Advanced Osteosarcoma Research Articles

SQLE promotes osteosarcoma progression via activating TGFβ/SMAD signaling pathway

BackgroundThe prognosis of advanced osteosarcoma (OS) has remained stagnant in last decades, requiring the identification of novel therapeutic targets. Recently, much attention was paid to the role of squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol metabolism, in the field of oncology, while the specific role of SQLE in OS has not been sufficiently elucidated. The present study aims to investigate the role of SQLE in the progression of OS and explore the potential mechanisms. MethodsThe expression levels of SQLE in OS tissues and adjacent normal tissues were compared using bioinformatic methods and experiments. Kaplan-Meier survival analysis and univariate and multivariate Cox analysis were performed to detect the association of SQLE expression and patient’ prognosis. Stably cell lines with SQLE knockdown or overexpression were constructed by lentivirus infection. CCK-8, colony formation, scratch healing, and Transwell invasion assays were carried out to explore the effect of SQLE knockdown or overexpression on the proliferation, migration, and invasion of OS cells. Gene set enrichment analysis was conducted to reveal signaling pathways associated with SQLE expression. The effect of SQLE on TGFβ/SMAD signaling pathway were explored by Western blot assay. ResultsHere, we found a notable rise of SQLE expression in OS tissues and cell lines. Survival analysis showed that individuals with high SQLE expression had a lower median overall survival time compared to those with low SQLE expression. Univariate and multivariate Cox regression analyses showed that SQLE might have the potency to serve as an independently prognostic biomarker in OS. Loss- and gain-of-function experiments indicated that silence of SQLE suppressed OS cell proliferation, migration, and invasion, while overexpression of SQLE exerted the opposite effects. Mechanistically, TGF-β signaling pathway was identified as the downstream pathway of SQLE through bioinformatic methods, and the results of Western blot assay showed that SQLE positively regulated the activity of TGFβ1/SMAD2/3 signaling in OS. Resue experiments demonstrated that SB431542, a small molecule that inhibits TGFβ/SMAD signaling, could partly reverse the promoting effects of SQLE on OS cell proliferation, migration, and invasion. ConclusionOur results provided preliminary evidences that SQLE was a tumor-promoting factor and prognosis predictor in OS. SQLE promoted OS cell proliferation, migration, and invasion via activating TGFβ/SMAD signaling and targeting SQLE might be a potential strategy for the treatment of OS.

Enhanced immune responses are accompanied by increased MAGEA expression in osteosarcoma metastases

ObjectiveOsteosarcoma is the most common primary bone sarcoma. About 50% of patients develop metastatic disease and their 5-year survival lingers at around 20%–30%. T cell checkpoint blockade immunotherapies have revolutionised...

Spatial multiplexed immunofluorescence analysis reveals coordinated cellular networks associated with overall survival in metastatic osteosarcoma

Patients diagnosed with advanced osteosarcoma, often in the form of lung metastases, have abysmal five-year overall survival rates. The complexity of the osteosarcoma immune tumor microenvironment has been implicated in clinical trial failures of various immunotherapies. The purpose of this exploratory study was to spatially characterize the immune tumor microenvironment of metastatic osteosarcoma lung specimens. Knowledge of the coordinating cellular networks within these tissues could then lead to improved outcomes when utilizing immunotherapy for treatment of this disease. Importantly, various cell types, interactions, and cellular neighborhoods were associated with five-year survival status. Of note, increases in cellular interactions between T lymphocytes, positive for programmed cell death protein 1, and myeloid-derived suppressor cells were observed in the 5-year deceased cohort. Additionally, cellular neighborhood analysis identified an Immune-Cold Parenchyma cellular neighborhood, also associated with worse 5-year survival. Finally, the Osteosarcoma Spatial Score, which approximates effector immune activity in the immune tumor microenvironment through the spatial proximity of immune and tumor cells, was increased within 5-year survivors, suggesting improved effector signaling in this patient cohort. Ultimately, these data represent a robust spatial multiplexed immunofluorescence analysis of the metastatic osteosarcoma immune tumor microenvironment. Various communication networks, and their association with survival, were described. In the future, identification of these networks may suggest the use of specific, combinatory immunotherapeutic strategies for improved anti-tumor immune responses and outcomes in osteosarcoma.

