Supplementary Figure S4: Identification of immune-related malignant osteosarcoma cell subpopulations and key SUMOylation genes at the single-cell level.

Osteosarcoma is an aggressive malignancy characterized by rapid proliferation and a propensity for metastasis. The endoplasmic reticulum (ER) chaperone GRP78, a critical regulator of osteosarcoma progression, represents a promising therapeutic target. In this study, we identified the natural compound ginkgetin (Gink) as a novel GRP78 inhibitor. Cellular thermal shift assays, surface plasmon resonance, and mutagenesis studies revealed that Gink directly binds to GRP78, with K296 serving as a key interaction site. In vitro, Gink suppressed osteosarcoma cell proliferation, migration, and invasion while inducing apoptosis and autophagy by activating the PERK-eIF2α-ATF4 pathway. Co-immunoprecipitation assays showed that Gink competitively disrupted GRP78-PERK interaction. In orthotopic and patient-derived xenograft models, Gink treatment markedly attenuated tumor growth and metastasis. Gink also reprogrammed the tumor immune microenvironment by reducing M2 macrophage polarization and synergizing with anti-PD1 therapy to enhance CD8+ T-cell activity. Accordingly, Gink could be developed as a GRP78-targeting agent that triggers ER stress and immune activation, offering a dual-pronged therapeutic strategy against osteosarcoma. Ginkgetin (Gink) directly binds to GRP78 in a competitive manner, disrupting the interaction between GRP78 and PERK. This leads to PERK activation and phosphorylation, which in turn phosphorylates eIF2α to trigger ATF4 transcription. Ultimately, this cascade induces apoptosis and autophagy, inhibiting cancer progression. Additionally, Gink suppresses M2 macrophage polarization and enhances CD8+ T cell cytotoxicity, both of which contribute to the prevention of cancer development.

Differentiated thyroid carcinoma (DTC) generally has a favorable prognosis; however, approximately one-third of advanced patients progress to radioiodine-refractory DTC (RAIR-DTC), which is associated with poor survival outcomes. BRAF V600E is the most prevalent oncogenic driver mutation in thyroid cancer. It is responsible not only for the aberrant activation of the mitogen-activated protein kinase (MAPK) signaling pathway but also serves as the core mechanism for tumor dedifferentiation and iodine resistance. Currently, multi-target tyrosine kinase inhibitors (MTKIs) serve as the first-line systemic therapy for RAIR-DTC. Although MTKIs can prolong progression-free survival, their "off-target" effects frequently lead to severe cardiovascular toxicities, which impact patient compliance and quality of life. In recent years, highly selective BRAF/MEK dual inhibitors (e.g., dabrafenib plus trametinib) have demonstrated high objective response rates and durable survival benefits in phase Ⅲ studies of RAIR-DTC, showing a more advantageous safety profile. Furthermore, targeted neoadjuvant therapy increases the opportunity for patients to achieve radical resection; meanwhile, short-term targeted blockade of the MAPK pathway can induce "redifferentiation" of thyroid cancer cells, restoring radioiodine uptake in the majority of patients. This article systematically reviews the oncogenic mechanisms of the BRAF V600E mutation and its latest clinical advancements in precision systemic therapy, neoadjuvant conversion therapy and redifferentiation therapy, aiming to provide a reference for the individualized management of thyroid cancer.

The tumor ecosystem heterogeneity of papillary thyroid carcinoma (PTC) is poorly characterized in children and young adult patients (CAYA-PTC). In this study, single-cell RNA sequencing is used to profile transcriptomes from the paratumor and tumor tissues of 11 patients. Compared to adult, CD4T_Tfh and CD8T_Tex cells are significantly more prevalent in CAYA-PTC patients. Three phenotypes are identified within the thyrocytes through differentiation trajectory analysis, including normal, BRAF-like, and Fusion-like. Notably, the data reveal that CAYA-PTC patients lack the "mild-state (BRAF-like)" malignant thyrocyte population. This variation in differentiation states indicates that PTC cells in CAYA patients rapidly develop into invasive and metastatic forms, whereas in adult patients, this progression occurs gradually over a longer period. Additionally, extracellular matrix cancer-associated fibroblasts (emCAFs_LAMP5) interact with endothelial cells and thyrocytes, promoting tumor angiogenesis and metastasis more prominently in CAYA patients. Fibroblast activation protein (FAP) expression is high in emCAF_LAMP5 and positively correlated with LAMP5 in CAYA-PTC tissues. Consequently, 68Ga-FAPI-PET emerges as a promising diagnostic method for CAYA patients who are not effectively diagnosed by traditional 18F-FDG-PET. Collectively, the findings provide insight into the CAYA-PTC ecosystem that suggests distinct diagnostic, prognostic, and therapeutic implications compared to adults.

