferative potential of osteosarcoma cells (143B).

Background: Bone sarcoma constitutes a heterogeneous group of aggressive malignancies associated with poor overall prognosis. Immune checkpoint inhibitor (ICI) therapy has emerged as a potential treatment for bone sarcoma, yet its overall efficacy, safety, and differential activity across pathological subtypes and combination regimens remain poorly defined. Methods: We performed literature searches across PubMed, Embase, Cochrane Library, and Web of Science from the inception of each database through 7 February 2026. Eligible publications included all clinical studies evaluating ICI-based treatment for bone sarcoma, irrespective of study design or the presence of a comparator arm. Objective response rate (ORR) was defined as the primary endpoint. Secondary endpoints included disease control rate (DCR), median progression-free survival (mPFS), median overall survival (mOS), and grade 3-5 treatment-related adverse events (G3-5 trAEs). All pooled analyses were conducted using a random-effects model. This meta-analysis was prospectively registered with PROSPERO (CRD420251125681). Findings: Overall pooled ORR and DCR were 11.7% (95% CI, 8.0%–16.1%) and 48.7% (95% CI, 35.6%–61.8%), with pooled mPFS of 3.78 months and pooled mOS of 10.41 months. Subgroup analyses stratified by treatment regimens revealed marked variability in clinical efficacy. ICIs combined with tumor-infiltrating lymphocyte therapy (TILs) demonstrated the highest anti-tumor activity, with an ORR of 35.9% (95% CI, 19.1%–54.6%). In addition, ICIs plus tyrosine kinase inhibitor (TKIs) also yielded favorable anti-tumor activity, with an ORR of 17.6% (95% CI, 9.3%–27.9%) and a DCR of 71.3% (95% CI, 56.0%–84.4%). Pathological subtype analyses revealed divergent treatment responses: chordoma exhibited the most favorable outcomes (ORR 19.4%, DCR 81.3%), followed by chondrosarcoma (ORR 17.8%, DCR 49.2%). Osteosarcoma, the largest subgroup, showed modest anti-tumor activity (ORR 9.4%, DCR 40.2%), and Ewing sarcoma produced the weakest clinical response (ORR 5.9%, DCR 19.5%). The pooled incidence of G3-5 trAEs was 30.7% (95% CI, 16.5%–47.0%) across 453 patients from 13 studies. Leave-one-out sensitivity analyses confirmed the robustness of our primary efficacy endpoints. Interpretation:This meta-analysis demonstrates that ICI-based regimens, especially when combined with TILs or TKIs, yield meaningful clinical activity in bone sarcoma. Notably, chordoma and chondrosarcoma exhibit particularly favorable responses to these therapies. Collectively, these findings highlight the transformative potential of ICIs in advancing treatment strategies for bone sarcoma.

Bone metastases (BM) frequently lead to skeletal-related events (SREs) and significant pain in lung cancer patients, requiring more effective supportive management strategies. We designed a prospective, multicenter, randomized, double-blind, placebo-controlled trial to evaluate Bawei Aiguning Granules (BWAGN) in combination with standard bone-targeted therapy. A total of 156 patients will be randomized across six centers. The primary endpoint is the 1-year incidence of SREs. Secondary endpoints include time to first SRE, pain assessment, quality of life, survival, Traditional Chinese Medicine symptom score, and bone turnover markers. Safety will be assessed according to Common Terminology Criteria for Adverse Events v5.0. Alongside the trial protocol, this manuscript presents currently available descriptive observations from a representative participant. During follow-up, no SREs were documented, and serial Electron Computed Tomography (ECT) examinations showed no obvious new imaging abnormalities suggestive of bone progression or SRE-related change. Functional status, symptom burden, and tumor markers showed favorable changes over time, while routine safety assessments remained generally stable, with no adverse events or serious adverse events recorded. These observations are descriptive only and should be interpreted with caution. Definitive conclusions regarding efficacy and safety will require completion of the protocol-defined follow-up and formal analysis of the full study cohort.

N6-methyladenosine (m⁶A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA) and serves as a key regulator of post-transcriptional gene expression. Methods for investigating m⁶A can be applied at multiple levels of resolution, including global quantification, nucleotide-specific detection, and analysis of particular transcripts of interest. Among these, the dot blot assay provides a straightforward, rapid, and cost-effective approach for semi-quantitative evaluation of global m⁶A modification levels. In this assay, cells are first processed to extract total RNA, which is then diluted and denatured. The RNA samples are spotted onto a nylon membrane, probed with an anti-m⁶A antibody, and visualized by chemiluminescence, with signal intensity indicating the relative abundance of methylation. Finally, quantification and comparison of signal intensities are performed by measuring grayscale values with ImageJ. Compared with sequencing or mass spectrometry-based methods, dot blot requires minimal instrumentation and technical expertise, making it particularly suited for routine screening, preliminary functional studies, and comparative analyses. This protocol provides a detailed and reproducible workflow for implementing the m⁶A dot blot assay in both basic research and translational applications.