Targeted anti-angiogenesis therapy for advanced osteosarcoma.

To date, despite extensive research, the prognosis of advanced osteosarcoma has not improved significantly. Thus, patients experience a reduced survival rate, suggesting that a reevaluation of current treatment strategies is required. Recently, in addition to routine surgery, chemotherapy and radiotherapy, researchers have explored more effective and safer treatments, including targeted therapy, immunotherapy, anti-angiogenesis therapy, metabolic targets therapy, and nanomedicine therapy. The tumorigenesis and development of osteosarcoma is closely related to angiogenesis. Thus, anti-angiogenesis therapy is crucial to treat osteosarcoma; however, recent clinical trials found that it has insufficient efficacy. To solve this problem, the causes of treatment failure and improve treatment strategies should be investigated. This review focuses on summarizing the pathophysiological mechanisms of angiogenesis in osteosarcoma and recent advances in anti-angiogenesis treatment of osteosarcoma. We also discuss some clinical studies, with the aim of providing new ideas to improve treatment strategies for osteosarcoma and the prognosis of patients.

Single-cell transcriptomic insights into chemotherapy-induced remodeling of the osteosarcoma tumor microenvironment

PurposeNeoadjuvant chemotherapy serves as an effective strategy for treating osteosarcoma (OS) not only by targeting cancerous cells but also by influencing the tumor's immune and stromal elements. Gaining insights into how chemotherapy reshapes the tumor's local environment is crucial for advancing OS treatment protocols.MethodsUsing single-cell RNA sequencing, this study analyzed tumor samples from patients with advanced osteosarcoma collected both before and after chemotherapy.ResultsThe results revealed that chemotherapy caused the remaining OS cells to express higher levels of genes associated with stemness. Additionally, this process enhances the presence of cancer-associated fibroblasts, increasing their ability to modify the extracellular matrix (ECM). Chemotherapy also increases the number of endothelial cells, albeit with compromised differentiation capabilities. Importantly, the treatment reduced the immune cell population, including myeloid and T/NK cells, particularly impacting the subpopulations with tumor-fighting capabilities.ConclusionThese findings highlight the complex reaction of the tumor environment to chemotherapy, providing valuable insights into how chemotherapy influences OS cells and the tumor microenvironment (TME). This knowledge is essential for understanding OS resistance mechanisms to treatments, potentially guiding the development of novel therapies for managing advanced OS.

Synergistic anti-osteosarcoma cell effects of the methotrex-ate/PI3K/mTOR inhibitor nanoparticle codelivery system

ABSTRACT Advanced osteosarcoma (OS) has low responsiveness to treatment regimens. Methotrexate (MTX), the first-line chemotherapeutic agent for advanced OS, frequently suffers from severe side effects that lead to chemotherapy interruption. To improve the efficacy and reduce the toxic side effects, we constructed a PF@MTX NP nano-delivery system using MTX-ss-γPGA polymer as a drug carrier loaded with PF. The average size of PF@MTX NPs was approximately 171.9 ± 2.13 nm with a zeta potential of approximately -32.91 ± 0.03 mV, uniformly distributed. We found that the nano showed a significant slowdown in MTX and PF release within 48 h and exhibited GSH-responsive pH sensitivity. In addition, the results of cellular uptake assay, toxicity, scratch and AO/EB staining assays showed that the PF@MTX NPs nanoparticles were able to enter the cells and induced 143B cell death and migration. Thus, the PF@MTX NPs nano-delivery system provides an effective strategy for the combined treatment of solid osteosarcoma.

Pulmonary Surgical Margins for Metastatic Osteosarcoma: Is Negative Margin Enough?

