Osteosarcoma Research Articles

CXCL6-CXCR2 axis-mediated PD-L2+ mast cell accumulation shapes the immunosuppressive microenvironment in osteosarcoma

Osteosarcoma (OS) is the most common primary bone malignancy and has a high propensity for local invasion and metastasis. The tumour microenvironment of OS is infiltrated by a large number of immune cells, which play a crucial role in its progression and prognosis. Mast cells are important innate immune cells in the tumour stroma and exhibit different phenotypes in diverse tumour microenvironments. However, the underlying mechanisms of mast cell accumulation and the phenotypic characteristics of mast cells in OS remain poorly understood. In this article, we found for the first time that mast cell accumulation in osteosarcoma tissue was modulated by the CXCL6-CXCR2 axis and that the number of infiltrating mast cells was significantly greater in tumour tissues than in adjacent nontumour tissues. These tumour-infiltrating mast cells express high levels of the immunosuppressive molecule PD-L2, and survival analyses revealed that patients in the PD-L2+ high-expression group had a worse prognosis. In vitro, mast cells were induced to express PD-L2 in a time- and dose-dependent manner using OS tissue culture supernatants to mimic the tumour microenvironment. Mechanistic studies revealed that tumour cell-derived G-CSF significantly induced mast cell PD-L2 expression by activating STAT3. Importantly, mast cells overexpressing PD-L2 inhibit tumour-specific CD8+ T-cell proliferation and tumour-killing cytokine secretion, which is reversed by blocking PD-L2 on mast cells. Therefore, our findings provide new insight into the immunosuppressive and tumorigenic roles of mast cells, as well as a novel mechanism by which PD-L2-expressing mast cells mediate immune tolerance.

Directing B7-H3 chimeric antigen receptor T cell homing through IL-8 induces potent antitumor activity against pediatric sarcoma

BackgroundAdvances in pediatric oncology have occurred for some cancers; however, new therapies for sarcoma have been inadequate. Cellular immunotherapy using chimeric antigen receptor (CAR) T cells has shown dramatic benefits...

In vitro Molecular Mechanisms of Anticancer Activity of Stevioside in Human Osteosarcoma Cell Lines (Sarcoma Osteogenic).

Osteosarcoma (OS) is a rare and aggressive form of bone cancer that primarily affects the long bones of the body, such as the arms and legs. It is characterized by the uncontrolled growth of malignant cells in the bone tissue, leading to the formation of abnormal and painful bone masses. Steviol glycosides have been widely used as natural noncalorie sweeteners and are the collective name of the sweet substances found naturally in the plant Stevia rebaudiana, which is commonly called Stevia. Our study aimed to analyze the anticancer activity of Stevioside in OS. Stevioside was applied to OS cells, and the levels of Bcl xL, Bcl-2, and Bax were then estimated. The results of three separate studies, each carried out in triplicate, were expressed as the mean ± standard errors of the mean (SEM). One-way ANOVA was used for statistical analysis. The findings showed that the effect of Stevioside on sarcoma osteogenic cells with mean ± SEM as 0.74 ± 0.05, 0.69 ± 0.09, 0.46 ± 0.09 for Bcl-xL gene, 0.98 ± 0.06, 0.58 ± 0.07, 0.5 ± 0.07 for Bcl-2 gene, and 1.2 ± 0.08, 1.45 ± 0.11, 1.67 ± 0.12 for Bax gene, respectively, when treated with untreated control cells. The study concludes its action against bone OS cells was significant with apoptotic induction. Stevia has a wide range of health benefits as well as being a plant-based diet it has less of side effects and promoting features even by intaking it daily along with other medicines.

MFAP2 induces epithelial-mesenchymal transformation of osteosarcoma cells by activating the Notch1 pathway.

