Osteosarcoma Tumor Research Articles

Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

The outcomes of metastatic and nonresponder pediatric osteosarcoma patients are very poor and have not improved in the last 30 years. These tumors harbor a highly immunosuppressive environment, making existing immunotherapies ineffective. Here, we evaluated the use of Semliki Forest virus (SFV) vectors expressing galectin-3 (Gal3) inhibitors as therapeutic tools, since both the inhibition of Gal3, which is involved in immunosuppression and metastasis, and virotherapy based on SFV have been demonstrated to reduce tumor progression in different tumor models. In vitro, inhibitors based on the Gal3 amino-terminal domain alone (Gal3-N) or fused to a Gal3 peptide inhibitor (Gal3-N-C12) were able to block the binding of Gal3 to the surface of activated T cells. In vivo, SFV expressing Gal3-N-C12 induced strong antitumor responses in orthotopic K7M2 and MOS-J osteosarcoma tumors, leading to complete regressions in 47% and 30% of mice, respectively. Pulmonary metastases were also reduced in K7M2 tumor-bearing mice after treatment with SFV-Gal3-N-C12. Both the antitumor and antimetastatic responses were dependent on modulation of the immune system, primarily including an increase in tumor-infiltrating lymphocytes and a reduction in the immunosuppressive environment inside tumors. Our results demonstrated that SFV-Gal3-N-C12 could constitute a potential therapeutic agent for osteosarcoma patients expressing Gal3.

Phase 2 study of anti-disialoganglioside antibody, dinutuximab, in combination with GM-CSF in patients with recurrent osteosarcoma: A report from the Children's Oncology Group

PurposeNovel effective therapies are urgently needed in recurrent osteosarcoma. GD2 is expressed in human osteosarcoma tumours and cell lines. This study evaluated the disease control rate (DCR) in patients with recurrent osteosarcoma treated with the anti-GD2 antibody dinutuximab plus cytokine therapy as compared to historical outcomes. MethodsAOST1421 was a single-arm Phase 2 study for patients with recurrent pulmonary osteosarcoma in complete surgical remission. Patients received up to five cycles of dinutuximab (70 mg/m2/cycle) with granulocyte-macrophage colony-stimulating factor (GM-CSF). Two different dinutuximab infusion schedules were studied: 35 mg/m2/day over 20 h (2 days) and 17.5 mg/m2/day over 10 h (4 days). Primary end point was DCR, defined as a proportion of patients event free at 12 months from enrolment. The historical benchmark was 12-month DCR of 20% (95% CI 10–34%). Dinutuximab would be considered effective if ≥ 16/39 patients remained event free. Secondary objectives included toxicity evaluation and pharmacokinetics. ResultsThirty-nine eligible patients were included in the outcome analysis. Dinutuximab did not demonstrate evidence of efficacy as 11/39 patients remained event free for a DCR of 28.2% (95% CI 15–44.9%). One of 136 administered therapy cycles met criteria for unacceptable toxicity when a patient experienced sudden death of unknown cause. Other ≥ Grade 3 toxicities included pain, diarrhoea, hypoxia, and hypotension. Pharmacokinetic parameters were similar in the two schedules. ConclusionsThe combination of dinutuximab with GM-CSF did not significantly improve DCR in recurrent osteosarcoma. Dinutuximab toxicity and pharmacokinetics in adolescent and young adult osteosarcoma patients were similar to younger patients. Other strategies for targeting GD2 in osteosarcoma are being developed.

Membrane-Anchored and Tumor-Targeted IL12 (attIL12)-PBMC Therapy for Osteosarcoma.

Chimeric antigen receptor (CAR) T-cell therapy has shown great promise for treating hematologic malignancies but requires a long duration of T-cell expansion, is associated with severe toxicity, and has limited efficacy for treating solid tumors. We designed experiments to address those challenges. We generated a cell membrane-anchored and tumor-targeted IL12 (attIL12) to arm peripheral blood mononuclear cells (PBMC) instead of T cells to omit the expansion phase for required CAR T cells. This IL12-based attIL12-PBMC therapy showed significant antitumor efficacy in both heterogeneous osteosarcoma patient-derived xenograft tumors and metastatic osteosarcoma tumors with no observable toxic effects. Mechanistically, attIL12-PBMC treatment resulted in tumor-restricted antitumor cytokine release and accumulation of attIL12-PBMCs in tumors. It also induced terminal differentiation of osteosarcoma cells into bone-like cells to impede tumor growth. In summary, attIL12-PBMC therapy is safe and effective against osteosarcoma. Our goal is to move this treatment into a clinical trial. Owing to the convenience of the attIL12-PBMC production process, we believe it will be feasible.

Reconstruction and Outcome of Foot Defects Following Oncological Resection-Experience from a Cancer Centre in North-East India.

