Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues.

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.

Functional and oncological outcomes of patients with proximal humerus osteosarcoma managed by limb salvage.

BACKGROUND: Osteosarcoma is the most common primary bone malignancy in skeletally immature patients. The proximal humerus is the third most common site of osteosarcoma. The literature shows a paucity of published data concerning the outcome of proximal humerus osteosarcoma managed by limb salvage. The purpose of this study was to answer the following questions: (1) do patients with proximal humerus osteosarcoma managed by limb salvage and neoadjuvant chemotherapy show good functional and oncological outcomes, and (2) are there any prognostic factors that are associated with better oncological and functional outcomes? MATERIALS AND METHODS: The study was a retrospective case series study assessing the overall outcome of 34 patients with proximal humerus osteosarcoma. Eighteen patients were males (53%) while 16 were females. Biological reconstruction was done in 15 patients (44%), while nonbiological reconstruction was done in 19 patients. Resections were mainly intraarticular (82%). Functional outcome was assessed using the Musculoskeletal Tumor Society (MSTS) score, while oncological outcome was assessed based on local recurrence and development of chest metastasis. Comparisons between quantitative variables were done using the nonparametric Mann-Whitney test. To compare categorical data, the chi-square (χ2) test was performed. The exact test was used instead when the expected frequency was less than 5. Correlations between quantitative variables were examined using the Spearman correlation coefficient. RESULTS: The mean MSTS score was 25.5 (range 23-29). A younger age was statistically correlated with a poorer MSTS score (P = 0.0016). Six patients out of 34 (17.6%) had local recurrence and four of them (67%) were treated by forequarter amputation. 41% of patients developed chest metastasis, and the majority of them were treated by chemotherapy (71%). In comparison with patients with osteosarcoma at other sites who were also managed in our institution, proximal humerus osteosarcoma patients showed higher incidence rates of local recurrence and chest metastasis along with lower 5-year patient and limb survivorships compared to distal femur, proximal tibia and proximal femur osteosarcoma patients. CONCLUSION: Treatment of osteosarcoma of the proximal humerus by limb salvage and chemotherapy yields a good functional outcome. The method of reconstruction does not impact the resultant function. The 5-year survivorship of these patients is 65%. Younger patients have a better oncological outcome and an inferior functional outcome. LEVEL OF EVIDENCE: Level IV therapeutic study.

CircPRMT5 promotes progression of osteosarcoma by recruiting CNBP to regulate the translation and stability of CDK6 mRNA.

Research has demonstrated that circular RNAs (circRNAs) exert critical functions in the occurrence and progression of numerous malignant tumors. CircPRMT5 was recently reported to be involved in the pathogenesis of cancers. However, the potential role of circPRMT5 in osteosarcoma needs further investigation. In present study, our results suggested that circPRMT5 was highly upregulated in osteosarcoma cells and mainly localizes in the cytoplasm. CircPRMT5 promoted the proliferation, migration and invasion capacities of osteosarcoma cells, and suppressed cell apoptosis. Knockdown of circPRMT5 exerted the opposite effects. Mechanically, circPRMT5 promoted the binding of CNBP to CDK6 mRNA, which enhanced the stability of CDK6 mRNA and facilitated its translation, thereby promoting the progression of osteosarcoma. Knockdown of CDK6 reversed the promoting effect of circPRMT5 on osteosarcoma cells. These findings suggest that circPRMT5 promotes osteosarcoma cell malignant activity by recruiting CNBP to regulate the translation and stability of CDK6 mRNA. Thus, circPRMT5 may represent a promising therapeutic target for osteosarcoma.

Identification of allograft inflammatory factor-1 suppressing the progression and indicating good prognosis of osteosarcoma.

