Figure S6. Differential pathway gene set enrichment analysis among DKOAA vs DKO in all cell types, visualized as networks via aPEAR.

Figure S20. DE across OS models, stratified by pathologic subtype for which samples were available (including Osteo and Fibro-like, but not chondro-like).

Table S9. Compositional analysis (differential abundance analysis) comparing proportions of cell subtypes from each genotype using the Propeller test from the Speckle package.

Table S8. Marker genes for all celltype subclusters.

Figure S1. Clustering and markers of transgenic OS tumors.

Supplementary Figure S1 shows results from toxicity evaluation of WAY262611, the effect of WAY262611 on the growth of the primary tumor and on mouse survival, as well as examples of metastatic lesions.

Figure S4. Leading edge genes from GSEA showing downregulation of invasive phenotypes in TKO and DKOAA relative to DKO.

Upregulated Myc target activity in TKO and DKOAA malignant cells. A, SCENIC transcription factor activity scores of E2F family transcription factors. B, Expression of apoptosis-related genes derived from the Reactome apoptosis gene set. C and D, GSEA plot of one of the “Hallmark Myc targets V1” gene set in TKO and DKOAA relative to DKO, respectively. E, Heatmap of GSEA leading-edge genes in the “Hallmark Myc targets V1” gene set. The intersect of leading-edge genes from the TKO and DKOAA enrichments is shown. F, Compositional analysis of Myc-high responder cells. G, Violin plot for malignant cells only of signature scores calculated from genes in the “Hallmark Myc targets V1” gene set. H, Violin plot for malignant cells of Ccna2, a Myc target gene. I, Violin plot of the Myc signature scores in all cells.

Supplementary Table S1: Top 10 GO Biological Processes Enriched in Treated vs Control

Table S3. Markers of celltypes after annotation. Wilcox test from Seurat (v5) FindAllMarkers function was used.

Abstract Osteosarcoma is the most common primary pediatric bone malignancy. One promising new target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase that targets p27 for proteasomal degradation, driving cellular proliferation. Knockout (KO) of Skp2 in an immunocompetent transgenic mouse model of osteosarcoma improved survival, drove apoptosis, and induced antitumor immunity. In this study, we applied single-cell RNA-sequencing (scRNA-seq) to primary osteosarcoma tumors from Osx-Cre conditional Rb1/Trp53 KO mice. We further compared with models of Skp2 disruption: Skp2 KO or disruption of the Skp2–p27 interaction (resulting in p27 overexpression). We report that murine osteosarcoma models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. Skp2 disruption led to reduction of T-cell exhaustion and upregulation of interferon (IFN) signaling, as well as induction of cell type–specific replicative and endoplasmic reticulum stress, which we validated with proteomics analysis. Furthermore, we showed that IFN induction was correlated with improved survival in patients with osteosarcoma. Additionally, our scRNA-seq analysis uncovered decreased expression of metastasis-related gene signatures in Skp2-disrupted osteosarcoma, which we validated by a strong reduction in lung metastasis in the Skp2 KO mice. Finally, we report several mechanisms potentially used by osteosarcoma to escape from Skp2 targeting, including upregulation of Myc targets, induction of genomic instability, overexpression of alternative E3 ligases, and lineage plasticity. These mechanistic insights into osteosarcoma tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, whereas the data and our comprehensive analysis may serve as a public resource for further big data–driven osteosarcoma research. Significance: Our single-cell study of murine osteosarcoma models uncovers Skp2 function in metastasis, genomic instability, and immune activation and reveals additional target pathways to overcome resistance to Skp2 disruptions.

Induced immune activation in the form of IFN pathway activity and reduction of T-cell exhaustion. A, Dotplot of Hallmarks gene sets significantly upregulated in TKO relative to DKO across cell types. B, GSEA plots showing enrichment of IFN response pathways in genes differentially expressed in TKO relative to DKO among immune cells. C, UMAP showing subclustering of T cells. D, Canonical and data-driven markers of T-cell subclusters. E, Signature gene scores of T-cell states derived from marker genes in a published meta-analysis of tumor-infiltrating T cells (47). F, Compositional analysis of T-cell subclusters across 3 osteosarcoma tumors. UMAP, Uniform Manifold Approximation and Projection.

Figure S15. E2f and Myc upregulation in TKO and DKOAA malignant cells.

Osteosarcoma is the most common primary pediatric bone malignancy. One promising new target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase that targets p27 for proteasomal degradation, driving cellular proliferation. Knockout (KO) of Skp2 in an immunocompetent transgenic mouse model of osteosarcoma improved survival, drove apoptosis, and induced antitumor immunity. In this study, we applied single-cell RNA-sequencing (scRNA-seq) to primary osteosarcoma tumors from Osx-Cre conditional Rb1/Trp53 KO mice. We further compared with models of Skp2 disruption: Skp2 KO or disruption of the Skp2-p27 interaction (resulting in p27 overexpression). We report that murine osteosarcoma models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. Skp2 disruption led to reduction of T-cell exhaustion and upregulation of interferon (IFN) signaling, as well as induction of cell type-specific replicative and endoplasmic reticulum stress, which we validated with proteomics analysis. Furthermore, we showed that IFN induction was correlated with improved survival in patients with osteosarcoma. Additionally, our scRNA-seq analysis uncovered decreased expression of metastasis-related gene signatures in Skp2-disrupted osteosarcoma, which we validated by a strong reduction in lung metastasis in the Skp2 KO mice. Finally, we report several mechanisms potentially used by osteosarcoma to escape from Skp2 targeting, including upregulation of Myc targets, induction of genomic instability, overexpression of alternative E3 ligases, and lineage plasticity. These mechanistic insights into osteosarcoma tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, whereas the data and our comprehensive analysis may serve as a public resource for further big data-driven osteosarcoma research. SIGNIFICANCE:Our single-cell study of murine osteosarcoma models uncovers Skp2 function in metastasis, genomic instability, and immune activation and reveals additional target pathways to overcome resistance to Skp2 disruptions.