Solid tumours around the foot are rare and include soft tissue sarcomas, skin and bone malignancies. Extended soft tissue defects due to oncological resection result in the loss of shock-absorbing and friction resistant tissue, which leads to altered walking patterns and pain. Replacement of plantar tissue requires soft tissue resistant to weight, pressure and shear stress. The other important desired goal of foot reconstruction is short wound healing time in order to allow adjuvant therapy at stipulated time. This is a retrospective study from March 2016 to October 2019. A total of twenty-one (n = 21) patients were operated for foot malignancies during this period in our institute and the resulting defects were reconstructed using various methods. Different reconstructive surgeries were performed depending on tumour size, location and general health status of patients. The length of hospitalization and the presence of local postoperative complications were assessed. Functional outcomes were measured in terms of MSTS score. Average age of the series is 53.1years. Sixty-six percent of the patients (n = 14) presented with tumour at the weight bearing areas and 33% patients (n = 7) at the non-weight bearing areas of the foot. Fifty-seven percent of patients (n = 12) presented with malignant melanoma of foot, squamous cell carcinoma was seen in 33% (n = 7) patients and 4% patients (n = 1 each) presented as osteosarcoma and malignant peripheral nerve sheath tumour respectively. The mean MSTS score in patients with weight bearing areas (location) is statistically significant (p = 0.031). There is a significant correlation between the surgical complications and follow up MSTS score (p = 0.046) which signifies that flap related complications result in lower MSTS score. The mean MSTS score was 22.71/30. Complications were observed in three cases which included partial flap necrosis, graft loss and foot stiffness. Simple skin grafts to local flaps maybe a viable option in a limited resource setting based on the location of defect. Free tissue transfer is the ideal choice in case of weight bearing areas to achieve acceptable outcomes.

Abstract 6188: Targeted inhibition of the E3 ligase SCFSkp2/Cks1 has survival benefit and antitumor activity in osteosarcoma

Abstract Background: Osteosarcoma (OS) is a highly aggressive bone malignancy for which treatment has remained unchanged for years. Our previous studies indicate that the F-box protein SKP2 is overexpressed in OS and acts as a proto-oncogene. We recently reported that abolishing the interaction between SKP2 and its downstream substrate p27 using a Thr187Ala knock-in mutation leads to impaired tumor growth in an RB1/TP53 deficient transgenic OS mouse model. These data suggest that inhibiting the function of SKP2 may yield therapeutic benefits in OS. However, the precise mechanisms by which SKP2 promotes tumorigenesis in OS are not fully understood. Purposes: To determine the role of SCFSkp2/Cks1 in OS tumorigenesis and as a potential therapeutic target. Methods: We generated a genetically engineered mouse model of OS with a double knockout of Rb1 and p53 only within cells of the osteoblastic lineage (Osx1-Cre;Rb1lox/lox;Trp53lox/lox, DKO). The SKP2-knockout OS mouse model was further developed in the DKO background (Osx1-Cre;Rb1lox/lox;Trp53lox/lox; Skp2-/-, TKO). Mice of both genotypes were monitored for tumor growth, overall survival, and in vivo drug response. Early passaged OS cells and OS organoid culture was derived from mice tumors and used for in vitro analysis. Western Blot and IHC were used for protein level determination. RT-qPCR and RNA-seq were used for transcriptional level determination. Two small-molecule SCFSkp2/Cks1 inhibitors (C1 and Pevonedistat) were used for in vivo and in vitro testing. Results: All genotypes were born at the expected ratios and developmentally normal. DKO animals developed OS tumors with nearly full penetrance. SKP2 deletion significantly prolongs the overall survival (p<0.0001) and reduced the average tumor occurrence in OS mice (p<0.001). The in vivo tumor proliferation was also significantly delayed in the SKP2 deletion OS tumorigenesis (p=0.007). A histological analysis comparing tumors from two groups showed a decrease in proliferation (PCNA) and an increase in pro-apoptotic markers (TUNEL,etc) expression in SKP2-deleted OS. RT-qPCR and RNA-seq revealed upregulation of E2F1 downstream apoptotic-related target genes in Skp2-/- OS tumors. The transcriptional level analysis also revealed inhibition of cancer stemness markers and pathways in SKP2 deleted tumors and further proved by functional analyses. Two SCFSkp2/Cks1 inhibitors not only showed anti-tumoral activities both in vivo (p<0.05) and in organoid culture, but also synergistic with chemotherapy agents (ZIP synergy Score:9.34±1.24). Conclusions: Using genetic and pharmacologic approaches, we show that inhibiting SKP2 leads to significant antitumor activities in a clinically relevant OS mouse model. The strong survival benefit highlights SKP2 as a downstream actionable target in TP53/RB1-deficient OS, for which the targeted therapies have been disappointing to date. Citation Format: Jichuan Wang, Alexander Ferrena, Ranxin Zhang, Swapnil Singh, Hongling Zhao, Deyou Zheng, Hasibagan Borjihan, Waleed Al-Harden, Janet Tingling, Rui Yang, David S. Geller, Bang H. Hoang. Targeted inhibition of the E3 ligase SCFSkp2/Cks1 has survival benefit and antitumor activity in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6188.