Osteosarcoma responds poorly to immune-checkpoint blockade (ICB), and the molecular drivers of its immune-cold state remain unclear. Using multi-omics analyses, we identified an immune-cold osteosarcoma subtype characterized by enrichment of protein SUMOylation and found the SUMO E1 subunit UBA2 as a key driver. UBA2 was anomalously upregulated in osteosarcoma cohorts and linked to worse outcomes. Functionally, UBA2 promoted autophagy, thereby exerting noncanonical protumor and immunosuppressive effects. Mechanistically, UBA2 catalyzed SUMO2-dependent SUMOylation of SESN2, enhancing autophagic flux. At the immune interface, UBA2 did not alter the expression of immune checkpoint molecules but reduced surface MHC-I through NBR1-mediated autophagy-lysosomal degradation, thereby limiting CD8+ T-cell infiltration. In vivo, pharmacologic UBA2 inhibition with ML-792 slowed tumor growth and sensitized tumors to ICB therapy. Together, these findings show that UBA2 links SUMOylation to autophagy-driven loss of antigen presentation and immune exclusion, highlighting the UBA2-autophagy-MHC-I axis as a therapeutic target and potential biomarker in osteosarcoma.

Supplementary Figure S3: Single-cell transcriptomic quality control, batch effect correction, clustering, and cell-type annotation.

Supplementary Figure S15. Representative flow cytometry plots of tumor-infiltrating immune cells.

PURPOSE:Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, yet its genetic etiology remains poorly understood. In this study, we described a family from the Shanghai General Hospital Osteosarcoma (SGH-OS) cohort in which two siblings developed osteosarcoma as their first primary malignancy, and we investigated the germline and somatic genetic basis underlying this familial presentation. METHODS:Whole-genome sequencing (WGS) was performed on tumors and matched germline DNA from the affected siblings, as well as germline DNA from their parents and unaffected sister. Comprehensive somatic and germline analyses were conducted to assess mutational profiles, copy-number alterations, and structural variants. RESULTS:Both affected siblings carried a paternally inherited RB1 splice-donor variant (NM_000321.3:c.539+1G>A), with biallelic inactivation confirmed by 13q14 loss in both tumors. Tumor WGS revealed additional somatic alterations, suggesting cooperation between germline and acquired mutations in osteosarcoma development. In addition, the carrier father and elder sister remained unaffected, consistent with incomplete penetrance. CONCLUSION:This study further supports the expanding spectrum of RB1-associated cancer predisposition, showing that low-penetrance RB1 variants may present with osteosarcoma without preceding retinoblastoma. In addition, our findings underscore the value of integrated WGS for characterizing inherited susceptibility in familial osteosarcoma.