Osteosarcoma is the most prevalent primary malignant bone tumor in children and adolescents. Despite advancements in adjuvant chemotherapy and surgical techniques, the overall survival rate of osteosarcoma patients remains suboptimal, particularly in cases of metastatic or recurrent disease. Programmed cell death (PCD), a highly regulated process that includes apoptosis, autophagy, ferroptosis, pyroptosis, and necroptosis, plays a pivotal role in determining cellular fate (survival and death). In osteosarcoma, PCD dysregulation enables malignant cells to evade cell death signals, thereby accelerating tumor development. Natural products derived from various medicinal plants and dietary components exhibit promising potential for osteosarcoma treatment by regulating PCD through different mechanisms. With advantages such as low cost, minimal side effects, and wide availability, natural products have attracted considerable attention for translational research. This review systematically synthesizes current knowledge on PCD subtypes in osteosarcoma and elucidates the molecular mechanisms by which natural products regulate PCD to inhibit the development of osteosarcoma. By integrating these insights, we aim to offer novel perspectives for developing targeted therapeutic strategies and improving clinical outcomes in osteosarcoma patients.

Background Bone metastasis is a major cause of morbidity and disease progression in breast cancer, but the cellular sources of patient-level metastatic risk programs remain incompletely defined. We aimed to connect bulk transcriptomic risk signals with specific microenvironmental cell states in breast cancer bone metastasis. Methods We integrated three public bulk transcriptomic cohorts to construct a bone metastasis-free survival (BMFS) risk model and evaluated it in an external validation cohort. The resulting risk program was projected onto single-cell RNA-sequencing data comprising 37,001 cells from 13 breast cancer bone-metastatic lesions. Cell-type scoring, perivascular stromal subclustering, ligand–receptor inference, functional module scoring and pseudotime analysis were used to characterize stromal states associated with osteoclast programs. Cox regression, Kaplan–Meier analysis, time-dependent receiver operating characteristic analysis and sample-level correlation testing were used to evaluate clinical and cross-lesion associations. Results The Cox-based model stratified BMFS in the training cohort and significantly stratified BMFS in the external validation cohort, although discriminatory performance was more moderate in the latter. Single-cell projection mapped high-risk program activity and prominent inferred outgoing communication to perivascular stromal cells. Reclustering identified an extracellular matrix (ECM)-remodeling stromal subcluster, C1, characterized by ECM organization, ossification-related pathways and matrix- and bone-associated regulon activity. C1 abundance correlated with osteoclast abundance across lesions, whereas other perivascular stromal states showed no comparable association. C1-associated ligand scores were positively associated with osteoclast functional programs. Ligand–receptor inference, partial correlation and in silico network-sensitivity analysis prioritized ITGAV within a candidate ECM–osteoclast interaction. When projected into the integrated bulk training cohort, higher C1 ligand and osteoclast function signature scores were associated with shorter BMFS. Conclusions This integrative analysis identifies an ECM-remodeling perivascular stromal state associated with osteoclast functional programs in breast cancer bone metastasis. These findings provide a clinically anchored framework for investigating stromal–osteoclast organization and prioritize an ECM–ITGAV interaction for subsequent spatial and functional validation.

RNA epitranscriptomic modification plays a critical role in the initiation and development of various cancers, among which N6-methyladenosine (m6A) is the most prevalent and functionally diverse modification. Here, we present a streamlined and reproducible protocol using MeRIP-qPCR to assess m6A modification levels of specific transcripts in osteosarcoma cells. In this assay, total RNA was extracted, followed by incubation with a specific anti-m6A antibody to immunocapture RNA of m6A modification. Then, RNA was fragmented, and enrichment of m6A-modified RNA fragments was achieved using protein G magnetic beads. The enriched RNA was released by proteinase K digestion and purified by RNA-binding beads. Lastly, RNA was subjected to reverse transcription and quantitative polymerase chain reaction (PCR) to assess m6A levels in candidate genes. The results have demonstrated that this method effectively distinguishes methylation differences and provides a reliable tool for exploring the functional mechanisms of m6A modification in osteosarcoma cells. In conclusion, MeRIP-qPCR offers a simple and highly specific approach for studying m6A modification in osteosarcoma cells. This protocol holds great potential for elucidating m6A regulatory networks and identifying novel therapeutic targets.