BackgroundIn advanced osteosarcoma, the lung is the most frequent site of distant metastasis, with metastasectomy often used for local disease control. The influence of pulmonary resection margin length on outcomes for osteosarcoma has not been well explored. This study sought to evaluate the impact of margin length relative to tumor size on local recurrence and survival in lung-limited metastatic osteosarcoma. MethodsPatients with metastatic osteosarcoma who underwent lung resection between 2000 and 2020 were identified from a single institution. Clinicopathologic variables were collected. The margin length-to-tumor size ratio (MTR) was calculated per nodule and classified relative to an MTR of 0.5. The primary outcome was development of local recurrence per nodule. Multivariate logistic regression was used to investigate covariates. ResultsA total of 142 patients with 689 nodules met inclusion criteria, with mean age of 35.6 years (interquartile range [IQR], 20.9-46.6 years). Patients were predominantly male (n = 87; 61.3%) and White (n = 106; 72.5%). Most nodules (n = 644; 93.5%) were resected through thoracotomy. The mean tumor size was 0.8 cm (IQR, 0.5-1.70 cm), with an average margin length of 0.3 cm (IQR, 0.1-0.7 cm). Among all nodules, 299 (43.4%) had an MTR >0.5. Systemic therapy was received by 94 patients (66.2%) preoperatively and by 100 patients (70.4%) postoperatively. Importantly, the study found that an MTR >0.5 conferred a protective effect against disease recurrence (hazard ratio, 0.67; 95% CI, 0.52-0.87; P = .003). ConclusionsIn resected pulmonary metastatic osteosarcoma, a margin length greater than one-half the size of the pulmonary nodule is associated with a lower incidence of local disease recurrence. This finding has implications for the subsequent need for additional therapy and disease-free status, thus meriting attentive intraoperative consideration.

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.

Identifying the Best Candidate for Primary Tumor Resection in Patients With Advanced Osteosarcoma.

Not all patients with stage III and IV osteosarcoma who undergo surgery to remove the primary tumor will benefit from surgery; therefore, we developed a nomogram model to test the hypothesis that only a subset of patients will benefit from surgery. 412 patients were screened from the Surveillance, Epidemiology and End Results (SEER) database. Subsequently, 1:1 propensity score matching (PSM) was used to screen and balance confounders. We first made the hypothesis that patients who underwent the procedure would benefit more. A multivariate Cox model was used to explore the independent influencing factors of CSS in two groups (benefit group and non-benefit group) and constructed nomograms with predicted prognosis. Finally, receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA) were used to verify the performance of the nomogram. Of these patients, approximately 110 did not undergo primary tumour resection. After passing PSM, they were divided into a surgical group and a non-surgical group. Age, primary site and chemotherapy as calculated independent factors were used to construct a nomogra. The predicted nomogram showed good consistency in terms of the ROC curve and the calibration curve, and the DCA curve showed a certain clinical utility. Finally, dividing the surgical patients into surgical beneficiaries and surgical non-beneficiaries, a Kaplan-Meier analysis showed that the nomogram can identify patients with osteosarcoma who can benefit from surgery. A practical predictive model was established to determine whether patients with stage III or IV osteosarcoma would benefit from surgery.

A meta-analysis: the clinical value of PD-1 inhibitor or protein tyrosine kinase inhibitors in the treatment of advanced osteosarcoma.