Abstract 801: Surprisingly conserved copy numbers from cell to cell within structurally complex tumors challenge the unstable genome hypothesis

Abstract Osteosarcoma is an aggressive disease that exhibits almost universal p53 inactivation and a high level of genomic complexity. The chaotic genome of most osteosarcomas has led many to assume that this complexity must accumulate under conditions of significant genomic/chromosomal instability. However, such conclusions have been drawn primarily from single-timepoint bulk-tumor analyses. More recent studies have questioned this assumption of chromosomal instability, hypothesizing that genomic complexity could arise from an early catastrophic event followed by faithful propagation of a highly complex, malignancy-promoting genome. To determine whether osteosarcoma tumors show evidence of instability, we performed single-cell whole-genome sequencing of >14,000 osteosarcoma cells obtained from 15 distinct lesions resected from 8 individual patients. Using the CHISEL algorithm, we inferred allele- and haplotype-specific copy numbers from this sequencing data. To evaluate how the SCNA profiles changed during the evolutionary pressures associated with metastatic dissemination and therapy, we compared the single-cell SCNA profiles of paired samples collected from both primary sites and metastases and from pre-treatment and relapsed specimens. We likewise compared profiles of patient tumors propagated orthotopically in mouse tibias to those grown within the lung to evaluate for tissue-dependent emergence of rare subclones. Despite extensive structural variations that give rise to highly complex patterns of SCNA, the cells within each tumor showed remarkably little heterogeneity. We found similar evidence of chromosomal stability when we reconstructed phylogenetic trees to identify the evolutionary relationships of cells collected from metastatic lesions or at relapse. Despite being evolutionarily distant, nearly all variants could be assigned to the trunk of the phylogenetic tree, with only modest changes occurring in the branches. This result suggests that nearly all SCNAs were acquired in an early event, followed by selection of advantageous patterns with subsequent preservation of that particular SCNA pattern. Analysis of bulk whole-genome sequencing from serially collected patient samples supports the preservation of SCNA profiles over time. This work demonstrates the power of combining single-cell DNA sequencing with an allele- and haplotype-specific CNV inference algorithm. Our approach clarifies longstanding questions about the genetics of osteosarcoma initiation and progression, calling into question previous assumptions of genomic instability inferred from single-timepoint bulk sequencing data. These results suggest that an isolated, early catastrophic event, rather than sustained genomic instability, gives rise to the complex genome that characterizes osteosarcoma tumors. Citation Format: Sanjana Rajan, Simone Zaccaria, Matthew Cannon, Maren Cam, Amy Gross, Benjamin Raphael, Ryan D. Roberts. Surprisingly conserved copy numbers from cell to cell within structurally complex tumors challenge the unstable genome hypothesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 801.

Abstract 2509: Cytokines derived from tumor initiating osteosarcoma cells mediate a novel self-seeding mechanism relevant to growth of primary and metastatic tumors

Abstract Osteosarcoma is the most common pediatric bone tumor. Death from osteosarcoma is almost always due to metastatic spread to the lungs, which largely occurs after the primary tumor has been resected and treated with chemotherapy. Others have suggested that the primary tumor elaborates factors that suppress metastasis, though mechanisms explaining this phenomenon have not been well-validated suggested that primary osteosarcoma tumors suppress metastasis by recruiting circulating tumor cells back to the tumor via self-seeding, though the mechanism(s) of recruitment remained poorly defined. We found that osteosarcoma cells induce chemotaxis of other osteosarcoma cells in vitro. We previously demonstrated that osteosarcoma cells produce IL6 and CXCL8. Here, we found that both cytokines induced chemotaxis of osteosarcoma cells. Conversely, inhibition of both cytokines prevented osteosarcoma-induced osteosarcoma chemotaxis. These results suggested a model wherein tumor cells attract other tumor cells via inflammatory cytokine signaling. Within a living organism, such mechanisms might have implications for therapy. We wondered whether resection of a primary osteosarcoma tumor might redirect circulating tumor cells from the primary tumor to the lung (the “next best” site). To test this hypothesis, we generated orthotopic tibial tumors in mice. One cohort of mice underwent amputation of primary tumor while another retained their primary tumors. At euthanasia, mice that underwent amputation had significantly increased tumor cell burden in the lungs. In separate experiments, lungs from mice with established primary tumors showed significantly less osteosarcoma lung burden than was seen in mice that did not have a primary tumor. Many of infiltrating (labeled) tumor cells were subsequently identified within the primary tumor, strongly supporting the self-seeding model. Finally, we asked whether this same self-seeding mechanism might drive events important to lung metastasis. To test this hypothesis, mice were first inoculated with green labelled osteosarcoma cells to induce lung metastasis. Fourteen days later, the same mice received inoculation with red labelled osteosarcoma cells. Strikingly, we found that lesions contained many more red- than green-labeled osteosarcoma cells. In addition, we have demonstrated that inhibition of IL6/CXCL8 blocks circulating tumor cell recruitment to established lung niches. This suggests circulating tumor cells preferentially target an established niche within the lung in an IL6/CXCL8 dependent manner in the absence of a primary tumor. Collectively, our data suggests that a loss of self-seeding in osteosarcoma on excision of the primary tumor increases metastatic burden. We are now exploring ways to leverage this biology in the development of novel therapies that prevent metastatic disease. Citation Format: Amy C. Gross, James B. Reinecke, Amanda Saraf, Helene LePommellet, Sophia Vatelle, Ryan D. Roberts. Cytokines derived from tumor initiating osteosarcoma cells mediate a novel self-seeding mechanism relevant to growth of primary and metastatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2509.

