Osteosarcoma (OS) is the most prevalent primary cancer of the bone. Metastasis and chemoresistance are the major obstacles to the improvement of OS prognosis, in which N6-methyladenosine (m6A) modification plays an important role, but the exact molecular mechanisms are still unclear. MeRIP-seq and RNA-seq were conducted on OS and paired adjacent normal tissue samples, which determined CACNA1E as a key m6A-modified molecule. In vitro and in vivo models were established to evaluate the function of CACNA1E on OS growth, metastasis, and methotrexate (MTX) resistance, and to explore the upstream regulators and downstream effectors of CACNA1E. CACNA1E exhibited notable m6A hypermethylation and upregulated expression in OS than adjacent normal tissues. CACNA1E knockdown effectively hindered OS growth, lung metastasis, and MTX resistance. METTL3, an m6A "writer" boosted the mRNA stability of CACNA1E through m6A modification, and this process was recognized and enhanced by IGF2BP2, an m6A "reader". WNT7B was identified as a downstream molecule of CACNA1E. CACNA1E facilitated OS progression and MTX resistance by enhancing the non-canonical Wnt/Ca2+ signaling through transcriptionally activating WNT7B. Furthermore, a novel combination treatment of targeted inhibition of CACNA1E with MTX had a synergistic effect on suppressing OS progression. Collectively, our findings uncover that METTL3-mediated m6A modification of CACNA1E contributes to OS progression and chemoresistance through enhancing WNT7B-mediated non-canonical Wnt/Ca2+ signaling. Targeted inhibition of CACNA1E in combination with MTX may be a promising alternative therapeutic strategy for patients with MTX-resistant OS.

Dual CDK4/6-PI3K/mTOR inhibition reinforces cytostatic programs and tumor control in preclinical models of primary and metastatic osteosarcoma.

Osteosarcoma (OS) exhibits substantial genomic complexity and inter-patient heterogeneity, necessitating longitudinal, patient-matched analyses to understand acquired features of tumor evolution. However, most published OS data is limited to primary tumor samples, limiting insight into patient-specific resistance mechanisms. To address this, we characterized the genomic landscape of paired primary and metastatic tumor samples from dogs with spontaneous OS. Whole-genome and single-cell RNA sequencing reveal mutation and gene expression profiles that are predominantly organized by patient identity. Mutational burden and pathway alterations such as those involving PI3K, NOTCH, TP53, MAPK, RAS and epigenetic regulation differ between primary and metastatic samples. Variants present in tumor tissue are readily detectable in paired cfDNA samples, demonstrating the utility of this assay for identifying tumor-specific alterations associated with treatment resistance. Analysis of bulk RNA-seq data to estimate cell-type composition shows greater immune cell representation in metastases, underscoring the importance of immune signaling pathways in OS. These findings exemplify the presence of patient-specific alterations in genomic architecture over the course of tumor progression, linking CNV amplification, pathway reprogramming, and immune evasion in metastatic OS.

PP01.34: Clinical Study on Thoracoscopic Talc Pleurodesis Combined with Subpleural Lesion Resection for Treating Pneumothorax Secondary to Pulmonary Metastasis of Osteosarcoma