BACKGROUND:Acquired lymphedema is a global health concern with limited treatment options. While vascular endothelial growth factor C (VEGF-C) administration has shown promise for the treatment of this patient population, no small-molecule compounds have hitherto been identified to improve lymphedema by stimulating VEGF-C expression and lymphangiogenesis. OBJECTIVE:This study investigated the therapeutic effect of notoginsenoside R1 (R1) on a mouse model of tail acquired lymphedema and explored the underlying mechanisms. METHODS:C57BL/6J mice and lymphatic endothelial cells (LECs) specific VEGFR-3 knockout transgenic mice underwent surgical induction of tail acquired lymphedema. Tail circumference, lymphatic drainage function, VEGF-C expression, and lymphangiogenesis were measured. LECs' function was assessed using wound healing and tube formation assays. Quantitative PCR (q-PCR) and western blot were conducted to measure VEGF-C expression levels. In addition, RNA sequencing analysis and western blot were performed to elucidate the signal pathways involved. Luciferase reporter assays assessed VEGF-C promoter activity. RESULTS:R1 treatment improved lymphedema, lymphatic function, and lymphangiogenesis in the mouse model. R1 enhanced migration, tube formation, and VEGF-C expression of LECs. These effects were abolished by VEGF-C siRNA and VEGFR-3 inhibitors. VEGFR3 knockout in LECs completely blocked R1's ability to promote lymphangiogenesis and lymphatic drainage while partially but significantly reducing its improvement on lymphedema. R1 activated the cAMP/PKA signaling pathway, leading to PKA and CREB phosphorylation. The PKA inhibitor and CREB siRNA inhibited R1-induced VEGF-C expression. Additionally, R1 activated VEGF-C promoter activity in a CREB-dependent manner. CONCLUSION:R1 emerges as the first reported small natural compound to promote VEGF-C expression. It reduces acquired lymphedema and enhances lymphangiogenesis via the cAMP/PKA/CREB signaling pathway. These findings suggest R1 as a potential novel oral medication for treating acquired lymphedema patients.

Mesenchymal stem cells (MSCs) are a population of stem cells that can self-renew and differentiate into multiple cell lineages. Among them, MSCs found in bone marrow, known as bone marrow mesenchymal stem cells (BMSCs), play a crucial role in osteogenesis, hematopoietic support, homing/migration, and immunosuppression. BMSCs are widely used in cell biology and tissue engineering research due to their potential for cellular therapeutic strategies, as well as their easy availability, genetic stability, and low immunogenicity. Hematopoietic stem cells (HSCs) and BMSCs coexist in bone marrow but have distinct progenitor cells. When extracting cells from bone marrow, multiple cell types are typically obtained, which can lead to heterogeneity in the cell population, potentially causing bias and not meeting high experimental standards. Therefore, it is crucial to develop effective techniques to improve the homogeneity and yield of BMSCs in vitro. This study presents a detailed protocol for the purification, expansion, and flow cytometry-based phenotyping of BMSCs obtained from BALB/c mice. The homogeneity and stem cell phenotype of third-passage BMSCs were characterized using flow cytometry, demonstrating positivity for CD29, CD44, and Sca-1, negativity for CD45, and CD31, and confirming their osteogenic and adipogenic differentiation ability. This cost-effective protocol provides an easy and efficient method to obtain a sufficient number of homogeneous BMSCs with high proliferation and differentiation potential, which is essential for advancing research in mouse models and extensive preclinical studies.