Abstract 2016: Preclinical evaluation of uPARAP (MRC2) antibody-drug conjugates (ADCE-003,010,011) in osteosarcoma pdx models

Abstract Introduction: Antibody-drug Conjugates (ADCs) targeting differentially expressed tumor cell-surface antigens show robust clinical activity in several solid tumor cancers, but none is yet available for osteosarcoma. Due to the high unmet medical need in this indication, there is an urgent requirement to identify respective osteosarcoma cell-surface antigens and evaluate the antitumor activity of an ADC that can deliver tailored cytotoxic payloads to osteosarcoma tumors expressing these targets. Methods: We used an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. uPARAP (MRC2) was found to be enriched in osteosarcoma at both protein and mRNA levels. The cell-surface expression of uPARAP (MRC2) was further validated by IHC and flow cytometry with osteosarcoma cell lines, PDX models, and patient tumor tissue microarray. As a proof of concept, three MRC2 targeted ADCs with different Microtubulin- and alkylating agent types of payloads were tested in 8 osteosarcoma PDX models. uPARAP (MRC2) ADC, control ADC, or vehicle control was administered IV to mice harboring flank tumors at a dose of 3mg/kg, weekly X 3 (ADCE-003), 2mg/kg on Day 1 (ADCE-010), and 10mg/kg weekly X 2 (ADCE-011). EFS for treatment (T) and control (C) groups, minimum relative tumor volume (minRTV), and objective response measures were analyzed. Results: Western blotting confirmed the expression of uPARAP (MRC2) in 8 osteosarcoma cell lines and 8 PDX models. Flow cytometry further validated cell-surface localization and expression levels of uPARAP (MRC2) in 7 osteosarcoma cell lines. We then performed immunohistochemistry (IHC) staining using an osteosarcoma tissue microarray from 37 patients and 19 PDX models. uPARAP (MRC2) was expressed in 97% of the patient samples and 95% of the PDXs. 51% of the patient samples and 39% of the PDXs had an overall H-score of 100 or higher. Mice tolerated ADCs well with minimal toxicity. All 3 ADCs significantly prolonged EFS in 6/8 osteosarcoma models. Complete response (CR) or maintained CR were observed in 2 models in ADCE-010 and ADCE-011 groups, respectively. Conclusions: uPARAP (MRC2) is highly expressed in most osteosarcoma samples, which makes it a viable target for respective antigen targeting ADC therapies. Three uPARAP (MRC2) targeting ADCs showed antitumor activities in osteosarcoma preclinical models which warrant further investigation of uPARAP targeting ADC therapies for osteosarcoma. Citation Format: Yifei Wang, Wendong Zhang, Zhongting Zhang, Xiangjun Tian, Rossana Lazcano, Pooja Hingorani, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Jing Wang, Niels Behrendt, Christoffer F. Nielsen, Lars H. Engelholm, Richard Gorlick. Preclinical evaluation of uPARAP (MRC2) antibody-drug conjugates (ADCE-003,010,011) in osteosarcoma pdx models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2016.

Abstract 6046: Targeting TBL1 inhibits nuclear β-catenin activity and enhances immune checkpoint inhibition efficacy in osteosarcoma

Abstract Background: Osteosarcoma (OS) is the most common bone malignancy in childhood and adolescence. Though the prognosis of OS patients has been improved by multimodal therapies, patients with metastatic disease still have a very poor prognosis. We previously reported a novel TBL1/β-catenin inhibitor, Tegavivint, suppresses OS primary tumor and lung metastasis using patient-derived cell lines. Wnt/β-catenin signaling is reported to influence intratumoral T cell infiltration and using murine syngeneic OS models we investigated the effects of Tegavivint on improving immune checkpoint inhibition and the OS tumor immune microenvironment. Methods: We used murine OS cell lines F331 and F420 for in vitro and in vivo studies. We performed cytotoxicity studies using the CCK-8 assay and western blots to investigate downstream β-catenin signaling perturbations secondary to Tegavivint. Intratibial injections were performed into C57BL/6 mice using both murine cell lines. Tegavivint and anti-PD-1 antibody was administered as monotherapy or in combination. After treatment, we compared the tumor volume in the control, monotherapy (Tegavivint or PD-1 only) and combination therapy groups (Tegavivint and PD-1). We assessed T cell tumor infiltration using immunohistochemistry and flow cytometry. Results: In vitro, Tegavivint suppresses the growth of F331 and F420 cell lines with IC50s of 9.52nM and 49.4nM, respectively after 72hours of treatment. In vivo, anti-PD1 monotherapy did not show any significant anti-tumor activity, but Tegavivint monotherapy significantly suppressed the growth of murine tumors (p<0.05) and the combination therapy further improved overall anti-tumor activity. IHC staining showed an increase in CD8-positive T cells in Tegavivint monotherapy and combination therapy treated tumors compared with the control tumors. Furthermore, flow cytometry validated the increase in CD8-positive T cell infiltration and also showed an increase in intratumoral NK cells in Tegavivint treated tumors. Conclusions: These results indicate that TBL1 inhibition, via Tegavivint, inhibits nuclear β-catenin activity and alters the tumor immune microenvironment and enhances immune checkpoint blockade in OS. Future directions include more comprehensive profiling and molecular analysis of the tumor and immune microenvironment, and evaluation of additional immune modulatory approaches for the treatment of OS. Citation Format: Kengo Nakahata, Bikesh K. Nirala, Ryan L. Shuck, Lyazat Kurenbekova, Jason T. Yustein. Targeting TBL1 inhibits nuclear β-catenin activity and enhances immune checkpoint inhibition efficacy in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6046.