BACKGROUND: Osteosarcoma is one of the most common cancers worldwide. Intense efforts have been made to elucidate the pathogeny, but the mechanisms of osteosarcoma are still not well understood. We aimed to investigate the potential biomarker, allograft inflammatory factor-1 (AIF1), affecting the progression and prognosis of osteosarcoma. METHODS: Three microarray datasets were downloaded from GEO datasets and one was obtained from the TCGA dataset. The differentially expressed genes (DEGs) were identified. GO and KEGG functional enrichment analyses of overlapped DEGs were performed. The PPI network of overlapped DEGs was constructed by STRING and visualized with Cytoscape. Overall survival (OS) and Metastasis free survival (MFS) were analyzed from GSE21257. Finally, the effect of the most relevant core gene affecting the progression of osteosarcoma was examined in vitro. RESULTS: One hundred twenty six DEGs were identified, consisting of 65 upregulated and 61 downregulated genes. Only AIF1 was significantly associated with OS and MFS. It was found that AIF1 could be enriched into the NF-κB signaling pathway. GSEA and ssGSEA analyses showed that AIF1 was associated with the immune invasion of tumors. Cell experiments showed that AIF1 was underexpressed in osteosarcoma cell lines, while the malignant propriety was attenuated after overexpressing the expression of AIF1. Moreover, AIF1 also affects the expression of the NF-κB pathway. CONCLUSION: In conclusion, DEGs and hub genes identified in the present study help us understand the molecular mechanisms underlying the carcinogenesis and progression of osteosarcoma, and provide candidate targets for diagnosis and treatment of osteosarcoma.

Combined SET7/9 and CDK4 inhibition act synergistically against osteosarcoma.

Osteosarcoma is the most common malignant bone tumor. It has a poor prognosis because of a lack of therapeutic targets and strategies. The SET domain-containing lysine-specific methyltransferase, SET7/9, has various functions in different cancer types in tissue-type and signaling context-dependent manners. The role of SET7/9 in osteosarcoma cells is currently controversial and its potential as a therapeutic candidate in osteosarcoma is unknown. In the present study, SET7/9 inhibition or ablation suppressed osteosarcoma cell proliferation by causing G1 arrest. Mechanistically, SET7/9 inhibition disrupted the interaction between cyclin-dependent kinase 4 (CDK4) and cyclin D1, which affected CDK4-cyclin D1 complex function, leading to decreased phosphorylation of retinoblastoma protein. CDK4 was overexpressed in osteosarcoma tissues and was closely related to a poor prognosis in patients with osteosarcoma. We therefore hypothesized that SET7/9 inhibition might increase the sensitivity of osteosarcoma cells to CDK4 inhibitors, potentially decreasing the risk of adverse effects of CDK4 inhibitors. The combination of SET7/9 and CDK4 inhibition enabled dose reductions of both inhibitors and had a synergistic effect against osteosarcoma growth in vivo. Collectively, these findings indicate that SET7/9 plays an oncogenic role in osteosarcoma by regulating CDK4-cyclin D1 complex interaction and function. The combination of SET7/9 and CDK4 inhibition may thus provide a novel effective therapeutic strategy for osteosarcoma with no significant toxicity.

Proximal Humerus Reconstruction for Bone Sarcomas: A Critical Analysis.

¼ The proximal humerus is a common location for primary bone tumors, and the goal of surgical care is to obtain a negative margin resection and subsequent reconstruction of the proximal humerus to allow for shoulder function.¼ The current evidence supports the use of reverse total shoulder arthroplasty over hemiarthroplasty when reconstructing the proximal humerus after resection of a bone sarcoma if the axillary nerve can be preserved.¼ There is a lack of high-quality data comparing allograft prosthetic composite (APC) with endoprosthetic reconstruction of the proximal humerus.¼ Reverse APC should be performed using an allograft with donor rotator cuff to allow for soft-tissue repair of the donor and host rotator cuff, leading to improvements in shoulder motion compared with an endoprosthesis.