Osteosarcoma is a highly malignant bone tumor affecting children and adolescents. Once metastasis occurs, the five-year survival rate drops to ~20%, emphasizing the need for new therapies. Fibroblast growth factor-23 (FGF-23), a bone-derived hormone, has been implicated in tumor progression, but its role in osteosarcoma remains unclear. Bioinformatics analysis using the R2 database revealed that elevated FGF-23 expression is associated with increased metastasis and reduced overall survival in osteosarcoma patients. Functional assays confirmed that FGF-23 enhances the migratory ability of osteosarcoma cells. Gene Expression Omnibus (GEO) analysis indicated that lysyl oxidase-like proteins-particularly LOXL1, LOXL2, and LOXL3-are overexpressed in osteosarcoma tissues compared to adjacent normal bone. In vitro experiments further showed that FGF-23 significantly upregulates LOXL2 expression in 143B and MG63 osteosarcoma cells, while LOXL2 knockdown via small interfering RNA (siRNA) markedly reduces cell migration. Moreover, pretreatment with ERK, p38, and JNK inhibitors or siRNAs targeting these pathways suppressed both FGF-23-induced LOXL2 expression and wound healing, indicating that FGF-23 promotes cell motility through ERK-, p38-, and JNK-dependent LOXL2 upregulation. FGF-23 stimulation also increased phosphorylation of ERK, p38, and JNK, and downregulated miR-4463. Inhibition of these pathways restored miR-4463 levels and suppressed LOXL2 expression. Taken together, these findings suggest that FGF-23 promotes osteosarcoma cell migration and may contribute to metastasis through coordinated regulation of the miR-4463/LOXL2 axis via ERK, p38, and JNK signaling. Targeting FGF-23 or its downstream signaling cascades may offer a promising therapeutic approach for metastatic osteosarcoma.

Osteosarcoma (OS) survival rates have remained unchanged for decades, while video-assisted thoracic surgery (VATS) for lung metastasis (LM) became common. We aimed to clarify clinical outcomes of post-relapse survival (PRS) for patients with OS based on LM-free survival (LMFS) across different eras. This single-centre retrospective study included 168 patients with OS without LM at initial diagnosis. Patients were categorised into three groups: non-LM (n = 89), synchronous LM (sLM: less than 1-year LMFS) (n = 40), and mLM (at least 1-year LMFS) (n = 39). We compared PRS in patients with sLM and mLM across periods 1 (1990-2005) and 2 (2006-2022). PRS for mLM was longer in period 2 than in period 1 (Hazard ratio: 0.37, 95% confidence interval: 0.12-0.97, p = 0.04), whereas no difference was observed for sLM. In mLM, ratios of surgery, radiotherapy, and chemotherapy were unchanged; the rate of VATS increased in period 2 (7/18 vs. 10/12, p = 0.01). The rate of reoperation remained unchanged (7/18 vs. 7/12, p = 0.39). In period 2, mLM had a better prognosis than in period 1. There was an increased use of VATS, while the rates of reoperation for LM remained unchanged. Conversely, the prognosis for sLM was not altered.

ABSTRACTIf lung function permits, resectioning recurrent pulmonary metastases can contribute to improved long‐term survival. Additionally, three‐dimensional computed tomography could be useful for lung function assessment.

High-dose methotrexate (MTX) is one of the key components of multimodal therapy for children and adolescents with osteosarcoma (OS). This study aimed to evaluate the prognostic significance of peak plasma concentrations of MTX (12 g/m²) at the end of the 4-hour infusion in newly diagnosed OS patients under 18 years of age who received comprehensive treatment at Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of the Russian Federation. We analyzed a total of 1,195 MTX concentration measurements in 136 patients with localized and metastatic OS. The mean peak plasma concentration was 1249 ± 337 μmol/L. Significantly higher mean peak MTX concentrations were observed in the patients demonstrating a good histological response to neoadjuvant chemotherapy (1274 μmol/L vs. 1154 μmol/L; p = 0.0056), as well as in those with localized disease compared to the patients with metastatic OS (1280 μmol/L vs. 1180 μmol/L; p = 0.0131). Survival analysis revealed higher 5-year event-free survival rates among the patients with mean peak MTX concentrations ≥1500 μmol/L (79.4% vs. 53.8%; p = 0.044). No statistically significant difference in overall survival was observed depending on peak MTX concentrations. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of the Russian Federation.