Osteosarcoma (OS) is a malignancy originating from mesenchymal tissue. Microfibril-associated protein 2 (MFAP2) plays a crucial role in cancer, notably promoting epithelial-mesenchymal transition (EMT). However, its involvement in OS remains unexplored. MFAP2 was silenced in U2OS cells using shRNA targeting MFAP2 (sh-MFAP2) and validated by quantitative real-time polymerase chain reaction (qRT-PCR). We extracted gene chip data of MFAP2 from multiple databases (GSE28424, GSE42572, and GSE126209). Correlation analyses between MFAP2 and the Notch1 pathway identified through the gene set variation analysis (GSVA) enrichment analysis were conducted using the Pearson correlation method. Cellular behaviors (viability, migration, and invasion) were assessed via the Cell Counting Kit-8 (CCK-8), wound healing, and Transwell assays. EMT markers (N-cadherin, vimentin, and β-catenin) and Notch1 levels were examined by western blotting and qRT-PCR. Cell morphology was observed microscopically to evaluate EMT. Finally, the role of MFAP2 in OS was validated through a xenograft tumor model. OS cell lines exhibited higher MFAP2 mRNA expression than normal osteoblasts. MFAP2 knockdown in U2OS cells significantly reduced viability, migration, and invasion, along with downregulation of N-cadherin and vimentin, as well as upregulation of β-catenin. MFAP2 significantly correlated with the Notch1 pathway in OS and its knockdown inhibited Notch1 protein expression. Furthermore, Notch1 activation reversed the inhibitory effects of MFAP2 knockdown on the malignant characteristic of U2OS cells. Additionally, MFAP2 knockdown inhibited tumor growth, expression levels of EMT markers, and Notch1 expression in OS tumor tissues. Our study revealed that MFAP2 was an upstream regulator of the Notch1 signaling pathway to promote EMT in OS. These findings suggested MFAP2 as a potential OS therapy target.

P53-armed oncolytic virotherapy induces abscopal effect in osteosarcoma by promoting immunogenic cell death

Osteosarcoma (OS), the most frequent primary malignant tumor of bone in children and adolescents, is refractory to immune checkpoint inhibitors due to poor antitumor immune response. Chemotherapy and virotherapy induce immunogenic cell death (ICD) and antitumor immune responses, leading to the abscopal effect in untreated tumors. We previously demonstrated the antitumor activity of the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 in human OS cells. Here, we show the therapeutic potential of chemotherapeutic drugs (doxorubicin, cisplatin) and telomerase-specific oncolytic adenoviruses (OBP-301, p53-armed OBP-702) to induce ICD in human OS cells (U2OS, MNNG/HOS, SaOS-2) and murine OS cells (NHOS). OBP-702 induced more profound ICD via secretion of adenosine triphosphate (ATP) and high-mobility group box protein B1 (HMGB1) compared with chemotherapy and OBP-301 in human OS cells. Murine NHOS cells were also more sensitive to OBP-702 than OBP-301. Subcutaneous NHOS tumor models demonstrated that intratumoral injection of OBP-702 significantly increased the tumor infiltration of cytotoxic CD8+ T cells and induced the abscopal effect against non-treated tumors compared with OBP-301. Our results suggest that OBP-702 is a promising antitumor reagent to induce ICD with secretion of ATP and HMGB1 and the abscopal effect against OS.

A Prospective Observational Cohort Study for Newly Diagnosed Osteosarcoma Patients in the UK: ICONIC Study Initial Results.

There has been little change to the standard treatment for osteosarcoma (OS) over the last 25 years and there is an unmet need to identify new biomarkers and novel therapeutic approaches if outcomes are to improve. Furthermore, there is limited evidence on the impact of OS treatment on patient-reported outcomes (PROs). ICONIC (Improving Outcomes through Collaboration in Osteosarcoma; NCT04132895) is a prospective observational cohort study recruiting newly diagnosed OS patients across the United Kingdom (UK) with matched longitudinal collection of clinical, biological, and PRO data. During Stage 1, which assessed the feasibility of recruitment and data collection, 102 patients were recruited at 22 sites with representation from patient groups frequently excluded in OS studies, including patients over 50 years and those with less common primary sites. The feasibility of collecting clinical and biological samples, in addition to PRO data, has been established and there is ongoing analysis of these data as part of Stage 2. ICONIC will provide a unique, prospective cohort of newly diagnosed OS patients representative of the UK patient population, with fully annotated clinical outcomes linked to molecularly characterised biospecimens, allowing for comprehensive analyses to better understand biology and develop new biomarkers and novel therapeutic approaches.