Single-cell RNA sequencing reveals differential expression of EGFL7 and VEGF in giant-cell tumor of bone and osteosarcoma.

Dysregulation of angiogenesis is associated with tumor development and is accompanied by altered expression of pro-angiogenic factors. EGFL7 is a newly identified antigenic factor that plays a role in various cancers such as breast cancer, lung cancer, and acute myeloid leukemia. We have recently found that EGFL7 is expressed in the bone microenvironment, but its role in giant-cell tumor of bone (GCTB) and osteosarcoma (OS) is unknown. The aims of this study are to examine the gene expression profile of EGFL7 in GCTB and OS and compare with that of VEGF-A-D and TNFSF11 using single-cell RNA sequencing data. In-depth differential expression analyses were employed to characterize their expression in the constituent cell types of GCTB and OS. Notably, EGFL7 in GCTB was expressed at highest levels in the endothelial cell (EC) cluster followed by osteoblasts, myeloid cells, and chondrocytes, respectively. In OS, EGFL7 exhibited highest expression in EC cell cluster followed by osteoblastic OS cells, myeloid cells 1, and carcinoma associated fibroblasts (CAFs), respectively. In comparison, VEGF-A is expressed at highest levels in myeloid cells followed by OCs in GCTB, and in myeloid cells, and OCs in OS. VEGF-B is expressed at highest levels in chondrocytes in GCTB and in OCs in OS. VEGF-C is strongly enriched in ECs and VEGF-D is expressed at weak levels in all cell types in both GCTB and OS. TNFSF11 (or RANKL) shows high expression in CAFs and osteoblastic OS cells in OS, and osteoblasts in GCTB. This study investigates pro-angiogenic genes in GCTB and OS and suggests that these genes and their expression patterns are cell-type specific and could provide potential prognostic biomarkers and cell type target treatment for GCTB and OS.

Cohort profile: The Golden Retriever Lifetime Study (GRLS).

The aim of this article is to provide a detailed description of the Golden Retriever Lifetime Study (GRLS), a prospective cohort study investigating nutritional, environmental, lifestyle, and genetic risk factors for cancer and other common diseases in dogs. Primary outcomes of interest include hemangiosarcoma, lymphoma, osteosarcoma, and high-grade mast cell tumors. Secondary outcomes of interest include other cancers, hypothyroidism, epilepsy, atopy, otitis externa, hip dysplasia, heart failure, and renal failure. A total of 3,044 United States Golden Retrievers aged 6 months to 2 years completed baseline enrollment from June 2012 to April 2015. As of May 31, 2021, 2,251 dogs remain engaged in the study, 352 have died, and 441 are lost to follow-up. Extensive annual questionnaires completed by owners and veterinarians gather information about lifestyle, environmental exposures, physical activity, reproductive history, behavior, diet, medications, and diagnoses. Dogs also have annual veterinary examinations and biospecimen collection (blood, serum, hair, nails, feces, urine) for biobanking. Additional reporting, including histology and tumor biobanking, is conducted for any malignancies or deaths. When an animal dies, full medical records are obtained, and necropsies are requested at owner discretion. Full or partial necropsies have been performed on 218 dogs. Questionnaire data are freely available to researchers with approved credentials who agree to a data use agreement. In addition, researchers can submit proposals to utilize biospecimens or obtain additional data.

Expression pattern and clinical significance of β-catenin gene and protein in patients with primary malignant and benign bone tumors.

This study is aimed to unravel the status of local and circulating β-catenin in different primary bone tumors and its relevance to tumor types, severity, and chemotherapy. The β-catenin mRNA expression level and the expression of the protein (intensity level) were evaluated in tumor tissue and peripheral blood mononuclear cells of 150 patients with different types of primary bone tumors (78 malignant and 72 benign tumors) using Real-Time PCR and immunohistochemistry. The β-catenin mRNA expression level and the expression of the protein were increased in bone tumors which was positively correlated with the tumor malignancy. Amongst osteosarcoma, Ewing's Sarcoma, chondrosarcoma, osteochondroma, Giant Cell Tumor, and exostosis tumors, the osteosarcoma, and Giant Cell Tumor groups showed the highest level of β-catenin expression. The β-catenin expression in malignant bone tumors was significantly correlated with tumor grade, size, metastasis, tumor recurrent, and the level of response to chemotherapy. A similar pattern of β-catenin gene expression and its association with tumor characteristics was detected in the patient's peripheral blood cells. The simultaneous increase in the expression of the β-catenin gene and protein in tumor tissue and in circulating blood cells and its relationship with tumor severity indicates the possible promoting role of β-catenin in primary bone tumor pathogenesis.

Licochalcone B Induced Apoptosis and Autophagy in Osteosarcoma Tumor Cells via the Inactivation of PI3K/AKT/mTOR Pathway.

