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Reconstruction of extra-articular shoulder resections is challenging. We present a novel method using the patient’s own pedicled proximal humerus and scapula, sterilized with liquid nitrogen cryoablation and recycled as an autograft. The technique was used for three patients with malignant tumors. We present the surgical technique and clinical outcomes. All patients had high-grade sarcomas of the proximal humerus requiring extra-articular resection. At surgery, the tumor-bearing shoulder girdle was exteriorized with wide margins and pedicled on the humerus via a medial scapula and clavicular osteotomy. The pedicled shoulder was then frozen in liquid nitrogen, the extraosseous tumor mass and non-viable muscle were removed and the residual shoulder girdle was re-implanted. Average follow up was 4.5 years. At the time of writing, all patients are alive without disease and have retained their original reconstructions. Scapulothoracic and elbow movements were preserved with stiff glenohumeral joints. One patient experienced an implant complication requiring surgery to remove a migrated Kirschner wire and to mesh reinforce the glenohumeral capsule. The flag technique is a reliable, low cost reconstructive option for extensive sarcomas of the shoulder girdle. A stiff glenohumeral joint with preserved scapulothoracic motion may be expected post-surgery.

The research aimed to develop an effective prognostic model for osteosarcoma. This study included 72 osteosarcoma cases from the Target OS database and 87 cases from the Shanghai General Hospital (SGH) Cohort. DNA methylation data and RNA-seq data collected from the Target OS database and the SGH Cohort were processed using synthetic minority over-sampling technique (SMOTE), Tomek links, convolutional neural network (CNN), and Random Forest models. Based on the prognosis of the patients, we divided the cases into a death/survival group and a recurrence/nonrecurrence group. A total of 10 times of model training and testing were performed separately, using the test results as model evaluation parameters. We evaluated the model performance by comprehensively assessing its precision, recall, accuracy, and F1 score. The results of this study offer a robust methodology for determining the prognosis and characteristics of osteosarcoma patients, applicable even to smaller data sets. This approach provides novel insights into the molecular data analysis of rare diseases.

Osteosarcoma (OS) is a type of bone cancer. Eighty percent of this tumor will metastasize to the lungs or liver, and as a result, patients generally need chemotherapy to improve survival possibility. Recently, antitumor activity has been reported in Ocimum gratissimum aqueous extract (OGE), which has been the focus of recent extensive studies on therapeutic strategies due to its antioxidant properties. We performed pharmacogenomics analyses for the effect of OGE on human osteosarcoma U2-OS and HOS cell growth. Cell viability, Western blot and flow cytometry analysis were performed before performing pharmacogenomics analyses for the effect of OGE on human osteosarcoma U2-OS and HOS cell growth, including cDNA microarray and RT-PCR assays. Cell viability assays revealed that OGE significantly and dose-dependently decreased the viability of U2-OS and HOS cells. Increases in cell shrinkage, Sub-G1 fragments and the activation of caspase 3 indicated that OGE induced cell apoptosis in U2-OS and HOS cells. There was no change in human osteoblast hFOS cells. cDNA microarray assay demonstrated that the expression of cell cycle regulators, apoptosis-related factors and cell proliferation markers were all modified by OGE treatment. RT-PCR analysis also confirmed the down-regulation of SKA2 and BUB1B, and the up-regulation of PPP1R15A, SQSTM1, HSPA1B, and DDIT4 by OGE treatment. The finding of anticancer activity in OGE and the identification of some potential target genes raise the expectation that OGE may become a useful therapeutic drug for human OS.

In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.

Paris polyphylla, a traditional antipyretic-detoxicate chinese medicinal herb, has been applied extensively in cancer treatments for nearly 2000 years. The purpose of the present study is to evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and to investigate its underlying mechanisms. The antiproliferation activity of PPEE was tested on 143B, MG-63, U-2 OS and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The antimigratory, anti-invasive and antivasculogenic mimicry (VM) effects of PPEE were investigated by wound healing, Transwell and 3D culture assays. Mouse xenograft model was used to examine its anti-osteosarcoma efficacy in vivo. Hematologic profiles and hepatorenal functions were evaluated to assess the toxicity of PPEE. PPEE evidently suppressed cell proliferation of 143B, MG-63 and U-2 OS with IC50 values of 10–60$\mu$g/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE promoted apoptosis in 143B cell via caspase activation, increased Bax/Bcl-2 ratio and PARP cleavage. It also induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1, CDK1, Cdc25C expression. Additionally, PPEE inhibited 143B cell migration, invasion and VM formation at noncytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP2 and MMP9. Finally, daily oral administration of PPEE for four weeks exhibits potent antitumor and anti-VM activity in 143B xenograft model with low toxicity. Taken together, these findings demonstrated PPEE possesses anti-osteosarcoma and anti-VM activity in vitro and in vivo, and therefore is a potential candidate for osteosarcoma treatment.