Objective: Osteosarcoma is a highly aggressive primary bone tumor that predominantly affects pediatric and adolescent populations. This study aims to explore the therapeutic potential and underlying mechanisms of melittin in treating osteosarcoma both in vitro and in vivo. Materials and methods: Two osteosarcoma cell lines, namely 143B and U-2 OS, were utilized to assess the impact of melittin on cellular proliferation, apoptosis, and cell cycle progression. The effect of melittin on cell viability was evaluated using the CCK-8 assay. Flow cytometry was employed to assess apoptosis and cell cycle progression, while Western blotting analyzed the expression of key proteins, including Cdc-2, c-Myc, and ꞵ-catenin. Specifically, we employed small interfering RNA (siRNA) to selectively knock down the expression of β-catenin in two osteosarcoma cell lines. In vivo studies, tumor growth in nude mice was evaluated through measurements of tumor weight, volume. Additionally, immunohistochemical analysis was performed to assess Ki-67 and active ꞵ-catenin expression in tumor tissues. Results: Melittin induced apoptosis in 143B and U-2 OS osteosarcoma cells in a concentration-dependent manner and caused S-phase cell cycle arrest as the drug concentration increased. Mechanistic investigations revealed that melittin's efficacy was associated with the inactivation of the Wnt/ꞵ-catenin signaling pathway. Specifically, melittin treatment reduced the expression of phosphorylated glycogen synthase kinase-3 beta (p-GSK-3ꞵ) and active ꞵ-catenin. Notably, silencing β-catenin in 143B and U-2 OS osteosarcoma cells significantly enhanced the anti-proliferative and pro-apoptotic effects of melittin. This was evidenced by reduced cell viability and increased apoptosis, thereby further confirming that the anti-tumor efficacy of melittin is dependent on the inhibition of the Wnt/β-catenin signaling pathway. In vivo studies confirmed that melittin significantly inhibited the growth of subcutaneously implanted tumors in nude mice. This inhibition was further supported by Ki-67 analyse and H&E staining, while immunohistochemical analysis revealed an obvious reduction in active ꞵ-catenin expression. Conclusions: Our results offer deeper mechanistic insights into the inhibitory impact of melittin on osteosarcoma progression, at least in part by suppressing the Wnt/β-catenin signaling pathway. We expand upon previous research by providing more comprehensive and robust evidences that underscore the potential of melittin as a viable and safe therapeutic agent for osteosarcoma.

Osteosarcoma is the most common bone malignancy in children and adolescents with a less than 30% overall survival rate for those with metastatic disease, particularly pulmonary metastases. Drug resistance hinders the effectiveness of current chemotherapies, making osteosarcoma a leading cause of mortality and an urgent requirement of new therapeutics in this population. Here, we developed a triptolide (TP)-loaded, pH-responsive and near-infrared light-activated nanoplatform (TP-TPBC-PEG) with photodynamic therapy (PDT) to target osteosarcoma cells both in vitro and in vivo. Our results demonstrated that PDT and chemotherapy of TP-TPBC-PEG synergistically reduced cell viability, colony proliferation, migration, and invasion in vitro, and inhibited osteosarcoma growth and pulmonary metastasis in vivo which the nano-micelles were intravenously injected to intratibia injection induced osteosarcoma mouse models, showing enhanced osteosarcoma cell killing by nanoparticle inflating in response to acidic endosomal pH and sensitized osteosarcoma cells to TP chemotherapy. Importantly, the nano-micelles did not exhibit organ toxicity in vivo. Dual-luciferase reporter gene and nuclear/ cytoplasm protein expression assays identified the involvement of HIPPO signaling pathway in mediating these effects. Overall, our study provides a promising therapeutic approach for treating primary osteosarcoma and preventing pulmonary metastases by activating the HIPPO signaling pathway.

OBJECTIVE:To investigate the synergistic effects of polyphyllin I(PPI)combined with tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)on the growth of osteosarcoma cells through downregulating the Wnt/β-catenin signaling pathway. METHODS:Cell viability,apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays.The morphology of cancer cells was observed with inverted phase contrast microscope.The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays.The expressions of poly(adenosine diphosphate-ribose)polymerase,C-Myc,Cyclin B1,cyclin-dependent kinases 1,N-cadherin,Vimentin,Active-β-catenin,β-catenin,p-glycogen synthase kinase 3β(GSK-3β)and GSK-3β were determined by Western blotting assay. RESULTS:PPI sensitized TRAIL-induced decrease of viability,migration and invasion,as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells.The synergistic effect of PPI with TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/β-catenin signaling pathway. CONCLUSION:The combination of PPI and TRAIL is potentially a novel treatment strategy of osteosarcoma.

Background: Osteosarcoma is the most common primary bone cancer in children and adolescents with high metastatic ability. Aim: This study aimed to explore the inhibitory effects of (S)-10-hydroxycamptothecin (HCPT) on osteosarcoma cell growth and metastasis as well as the underlying mechanism. Methods: The osteosarcoma cells of 143B and U-2 OS (U-2), treated with HCPT (20, 100, or 300 nM), underwent detections, such as CCK-8, flow cytometry, Transwell, wound healing, and immunoblotting. EMT-related key proteins, like N-cadherin, Snail, and Vimentin, were found to be down-regulated, while E-cadherin was up-regulated dose-dependently in HCPT-exposed 143B and U-2 cells. Additionally, incubation of 143B and U-2 cells with HCPT for 3 hours dosedependently reduced the expression ratios of p-LATS1/LATS1, p-MST1/MST1, p-YAP/YAP, and p-TAZ/TAZ. Results: Taken together, our study has demonstrated HCPT to inhibit osteosarcoma growth and metastasis potentially by activating the HIPPO signaling pathway and reversing EMT. Conclusion: HCPT might be a candidate agent for the prevention and treatment of osteosarcoma