PD-1 inhibitors and TKIs have been used to treat advanced osteosarcoma, but there is still a lack of intuitive data for the comparison of their efficacy. We conducted a meta-analysis to evaluate their therapeutic benefits. A systematic methodological search of five primary electronic databases was performed. Studies with a randomized design of any type about PD-1 inhibitors or TKIs for the treatment of advanced osteosarcoma were included. The primary outcomes mainly included CBR, PFS, OS and ORR, The CR, PR, SD and AEs were the secondary outcomes. The survival period (months) of patients was taken as the main analysis data. Random-effects models were used for meta-analysis. Eight immunocheckpoint inhibitors in 327 patients from 10 clinical trials were finally evaluated. For OS, TKIs [11.67 months (95% CI, 9.32-14.01)] show more obvious advantages than PD-1 inhibitors [6.37 months (95% CI, 3.96-8.78)]. For PFS, TKIs [4.79 months (95% CI, 3.33-6.24)] are longer than PD-1 inhibitors [1.46 months (95% CI, 1.23-1.69)]. Although there was no fatal event, attention should still be paid, especially during the combined application of PD-1 inhibitors with TKIs since their obvious AEs. The findings of this study suggest that patients with advanced osteosarcoma, TKIs may be more beneficial than PD-1 inhibitors. TKIs combined with PD-1 inhibitors has a bright future in the treatment of advanced osteosarcoma, but we should always pay attention to the strong side effects.

Longitudinal characterization of primary osteosarcoma and derived subcutaneous and orthotopic relapsed patient-derived xenograft models.

Osteosarcoma is a rare bone cancer in adolescents and young adults with a dismal prognosis because of metastatic disease and chemoresistance. Despite multiple clinical trials, no improvement in outcome has occurred in decades. There is an urgent need to better understand resistant and metastatic disease and to generate in vivo models from relapsed tumors. We developed eight new patient-derived xenograft (PDX) subcutaneous and orthotopic/paratibial models derived from patients with recurrent osteosarcoma and compared the genetic and transcriptomic landscapes of the disease progression at diagnosis and relapse with the matching PDX. Whole exome sequencing showed that driver and copy-number alterations are conserved from diagnosis to relapse, with the emergence of somatic alterations of genes mostly involved in DNA repair, cell cycle checkpoints, and chromosome organization. All PDX patients conserve most of the genetic alterations identified at relapse. At the transcriptomic level, tumor cells maintain their ossification, chondrocytic, and trans-differentiation programs during progression and implantation in PDX models, as identified at the radiological and histological levels. A more complex phenotype, like the interaction with immune cells and osteoclasts or cancer testis antigen expression, seemed conserved and was hardly identifiable by histology. Despite NSG mouse immunodeficiency, four of the PDX models partially reconstructed the vascular and immune-microenvironment observed in patients, among which the macrophagic TREM2/TYROBP axis expression, recently linked to immunosuppression. Our multimodal analysis of osteosarcoma progression and PDX models is a valuable resource to understand resistance and metastatic spread mechanisms, as well as for the exploration of novel therapeutic strategies for advanced osteosarcoma.

A phase II study of surufatinib in patients with osteosarcoma and soft tissue sarcoma who have experienced treatment failure with standard chemotherapy.

e23540 Background: Surufatinib is a multi-targeted, small-molecule tyrosine kinase inhibitor (TKI) that shows strong inhibitory effect on the activity of VEGFR-1, 2, 3, FGFR1 and CSF-1R. Here we conducted this trial to explore the efficacy and safety of surufatinib in the treatment for osteosarcoma and soft tissue sarcoma (STS) patients (pts) who have failed in standard chemotherapy. Methods: Pts with advanced osteosarcoma and STS (unresectable or metastatic), 14-70 years, and ECOG PS 0-1, are eligible. Surufatinib 300 mg q.d. is given in a 21-days schedule. Progression-free rate at 12 weeks (PFR12weeks) was the primary endpoint. Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), overall survival (OS) and safety (NCT05106777). Results: As of December 30, 2022, 19 pts (male, n=12; median age, 44 years [range, 23-67]; median lines of prior therapy, 2 [range, 2-5]) were enrolled. The most common histological subtypes included 3 leiomyosarcoma, 3 liposacroma, 3 epithelioid hemangioendothelioma, 2 fibrosarcoma and other. The interim analysis revealed 7 pts with stable disease (SD) and 1 pt with partial response (PR) at 12 weeks in stage 1. Recruitment was continued in stage 2 as initially planned. With 15 pts for efficacy analysis, PFR12weeks was 60% (9 pts). Best objective response by RECIST 1.1 was complete response (CR) in 1 pt (ORR of 6.7%) and SD in 11 pts (DCR of 80%). With a median follow-up of 6.97 months (95%CI: 5.28-8.65), the median PFS was 5.7 months (95%CI: 1.07-10.30). Treatment emergent adverse events (TEAEs) were mostly mild (grade 1-2), and the most common were hypertension (80%), proteinuria (73%), hypertriglyceridemia (53%), diarrhea (47%) and hyperbilirubinemia (47%). Grade 3-4 TEAEs were recorded in 4 pts including hypertension, proteinuria, hypertriglyceridemia and hypermagnesemia. Conclusions: Surufatinib is well tolerated and have some clinical activity in advanced osteosarcoma and STS who failed standard chemotherapy. Enrollment is ongoing and updated data will be presented in the future. Clinical trial information: NCT05106777 .