Carnosol is an anti-oxidant and anti-inflammatory compound from rosemary. In this paper, we investigated antitumor activity of carnosol against human osteosarcoma cells. We found the viability of human osteosarcoma MG-63 cells was significantly decreased in the presence of carnosol (cell viabilities: 17.2% for 20μg/ml of CS vs. 100% for control, $p<0.05$). Carnosol induced apoptosis and cell cycle arrest in a dose-dependent manner in MG-63 cells. Furthermore, carnosol exposure increased the levels of reactive oxygen species (ROS). The pre-treatment of NAC, the ROS scavenger, blocked the inhibition of cell viability in the carnosol treatment, indicating that ROS is important in the antiproliferation effect. Moreover, we demonstrated that carnosol significantly induced autophagy and co-administration of autophagy inhibitor reduced the antiproliferating effect of carnosol. This result exhibited the cytotoxic effect of autophagy induced by carnosol in MG-63 cells. Interestingly, the treatment of NAC decreased carnosol-induced autophagy. Collectively, these data indicate that carnosol suppresses the viability of human osteosarcoma MG-63 cells by upregulation of apoptosis and autophagy, which are both mediated by ROS. Thus, carnosol might serve as a potential therapeutic agent against osteosarcoma.

Neohesperidin has anti-oxidative and anti-inflammatory properties and exerts extensive therapeutic effects on various cancers. In this study, the osteosarcoma cell lines were exposed to different concentrations of neohesperidin. Cell proliferation and viability were assessed by CCK-8 and colony-formation assays. The role of neohesperidin in cell cycle progression and apoptosis were analyzed by flow cytometry and western blotting. To identify autophagosomes and autolysosomes, we used a tandem GFP-mRFP-LC3B lentiviral construct. In addition, autophagy was evaluated by examining autophagosome formation using transmission electron microscopy. Intracellular reactive oxygen species (ROS) production was detected by fluorescence microscopy and flow cytometry. Subsequently, the activation of the ROS/JNK signaling pathway was investigated. Neohesperidin could inhibit proliferation and induce apoptosis in SJSA and HOS cells. The formation of autophagosomes indicated that autophagy occurred in neohesperidin-treated cells and the apoptotic effect of neohesperidin was significantly increased after the use of autophagy inhibitors. Subsequently, we found that neohesperidin-induced apoptosis and autophagy were related to the increase in ROS generation and were significantly inhibited by GSH. Moreover, neohesperidin induced activation of the c-Jun N-terminal kinase (JNK) signaling pathway and inhibition of JNK with SP600125 attenuated neohesperidin-induced apoptosis and autophagy simultaneously. Our data indicated that neohesperidin caused G2/M phase arrest and induced apoptosis and autophagy by activating the ROS/JNK pathway in human osteosarcoma cells, suggesting that neohesperidin is a potential drug candidate for the treatment of osteosarcomas.

Osteosarcoma is the most common malignant bone-forming tumor, wherein most patients with high grade osteosarcomas are treated with chemotherapy. Despite this, survival for metastatic or relapsed osteosarcoma patients has remained at an overall 5-year survival rate of 20%. In particular, the extracts of Corylopsis coreana (Korean winter hazel), a cultivated woody plant in South Korea, have shown beneficial anti-inflammatory, anti-oxidative, anti-osteoclastic, and antihyperuricemic properties. Therefore, this study aimed to demonstrate the antitumor activities and underlying mechanism of 11-O-Galloyl bergenin (OGAL) isolated from Corylopsis coreanas leaves in human osteosarcoma cells. Herein, we found that OGAL inhibited MG63 cell proliferation and induced cellular apoptosis as evidenced by cleaved-PARP, cleaved-caspase 3, TUNEL-positive cells, and Annexin V-positive cells. Specifically, OGAL-induced apoptosis was accompanied by p53 and p21 upregulation, BAX expression, and decreased Bcl-2 and cdk2. Moreover, OGAL induced autophagy via AKT inactivation, LC3II upregulation, and MG63 cell autophagosome formation. OGAL-induced autophagy was also accompanied by increased p38 phosphorylation, whereas JNK and ERK1/2 activities were found to be unaffected upon examining the MAPK signaling pathway. Furthermore, wound healing and Boyden chamber assays showed that OGAL suppressed MG63 cell migration and invasion. Given these findings, this study provided evidence that OGAL has antitumor effects by apoptosis and autophagy enhancement through increased p53, AKT, and p38 signaling, suggesting that OGAL may be a potential therapeutic strategy for osteosarcoma treatment.