A deep learning model for accurately predicting cancer-specific survival in patients with primary bone sarcoma of the extremity: a population-based study

Purpose Primary bone and joint sarcomas of the long bone are relatively rare neoplasms with poor prognosis. An efficient clinical tool that can accurately predict patient prognosis is not available. The current study aimed to use deep learning algorithms to develop a prediction model for the prognosis of patients with long bone sarcoma. Methods Data of patients with long bone sarcoma in the extremities was collected from the Surveillance, Epidemiology, and End Results Program database from 2004 to 2014. Univariate and multivariate analyses were performed to select possible prediction features. DeepSurv, a deep learning model, was constructed for predicting cancer-specific survival rates. In addition, the classical cox proportional hazards model was established for comparison. The predictive accuracy of our models was assessed using the C-index, Integrated Brier Score, receiver operating characteristic curve, and calibration curve. Results Age, tumor extension, histological grade, tumor size, surgery, and distant metastasis were associated with cancer-specific survival in patients with long bone sarcoma. According to loss function values, our models converged successfully and effectively learned the survival data of the training cohort. Based on the C-index, area under the curve, calibration curve, and Integrated Brier Score, the deep learning model was more accurate and flexible in predicting survival rates than the cox proportional hazards model. Conclusion A deep learning model for predicting the survival probability of patients with long bone sarcoma was constructed and validated. It is more accurate and flexible in predicting prognosis than the classical CoxPH model (AU)

USP3 promotes osteosarcoma progression via deubiquitinating EPHA2 and activating the PI3K/AKT signaling pathway.

Ubiquitin-specific protease 3 (USP3) plays an important role in the progression of various tumors. However, the role of USP3 in osteosarcoma (OS) remains poorly understood. The aim of this study was to explore the biological function of USP3 in OS and the underlying molecular mechanism. We found that OS had higher USP3 expression compared with that of normal bone tissue, and high expression of USP3 was associated with poor prognosis in patients with OS. Overexpression of USP3 significantly increased OS cell proliferation, migration, and invasion. Mechanistically, USP3 led to the activation of the PI3K/AKT signaling pathway in OS by binding to EPHA2 and then reducing its protein degradation. Notably, the truncation mutant USP3-F2 (159-520) interacted with EPHA2, and amino acid 203 was found to play an important role in this process. And knockdown of EPHA2 expression reversed the pro-tumour effects of USP3-upregulating. Thus, our study indicates the USP3/EPHA2 axis may be a novel potential target for OS treatment.

Protein Stability Regulation in Osteosarcoma: The Ubiquitin-like Modifications and Glycosylation as Mediators of Tumor Growth and as Targets for Therapy.

The identification of new therapeutic targets and the development of innovative therapeutic approaches are the most important challenges for osteosarcoma treatment. In fact, despite being relatively rare, recurrence and metastatic potential, particularly to the lungs, make osteosarcoma a deadly form of cancer. In fact, although current treatments, including surgery and chemotherapy, have improved survival rates, the disease's recurrence and metastasis are still unresolved complications. Insights for analyzing the still unclear molecular mechanisms of osteosarcoma development, and for finding new therapeutic targets, may arise from the study of post-translational protein modifications. Indeed, they can influence and alter protein structure, stability and function, and cellular interactions. Among all the post-translational modifications, ubiquitin-like modifications (ubiquitination, deubiquitination, SUMOylation, and NEDDylation), as well as glycosylation, are the most important for regulating protein stability, which is frequently altered in cancers including osteosarcoma. This review summarizes the relevance of ubiquitin-like modifications and glycosylation in osteosarcoma progression, providing an overview of protein stability regulation, as well as highlighting the molecular mediators of these processes in the context of osteosarcoma and their possible targeting for much-needed novel therapy.

Identification of an early survival prognostic gene signature for localized osteosarcoma patients.