Osteosarcoma is a highly heterogeneous cancer, and molecular subtyping has the potential to increase diagnostic precision and facilitate targeted therapies. In our previous study, we classified osteosarcoma into four distinct subtypes. Here, we conducted a genomic subtype-guided pilot umbrella trial (ChiCTR2000036618, 2020/08/24) to evaluate the efficacy and safety of multiple precision therapies. Nineteen patients with refractory metastatic osteosarcoma were enrolled and stratified using whole-exome sequencing (WES) and immunohistochemistry (IHC). Patients were assigned to three arms: (A) PD-1 antibody plus gemcitabine and docetaxel; (B) PARP inhibitor combined with temozolomide; or (C) tinengotinib (TT-00420), a small-molecule aurora kinase inhibitor currently in clinical trials. The median progression-free survival and overall survival were 50 days (95% CI: 33–89) and 149 days (95% CI: 90–185). Tinengotinib has shown promising efficacy in our preclinical studies, suggesting its potential for clinical use, particularly in combination with immunotherapy. Additionally, we found that patients with MYC amplification presented increased tumor purity and ploidy, homologous recombination deficiency scores, and an immunosuppressive microenvironment. This study demonstrated the feasibility of using genomic molecular subtyping to guide the precise treatment of osteosarcoma. We also revealed that the abnormal genomic and transcriptomic profiles caused by MYC amplification could be suppressed by tinengotinib.

Photocatalytic therapy holds great prospects in high-performance tumor treatment owing to its advantages of noninvasiveness and oxygen nonreliance. However, there is still a lack of ideal photocatalyst materials that respond to near-infrared (NIR) light, especially with organelle-specific features. Here, a mitochondria-biomimetic organic semiconductor photocatalyst was designed for NIR-activatable photocatalytic immunotherapy of recurrent and metastatic tumor. With biomimetic of mitochondrial membrane, a NIR-responsive conjugated polymer YBSe-SS was prepared into biomimetic nano-photocatalysts (Mito-NPs). Upon NIR-light irradiation, the Mito-NPs achieved effective photocatalytic oxidation of reduced nicotinamide adenine dinucleotide and mitochondria dysfunction, benefiting from the mitochondria-targeted performance. Also, relatively high multiphototheranostics were demonstrated by the Mito-NPs. Subsequently, with Mito-NP–regulated mitochondrial function and metabolism, the in vivo experiments proved that the photocatalytic Mito-NPs combined with immune checkpoint therapy achieved photocatalytic immunotherapy for suppressing primary and pulmonary metastasis of osteosarcoma. This work offers a practical paradigm for NIR-activatable organic photocatalytic materials for high-performance tumor therapy.

This study aims to unravel the underlying mechanisms of osteosarcoma (OS) metastasis by single-cell RNA-sequencing. Ten pediatrics OS samples were analyzed using scRNA-seq and divided into three groups: primary tumor without lung metastasis (Pri_non_MT), primary tumor with lung metastasis (Pri_MT), and lung metastasis site (MT). Candidate genes associated with metastasis were identified by bioinformatics analysis and was confirmed at mRNA and protein levels in OS tissues. The function of candidate gene was identified in vitro and in vivo. Bulk RNA sequencing was used to explore downstream mechanism after candidate gene silenced. A total of 17 cell clusters were identified. ENO1 was selected as the candidate gene and was significantly expressed in Pri_MT and MT groups. In OS tissues, ENO1 significantly overexpressed in patients with lung metastasis compared to those without. Knocking down ENO1 resulted in a marked decrease in migration and invasion in vitro and a reduction in lung metastasis in vivo. Additionally, ENO1 suppression resulted in shifting the primary ATP production pathway from glycolysis to oxidative phosphorylation. Our findings highlight ENO1 as a key regulator of glycolysis and metastasis in osteosarcoma, offering a novel therapeutic target for OS treatment.

Demethylase, ALKBH1, regulates osteosarcoma chemosensitivity through FZD8/Wnt/β-catenin signaling pathway in the m7G manner.

NSUN2 promotes osteosarcoma metastasis via stabilizing UBE2S mRNA in an m5C-dependent manner.