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by trans-oral administration, which is characterized by quick effect, easy absorption, and individualized treatment based on the specific syndromes of patients. The quality of the decoction formula is directly responsible for the clinical efficacy of traditional Chinese medicine; therefore, the standardization process of the decoction formula is important to avoid differences in decoction quality caused by subjective factors. Meanwhile, due to the limitations of performing clinical experiments, small animals bearing human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions, including the decoction formula for traditional Chinese medicine. Consequently, as an important trans-oral administration method, the skilled gavage technique is particularly important to avoid potential esophagus damage and drug spillage, which will ensure an equal amount of medicine being administered to each model animal, leading to accurate experimental results. Furthermore, the standardized method of decoction formula preparation and skilled gavage strategy are necessary to protect animal welfare and minimize the number of animals used. Here, we reported a detailed standardization process of the decoction formula and gavage technique with Yiqi Jiedu decoction in osteosarcoma mouse model as an example. The efficacy was evaluated by the tumor volume. This protocol will maximize animal protection and improve the reliability of research data, therefore providing effective strategies for future investigating therapeutic efficacy and molecular mechanisms of decoction formula for traditional Chinese medicine in vivo.

Osteosarcoma is the most common malignant bone tumor and is frequently diagnosed in juvenile. Cellular senescence is a fundamental hallmark of osteosarcoma and plays a vital role in the initiation and progression of aging and tumorigenesis. Long non-coding RNAs (lncRNAs) are implicated in tumorigenesis. In this study, six cellular senescence-related lncRNAs with independent prognostic significance in juvenile osteosarcoma patients were identified through univariate Cox regression analysis, least absolute shrinkage and selection operator (LASSO) regression analysis, and multivariate Cox regression analysis. Prognostic significance was further confirmed by Kaplan–Meier (KM) survival curves, co-expression interaction networks, and sankey diagrams. A prognostic model of cellular senescence-related genes in juvenile osteosarcoma patients was then constructed using multivariate Cox regression analysis based on these six genes. High- and low-risk groups were identified according to the median risk score calculated by the prognostic model. The favorable prognostic significance of this model was demonstrated through survival curves, receiver operating characteristic (ROC) curves, distribution scatter plots and lncRNA expression heatmaps. Furthermore, cellular senescence-related lncRNAs were validated by enrichment analysis, immunological correlation analysis, m6A correlation analysis, and drug sensitivity correlation analysis. These findings are important for improving the prognosis of juvenile osteosarcoma patients and understanding the mechanisms underlying cellular senescence in juvenile osteosarcoma development.

Background and study aimsThe prognosis of colorectal cancer (CRC) is related to natural killer (NK) cells, but the molecular subtype features of CRC based on NK cells are still unknown. This study aimed to identify NK cell-related molecular subtypes of CRC and analyze the survival status and immune landscape of patients with different subtypes.Patients/material and methodsmRNA expression data, single nucleotide variant (SNV) data, and clinical information of CRC patients were obtained from The Cancer Genome Atlas. Differentially expressed genes (DEGs) were obtained through differential analysis, and the intersection was taken with NK cell-associated genes to obtain 103 NK cell-associated CRC DEGs (NCDEGs). Based on NCDEGs, CRC samples were divided into three clusters through unsupervised clustering analysis. Survival analysis, immune analysis, Gene Set Enrichment Analysis (GSEA), and tumor mutation burden (TMB) analysis were performed. Finally, NCDEG-related small-molecule drugs were screened using the CMap database.ResultsSurvival analysis revealed that cluster2 had a lower survival rate than cluster1 and cluster3 (p < 0.05). Immune infiltration analysis found that the immune infiltration levels and immune checkpoint expression levels of cluster1_3 were substantially higher than those of cluster2, and the tumor purity was the opposite (p < 0.05). GSEA presented that cluster1_3 was significantly enriched in the chemokine signaling pathway, ECM receptor interaction, and antigen processing and presentation pathways (p < 0.05). The TMB of cluster1_3 was significantly higher than that of cluster2 (p < 0.05). Genes with the highest mutation rate in CRC were APC, TP53, TTN, and KRAS. Drug prediction results showed that small-molecule drugs that reverse the upregulation of NCDEGs, deoxycholic acid, dipivefrine, phenformin, and other drugs may improve the prognosis of CRC.ConclusionNK cell-associated CRC subtypes can be used to evaluate the tumor characteristics of CRC patients and provide an important reference for CRC patients.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patientderived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.