Metastasis of osteosarcoma is an important adverse factor affecting patients’ survival, and cancer stemness is the crucial cause of distant metastasis. Capsaicin, the main component of pepper, has been proven in our previous work to inhibit osteosarcoma proliferation and enhance its drug sensitivity to cisplatin at low concentrations. This study aims to further explore the anti-osteosarcoma effect of capsaicin at low concentrations (100$\mu$M, 24h) on stemness and metastasis. The stemness of human osteosarcoma (HOS) cells was decreased significantly by capsaicin treatment. Additionally, the capsaicin treatment’s inhibition of cancer stem cells (CSCs) was dose-dependent on both sphere formation and sphere size. Meanwhile, capsaicin inhibited invasion and migration, which might be associated with 25 metastasis-related genes. SOX2 and EZH2 were the most two relevant stemness factors for capsaicin’s dose-dependent inhibition of osteosarcoma. The mRNAsi score of HOS stemness inhibited by capsaicin was strongly correlated with most metastasis-related genes of osteosarcoma. Capsaicin downregulated six metastasis-promoting genes and up-regulated three metastasis-inhibiting genes, which significantly affected the overall survival and/or disease-free survival of patients. In addition, the CSC re-adhesion scratch assay demonstrated that capsaicin inhibited the migration ability of osteosarcoma by inhibiting its stemness. Overall, capsaicin exerts a significant inhibitory effect on the stemness expression and metastatic ability of osteosarcoma. Moreover, it can inhibit the migratory ability of osteosarcoma by suppressing its stemness via downregulating SOX2 and EZH2. Therefore, capsaicin is expected to be a potential drug against osteosarcoma metastasis due to its ability to inhibit cancer stemness.

Solasonine (SS) is a natural glycoalkaloid compound that has been reported to possess a significant anticancer function. However, its anticancer effects and related mechanisms in osteosarcoma (OS) have not been studied. This study sought to investigate the impact of SS on the growth of OS cells. OS cells were treated with different concentrations of SS for 24h, and the results showed that SS attenuated the survival of OS cells in a dose-dependent manner. Additionally, SS suppressed cancer stem-like properties and epithelial–mesenchymal transition (EMT) by inhibiting aerobic glycolysis in OS cells in an ALDOA-dependent manner. Additionally, SS reduced the levels of Wnt3a, $\beta$-catenin, and Snail in OS cells in vitro. Furthermore, Wnt3a activation reversed the SS-induced inhibition of glycolysis in OS cells. Collectively, this study discovered a novel effect of SS in inhibiting aerobic glycolysis, in addition to cancer stem-like features and EMT, implying that SS could be a therapeutic candidate for OS treatment.

Introduction: We have analyzed the clinical features and outcomes of malignant tumors of foot. Methods: Between January 2010 to January 2017, 21 patients of malignant tumors of calcaneum and talus were treated. These included 14 cases of osteosarcoma, 7 cases of Ewing’s sarcoma. Talus was involved in 8 cases and calcaneum involved in 11 cases. Mean follow-up for all patients was 30 months (range 24–70 months). Results: In osteosarcoma of calcaneum and talus the presenting symptom was local pain in 63% and swelling in 37%. Patients with Ewing’s sarcoma presented with swelling in 100% cases. The mean delay in diagnosis for osteosarcoma and Ewing’s sarcoma was 8.8 months (range 6–12 months) and 9.5 months (range 6–12 months), respectively. Patients with delay in diagnoses of more than six months and less than six months had disease free survival of 11.5 months and 15 months, respectively and $p$ value 0.3. Patients with delay in diagnoses of more than six months and less than six months had metastases in 5 and 1cases, respectively. The mean disease free interval (DFI) for osteosarcoma and Ewing’s sarcoma was 16 months and 5.5 months, respectively. The correlation between osteosarcoma and Ewing’s sarcoma of calcaneum and foot with delay in diagnosis and DFI did not reach statistical significance ($p$ value 0.93 and 0.14, respectively). Conclusion: An index of suspicion, proper examination with close scrutiny of the radiographs is required in patients presenting malignant of bones of foot. Osteosarcoma of calcaneum and talus most commonly presented with local pain and Ewing’s sarcoma present with swelling. The time interval between the onset of symptoms and diagnosis (delay in diagnosis) for calcaneum and talus osteosarcoma was similar to Ewing’s sarcoma.

Pain around the foot and ankle joint is often misdiagnosed as plantar fasciitis and may lead to delayed diagnosis of osteosarcoma in foot. Delay in diagnosis can lead to adverse prognosis and poor survival outcomes. We present a patient who had primary osteosarcoma of the calcaneum that was initially diagnosed and treated for plantar fasciitis, resulting in a delay of diagnosis. The patient eventually was treated with neo-adjuvant chemotherapy followed by below-knee amputation and adjuvant chemotherapy. This case highlights the need to maintain an index of suspicion with close scrutiny of the radiographs. We suggest few clinical recommendations and need for a great suspicion while evaluating a mass arising from the calcaneum.