Osteosarcoma is the most prevalent bone tumor in pediatric patients. Neoadjuvant chemotherapy has improved osteosarcoma patient survival, however the 5-year survival rate for localized osteosarcoma is 75% with a 30-50% recurrence rate. We, therefore, sought to identify a prognostic gene signature which could predict poor prognosis in localized osteosarcoma patients. Using the TARGET osteosarcoma transcriptomic dataset, we identified a 13-hub gene signature associated with overall survival and time to death of localized osteosarcoma patients, with the high-risk group showing a 22% and the low-risk group showing 100% overall survival. Furthermore, network analysis identified five modules of co-expressed genes that significantly correlated with survival, and identified 65 pathways enriched across 3 modules, including Hedgehog signaling, which includes 2 of the 13 genes, IHH and GLI1. Subsequently, we demonstrated that GLI antagonists inhibited growth of a recurrent localized PDX-derived cell line with elevated IHH and GLI1 expression, but not a non-relapsed cell line with low pathway activation. Finally, we show that our signature outperforms previously reported signatures in predicting poor prognosis and death within 3 years in patients with localized osteosarcoma.

Osteosarcoma cells exhibit functional interactions with stromal cells, fostering a lung microenvironment conducive to the establishment of metastatic tumor cells.

BACKGROUND: Osteosarcoma (OS) stands out as the most common bone tumor, with approximately 20% of the patients receiving a diagnosis of metastatic OS at their initial assessment. A significant challenge lies in the frequent existence of undetected metastases during the initial diagnosis. Mesenchymal stem cells (MSCs) possess unique abilities that facilitate tumor growth, and their interaction with OS cells is crucial for metastatic spread. METHODS AND RESULTS: We demonstrated that, in vitro, MSCs exhibited a heightened migration response toward the secretome of non-metastatic OS cells. When challenged to a secretome derived from lungs preloaded with OS cells, MSCs exhibited greater migration toward lungs colonized with metastatic OS cells. Moreover, in vivo, MSCs displayed preferential migratory and homing behavior toward lungs colonized by metastatic OS cells. Metastatic OS cells, in turn, demonstrated an increased migratory response to the MSCs' secretome. This behavior was associated with heightened cathepsin D (CTSD) expression and the release of active metalloproteinase 2 (MMP2) by metastatic OS cells. CONCLUSIONS: Our assessment focused on two complementary tumor capabilities crucial to metastatic spread, emphasizing the significance of inherent cell features. The findings underscore the pivotal role of signaling integration within the niche, with a complex interplay of migratory responses among established OS cells in the lungs, prometastatic OS cells in the primary tumor, and circulating MSCs. Pulmonary metastases continue to be a significant factor contributing to OS mortality. Understanding these mechanisms and identifying differentially expressed genes is essential for pinpointing markers and targets to manage metastatic spread and improve outcomes for patients with OS.

Targeting PERK-ATF4-P21 axis enhances the sensitivity of osteosarcoma HOS cells to Mppα-PDT.

Osteosarcoma (OS) is the most prevalent type of malignant bone tumor in adolescents. The overall survival of OS patients has reached a plateau recently. Thus, there is an urgent need to develop approaches to improve the sensitivity of OS to therapies. Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPα-PDT) is a new type of tumor therapy, and elucidating its mechanism is helpful to improve its anti-tumor efficacy. Here, we investigated how PERK signaling promotes the human OS (HOS) cell survival induced by MPPα-PDT, as overcoming this may enhance sensitivity to MPPα-PDT. We found that MPPα-PDT combined with PERK inhibitor GSK2656157 enhanced HOS cell apoptosis by suppressing autophagy and p21. Autophagy inhibition and p21 depletion enhanced cell death, indicating pro-survival effects in MPPα-PDT. Notably, p21 was found to be an effector of the PERK-Atf4 pathway, which could positively regulate autophagy mediated by MPPα-PDT. In conclusion, we found that the combination of MPPα-PDT and GSK2656157 enhanced apoptosis in HOS cells by inhibiting autophagy. Mechanistically, this autophagy is p21-dependent and can be suppressed by GSK2656157, thereby enhancing sensitivity to MPPα-PDT.