Cyclin L1 participates in Adriamycin resistance and progression of osteosarcoma via PI3K/AKT-mTOR pathway.

Chemoresistance is a common and thorny problem in the treatment of osteosarcoma (OS), which obstructs the response of relapse or metastasis of OS to chemotherapy and leads to the unfavorable prognosis of OS patients. Cyclin L1 (CCNL1) is a non-canonical cyclin that plays an important role in the regulation of tumor cell proliferation and lymph node metastasis. In this work, we explored the impact of CCNL1 expression levels on proliferation, migration, and Adriamycin (ADM) resistance in OS and related mechanisms. We found that CCNL1 expression levels were significantly associated with clinical prognosis of patients with OS and CCNL1 could promote OS proliferation and migration. In addition, we also revealed that cellular CCNL1 was significantly increased in ADM-resistant OS cells and promoted ADM resistance. The PI3K/AKT-mTOR pathway is involved in CCNL1-mediated ADM resistance in OS. In summary, CCNL1 is involved in the progression of ADM resistance and OS through the PI3K/AKT-mTOR pathway, which will provide a new clue to the mechanism of ADM resistance and a potential target for the treatment of ADM-resistant OS.

Myc upregulates Ggct, γ-glutamylcyclotransferase to promote development of p53-deficient osteosarcoma.

Osteosarcoma (OS) in humans is characterized by alterations in the TP53 gene. In mice, loss of p53 triggers OS development, for which c-Myc (Myc) oncogenicity is indispensable. However, little is known about which genes are targeted by Myc to promote tumorigenesis. Here, we examined the role of γ-glutamylcyclotransferase (Ggct) which is a component enzyme of the γ-glutamyl cycle essential for glutathione homeostasis, in human and mouse OS development. We found that GGCT is a poor prognostic factor for human OS, and that deletion of Ggct suppresses p53-deficient osteosarcomagenesis in mice. Myc upregulates Ggct directly by binding to the Ggct promoter, and deletion of a Myc binding site therein by genome editing attenuated the tumorigenic potential of p53-deficient OS cells. Taken together, these results show a rationale that GGCT is widely upregulated in cancer cells and solidify its suitability as a target for anticancer drugs.

Conversion of the primary aneurysmal bone cyst into osteogenic sarcoma following treatment with percutaneous sclerotherapy using polidocanol injection

Conversion of the primary aneurysmal bone cyst into osteogenic sarcoma following treatment with percutaneous sclerotherapy using polidocanol injection

TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Cargo-eliminated osteosarcoma-derived small extracellular vesicles mediating competitive cellular uptake for inhibiting pulmonary metastasis of osteosarcoma

Osteosarcoma (OS) derived small extracellular vesicles (OS-sEVs) have been shown to induce the formation of cancer-associated fibroblasts (CAFs), characterized by elevated pro-inflammatory factor expression and enhanced migratory and contractile abilities. These CAFs play a crucial role in priming lung metastasis by orchestrating the pre-metastatic niche (PMN) in the lung. Disrupting the communication between OS-sEVs and lung fibroblasts (LFs) emerges as a potent strategy to hinder OS pulmonary metastasis. Our previously established saponin-mediated cargo-elimination strategy effectively reduces the cancer-promoting ability of tumor-derived small extracellular vesicles (TsEVs) while preserving their inherent targeting capability. In this study, we observed that cargo-eliminated OS-sEVs (CE-sEVs) display minimal pro-tumoral and LFs activation potential, yet retain their ability to target LFs. The uptake of OS-sEVs by LFs can be concentration-dependently suppressed by CE-sEVs, preventing the conversion of LFs into CAFs and thus inhibiting PMN formation and pulmonary metastasis of OS. In summary, this study proposes a potential strategy to prevent LFs activation, PMN formation in the lung, and OS pulmonary metastasis through competitive inhibition of OS-sEVs’ function by CE-sEVs.

