Subcutaneous Osteosarcoma Research Articles

Targeting the mitochondrial protein YME1L to inhibit osteosarcoma cell growth in vitro and in vivo

Exploring novel diagnostic and therapeutic biomarkers is extremely important for osteosarcoma. YME1 Like 1 ATPase (YME1L), locating in the mitochondrial inner membrane, is key in regulating mitochondrial plasticity and metabolic activity. Its expression and potential functions in osteosarcoma are studied in the present study. We show that YME1L mRNA and protein expression is significantly elevated in osteosarcoma tissues derived from different human patients. Moreover, its expression is upregulated in various primary and immortalized osteosarcoma cells. The Cancer Genome Atlas database results revealed that YME1L overexpression was correlated with poor overall survival and poor disease-specific survival in sarcoma patients. In primary and immortalized osteosarcoma cells, silencing of YME1L through lentiviral shRNA robustly inhibited cell viability, proliferation, and migration. Moreover, cell cycle arrest and apoptosis were detected in YME1L-silenced osteosarcoma cells. YME1L silencing impaired mitochondrial functions in osteosarcoma cells, causing mitochondrial depolarization, oxidative injury, lipid peroxidation and DNA damage as well as mitochondrial respiratory chain complex I activity inhibition and ATP depletion. Contrarily, forced YME1L overexpression exerted pro-cancerous activity and strengthened primary osteosarcoma cell proliferation and migration. YME1L is important for Akt-S6K activation in osteosarcoma cells. Phosphorylation of Akt and S6K was inhibited after YME1L silencing in primary osteosarcoma cells, but was strengthened with YME1L overexpression. Restoring Akt-mTOR activation by S473D constitutively active Akt1 mitigated YME1L shRNA-induced anti-osteosarcoma cell activity. Lastly, intratumoral injection of YME1L shRNA adeno-associated virus inhibited subcutaneous osteosarcoma xenograft growth in nude mice. YME1L depletion, mitochondrial dysfunction, oxidative injury, Akt-S6K inactivation, and apoptosis were detected in YME1L shRNA-treated osteosarcoma xenografts. Together, overexpressed YME1L promotes osteosarcoma cell growth, possibly by maintaining mitochondrial function and Akt-mTOR activation.

Pramlintide: A Novel Therapeutic Approach for Osteosarcoma through Metabolic Reprogramming.

Simple SummaryOutcomes for patients with osteosarcoma have remained stagnant for more than 3 decades. Novel therapeutic agents are desperately needed. Osteosarcoma cells often express abnormal form of the p53, p63, and p73 genes, which promote glucose uptake, cell glycolysis, and rapid proliferation. Pramlintide, an FDA-approved drug for type 2 diabetes, interfered with tumor glycolysis leading to decreased cell growth and increased cell death. When we injected Pramlintide into osteosarcoma tumor nodules of the mice, the tumors were significantly smaller after 21 days while the control tumors continued to grow. Tumor hypoxia was also decreased. This is the first report showing the potential efficacy of Pramlintide against osteosarcoma, indicating that Pramlintide may be a novel therapeutic approach for patients with relapsed osteosarcoma.Despite aggressive combination chemotherapy and surgery, outcomes for patients with osteosarcoma have remained stagnant for more than 25 years, and numerous clinical trials have identified no new therapies. p53 deletion or mutation is found in more than 80% of osteosarcoma tumors. In p53-deficient cancers with structurally altered p63 and p73, interfering with tumor cell metabolism using Pramlintide (an FDA-approved drug for type 2 diabetes) results in tumor regression. Pramlintide response is mediated through upregulation of islet amyloid polypeptide (IAPP). Here, we showed that osteosarcoma cells have altered p63, p73, and p53, and decreased IAPP expression but have the two main IAPP receptors, CalcR and RAMP3, which inhibit glycolysis and induce apoptosis. We showed that in osteosarcoma cells with high- or mid-range glycolytic activity, Pramlintide decreased cell glycolysis, resulting in decreased proliferation and increased apoptosis in vitro. In contrast, Pramlintide had no effect in osteosarcoma cells with low glycolytic activity. Using a subcutaneous osteosarcoma mouse model, we showed that intratumoral injection of Pramlintide-induced tumor regression. Tumor sections showed increased apoptosis and a decrease in Ki-67 and HIF-1α. These data suggest that in osteosarcoma cells with altered p53, p63, and p73 and a high glycolytic function, Pramlintide therapy can modulate metabolic programming and inhibit tumor growth.