Licochalcone B (LicB) is a flavonoid derived from the Chinese medicinal herb Glycyrrhiza uralensis Fisch. Several previous studies have demonstrated the wide range of pharmacological activities shown by LicB. In this study, we investigated the anticancer effects of LicB in osteosarcoma (OS) tumor cells and its underlying mechanisms. According to the Cell Counting Kit-8 (CCK8) analysis and 5-ethynil-2'-deoxyuridine (EdU) staining results, we found that LicB suppresses OS cells (MG-63 and U2OS) growth depending on its concentration. Furthermore, flow cytometry and Western blot revealed that LicB promoted autophagy and apoptosis in OS cells in a dose-dependent manner. LicB treatment not only decreased the levels of Bcl-2, p62, Caspase-3, and Ki67 protein in MG-63 and U2OS cell lines but also increased the levels of Cleaved Caspase-3, Beclin1, Bax, Atg7, and LC3B. Mechanistically, LicB induced cell apoptosis by promoting the apoptosis-related cleavage of Caspase-3, while suppressing the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway to induce autophagy. The present work is the first to illustrate that LicB can serve as a potential drug candidate for tumor treatment owing to its ability to enhance autophagy and apoptosis, and suppress OS proliferation by inactivating the PI3K/AKT/mTOR pathway.

SGLT2 inhibitor activates the STING/IRF3/IFN-β pathway and induces immune infiltration in osteosarcoma

SGLT2 (sodium-glucose cotransporter 2) is an important mediator of epithelial glucose transport and has been reported that SGLT2, robustly and diffusely expressed in malignant cancer cells, was overexpressed in various tumors, and inhibiting the SGLT2 expression significantly inhibited tumor progression. By blocking the functional activity of SGLT2, SGLT2 inhibitors have shown anticancer effects in several malignant cancers, including breast cancer, cervical cancer, hepatocellular cancer, prostate cancer, and lung cancer. However, the anticancer effect of SGLT2 inhibitors in osteosarcoma and the specific mechanism are still unclear. In the present study, we found that SGLT2 was overexpressed at the protein level in osteosarcoma. Furthermore, our results showed that the SGLT2 inhibitor significantly inhibited osteosarcoma tumor growth and induced infiltration of immune cells in vivo by upregulating STING expression and activating the IRF3/IFN-β pathway, which could attribute to the suppression of AKT phosphorylation. In addition, the combined treatment with SGLT2 inhibitor and STING agonist 2’3’-cGAMP exerted synergistic antitumor effects in osteosarcoma. Furthermore, the overexpression of SGLT2 at the protein level was correlated with the degradation of SGLT2 induced by TRIM21. This result demonstrated that SGLT2 is a novel therapeutic target of osteosarcoma, and that the SGLT2 inhibitor, especially in combination with 2’3’-cGAMP, is a potential therapeutic drug.

Characteristics and significance of T lymphocyte subsets in peripheral blood of osteosarcoma mice.

BackgroundOsteosarcoma (OS) is the most common malignant bone tumor, which often has lung metastasis. The survival rate after tumor metastasis is very low. Treatments including the antitumor immunocompetence of innate immune cells are found favorable for OS tumor. However, as a major means of early tumor detection, there are still few studies on T cells in peripheral blood of OS patients.MethodsOS cells were implanted into the femoral bone marrow cavity of C3H/HeN mice to construct OS model. The proportion of T lymphocyte subsets in peripheral blood of mice was detected by flow cytometry on the 14th and 21st days after modeling.ResultsCompared with sham operation group, on the 14th day after operation, the proportion of γδT cells and CD4+ regulatory T cells (Tregs) showed significantly higher level while other subsets didn’t have obvious difference. On the 21st day after operation, the proportion of CD3+ T cells, γδ T cells, CD4+ Tregs and Tregs/Th17 ratio were obviously higher than that of sham operation group. Meanwhile, while CD4+ T cells and CD8+ T cells were lower, suggesting the happen of peripheral immunosuppression. Comparing the T lymphocytes subsets on the 14th and 21st day after modeling in the tumor group, it was found that CD3+ T cells and CD4+ Tregs on the 21st day after modeling were significantly higher than those on the 14th day while CD4+ T cells, CD8+ T cells decreased significantly, indicating the changes of T lymphocytes in peripheral blood of OS mice are related to tumor development.ConclusionsThe formation and development of femoral OS is accompanied by the disorder of peripheral blood T lymphocyte subsets and peripheral immunosuppression.

A phase 1/2 dose-escalation and dose-expansion study of ZN-c3 in combination with gemcitabine in adult and pediatric subjects with relapsed or refractory osteosarcoma.