Comprehensive transcriptomic analysis identifies SLC25A4 as a key predictor of prognosis in osteosarcoma.

Osteosarcoma (OS) is highly malignant and prone to local infiltration and distant metastasis. Due to the poor outcomes of OS patients, the study aimed to identify differentially expressed genes (DEGs) in OS and explore their role in the carcinogenesis and progression of OS. RNA sequencing was performed to identify DEGs in OS. The functions of the DEGs in OS were investigated using bioinformatics analysis, and DEG expression was verified using RT-qPCR and Western blotting. The role of SLC25A4 was evaluated using gene set enrichment analysis (GSEA) and then investigated using functional assays in OS cells. In all, 8353 DEGs were screened. GO and KEGG enrichment analyses indicated these DEGs showed strong enrichment in the calcium signaling pathway and pathways in cancer. Moreover, the Kaplan-Meier survival analysis showed ten hub genes were related to the outcomes of OS patients. Both SLC25A4 transcript and protein expression were significantly reduced in OS, and GSEA suggested that SLC25A4 was associated with cell cycle, apoptosis and inflammation. SLC25A4-overexpressing OS cells exhibited suppressed proliferation, migration, invasion and enhanced apoptosis. SLC25A4 was found to be significantly downregulated in OS patients, which was associated with poor prognosis. Modulation of SLC25A4 expression levels may be beneficial in OS treatment.

Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

Lung metastases are the primary cause of death for osteosarcoma (OS) patients. We recently validated interleukin-11 receptor α (IL-11Rα) as a molecular target for the inhibition of OS lung metastases. Since there is no clinically approved antibody against this receptor, we sought to identify downstream targets that mediate the effects of IL-11Rα signaling. We used shRNA to deplete IL-11Rα from OS cells; as a complementary approach, we added IL-11 exogenously to OS cells. The resulting changes in gene expression identified EZH2 as a downstream candidate. This was confirmed by knockdown of IL-11Rα in OS cells, which led to increased expression of genes repressed by histone methyltransferase EZH2, including members of the WNT pathway, a known target pathway of EZH2. Exogenous IL-11 increased the global levels of histone H3 lysine 27 trimethylation, evidence of EZH2 activation. Treatment with the EZH2 inhibitor GSK126 significantly reduced in vitro proliferation and increased cell-cycle arrest and apoptosis, which were partially mediated through the WNT pathway. In vivo, treatment of an orthotopic nude mouse model of OS with GSK126 inhibited lung metastatic growth and prolonged survival. In addition, significantly shorter recurrence-free survival was seen in OS patients with high levels of EZH2 in their primary tumors (P < .05). This suggests that IL-11Rα promotes OS lung metastasis via activation of EZH2. Thus, blocking EZH2 activity may be an effective strategy for inhibiting OS lung metastasis and improving prognosis.

Enhanced Osteosarcoma Immunotherapy via CaCO3 Nanoparticles: Remodeling Tumor Acidic and Immune Microenvironment for Photodynamic Therapy.