Non-Thermal Histotripsy Ablation of Osteosarcoma Causes a Local Acute Immune Response

Abstract While surgery, chemotherapy, and radiotherapy are available as treatments for osteosarcoma, there is only a 60% 5 year overall survival rate. To address this need, novel therapies are being developed and investigated. Histotripsy is a non-invasive, non-thermal focused-ultrasound ablation therapy that uses acoustic cavitation to mechanically lyse targeted cells. Recent evidence suggests that it is immunomodulatory, and can shift the tumor microenvironment to being more pro-inflammatory. However, given histotripsy’s mechanical mechanism, it is not established to be able to treat stronger tissues such as osteosarcomas at a dose that will allow for the preservation of immune stimulating molecules. In this study, mice with subcutaneous osteosarcoma DLM8 tumors were treated with histotripsy and acute changes to the local tumor immune response were analyzed 72 hours later. Using partial ablation to avoid off-target damage, a 40% average reduction in tumor size was achieved with targeted tissues being fully ablated. Through rtPCR, the levels of various cytokines associated with wound healing were significantly increased in the treated tumors following histotripsy treatment, with the greatest fold change observed in the concentrations for IL-6 and IL-13 over a given period of time. An increase in fold change for CD274, also known as PD-L1, was also observed, implicating the potential to pair histotripsy with checkpoint inhibitors for osteosarcoma therapy. The overall results of the study suggest that histotripsy is able to shift the tumor microenvironment following partial-ablation treatment. Thus, upon activation of the immune system, wound and tissue repair mechanisms are subsequently activated and responses are regulated.

A Humanized Bone Niche Model Reveals Bone Tissue Preservation Upon Targeting Mitochondrial Complex I in Pseudo-Orthotopic Osteosarcoma.

A cogent issue in cancer research is how to account for the effects of tumor microenvironment (TME) on the response to therapy, warranting the need to adopt adequate in vitro and in vivo models. This is particularly relevant in the development of strategies targeting cancer metabolism, as they will inevitably have systemic effects. For example, inhibition of mitochondrial complex I (CI), despite showing promising results as an anticancer approach, triggers TME-mediated survival mechanisms in subcutaneous osteosarcoma xenografts, a response that may vary according to whether the tumors are induced via subcutaneous injection or by intrabone orthotopic transplantation. Thus, with the aim to characterize the TME of CI-deficient tumors in a model that more faithfully represents osteosarcoma development, we set up a humanized bone niche ectopic graft. A prominent involvement of TME was revealed in CI-deficient tumors, characterized by the abundance of cancer associated fibroblasts, tumor associated macrophages and preservation of osteocytes and osteoblasts in the mineralized bone matrix. The pseudo-orthotopic approach allowed investigation of osteosarcoma progression in a bone-like microenvironment setting, without being invasive as the intrabone cell transplantation. Additionally, establishing osteosarcomas in a humanized bone niche model identified a peculiar association between targeting CI and bone tissue preservation.

Targeting tumour associated antigens expressed in osteosarcoma and ewing's sarcoma with CAR-T cells