Can conventional magnetic resonance imaging at presentation predict chemoresistance in osteosarcoma?

OBJECTIVES: Histological tumour necrosis is the current indicator for the response of osteosarcoma after neoadjuvant chemotherapy. Chemoresistant tumours require close monitoring and adjustment of treatment. Characteristics of tumours on baseline MRI may be able to predict response to chemotherapy. The aim is to identify which baseline MRI findings can help predict chemoresistant osteosarcoma. METHODS: Baseline MRI before giving neoadjuvant chemotherapy of 95 patients during 2008-2021 was reviewed by 2 musculoskeletal radiologists. Histological necrosis from surgical specimens was the reference standard. MRIs were reviewed for tumour characteristics (tumour volume, maximum axial diameter, central necrosis, haemorrhage, fluid-fluid level), peritumoural bone and soft tissue oedema, and other parameters including intra-articular extension, epiphyseal involvement, neurovascular involvement, pathologic fracture, and skip metastasis. The cut-off thresholds were generated by receiver operating characteristic curves which then tested for diagnostic accuracy. RESULTS: Two-third of patients were chemoresistance (histological necrosis <90%). Tumour volume >150 mL, maximum axial diameter >7.0 cm, area of necrosis >50%, presence of intra-articular extension, and peritumoural soft tissue oedema >6.5 cm significantly predicted chemoresistance, particularly when found in combination. Tumour volume >150 mL and maximum axial diameter >7.0 cm could be used as an independent predictor (multivariable analysis, P-value = .025, .045). CONCLUSIONS: Findings on baseline MRI could help predicting chemoresistant osteosarcoma with tumour size being the strongest predictor. ADVANCES IN KNOWLEDGE: Osteosarcomas with large size, large cross-sectional diameter, large area of necrosis, presence of intra-articular extension, and extensive peritumoural soft tissue oedema were most likely to have a poor response to neoadjuvant chemotherapy.

Dipsacus Asperoides-Derived Exosomes-Like Nanoparticles Inhibit the Progression of Osteosarcoma via Activating P38/JNK Signaling Pathway.

Introduction: Osteosarcoma is a prevalent and highly malignant primary bone tumor. However, current clinical therapeutic drugs for osteosarcoma are not suitable for long-term use due to significant side effects. Therefore, there is an urgent need to develop new drugs with fewer side effects. Dipsacus asperoides C. Y. Cheng et T. M. Ai, a traditional Chinese medicine, is commonly used for its anti-inflammatory, anti-pain, bone fracture healing, and anti-tumor effects. In this study, we investigated the effects of exosome-like nanoparticles derived from Dipsacus asperoides (DAELNs) on osteosarcoma cells in vitro and in vivo. Methods: DAELNs were isolated and purified from Dipsacus asperoides and their physical and chemical properties were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The cellular uptake of DAELNs in osteosarcoma cells was analyzed by PKH26 staining. The proliferation, invasion, migration, and apoptosis of osteosarcoma cells were assessed using CCK8 assay, EdU assay, colony-formation assay, transwell assay, wound healing assay, and mitochondrial membrane potential measurement, respectively. The regulatory mechanism of DAELNs inhibiting the progression of osteosarcoma via activating P38/JNK signaling pathway was investigated using Western blotting and immunohistochemistry. Moreover, the therapeutic effects of DAELNs were evaluated using in vivo small animal imaging assay, HE staining, and immunohistochemistry. Results: Our results showed that DAELNs inhibited the proliferation, invasion, migration, and fostered the apoptosis of osteosarcoma cells in vitro and suppressed the tumor growth of osteosarcoma cells in a xenograft nude mouse model. Furthermore, the bio-distribution of DiD-labeled DAELNs showed preferential targeting of osteosarcoma tumors and excellent biosafety in histological analysis of the liver and kidney. Mechanistically, DAELNs activated the P38/JNK signaling pathway-induced apoptosis. Conclusion: Taken together, DAELNs are novel, natural, and osteosarcoma-targeted agents that can serve as safe and effective therapeutic approaches for the treatment of osteosarcoma.