BACKGROUND:Hypoparathyroidism is one of the most common postoperative complications of total thyroidectomy (TT) in children and young adolescents differentiated thyroid cancer (CAYA-DTC). Due to lack of data in Chinese population, we aimed to investigate their incidence of transient/permanent hypoparathyroidism and relevant clinical risk factors. METHODS:A multicenter retrospective cohort of CAYA-DTC patients who underwent TT was launched in three tertiary hospitals in China. Serum PTH and calcium level were measured on the first day after surgery (post-operation Day 1, POD1) and around 6 months during follow-ups. The occurrence of transient hypoparathyroidism was treated as a competing risk factor to the end-point of permanent hypoparathyroidism. RESULTS:Among all 104 enrolled patients, the average age was 15.26 ± 2.52 years old. Symptoms of post-operation hypocalcemia were seen in one third. The incidence rate of transient and permanent hypoparathyroidism was 24.0 % and 6.7 %, respectively. Patients with large tumor size, lateral lymph node metastasis (LNM), bilateral involvement or radioactive iodine (RAI) treatment were more likely to develop hypoparathyroidism after surgery (p < 0.05), while only low POD1 PTH level <8 pg/mL and RAI treatment were independence risk factors for permanent hypoparathyroidism (p < 0.01). Puberty (age <14 years old) displayed no significant impact on the incidence of hypoparathyroidism. CONCLUSIONS:This is the first report on hypoparathyroidism after TT among Chinese CAYA-DTC. Patients who had low POD1 PTH or underwent postoperative RAI treatment were more likely to develop permanent hypoparathyroidism. Our study provides clinical references for updating post-operation management guidelines in CAYA-DTC in China.

Purpose: This study aimed to describe the ultrasound characteristics of papillary thyroid carcinoma (PTC) harboring RET gene fusion and explore its clinical significance. Methods: A retrospective study was conducted on 209 patients with PTC diagnosed between August 2021 and January 2024. All patients underwent ultrasound examination and were confirmed to be positive for RET fusion or BRAFV600E by pathological results. The differences in clinical characteristics and ultrasonography features between the 2 groups were analyzed. Results: Among all PTCs (n = 209), we detected 30 RET fusions and 179 BRAFV600E. RET-fusion PTCs showed younger age [38.5 (16.0-69.0) vs 42.9 (20.0-74.0) years, P < .05], larger tumor size [1.09 (0.5-4.0) vs 0.77 (0.1-4.0) cm, P < .005], and more advanced N stage (P < .001) than BRAFV600E PTCs. RET-fusion PTCs were mainly classical and diffuse sclerosing subtypes. In terms of ultrasound performance, RET-fusion PTCs were mainly manifested as heterogeneous echogenicity (43.3%), ill-defined tumor margin (90.0%), irregular shape (83.3%), and intranodular microcalcification (83.3%), characterized by scattered microcalcification around the tumor/within thyroid gland (40.0%). In comparison, BRAFV600E PTCs were mainly characterized by hypoechogenicity (95.5%), round/oval shape (80.4%), and intranodular noncalcification (54.2%). Multivariate logistic regression analysis revealed that scattered microcalcification around the tumor/within the normal gland was an independent risk factor for lateral lymph node metastasis (LLNM) in RET-fusion PTCs (odds ratio 9.79, 95% confidence interval 1.31-72.93, P = .026). Conclusion: Patients diagnosed with PTC harboring RET fusion presented with distinctive clinical characteristics and sonographic patterns, underscoring the unique diagnostic value of ultrasound examination. It can provide a preoperative noninvasive primary screening method for RET-fusion diagnosis, thus facilitating targeted patients with purposeful molecular sequencing to improve treatment outcomes.

PURPOSE:Osteosarcoma (OS) is the most prevalent primary malignant bone sarcoma, characterized by its high rates of metastasis and mortality. In our previous multiomics analysis of the Shanghai General Hospital OS (SGH-OS) cohort, we identified four distinct OS subtypes, each with unique molecular characteristics and clinical outcomes. Of particular importance was the identification of the MYC-driven subtype, which exhibited the poorest prognosis and was referred to as high-risk OS. A diagnostic tool is needed for clinicians to identify high-risk OS in advance. The purpose of this study is to develop a classifier capable of accurately predicting the high-risk OS subtype using transcriptome and methylation data. METHODS:In this study, using eXtreme Gradient Boosting (XGBoost) with Bayesian optimization, we developed a classification model by integrating transcriptome and methylation data from our internal SGH-OS cohort. We further validated the model's predictive performance with the external TARGET-OS cohort. RESULTS:Using the XGBoost algorithm, we developed a classifier incorporating nine genes (ARHGAP9, CADM1, CPE, DUSP3, FGFR1, GALNT3, IGF2BP3, KIF26A, ZFP3). In our internal cohort, the classifier exhibited excellent predictive performance, with an area under the receiver operating characteristics curve (AUC) of 0.999 and an overall accuracy of 0.989. Furthermore, the classifier successfully stratified two groups with distinct survival outcomes in the external TARGET-OS cohort. Notably, our analysis revealed a positive correlation between IGF2BP3 and MYC signaling pathways, highlighting IGF2BP3 as a potential therapeutic target in high-risk OS. CONCLUSION:Our classifier demonstrated excellent predictive performance in identifying patients with high-risk OS, offering the potential to enhance treatment decision making and optimize patient management strategies.