Background & Aim The establishment of Chimeric Antigen Receptor (CAR) T cell immunotherapy as a treatment for refractory and relapsed B cell malignancies demonstrates that genetic modification of immune cells to circumvent tumour cell immune escape is a powerful strategy to pursue in the context of other paediatric cancers. However, efforts to develop effective CAR T cell therapies for solid tumours face a number of challenges; establishing an effective dose, route of administration, tumour cell mediated dampening of immune cell function, and overcoming heterogeneous antigen expression across the tumour cell population. The focus of our work is to translate the therapeutic potential of CAR-T cell therapy into an effective treatment for children with recurrent or refractory bone tumours. In addition, we are seeking to ensure effective targeting of metastatic spread of these tumours, since metastasis at the time of diagnosis is a significant problem in this patient population. Methods, Results & Conclusion We have developed reagents and methodology to generate CAR T cells specific for the protein Erythropoietin-producing Hepatocellular receptor tyrosine kinase class A2 (EphA2), which is expressed on a range of paediatric solid tumours. We can show that EphA2 CAR T cells specifically target and kill EphA2+ osteosarcoma and Ewing's sarcoma cell lines in vitro, alongside secretion of cytokines indicative of effector T cell activation. In in vivo sub-cutaneous osteosarcoma and Ewing's sarcoma xenograft models, EphA2 CAR T cells effectively mediate complete tumour regression in a dose and route dependent manner, with direct injection into established tumours resulting in extended survival. Systemic delivery of CAR T cells was also effective at mediating tumour regression and extending survival, but required a higher cell dose. Studies testing EphA2 CAR T cell efficacy against metastatic osteosarcoma deposits are currently ongoing. Further work focussing on safety and possible off-target effects, design of alternative gene therapy vectors and combinatorial testing of EphA2 CAR T cells with CAR T cells targeting alternative antigens, or immune checkpoint blockade, will elucidate a way forward in designing an effective CAR T cell therapy for osteosarcoma and Ewing's sarcoma.

Theranostics of osteosarcoma and lung metastasis with new integrin α vβ 3 receptor targeted radiotracers

To investigate the value of integrin α vβ 3 targeted microPET/CT imaging with 68Ga-NODAGA-RGD 2 as radiotracer for the detection of osteosarcoma and theranostics of osteosarcoma lung metastasis. The 68Ga-NODAGA-RGD 2 and 177Lu-NODAGA-RGD 2 were prepared via one-step method and their stability and integrin α vβ 3 binding specificity were investigated in vitro. Forty-one nude mice were injected with human MG63 osteosarcoma to established the animal model bearing subcutaneous osteosarcoma ( n=21), osteosarcoma in tibia ( n=5), and osteosarcoma pulmonary metastatic ( n=15). The microPET-CT imaging was carried out in 3 animal models at 1 hour after tail vein injection of 68Ga-NODAGA-RGD 2. Biodistribution study of 68Ga-NODAGA-RGD 2 was performed in animal model bearing subcutaneous osteosarcoma at 10, 60, and 120 minutes. The animal model bearing pulmonary metastatic osteosarcoma was injected with 177Lu-NODAGA-RGD 2 at 7 weeks after model establishment to observe the therapeutic effect of pulmonary metastatic osteosarcoma. Histological and immunohistochemistry examinations were also done to confirm the establishment of animal model and integrin β 3 expression in animal models bearing subcutaneous osteosarcoma and bearing pulmonary metastatic osteosarcoma. 68Ga-NODAGA-RGD 2 and 177Lu-NODAGA-RGD 2 had good stability in vitro with the 50% inhibitory concentration value of (5.0±1.1) and (6.5±0.8) nmol/L, respectively. The radiochemical purity of 68Ga-NODAGA-RGD 2 at 1, 4, and 8 hours was 98.5%±0.3%, 98.3%±0.5%, and 97.9%±0.4%; while the radiochemical purity of 177Lu-NODAGA-RGD 2 at 1, 7, and 14 days was 99.3%±0.7%, 98.7%±1.2%, and 96.0%±2.8%. 68Ga-NODAGA-RGD 2 microPET-CT showed that the accumulation of 68Ga-NODAGA-RGD 2 in animal models bearing subcutaneous osteosarcoma and osteosarcoma in tibia and in lung metastasis as small as 1-2 mm in diameter of animal model bearing pulmonary metastatic osteosarcoma. Biodistribution study of 68Ga-NODAGA-RGD 2 in animal model bearing subcutaneous osteosarcoma revealed rapid clearance from blood with tumor peak uptake of (3.85±0.84) %ID/g at 120 minutes. The distribution of 177Lu-NODAGA-RGD 2 in lung metastasis was similar with 68Ga-NODAGA-RGD 2. The number and size of osteosarcoma metastasis decreased at 2 weeks after 177Lu-NODAGA-RGD 2 administration and integrin targeting specificity was confirmed by pathology examination. 68Ga-NODAGA-RGD 2 was potential for positive imaging and early detection of osteosarcoma and metastasis. Targeted radiotherapy with 177Lu-NODAGA-RGD 2 was one potential alternative for osteosarcoma lung metastasis.