Comparative Analysis of Skip Metastasis in Pediatric Osteosarcoma: Clinical Features and Outcomes.

BACKGROUND: Skip metastasis (SM) is a synchronous regional bone metastasis. Using new imaging modalities, the detection of SM is easier and possibly more common. We reviewed patients with SM and compared their characteristics and outcomes to other patients with osteosarcoma treated at our center. METHODS: We reviewed retrospectively children (<18 years) with newly diagnosed osteosarcoma who presented from June 2006 to March 2022. Patients' characteristics, treatment modalities, and outcomes were analyzed. All cases were discussed in a multidisciplinary clinic that included 2 experienced radiologists. RESULTS: We identified 155 patients with osteosarcoma, among which 13 (8.3%) patients had SM detected by MRI. Patients with SM had a median age at diagnosis of 11.2 years (range 7 to 17). Three patients had lung metastasis at diagnosis. Bone scan was positive for the SM in 8 patients (62%). All patients underwent primary tumor resection after neoadjuvant chemotherapy (amputation in 5, limb salvage surgery in 8). Five had postchemotherapy necrosis ≥90% in primary tumor. Seven patients relapsed/progressed (1 local and 6 in the lung), all relapsed patients died of disease. Compared to the rest of the patients, those with SM had similar clinical features to patients without SM; outcomes were similar with no significant differences in event-free survival and overall survival ( P =0.7 and 0.3, respectively). CONCLUSION: In this study, we observed a percentage of patients with SM comparable to previous reports. Patients with SM exhibited clinical features akin to the rest of our patients. Thorough evaluation of imaging studies and multidisciplinary care, coupled with meticulous surgical planning, are crucial for achieving a cure, which remained unjeopardized in our patients with SM.

MicroRNA-329-3p inhibits the Wnt/ß-catenin pathway and proliferation of osteosarcoma cells by targeting transcription factor 7-like 1.

An important factor in the emergence and progression of osteosarcoma (OS) is the dysregulated expression of microRNAs (miRNAs). Transcription factor 7-like 1 (TCF7L1), a member of the T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor family, interacts with the Wnt signaling pathway regulator ß-catenin and acts as a DNA-specific binding protein. This study sought to elucidate the impact of the interaction between miR-329-3p and TCF7L1 on the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches. MiR329-3p was significantly downregulated, while TCF7L1 was considerably up-regulated in all examined OS cell lines. Additionally, a clinical comparison study was performed using the TCGA database. Subsequently, the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments. When miR-329-3p was transfected into the OS cell line, the expression of TCF7L1 decreased, the proliferation of OS cells was inhibited, the cytoskeleton disintegrated, and the nucleus condensed to form apoptotic bodies. The expression of proteins that indicate apoptosis increased simultaneously. The cell cycle was arrested in the G0/G1 phase, and the G1/S transition was blocked. The introduction of miR-329-3p also inhibited downstream Cyclin D1 of the Wnt pathway. Xenograft experiments indicated that the overexpression of miR-329-3p significantly inhibited the growth of OS xenografts in nude mice, and the expression of TCF7L1 and c-Myc in tumor tissues decreased. MiR-329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo. Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7L1 by miR-329-3p. Summarizing these results, it can be inferred that miR-329-3p exerts anticancer effects in osteosarcoma by inhibiting TCF7L1.

RANK/RANKL axis promotes migration, invasion, and metastasis of osteosarcoma via activating NF-κB pathway.