TPS11584 Background: Osteosarcoma (OS) is the most common primary bone malignancy of childhood and adolescence with 5-year survival rates of 65-70% for localized disease and < 30% for de novo metastatic disease or recurrent disease. Pooled analysis of previous phase 2 trials by the Children’s Oncology Group has determined a 4-month event-free survival (EFS) of 12%. The Wee1 kinase helps regulate DNA damage repair at the G2-M checkpoint. In the presence of DNA damage, the Wee1 kinase is activated, arresting cells in the G2 phase and preventing entry into the M phase. Inhibition of the Wee1 kinase abrogates the G2-M checkpoint, forcing cancer cells to undergo unscheduled mitosis even in the presence of DNA damage, leading to mitotic catastrophe. However, the Wee1 kinase is often upregulated in OS, preserving the G2-M checkpoint and allowing tumor growth and metastases. Additionally, up to 90% of OS tumors have alterations in p53, a critical protein in the regulation of the G1-S checkpoint, especially in relapsed or refractory cases. With a dysfunctional G1-S checkpoint, cancer cells further rely on G2-M checkpoint to repair DNA damage and preserve genomic integrity. Prior studies have demonstrated that pharmacologic inhibition of the Wee1 kinase produced cell death in OS cell lines and patient-derived xenografts. While p53 mutational status appeared to modulate efficacy of the Wee1 kinase inhibitor, activity was observed in p53 wild type, mutant and null cell lines. Combination therapy studies have also been performed, demonstrating potential synergism with gemcitabine. As expected, by precipitating DNA damage, susceptibility to inhibition of the G2-M checkpoint is further increased. Methods: NCT04833582 is an ongoing, open label, multicenter, phase 1/2 clinical trial to evaluate the activity of ZN-c3, an oral Wee1 inhibitor, in combination with gemcitabine in subjects ≥12 years and ≥40 kg, with relapsed, refractory OS. Subjects are dosed once daily, continuously with ZN-c3 and receive gemcitabine 1000 mg/m2 on days 1 and 8 of 21-day cycles. Up to 18 subjects are expected to enroll in the phase 1 portion based on a typical 3 + 3 escalation design; ̃60 subjects will be enrolled in the phase 2 portion, consisting of three stages: futility, promising clinical activity, and improved precision for clinical activity. The first two stages follow a Simon two-stage optimal design with 30 subjects, to differentiate an EFS rate at 18 weeks between 12% and 36% (which may be considered a more suitable endpoint for OS, compared with radiographic response). Tumor and skin punch biopsies are incorporated into the trial to identify potential biomarkers of treatment response. Subjects must be able to swallow oral tablets and have measurable disease by RECIST v1.1; prior exposure to gemcitabine is allowed. Global enrollment began August 1, 2021, and is ongoing. Clinical trial information: NCT04833582.

Immune contexture of paediatric cancers

BackgroundThe clinical development of immune checkpoint–targeted immunotherapies has been disappointing so far in paediatric solid tumours. However, as opposed to adults, very little is known about the immune contexture of paediatric malignancies. MethodsWe investigated by gene expression and immunohistochemistry (IHC) the immune microenvironment of five major paediatric cancers: Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), medulloblastoma (MB) and neuroblastoma (NB; 20 cases each; n = 100 samples total), and correlated them with overall survival. ResultsNB and RMS tumours had high immune cell gene expression values and high T-cell counts but were low for antigen processing cell (APC) genes. OS and ES tumours showed low levels of T-cells but the highest levels of APC genes. OS had the highest levels of macrophages (CSF1R, CD163 and CD68), whereas ES had the lowest. MB appeared as immune deserts. Tregs (FOXP3 staining) were higher in both RMS and OS. Most tumours scored negative for PD-L1 in tumour and immune cells, with only 11 of 100 samples positive for PD-L1 staining. PD-L1 and OX40 levels were generally low across all five indications. Interestingly, NB had comparable levels of CD8 by IHC and by gene expression to adult tumours. However, by gene expression, these tumours were low for T-cell cytotoxic molecules GZMB, GZMA and PRF1. Surprisingly, the lower the level of tumour infiltrative CD8 T-cells, the better the prognosis was in NB, RMS and ES. Gene expression analyses showed that MYCN-amplified NB have higher amounts of immune suppressive cells such as macrophages, myeloid-derived suppressor cells and Tregs, whereas the non-MYCN-amplified tumours were more infiltrated and had higher expression levels of Teff. ConclusionsOur results describe the quality and quantity of immune cells across five major paediatric cancers and provide some key features differentiating these tumours from adult tumour types. These findings explain why anti-PD(L)1 might not have had single agent success in paediatric cancers. These results provides the rationale for the development of biologically stratified and personalised immunotherapy strategies in children with relapsing/refractory cancers.

Development of a Chemoresistant Risk Scoring Model for Prechemotherapy Osteosarcoma Using Single-Cell Sequencing.