Genome instability in osteosarcoma malignant cells. A, Heatmap showing HMM results of CNVs as determined by InferCNV. The top heatmap shows CNV states in macrophages (reference nonmutated cells), whereas the bottom heatmap shows malignant cells from each sample (rows). The columns represent chromosomes. B, Violin plots showing the number of genes affected by “extreme CNVs” (2× deletions or 2×> amplification). The left shows all extreme CNVs, the top-right shows extreme amplifications, and the bottom-right shows extreme deletions. C, PCA plot of malignant cells using inferred CNVs. D, Same PCA split but by OS models and colored by samples. E, Venn Diagram showing comparison of two differential CNV identification methods with differential expressed genes of TKO vs. DKO. F, Venn diagram showing comparison of two differential CNV identification methods with differential expressed genes of DKOAA vs. DKO. G, Heatmap showing expression patterns of significantly DNA copy number–amplified genes that were also significantly overexpressed in TKO and DKOAA.

Pediatric bone sarcomas continue to present substantial therapeutic challenges in the metastatic, relapsed, or refractory setting, where clinical outcomes have remained largely static for decades despite advances in multimodal therapy. This review summarizes emerging immunotherapeutic strategies aimed at overcoming the immunosuppressive tumor microenvironment, low mutational burden and limited targetable antigens characteristic of these tumors. Current clinical investigations span multiple immunotherapeutic modalities, including adoptive natural killer cell therapies, cytokine-based immune priming, CAR T cell approaches and a growing array of RNA-based therapeutics. Checkpoint inhibitors are also under evaluation, particularly in combination with immunomodulatory or targeted agents. Collectively, these trials highlight both the promise and limitations of immunotherapy in pediatric sarcomas and underscore the need for deeper understanding of sarcoma immune biology to guide future, more effective therapeutic strategies.

Pathologic subtype and lineage infidelity in mouse osteosarcoma tumor. A and B, UMAP showing malignant cells from OS tumors, colored by sample (A) and pathologic subtype (B) as inferred via label transfer from a murine nonmalignant bone atlas dataset. C, The same UMAP, colored by label transfer scores. D, Violin plots of signature scores computed for markers of various bone cells. E, Dot plot of markers of malignant subtype classifications. F, Bar plot showing proportions of malignant cells annotated to each pathologic subtype in each sample. The subtype with the highest proportion is considered the inferred pathologic classification of the indicated tumor. G, UMAP of all OS tumor cells similar to Fig. 1B but split according to sample-wise inferred pathologic classification. H, Proportion of cell types in each pathologic subtype. I, Expression patterns of genes related to macrophage and osteoclast differentiation among malignant cells from each subtype. UMAP, Uniform Manifold Approximation and Projection.

Table S4. Cell metadata and celltype annotations.

Background: Hepatic veno-occlusive disease (VOD) is an endothelial injury seen after stem cell transplantation that can also occur with conventional chemotherapies. Morbidity can be significant, highlighting the need for effective supportive care. Case Report: We present a pediatric patient with Wilms tumor who developed VOD after dactinomycin and vincristine. Management included a molecular adsorbent recirculating system (MARS) for acute liver failure and irinotecan with vincristine (VI) as bridging chemotherapy before gradual reintroduction of dactinomycin. Conclusion: The patient remains in remission without long-term VOD complications. MARS facilitated hepatic recovery, and disease control was maintained with VI serving as a low-hepatotoxic bridging regimen before rechallenging with dactinomycin.

Abstract Ewing sarcoma is an aggressive bone and soft-tissue cancer affecting adolescents and young adults. In vitro and in vivo models of Ewing sarcoma have been instrumental in advancing our understanding of Ewing sarcoma biology and essential in evaluating potential therapies, particularly for metastatic or relapsed disease for which effective treatment options remain limited. Through an international collaborative effort between the Children’s Oncology Group Bone Tumor Committee and the Euro Ewing Consortium, we review the current landscape of preclinical modeling used in Ewing sarcoma research encompassing both in vitro (cell lines and tumor organoids) and in vivo (mouse and nonmammalian xenografts) model systems. We discuss factors that can influence experimental results, provide testing considerations for both in vitro and in vivo studies, and descriptions of existing preclinical data repositories. We highlight current needs in Ewing sarcoma modeling and the importance of enhanced international cooperative research and patient advocacy efforts which will be critical in expanding our resources of biologically relevant Ewing sarcoma models to enable translation of preclinical findings into effective therapeutic strategies for patients with Ewing sarcoma.