Osteosarcoma (OS) is one of the most prevalent primary bone tumors with a high degree of metastasis and poor prognosis. Epithelial-to-mesenchymal transition (EMT) is a cellular mechanism that contributes to the invasion and metastasis of cancer cells, and OS cells have been reported to exhibit EMT-like characteristics. Our previous studies have shown that the interaction between tumor necrosis factor superfamily member 11 (TNFRSF11A; also known as RANK) and its ligand TNFSF11 (also known as RANKL) promotes the EMT process in breast cancer cells. However, whether the interaction between RANK and RANKL enhances aggressive behavior by inducing EMT in OS cells has not yet been elucidated. In this study, we showed that the interaction between RANK and RANKL increased the migration, invasion, and metastasis of OS cells by promoting EMT. Importantly, we clarified that the RANK/RANKL axis induces EMT by activating the nuclear factor-kappa B (NF-κB) pathway. Furthermore, the NF-κB inhibitor dimethyl fumarate (DMF) suppressed migration, invasion, and EMT in OS cells. Our results suggest that the RANK/RANKL axis may serve as a potential tumor marker and promising therapeutic target for OS metastasis. Furthermore, DMF may have clinical applications in the treatment of lung metastasis in patients with OS.

Inhibition of anlotinib-induced autophagy attenuates invasion and migration by regulating epithelial-mesenchymal transition and cytoskeletal rearrangement through ATG5 in human osteosarcoma cells.

The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.

Investigating the synergy of Shikonin and Valproic acid in inducing apoptosis of osteosarcoma cells via ROS-mediated EGR1 expression.

BACKGROUND: Osteosarcoma is the most prevalent malignant bone tumour with a poor prognosis. Shikonin (SHK) is derived from the traditional Chinese medicine Lithospermum that has been extensively studied for its notable anti-tumour effects, including for osteosarcoma. However, its application has certain limitations. Valproic acid (VPA) is a histone deacetylase inhibitor (HDACI) that has recently been employed as an adjunctive therapeutic agent that allows chromatin to assume a more relaxed state, thereby enhancing anti-tumour efficacy. PURPOSE: This study was aimed to investigate the synergistic anti-tumour efficacy of SHK in combination with VPA and elucidate its underlying mechanism. METHODS/STUDY DESIGN: CCK-8 assays were utilized to calculate the combination index. Additional assays, including colony formation, acridine orange/ethidium bromide double fluorescent staining, and flow cytometry, were employed to evaluate the effects on osteosarcoma cells. Wound healing and transwell assays were utilized to assess cell mobility. RNA sequencing, PCR, and Western blot analyses were conducted to uncover the underlying mechanism. Rescue experiments were performed to validate the mechanism of apoptotic induction. The impact of SHK and VPA combination treatment on primary osteosarcoma cells was also assessed. Finally, in vivo experiments were conducted to validate its anti-tumour effects and mechanism. RESULTS: The combination of SHK and VPA synergistically inhibited the proliferation and migration of osteosarcoma cells in vitro and induced apoptosis in these cells. Through a comprehensive analysis involving RNA sequencing, PCR, Western blot, and rescue experiments, we have substantiated our hypothesis that the combination of SHK and VPA induced apoptosis via the ROS-EGR1-Bax axis. Importantly, our in vivo experiments corroborated these findings, demonstrating the potential of the SHK and VPA combination as a promising therapeutic approach for osteosarcoma. CONCLUSION: The combination of SHK and VPA exerted an anti-tumour effect by inducing apoptosis through the ROS-EGR1-Bax pathway. Repurposing the old drug VPA demonstrated its effectiveness as an adjunctive therapeutic agent for SHK, enhancing its anti-tumour efficacy and revealing its potential value. Furthermore, our study expanded the application of natural compounds in the anti-tumour field and overcame some of their limitations through combination therapy. Finally, we enhanced the understanding of the mechanistic pathways linking reactive oxygen species (ROS) accumulation and apoptosis in osteosarcoma cells. Additionally, we elucidated the role of EGR1 in osteosarcoma cells, offering novel strategies and concepts for the treatment of osteosarcoma.