Our research observed the condition and potential surgical risks of patients who have previously contracted coronavirus disease 2019 (COVID-19) and subsequently underwent thyroid surgery. A research cohort of 140 patients undergoing thyroid surgery within our institution between October 2022 and March 2023. Patients were categorized into 4 distinct groups, factoring in varying stages post their COVID-19 infection. The study documented various pre- and postoperative clinical data, as well as the patients' subjective experiences. There existed no statistically significant distinction in the incidence of postoperative adverse reactions (P = .81) or the pharyngalgia score (P = .57) among the different patient groups. The statistical analysis revealed the severe acute respiratory syndrome-CoV-2 Immunoglobulin G (IgG) levels emerged as a critical positive correlation factor, signifying an escalated probability of postoperative adverse events (r = 0.31, P = .02). However, the levels of IgG are not affected by the extent of vaccination, and it do not exert a significant impact on postoperative pharyngalgia, pulmonary focal exudate, or tumor progression. Furthermore, the probability of postoperative adverse reactions heightened when patients still exhibited incomplete absorption of lung infection lesions on preoperative lung CT (r = 0.11, P = .04). With a thorough preoperative assessment, early thyroid surgery following COVID-19 infection is feasible. It is recommended to incorporate IgG testing as an indicator for assessing adverse surgical events.

Osteosarcoma (OS), a malignant bone tumor with limited treatment options, exhibits low sensitivity to immune checkpoint therapy (ICT). Through genomics and transcriptomics analyses, we identify a subgroup of OS with methylthioadenosine phosphorylase (MTAP) deletion, which contributes to ICT resistance, leading to a "cold" tumor microenvironment. MTAP-deleted OS relies on methionine metabolism and is sensitive to methionine intervention, achieved through either dietary restriction or inhibition of methionine adenosyltransferase 2a (MAT2A), a key enzyme in methionine metabolism. We further demonstrate that methionine intervention triggers programmed death-ligand 1 (PD-L1) transcription factor IKAROS family zinc finger 1 (IKZF1) and enhances PD-L1 expression in MTAP-deleted OS cells. Methionine intervention also activates the immune-related signaling pathways in MTAP-deleted OS cells and attracts CD8+ T cells, thereby enhancing the efficacy of ICT. Combining methionine intervention with ICT provides a significant survival benefit in MTAP-deleted OS murine models, suggesting a rationale for combination regimens in OS ICT.

Background:Osteosarcoma (OS) is a highly aggressive primary bone tumor with poor outcomes, particularly in metastatic or recurrent cases. Methionine metabolism and histone methylation, such as H3K27me3, play crucial roles in OS progression. Methods:We analyzed single-cell RNA sequencing (scRNA-seq) data to identify histone methylation and related pathways associated with malignant proliferation OS cells. A high-throughput compound screen was performed to evaluate potential metabolic and epigenetic targets. In vitro and in vivo experiments were conducted to assess the therapeutic potential of MAT2A inhibition, methionine restriction, and EZH2 inhibition. Results:MAT2A inhibition or methionine restriction reduced H3K27me3 levels, induced DNA damage, and suppressed OS cell growth. Combining MAT2A and EZH2 inhibitors demonstrated synergistic effects in reducing H3K27me3 levels, enhancing DNA damage, and inhibiting OS growth both in vitro and in vivo. Conclusion:The combination of MAT2A and EZH2 inhibition significantly reduces intracellular H3K27me3 levels by depleting S-adenosylmethionine (SAM) and inhibiting synthetic enzyme activity, thereby inducing DNA damage in osteosarcoma (OS). Methionine-restricted diet combined with EZH2 inhibition effectively suppresses osteosarcoma growth in vivo. The translational potential of this article:This study highlights the potential of integrating metabolic and epigenetic interventions in OS therapy. Our findings might present a promising therapeutic strategy for chemotherapy-resistance OS.