BackgroundChemoresistance is one of the leading causes that severely limits the success of osteosarcoma treatment. Evaluating chemoresistance before chemotherapy poses a new challenge for researchers. We established an effective chemoresistance risk scoring model for prechemotherapy osteosarcoma using single-cell sequencing.MethodsWe comprehensively analyzed osteosarcoma data from the bulk mRNA sequencing dataset TARGET-OS and the single-cell RNA sequencing (scRNA-seq) dataset GSE162454. Chemoresistant tumor clusters were identified using enrichment analysis and AUCell scoring. Its differentiated trajectory was achieved with inferCNV and pseudotime analysis. Ligand–receptor interactions were annotated with iTALK. Furthermore, we established a chemoresistance risk scoring model using LASSO regression based on scRNA-seq-based markers of chemoresistant tumor clusters. The TARGET-OS dataset was used as the training group, and the bulk mRNA array dataset GSE33382 was used as the validation group. Finally, the performance was verified for its discriminatory ability and calibration.ResultsUsing bulk RNA data, we found that osteogenic expression was upregulated in chemoresistant osteosarcoma as compared to chemosensitive osteosarcoma. Then, we transferred the bulk RNA findings to scRNA-seq and noticed osteosarcoma tumor clusters C14 and C25 showing osteogenic cancer stem cell expression patterns, which fit chemoresistant characteristics. C14 and C25 possessed bridge roles in interactions with other clusters. On the one hand, they received various growth factor stimulators and could potentially transform into a proliferative state. On the other hand, they promote local tumor angiogenesis, bone remodeling and immunosuppression. Next, we identified a ten-gene signature from the C14 and C25 markers and constructed a chemoresistant risk scoring model using LASSO regression model. Finally, we found that chemoresistant osteosarcoma had higher chemoresistance risk score and that the model showed good discriminatory ability and calibration in both the training and validation groups (AUCtrain = 0.82; AUCvalid = 0.84). Compared with that of the classic bulk RNA-based model, it showed more robust performance in validation environment (AUCvalid-scRNA = 0.84; AUCvalid-bulk DEGs = 0.54).ConclusionsOur work provides insights into understanding chemoresistant osteosarcoma tumor cells and using single-cell sequencing to establish a chemoresistance risk scoring model. The model showed good discriminatory ability and calibration and provided us with a feasible way to evaluate chemoresistance in prechemotherapy osteosarcoma.

Abstract A020: Multi-species regulatory analysis of osteosarcoma to reduce clinical trial failure rates

Abstract Osteosarcoma is a rare pediatric cancer characterized by poor prognosis and a high likelihood of metastasis, for which no systemic treatments have been successfully developed in the past 30 years [1]. The lack of effective biologically relevant osteosarcoma tumor models is a significant obstacle to the development of effective therapeutics [2]. Therefore, developing a framework that addresses this issue is essential in further advancing therapeutics studies. We postulate that a comparative genomics approach, evaluating whole-transcriptome osteosarcoma response in species closely related to humans, would highlight immune and other differences in each species that will better guide the design of human therapeutics. To investigate this, we compile RNA-seq data from studies of mice, human, and canine osteosarcoma [3], and perform an integrated analysis of disease in these three species. We build a tri-species multi-layer network of regulatory interactions in osteosarcomas to identify evolutionary links between osteosarcomas. Each layer of our network represents the interactions in one species, and the integrated network captures interactions preserved across species. We perform enrichment analysis on these networks and statistically evaluate differences between the topologies of the regulatory networks of these three species, investigating the functional impact of species-specific interactions and how each affects drug response. Additionally, we survey the literature to identify mutations in osteosarcomas from each species, and calculate diffusion scores for mutated genes in each layer of the network compared to genes mutated in osteosarcoma. As future work, we aim to utilize our comparative genomics approach and leverage the differences between interaction networks of related species to compute the likelihood of identifying early-stage therapeutic trials that will fail in later stages, thus avoiding costly experiments and guiding drug development. Citation Format: Tina Salehi Torabi, Soumya Gottipati, Tamer Kahveci, Kiley Graim. Multi-species regulatory analysis of osteosarcoma to reduce clinical trial failure rates [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A020.

Abstract A039: Transcriptomic analysis of canine osteosarcoma from a precision medicine perspective reveals limitations of differential gene expression studies

Abstract Despite significant advances in cancer diagnosis and treatment, osteosarcoma (OSA), an aggressive primary bone tumor, has eluded attempts at improving patient survival for many decades. Spontaneously occurring OSA in the dog shares remarkably similar clinical, histological, and molecular characteristics and therefore serves as an excellent model for the disease. The difficulty in managing OSA lies in its extreme genetic complexity, drug-resistance, and heterogeneity, making it improbable that a single-target treatment would be beneficial for the majority of affected individuals. Precision medicine seeks to fill this gap by addressing the intra- and inter-tumoral heterogeneity to improve patient outcome and survival. Characterization of differentially expressed genes (DEGs) provides insight into the phenotype and can be useful for informing appropriate therapies as well as development of novel treatments. To identify relevant DEGs, RNA profiles of primary tumor should be contrasted with that of normal tissue derived from the same cell-of-origin as the tumor prior to chemotherapy, as convoluted drug-resistant pathways make interpretation difficult. Traditional DEG analysis combines patient data to derive statistically inferred genes that are dysregulated in the group; however, the results from this approach are not necessarily consistent across individual patients, thus contradicting the basis of precision medicine. In this preliminary study, we use transcriptomic sequencing of RNA isolated from primary canine OSA tumor and patient-matched normal bone from seven dogs prior to chemotherapy to identify DEGs in the group. We then evaluate the universality of these changes in transcript levels among patients by deriving individual-level fold-change values using strict filtering parameters. The results from this study can be useful for reframing our perspective of transcriptomic analysis from a precision medicine perspective by identifying variations in DEGs among individuals. Citation Format: Rebecca L. Nance, Sara Cooper, Dmytro Starenki, Xu Wang, Maninder Sandey, Jey Koehler, Payal Agarwal, Brad Matz, Stephanie Lindley, Annette Smith, Ashley Smith, Noelle Bergman, Bruce F. Smith. Transcriptomic analysis of canine osteosarcoma from a precision medicine perspective reveals limitations of differential gene expression studies [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A039.