Osteosarcoma is the most common primary bone tumor with high malignancy. It is particularly necessary to achieve rapid and accurate diagnosis in its intraoperative examination and early diagnosis. Accordingly, the multimodal microscopic imaging diagnosis system constructed by bright field, spontaneous fluorescence and polarized light microscopic imaging was used to study the pathological mechanism of osteosarcoma from the tissue microenvironment level and achieve rapid and accurate diagnosis. First, the multimodal microscopic images of normal and osteosarcoma tissue slices were collected to characterize the overall morphology of the tissue microenvironment of the samples, the arrangement structure of collagen fibers and the content and distribution of endogenous fluorescent substances. Second, based on the correlation and complementarity of the feature information contained in the three single-mode images, combined with convolutional neural network (CNN) and image fusion methods, a multimodal intelligent diagnosis model was constructed to effectively improve the information utilization and diagnosis accuracy. The accuracy and true positivity of the multimodal diagnostic model were significantly improved to 0.8495 and 0.9412, respectively, compared to those of the single-modal models. Besides, the difference of tissue microenvironments before and after cancerization can be used as a basis for cancer diagnosis, and the information extraction and intelligent diagnosis of osteosarcoma tissue can be achieved by using multimodal microscopic imaging technology combined with deep learning, which significantly promoted the application of tissue microenvironment in pathological examination. This diagnostic system relies on its advantages of simple operation, high efficiency and accuracy and high cost-effectiveness, and has enormous clinical application potential and research significance.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is the rapid acquisition of serial MRI images before, during, and after the administration of an MR contrast agent. Unlike conventional enhanced MRI, which simply provides a snapshot of enhancement at one point in time, DCE-MRI permits a fuller depiction of the wash-in and wash-out contrast kinetics, and thus provides insight into the microcirculation of the studied tissues or lesions. With the dynamic signal intensity change post-contrast agent injection, empirical measures such as maximal signal intensity enhancement and initial enhancement slope can be easily obtained. Such data are also amenable to two-compartment pharmacokinetic modeling, from which parameters based on the rates of exchange between the compartments can be generated. DCE-MRI can be used to characterize masses, stage tumors, and noninvasively monitor therapy. Measures of contrast uptake by dynamic MRI have demonstrated a convincing ability to aid in diagnosing the presence of viable tumors and to measure the response for arange of human tumors. While questions remain about how to best extract noninvasive pharmacokinetic measures of drug access from these novel dynamic imaging methods, scientists and clinicians are optimistic that these methods can provide important new clinical measures which reflect the range of biological variations within and between naturally occurring solid tumors. Efforts to standardize DCE-MRI acquisition, analysis, and reporting methods will allow wider dissemination of this useful functional imaging technique.

Osteosarcoma is one of the most common types of bone cancer in children. To gauge the extent of cancer treatment response in the patient after surgical resection, the H&E stained image slides are manually evaluated by pathologists to estimate the percentage of necrosis, a time consuming process prone to observer bias and inaccuracy. Digital image analysis is a potential method to automate this process, thus saving time and providing a more accurate evaluation. The slides are scanned in Aperio Scanscope, converted to digital Whole Slide Images (WSIs) and stored in SVS format. These are high resolution images, of the order of 10⁹ pixels, allowing up to 40X magnification factor. This paper proposes an image segmentation and analysis technique for segmenting tumor and non-tumor regions in histopathological WSIs of osteosarcoma datasets. Our approach is a combination of pixel-based and object-based methods which utilize tumor properties such as nuclei cluster, density, and circularity to classify tumor regions as viable and non-viable. A K-Means clustering technique is used for tumor isolation using color normalization, followed by multi-threshold Otsu segmentation technique to further classify tumor region as viable and non-viable. Then a Flood-fill algorithm is applied to cluster similar pixels into cellular objects and compute cluster data for further analysis of regions under study. To the best of our knowledge this is the first comprehensive solution that is able to produce such a classification for Osteosarcoma cancer. The results are very conclusive in identifying viable and non-viable tumor regions. In our experiments, the accuracy of the discussed approach is 100% in viable tumor and coagulative necrosis identification while it is around 90% for fibrosis and acellular/hypocellular tumor osteoid, for all the sampled datasets used. We expect the developed software to lead to a significant increase in accuracy and decrease in inter-observer variability in assessment of necrosis by the pathologists and a reduction in the time spent by the pathologists in such assessments.