Clinical Targeted Next-Generation Panel Sequencing Reveals MYC Amplification Is a Poor Prognostic Factor in Osteosarcoma.

Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS (P = .04). The most frequently altered genes in the primary cohort were TP53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort (P = .015) and the validation cohort (P = .012). The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Real-World Data on Cabozantinib in Advanced Osteosarcoma and Ewing Sarcoma Patients: A Study from the Hellenic Group of Sarcoma and Rare Cancers.

Advanced osteosarcomas (OSs) and Ewing sarcomas (ESs) tend to have poor prognosis with limited therapeutic options beyond first-line therapy. Aberrant angiogenesis and MET signaling play an important role in preclinical models. The anti-angiogenic drug cabozantinib was tested in a phase 2 trial of advanced OS and ES and was associated with clinical benefits. We retrospectively analyzed the off-label use of cabozantinib in adult patients with advanced OS and ES/primitive neuroectodermal tumors (PNETs) in three centers of the Hellenic Group of Sarcoma and Rare Cancers (HGSRC). Between April 2019 and January 2022, 16 patients started taking 60 mg of cabozantinib for advanced bone sarcoma or PNET. Median age at cabozantinib initiation was 31 years (17-83). All patients had received peri-operative chemotherapy for primary sarcoma and between 0 and 4 lines of treatment (median; 2.5) for advanced disease. The most common adverse effects included fatigue, anorexia, hypertransaminasemia, weight loss, and diarrhea. One toxic death was noted (cerebral hemorrhage). Dose reduction to 40 mg was required in 31.3% of the patients. No objective response was noted, and 9/16 patients exhibited stable disease outcomes. Progression-free survival varied from 1 to 8 (median; 5) months. Our study demonstrates that cabozantinib has antitumor activity in this population. In the real-life setting, we observed similar adverse events as in the CABONE study and in other neoplasms.

Retrospective review of safety and efficacy of anlotinib in advanced osteosarcoma with metastases after failure of standard multimodal therapy.

To study the safety and efficacy of anlotinib, a multitargeted tyrosine kinase inhibitor, in the treatment of advanced osteosarcoma (OSS) with metastases. We retrospectively studied patients with advanced OSS and metastases who received anlotinib treatment in our hospital from June 2018 to April 2020. All patients had received standard multimodal therapies, before taking anlotinib. Therapeutic doses of anlotinib were 12mg for adults and 10mg for children and adolescents once a day for 2 consecutive weeks, followed by a week of withdrawal. This 3-week cycle of treatment was continued until the tumor progressed rapidly or the patients failed to tolerate the side effects. Adverse drug reactions were recorded, and therapeutic efficacy was evaluated based on progression-free survival (PFS), disease control rate (DCR), overall survival (OS), and objective response rate (ORR). The median PFS was 9.8±.9 months, and the 6- and 10-month PFS rates were 73% and 33%, respectively. The median OS was 11.4±.6 months. No patients achieved complete response. After 6 months of treatment, the DCR and ORR were 80% and 13%, respectively. No drug-related deaths or Grade 4 adverse events occurred in the patients. Five patients (33%) had Grade 3 adverse events. The most common drug-related adverse events were hand-food syndrome, fatigue, high blood pressure, anorexia, and pneumothorax. Anlotinib had a modest therapeutic effect in patients with advanced OSS after the failure of standard treatment. The adverse events were mostly tolerable or relieved after treatment.