BACKGROUND:Salivary duct carcinoma (SDC) is an aggressive malignancy with limited treatment options, and immune checkpoint inhibitors may offer novel therapeutic alternatives. PURPOSE:The purpose of this study was to measure the association between immune checkpoint regulators and survival among patients with SDC. STUDY DESIGN, SETTING, AND SAMPLE:This retrospective cohort study was performed at Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital and Fudan University Shanghai Cancer Center between April 2006 and November 2016. Subjects were SDC patients meeting the inclusion/exclusion criteria. PREDICTOR VARIABLE:The predictor variable was the expression level of 5 immune checkpoint regulators (positive vs negative): programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and T-cell immunoglobulin and mucin domain 3 (TIM-3). MAIN OUTCOME VARIABLE(S):The primary and secondary outcomes were disease-free survival (DFS) and overall survival. COVARIATES:The covariates were subjects' demographics, tumor characteristics, and androgen receptor (AR) and human epidermal growth factor receptor 2 (HER2) status. ANALYSES:Survival analysis was performed using Kaplan-Meier curves and Cox regression models. Subtype comparisons were made using Pearson χ2 or Fisher's exact test. Statistical analyses were performed using SPSS v.26. Statistical significance was P < .05. RESULTS:The sample composed 54 subjects with a mean age of 59.39 years (SD 13.35) and 81% were male. PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, AR, and HER2 positivity rates were 37, 31, 33, 15, 44, 67, and 24%, respectively (P < .001). The DFS was worse in subjects positive for HER2, PD-1, PD-L1, or CTLA-4 (all P < .05), whereas positivity for LAG-3 or TIM-3 was not associated with DFS. Multivariate analysis identified PD-1/PD-L1 co-positivity as an independent negative prognostic factor for DFS (HR = 2.69, P = .02). HER2 positivity was also an independent predictor of poorer overall survival (P = .003). No significant differences in immune checkpoint regulator expression were observed across subtypes. CONCLUSIONS AND RELEVANCE:PD-1, PD-L1, CTLA-4, and HER2 positivity are associated with unfavorable clinical outcomes in SDC. Immune checkpoint regulator expression was comparable among AR/HER2 subtypes. PD-1, PD-L1, and CTLA-4 are potential therapeutic targets for SDC, particularly for the HER2/AR double-negative subtype.

Background Osteosarcoma patients with high propensity for metastasis and recurrence generally encounter a poor prognosis. Despite the extensive exploration of immunotherapy, particularly the anti-programmed cell death protein 1 (anti-PD-1) antibody, in clinical trials, the efficacy remains unsatisfactory. A more profound comprehension of the resistance mechanisms and the development of innovative therapeutic strategies is imperative.Methods A screening was performed for drugs capable of upregulating major histocompatibility class I (MHC I) expression among clinically common drugs. The effects of the drug on both T cells and tumor cells, as well as its combination efficacy with anti-PD-1 antibody, were studied in vitro and in vivo osteosarcoma models. The molecular mechanisms underlying these biological processes were explored via RNA sequencing analysis.Results Etoposide was shown to upregulate the MHC I expression in osteosarcoma cells, thereby enhancing the cytotoxicity of CD8+ T cells. Interleukin-33 (IL-33) played a dominant role in etoposide-activated anti-tumor immune response. Etoposide promoted the secretion of IL-33 and augmented the expression of IL-33 binding suppression of tumorigenicity 2 (ST2) receptor, which activated the nuclear factor kappa-B signaling pathway and resulted in MHC I upregulation. Furthermore, etoposide was demonstrated to improve the therapeutic efficacy of anti-PD-1 antibody.Conclusions This study revealed the molecular mechanism underlying etoposide-activated CD8+ T cell anti-tumor immunity. The combination of Etoposide and anti-PD-1 antibody has the potential to benefit patients with advanced osteosarcoma.