Osteosarcoma (OS) is a "cold" tumor enriched in noninflammatory M2 phenotype tumor-associated macrophages (TAMs), which limits the efficacy of immunotherapy. The acidic tumor microenvironment (TME), generated by factors such as excess hydrogen (H+) ions and high lactate levels, activates immunosuppressive cells, further promoting a suppressive tumor immune microenvironment (TIME). Therefore, a multitarget synergistic combination strategy that neutralizes the acidic TME and reprograms TAMs can be beneficial for OS therapy. Here, a calcium carbonate (CaCO3)/polydopamine (PDA)-based nanosystem (A-NPs@(SHK+Ce6)) is developed. CaCO3 nanoparticles are used to neutralize H+ ions and alleviate the suppressive TIME, and the loaded SHK not only synergizes with photodynamic therapy (PDT) but also inhibits lactate production, further reversing the acidic TME and repolarizing TAMs to consequently lead to enhanced PDT-induced tumor suppression and comprehensive beneficial effects on antitumor immune responses. Importantly, A-NPs@(SHK+Ce6), in combination with programmed cell death protein 1 (PD-1) checkpoint blockade, shows a remarkable ability to eliminate distant tumors and promote long-term immune memory function to protect against rechallenged tumors. This work presents a novel multiple-component combination strategy that coregulates the acidic TME and TAM polarization to reprogram the TIME.

Plumbagin induces G2/M arrest and apoptosis and ferroptosis via ROS/p38 MAPK pathway in human osteosarcoma cells

Plumbagin (PLB), a naphthoquinone compound, has been proven to have anti-cancer properties in different types of cancers. However, the anti-tumor actions as well as molecular processes in osteosarcoma (OS) remain unclear. This study investigates the underlying mechanisms of PLB in human OS cells in vitro. Our study showed that PLB significantly inhibited the proliferation of human OS cells in a time- and dosage-dependent manner. RNA sequencing data revealed that the efficacy of PLB is related to cell cycle, cell death, and p38 MAPK signaling pathway in KHOS-240S cells. Further studies indicated that PLB triggers G2/M phase arrest, mitochondrial-dependent apoptosis, as well as ferroptosis in human OS cells. Mechanistically, PLB treatment increased ROS generation that activates the p38 MAPK signaling pathway. BIRB 796, a p38 MAPK inhibitor, partly reversed PLB-induced phase arrest of G2/M and cell death. Furthermore, N-acetylcysteine (NAC), a ROS scavenger, significantly decreased G2/M phase arrest and cell death and reversed p38 MAPK activation caused by PLB. In conclusion, PLB induces arrest G2/M arrest, apoptosis as well as ferroptosis in human OS cells via ROS generation and p38 MAPK activation. There is potential for PLB to be an effective treatment for OS.

OSteosarcoma of Ethmoid Sinus-A Rare Entity.

Osteogenic sarcoma of the Craniofacial region are uncommon.They have been mainly reported in the mandible and the maxilla, Primary osteosarcoma arising from ethmoid sinus is an extremely rare condition. We report a case of 34 year old male, presented with gradually increasing swelling over the right medial canthus with history of double vision for one year. Clinically we thought it was a mucous retention cyst in the ethmoid area. After doing Contrast enhanced CT, we proceeded with transnasal endoscopic removal of the cyst. Histopathological examination of the specimen surprisingly turned out to be a low grade Osteogenic sarcoma. Patient was treated with adjuvant chemotherapy.

EYA4 reduces chemosensitivity of osteosarcoma to doxorubicin through DNA damage repair

Previous studies have demonstrated that Eyes Absent 4 (EYA4) influences the proliferation and migration of tumor cells. Notably, studies have established that EYA4 can also limit tumor sensitivity to chemotherapeutic agents. The objective of this study was to investigate the effect of EYA4 in conferring drug resistance in osteosarcoma (OS). Bioinformatics, histological, and cellular analyses revealed that the expression level of EYA4 was higher in OS tissues than in healthy tissues/cells and in resistant tissues/cells compared with sensitive tissues/cells. In vitro and in vivo experiments demonstrated that EYA4 knockdown increased the sensitivity of OS to doxorubicin (DOX). Conversely, overexpression of EYA4 decreased the sensitivity of OS to DOX. Exploration of the resistance mechanism exposed that EYA4 facilitates DNA double-strand break (DSB) repair, a typical mode of DNA damage repair (DDR). Subsequently, our findings indicated that EYA4 could directly interact with histone H2AX to activate the DDR pathway. Taken together, our observations indicated that EYA4 may serve as a target molecule for reversing drug resistance in OS patients.