Niclosamide is a potential candidate for the treatment of chemo-resistant osteosarcoma

Chemotherapy is the main treatment option for advanced osteosarcoma, which is the most common type of primary bone malignancy. However, patients develop resistance rapidly and many succumb to the disease. Niclosamide, an anthelmintic drug, has been recently identified to display potent and selective anti-cancer activity. In this work, we show that niclosamide at sub-micromolar concentrations inhibits proliferation and migration, and induces apoptosis in both parental and chemo-resistant osteosarcoma cells, with much less toxicity in normal osteoblastic cells. Interestingly, chemo-resistant osteosarcoma cells are more sensitive to niclosamide compared to parental cells. We further identify that inhibition of β-catenin is the underlying mechanism of niclosamide's action in osteosarcoma cells. In addition, we reveal that chemo-resistant osteosarcoma cells display increased β-catenin activity compared to parental cells, which might explain the hypersensitivity of chemo-resistant cells to niclosamide. Our work provides pre-clinical evidence that niclosamide can be repurposed for treating osteosarcoma. Our findings also suggest the therapeutic value of β-catenin to overcome osteosarcoma chemo-resistance.

Expression and Clinical Significance of Various Checkpoint Molecules in Advanced Osteosarcoma: Possibilities for Novel Immunotherapy

The fact that studies on anti-programmed cell death 1 (PD-1) or its relevant ligand 1 (PD-L1) have yielded such few responses greatly decreases the confidence in immunotherapy with checkpoint inhibitors for advanced osteosarcoma. We intended to characterize the expression of various checkpoint molecules with immunohistochemistry in osteosarcoma specimens and analyzed the relationship of the expression of these checkpoint molecules with patients' clinical courses. This study was a retrospective non-intervention study from August 1st 2017 to March 1st 2020. Immunohistochemistry for B7-H3 (CD276, Cluster of Differentiation 276), CD47 (Cluster of Differentiation 47), PD-L1 (programmed cell death ligand 1), TIM3 (mucin-domain containing-3), TGF-β (TransformingGrowth Factor β), CXCR 4 (Chemokine Receptor 4), CD27 (Cluster of Differentiation 27), IDO1 (Indoleamine 2,3-dioxygenase 1), KIRs (Killer cell Immunoglobulin-like Receptors), and SDF-1 (Stromal cell-Derived Factor-1) was performed on 35 resected osteosarcoma specimens. Patients progressed upon first-line chemotherapy with evaluable lesions were qualified for this study, and their specimens previously stored in the pathological department repository would be retrieved for analysis. Associations between the immunohischemistry markers and clinicopathological variables and survival were evaluated by the χ2 displayed by cross-table, Cox proportional hazards regression model, and Kaplan-Meier plots. The positive rates of B7-H3, CD47, PD-L1, TIM3, and TGF-β expression in this sample of 35 heavily treated osteosarcomas were 29% (10/35), 15% (5/35), 9% (3/35), 6% (2/35), and 6% (2/35), respectively, and diverse staining intensities were observed. Among these advanced patients, 15/35 (43%) had positive checkpoint expression, of which 33% (5/15) showed evidence of the co-expression of more than one checkpoint molecule. We did not find any obvious correlation with clinicopathological characteristics and the positive expression of these molecules. The present study highlights that only a small subset of progressive osteosarcomas, which had been heavily-treated, expressed tumor immune-associated checkpoint molecules, of which B7-H3 was the most positively expressed checkpoint and might be a promising target for further osteosarcoma investigation.