PURPOSE:Osteosarcoma, a highly malignant primary bone tumor primarily affecting adolescents, frequently develops resistance to initial chemotherapy, leading to metastasis and limited treatment options. Our study aims to uncover novel therapeutic targets for metastatic and recurrent osteosarcoma. METHODS:In this study, we proved the potential of modulating the YAP1-regulated glutamine metabolic pathway to augment the response of OS to DFMO. We initially employed single-cell transcriptomic data to gauge the activation level of polyamine metabolism in MTAP-deleted OS patients. This was further substantiated by transcriptome sequencing data from recurrent and non-recurrent patient tissues, confirming the activation of polyamine metabolism in progressive OS. Through high-throughput drug screening, we pinpointed CIL56, a YAP1 inhibitor, as a promising candidate for a combined therapeutic strategy with DFMO. In vivo, we utilized PDX and CDX models to validate the therapeutic efficacy of this drug combination. In vitro, we conducted western blot analysis, qPCR analysis, immunofluorescence staining, and PuMA experiments to monitor alterations in molecular expression, distribution, and tumor metastasis capability. We employed CCK-8 and colony formation assays to assess the proliferative capacity of cells in the experimental group. We used flow cytometry and reactive oxygen probes to observe changes in ROS and glutamine metabolism within the cells. Finally, we applied RNA-seq in tandem with metabolomics to identify metabolic alterations in OS cells treated with a DFMO and CIL56 combination. This enabled us to intervene and validate the role of the YAP1-mediated glutamine metabolic pathway in DFMO resistance. RESULTS:Through single-cell RNA-seq data analysis, we pinpointed a subset of late-stage OS cells with significantly upregulated polyamine metabolism. This upregulation was further substantiated by transcriptomic profiling of recurrent and non-recurrent OS tissues. High-throughput drug screening revealed a promising combination strategy involving DFMO and CIL56. DFMO treatment curbs the phosphorylation of YAP1 protein in OS cells, promoting nuclear entry and initiating the YAP1-mediated glutamine metabolic pathway. This reduces intracellular ROS levels, countering DFMO's anticancer effect. The therapeutic efficacy of DFMO can be amplified both in vivo and in vitro by combining it with the YAP1 inhibitor CIL56 or the glutaminase inhibitor CB-839. This underscores the significant potential of targeting the YAP1-mediated glutamine metabolic pathway to enhance efficacy of DFMO. CONCLUSION:Our findings elucidate YAP1-mediated glutamine metabolism as a crucial bypass mechanism against DFMO, following the inhibition of polyamine metabolism. Our study provides valuable insights into the potential role of DFMO in an "One-two Punch" therapy of metastatic and recurrent osteosarcoma.

Osteosarcoma (OS) exhibit intra- and inter- heterogeneity, complicating the exploration of effective therapeutic strategies. Traditional in vitro and in vivo models are limited in inheriting biological and genomic heterogeneities of OS patients, even in inheriting the features on tumor microenvironment. The prolonged generation time of current models makes the drug development of OS slow and is not suitable to clinically rapid timing. Here, we introduce methods for generating and biobanking patient/PDX-derived osteosarcoma organoids (OS PD(X)Os) that recapitulate the histological, biological and genomic features of their paired OS patients. OS PD(X)Os can be generated quickly with high reliability in vitro or transplanted to immunodeficient mice. We further demonstrate an immune-featured OS PD(X)O (named iOS) model and its method for testing personalized chemotherapy response, personalized immune therapeutic strategy and target drug development, such as a novel PRMT5MTA inhibitor ARPN2169 on MTAP-deleted OS. Our studies show that iOS models maintain many typical features of OS and could be rapidly employed to investigate patient-specific therapeutic strategies. Additionally, our biobank establishes a rich resource for basic, translational and even clinical OS researches. ### Competing Interest Statement The authors have declared no competing interest.