Hsa_circ_0064636 regulates voltage dependent anion channel 1/ubiquitination factor E4A through miR‑326/miR‑503‑5 in osteosarcoma.

Circular RNAs (circRNAs) are a subclass of non-coding RNAs that are important for the regulation of gene expression in eukaryotic organisms. CircRNAs exert various regulatory roles in cancer progression. However, the role of hsa_circ_0064636 in osteosarcoma (OS) remains poorly understood. In the present study, the expression of hsa_circ_0064636 in OS cell lines was measured by reverse transcription-quantitative PCR (RT-qPCR). Differentially expressed mRNAs and microRNAs (miRNA or miRs) were screened using mRNA(GSE16088) and miRNA(GSE65071) expression datasets for OS. miRNAs that can potentially interact with hsa_circ_0064636 were predicted using RNAhybrid, TargetScan and miRanda. Subsequently, RNAhybrid, TargetScan, miRanda, miRWalk, miRMap and miRNAMap were used for target gene prediction based on the overlapping miRNAs to construct a circ/miRNA/mRNA interaction network. Target genes were subjected to survival analysis using PROGgeneV2, resulting in a circRNA/miRNA/mRNA interaction sub-network with prognostic significance. miRNA and circRNA in the subnetwork may also have survival significance, but relevant data are lacking and needs to be further proved. RT-qPCR demonstrated that hsa_circ_0064636 expression was significantly increased in OS cell lines. miR-326 and miR-503-5p were identified to be target miRNAs of hsa_circ_0064636. Among the target genes obtained from the miR-326 and miR-503-5p screens, ubiquitination factor E4A (UBE4A) and voltage dependent anion channel 1 (VDAC1) were respectively identified to significantly affect prognosis; only miR-326 targets UBE4A and only miR-503 targets VDAC1. To conclude, these aforementioned findings suggest that hsa_circ_0064636 may be involved in the development of OS by sponging miR-503-5p and miR-326to inhibit their effects, thereby regulating the expression of VDAC1 and UBE4A.

CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS. Murine NK cells were activated and expanded ex vivo with soluble IL-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, GVHD and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray. AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Treatment with combination CD155 and anti-DNAM-1 pretreated alloNK ameliorated survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS. These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT. Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against OS and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

Comparative responses to demethylating therapy in animal models of osteosarcoma.

Background The demethylating agent decitabine (DAC) effectively inhibits tumor growth and metastasis by targeting ESR1 methylation to restore estrogen receptor alpha (ERα) signaling and promoting cellular differentiation in models of human osteosarcoma (OSA). Whether this pathway can be targeted in canine OSA patients is unknown. Methods Canine OSA tumor samples were tested for ERα expression and ESR1 promoter methylation. Human (MG63.3) and canine (MC-KOS) OSA cell lines and murine xenografts were treated with DAC in vitro and in vivo , respectively. Samples were assessed using mRNA sequencing and tissue immunohistochemistry. Results ESR1 is methylated in a subset of canine OSA patient samples and the MC-KOS cell line. DAC treatment led to enhanced differentiation as demonstrated by increased ALPL expression, and suppressed tumor growth in vitro and in vivo . Metastatic progression was inhibited, particularly in the MG63.3 model, which expresses higher levels of DNA methyltransferases DNMT1 and 3B. DAC treatment induced significant alterations in immune response and cell cycle pathways. Conclusion DAC treatment activates ERα signaling, promotes bone differentiation, and inhibits tumor growth and metastasis in human and canine OSA. Additional DAC-altered pathways and species- or individual-specific differences in DNMT expression may also play a role in DAC treatment of OSA.