409P Tertiary lymphoid structures are scarce but associated with BCR clonal expansion, B cell activity and checkpoint inhibitor response in advanced osteosarcoma

Tertiary lymphoid structures (TLS) have been associated with tumor response to checkpoint inhibitors (ICIs) in soft tissue sarcoma as well as several epithelial cancers. However, its role in bone sarcoma has not been thoroughly investigated to date.

Characterization of the tumour microenvironment phenotypes in malignant tissues and pleural effusion from advanced osteoblastic osteosarcoma patients.

Malignant pleural effusion (MPE) is an adverse prognostic factor in patients with osteoblastic osteosarcoma; however, the cellular contexts of MPE are largely unknown. We performed single-cell RNA-sequencing (scRNA-seq) on 27260 cells from seven MPE samples and 91186 cells from eight osteosarcoma tissues, including one recurrent, one lung metastasis and six primary tumour (PT) samples, to characterize their tumour microenvironment. Thirteen main cell groups were identified in osteosarcoma tumour and MPE samples. Immune cells dominate the cellular contexts in MPE with more T/NK cells and less osteoclasts compared to PT samples. Of T/NK cells, CD8+ GNLY+ , CD8+ KLRC2+ T cells and FCGR3A+ NK cells were enriched in MPE but CD4+ FOXP3+ Tregs were enriched in PT samples. Naïve IGHD+ B and immune regulatory IGHA1+ B cells were largely identified in MPE, whereas bone metabolism-related CLEC11A+ B cells were significantly enriched in osteosarcoma PT. M2-type TAMs, including CLEC11A_TAM, C1QC_TAM and Prolif_TAMs, among myeloid cells were enriched in PT, which may suppress cytotoxicity activities of T cells through multiple ligand-receptor interactions. Mature LAMP3+ DCs were transformed from CD1C+ DC and CLEC9A+ DC sub-clusters when exposure to tumour alloantigens, which may improve T cell cytotoxicity activities on tumour cells under anti-PD-L1 treatments. In further, immune cells from MPE usually present up-regulated glycolysis and down-regulated oxidative phosphorylation and riboflavin metabolism activities compared to those in PT samples. Our study provided a novel cellular atlas of MPE and PT in patients with advanced osteosarcoma, which may provide potential therapeutic targets in the future.

Comprehensive analysis of potential cellular communication networks in advanced osteosarcoma using single-cell RNA sequencing data.

Osteosarcoma (OS) is a common bone cancer in children and adolescents, and metastasis and recurrence are the major causes of poor treatment outcomes. A better understanding of the tumor microenvironment is required to develop an effective treatment for OS. In this paper, a single-cell RNA sequencing dataset was taken to a systematic genetic analysis, and potential signaling pathways linked with osteosarcoma development were explored. Our findings revealed 25 clusters across 11 osteosarcoma tissues, with 11 cell types including “Chondroblastic cells”, “Osteoblastic cells”, “Myeloid cells”, “Pericytes”, “Fibroblasts”, “Proliferating osteoblastic cells”, “Osteoclasts”, “TILs”, “Endothelial cells”, “Mesenchymal stem cells”, and “Myoblasts”. The results of Cell communication analysis showed 17 potential cellular communication networks including “COLLAGEN signaling pathway network”, “CD99 signaling pathway network”, “PTN signaling pathway network”, “MIF signaling pathway network”, “SPP1 signaling pathway network”, “FN1 signaling pathway network”, “LAMININ signaling pathway network”, “FGF signaling pathway network”, “VEGF signaling pathway network”, “GALECTIN signaling pathway network”, “PERIOSTIN signaling pathway network”, “VISFATIN signaling pathway network”, “ITGB2 signaling pathway network”, “NOTCH signaling pathway network”, “IGF signaling pathway network”, “VWF signaling pathway network”, “PDGF signaling pathway network”. This research may provide novel insights into the pathophysiology of OS’s molecular processes.