Osteosarcoma, one of the most common malignant bone tumors in children, carries a mortality rate of 8.6%. Its highly aggressive and metastatic nature, coupled with the severe bone marrow suppression, organ toxicity, and drug resistance associated with conventional radiotherapy, underscores the need for improved therapies. Degradation products from magnesium alloys can modulate the tumor microenvironment and exhibit potential antitumor properties. Consequently, this study examined the application of magnesium-based metallic materials for osteosarcoma treatment, specifically assessing the impact of a magnesium-calcium alloy/mineralized collagen composite on osteosarcoma cell biological behavior. Results demonstrated that the magnesium-calcium alloy/mineralized collagen composite significantly inhibited osteosarcoma cell proliferation, invasion, and metastasis while promoting apoptosis. In vivo experiments further corroborated these findings, revealing that the composite group exhibited the smallest tumor volume, confirming its potent antitumor efficacy against osteosarcoma. Collectively, these findings demonstrate that this biomaterial not only holds significant potential for localized antitumor therapy but also provides a foundational framework that paves the way for novel treatment strategies against osteosarcoma.

Osteosarcoma is the most prevalent primary malignant bone tumor in children and adolescents. However, its underlying pathogenesis and mechanisms driving metastasis remain poorly understood. Here, we identified a novel super-enhancer-associated long noncoding RNA (SE-lncRNA), Zinc Finger MIZ-Type Containing 1 Antisense RNA 1 (ZMIZ1-AS1), which is highly expressed in osteosarcoma and promoted tumor cell proliferation, migration, and invasion. Mechanistically, the m⁶A demethylase ALKBH5 post-transcriptionally stabilized ZMIZ1-AS1 through m⁶A demethylation. Furthermore, ZMIZ1-AS1 directly bound to the RNA-binding protein Polypyrimidine Tract Binding Protein 1 (PTBP1), facilitating the translocation of PTBP1 from the nucleus to the cytoplasm. The relocalized PTBP1 then bound to and stabilized fibroblast growth factor receptor 1 (FGFR1) mRNA. In nude mouse models, ZMIZ1-AS1 overexpression promoted tumor growth and lung metastasis. Notably, combined inhibition of ALKBH5 (using ALKBH5-IN-5) and FGFR1 (using BGJ398/infigratinib) synergistically suppressed ZMIZ1-AS1-driven oncogenesis in vivo. Our study establishes the ALKBH5/ZMIZ1-AS1/PTBP1/FGFR1 signaling axis as a key driver of osteosarcoma progression and a promising target for therapeutic intervention.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05969-2.

WGCNA-identified COL13A1 drives osteosarcoma metastasis and progression via TGF-β signaling

Osteosarcoma is a prevalent and aggressive bone malignancy primarily affecting young adults. Despite advances in standard treatment, the 5-year survival rate remains low, highlighting the need for new chemotherapy options. Mycophenolate mofetil, an immunosuppressant used post-transplant, has shown promising preclinical anticancer effects in osteosarcoma, warranting further investigation for potential repurpose in humans. We conducted this proof-of-concept phase II clinical trial to assess the efficacy and safety of mycophenolate mofetil in high-grade locally advanced or metastatic osteosarcoma. Patients received 3-5g/day mycophenolate mofetil for four 28-day cycles or until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS) at 16weeks. Secondary endpoints included overall survival (OS), overall response rate, safety, pharmacokinetics, pain score, and quality of life. Fifteen patients were enrolled. Due to intolerable toxicity at higher doses, most patients received 3g/day of mycophenolate mofetil. At 16weeks, the PFS rate was 40%, with only one patient (6.7%) achieving stable disease. Median PFS and OS were 70 and 301days, respectively. The most common grade 3-4 adverse events were anemia and fatigue, each occurring in 20% of patients. Pharmacokinetic analysis revealed high interindividual variability. Although mycophenolate mofetil at 3g/day was well tolerated, it did not meet expectations for anticancer efficacy in patients with high-grade locally advanced or metastatic osteosarcoma. This study should be considered a negative trial. Further research is needed to explore the therapeutic role of mycophenolate mofetil through alternative strategies or identify more effective interventions for osteosarcoma.

LINC00673 promotes osteosarcoma progression through the miR-92b-3p/DUSP1 axis.

Stereotactic Body Radiation Therapy Augmented Checkpoint Inhibitor Immunotherapy Response in Heavily Pretreated Metastatic Osteosarcoma.

Osteocyte-like differentiation of osteosarcoma by inorganic phosphate.