Is Exercise Treadmill Time or Reduction in Myocardial Ischemia the Appropriate Primary Endpoint to Assess Success of Percutaneous Coronary Intervention in Stable Angina (ORBITA)?

1 Objectives: In vivo fluorescence imaging in the second near-infrared window region (NIR-II, 1,000-1,700 nm) holds great promise for providing deeper tissue penetration, higher spatial resolution and signal-to-noise ratios than those obtained at the conventional near-infrared window I region (NIR-I, 650-900 nm), because of its reduced tissue autofluorescence and photon scattering, and low levels of photon absorption of long-wavelength photons. Developing semiconducting polymer nanoparticles (NPs) with a strong absorption peak at 808 nm, excellent photostability and biocompatibility will be highly important and find broad uses for efficient in vivo NIR II imaging. Methods: A diketopyrrolopyrrole-based semiconducting polymer NP (PDFT1032) as a novel NIR-II fluorescent probe has been developed for the first time. The versatile use of PDFT1032 for several important biomedical applications in NIR-II window have been demonstrated, including NIR-II optical imaging of tumor using subcutaneous osteosarcoma model, assessing of vascular embolization therapy of tumor, and sentinel lymph node biopsy (SLNB) with high spatial and temporal resolution. Results: Hydrophobic PDFT were encapsulated with the amphiphilic PEGylated phospholipid to provide excellent water-soluble and biocompatible PDFT1032 NP, with high monodispersity, homogeneity, particle size 68 nm in PBS, a unique absorption peak at 809 nm, and fluorescence emission peak at 1,032 nm. No apparent cytotoxicity of PDFT1032 to NIH3T3 cells was observed even with the concentration up to 150 μg/mL. In addition, the H&E staining of the major organs, including heart, liver, spleen, lung, and kidney, at 21 days after intravenous injection of PDFT1032 (1 mg/kg) demonstrated no obvious damages and necrotic lesions. Further NIR-II imaging showed that the nanoprobe clearly visualized the subcutaneous osteosarcoma up to 3 days with high tumor-to-background ratio (TBR, up to 3.4 ± 0.16 for lateral position and 2.4 ± 0.15 for prone position). Ex vivo fluorescent images also demonstrated the favorable contrast between tumor and skin because of the low fluorescence signal of skin. Lastly, with the help of NIR-II imaging and PDFT1032 nanoprobe, the axillary lymph node was clearly identified within short time and dissected from the ambient tissue and then confirmed by histological analysis. Conclusions: Excellent biocompatibility, favorable hydrophilicity, and desirable chemical and optical properties render NIR-II PDFT1032 a highly promise probe with a potential of being widely applicable in clinical imaging and surgical treatment of malignancy.

Fangchinoline suppresses the proliferation, invasion and tumorigenesis of human osteosarcoma cells through the inhibition of PI3K and downstream signaling pathways.

Osteosarcoma is the most common malignant bone tumor. Most patients diagnosed with osteosarcoma are less than 20 years of age. Osteosarcoma cells proliferate rapidly and invade other tissues. At present, neoadjuvant chemotherapy is the primary pharmacodynamic strategy to prevent the progression of osteosarcoma. However, adverse effects of this strategy limit its long-term application. Previous research has shown that fangchinoline exerts antitumor effects on several types of tumor cells; however, its effect on osteosarcoma cells remains unknown. The present study evaluated the effects of fangchinoline on the proliferation, apoptosis, migration and invasion of osteosarcoma cells in vitro and on their tumorigenesis in vivo and determined the possible underlying mechanism of action. Fangchinoline-treated MG63 and U20S cells showed significantly decreased proliferation and significantly increased apoptosis. Fangchinoline markedly suppressed the migration and invasion of the MG63 cells. Fangchinoline-treated MG63 cells showed significantly decreased expression of phosphoinositide 3-kinase (PI3K) and Aktp-Thr308. Moreover, fangchinoline-treated MG63 cells showed downregulated expression of cyclin D1 and matrix metalloproteinase 2 and 9, which act downstream of PI3K, and upregulated expression of caspase-3 and caspase-8. Furthermore, fangchinoline suppressed the growth of subcutaneous osteosarcoma tumors in Balb/c mice subcutaneously injected with osteosarcoma cells. These findings suggest that fangchinoline inhibits the progression of osteosarcoma by suppressing the proliferation, migration and invasion and by accelerating the apoptosis of osteosarcoma cells. In addition, our results suggest that the mechanism underlying the antitumor effects of fangchinoline involve the inhibition of PI3K and its downstream signaling pathways.

Subcutaneous osteosarcoma of the upper arm: A case report

Subcutaneous osteosarcoma of the upper arm: A case report

Antitumour effects of Yangzheng Xiaoji in human osteosarcoma: The pivotal role of focal adhesion kinase signalling

The present study examined, in vitro and in vivo, the potential antitumour effects of Yangzheng Xiaoji (YZXJ), a traditional Chinese medical formula used in cancer treatment, on osteosarcoma, a tumour type recently found to be sensitive to YZXJ. The human osteosarcoma cell line MG63 was used in cell-matrix adhesion and cell growth assays. The same cell line was used in an in vivo tumour model by establishing subcutaneous osteosarcoma xenografts. Oral and intraperitoneal routes were used to deliver the YZXJ extract. The effect of YZXJ on the activation of focal adhesion kinase (FAK) and paxillin was evaluated by immunofluorescence methods. It was found that YZXJ exhibited a significant inhibitory effect on cell-matrix adhesion as demonstrated by a cell-based assay and electric cell-substrate impedance sensing (ECIS) analysis. The effect was observed together with a reduction in phospho-FAK and phospho-paxillin in the cells when treated with YZXJ. In the in vivo tumour model, YZXJ was found to significantly inhibit the growth of osteosarcoma with a sustained effect observed when YZXJ was delivered intraperitoneally. YZXJ sensitized cells to the effect of FAK inhibitor in vitro and in vivo. It is concluded that Yangzheng Xiaoji plays a significant role in cell-matrix adhesion and tumour growth, likely by inhibiting the activation of the FAK pathway. The therapeutic role of Yangzheng Xiaoji in osteosarcoma warrants further investigation.

Subcutaneous extraskeletal osteosarcoma of the thigh with lymph node and lung metastasis: a case report

Background Extraskeletal osteosarcoma (ESOS) is a rare soft tissue tumour which accounts for approximately 1% of soft tissue sarcomas. In particular, subcutaneous osteosarcoma is extremely rare, occurring in less than 10% of ESOS cases. 1 Case report The case of a 71-year-old-woman with a 30-mm mass arising in the subcutaneous tissue of the thigh is presented. Radiological investigations showed spotty calcification in the mass without any attachment to the underlying bone. Wide surgical excision was performed. Microscopic examination revealed a pleomorphic spindle cell neoplasia with abnormal osteoid, some with ossification. It confirmed the diagnosis of subcutaneous ESOS with extension to the dermis and one lateral subcutaneous margin. Radiotherapy was commenced after a wide re-excision. Five months after the primary surgery, she developed metas-tases to the ipsilateral inguinal and pelvic lymph nodes and 5 months later she was diagnosed with inoperable pulmonary metastasis. Unfortunately she died 25 months after her first presentation. Discussion ESOS has been most often described in the deep soft tissues of the extremities. Subcutaneous tissue as a primary site is rare. This case emphasises the poor prognosis in patients with ESOS and the importance of aggressive treatment even in the cases with smaller size and more superficial location.

Extraskeletal subcutaneous osteosarcoma of the upper arm: A case report

Extraskeletal osteosarcoma (ESOS) occurs in approximately 1% of soft tissue sarcomas and 2-4% of all osteosarcomas. In particular, subcutaneous osteosarcoma is extremely rare, occurring in less than 10% of ESOS cases. This report presents a case of a subcutaneous tumor in the upper arm of a 79-year-old male. Imaging and pathological findings led to the conclusion that the soft tissue tumor should be diagnosed as subcutaneous osteosarcoma. Additionally, this case report documented the clinicopathological findings of the extraskeletal subcutaneous osteosarcoma in this case and discussed its clinical features by reviewing cases previously described in the literature.