Inhibition of anlotinib-induced autophagy attenuates invasion and migration by regulating epithelial-mesenchymal transition and cytoskeletal rearrangement through ATG5 in human osteosarcoma cells

Abstract The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.

Effect and mechanism of berberine-induced ferroptosis in osteosarcoma cells / 中国药房

OBJECTIVE To investigate the effect of berberine on ferroptosis in MG63 osteosarcoma cells and its mechanism. METHODS Using cells without drug treatment as control， the cell viability， proliferation， the related indexes of ferroptosis [nuclear proliferation associated-antigen （Ki67）， mitochondrial ultrastructure， ferric ion （Fe2+）， reactive oxygen species （ROS）， malondialdehyde （MDA）， and glutathione （GSH）]， the protein expression of signal transducer and activator of transcription 3 （STAT3）， tumor protein 53 （p53）， and solute carrier family 7 member 11 （SLC7A11） were detected after being treated with different concentrations of berberine. Cells were transfected with p53 siRNA and then assigned to the control group， p53 siRNA group， berberine group， and p53 siRNA+berberine group to explore the role of p53 in berberine-induced ferroptosis. After 24 h incubation with 10.0 μmol/L berberine， the protein expressions of p53 and SLC7A11， the levels of mitochondrial membrane potential， GSH， and MDA content were determined. Cells were transfected with STAT3 overexpressed plasmid and then assigned to the control group， berberine group， STAT3 group， and STAT3+berberine group to explore the effect of STAT3 on the regulation of the p53/SLC7A11 pathway. After 24 h incubation with 10 μmol/L berberine， the protein expressions of p-STAT3， STAT3， p53， and SLC7A11 were detected. RESULTS Compared with the control cell， the concentrations of 2.5， 5.0 and 10.0 μmol/L berberine could reduce the cell viability and expression of Ki67， and induce the morphological changes in ferroptosis-related mitochondria， increase the levels of Fe2+， ROS and MDA， and the protein expression of p53， reduce the level of GSH， the binding activity of STAT3 with DNA， and the protein expressions of p-STAT3 and SLC7A11； the above differences were statistically significant （P＜ 0.05 or P＜0.01）. Compared with the berberine group，significantly down-regulated p53 protein expression and MDA level， up-regulated SLC7A11 protein expression， and increased mitochondrial membrane potential and GSH level were observed in the p53 siRNA+berberine group （P＜0.01）. Compared with the berberine group， the protein expressions of p-STAT3， STAT3， and SLC7A11 in the STAT3+berberine group were significantly increased （P＜0.01）， while the protein expression of p53 was significantly decreased （P＜0.01）. CONCLUSIONS Berberine can induce the ferroptosis of MG63 cells by mediating STAT3/p53/SLC7A11 signaling pathway.

Mechanism of oxymatrine promoting the apoptosis of osteosarcoma MG63 cell line through mitophagy mediated by COX-2/PINK1/Parkin signaling pathway / 中国药房

OBJECTIVE To study the mechanism of oxymatrine inducing apoptosis of osteosarcoma MG63 cell line based on mitophagy mediated by cyclooxygenase-2 （COX-2）/PTEN-induced putative kinase-1 （PINK1）/Parkinson disease protein-2 （Parkin） signaling pathway. METHODS MG63 cells were treated with 2.0， 4.0， 8.0 mg/mL oxymatrine and 6 μmol/L 5-fluorouracil， then the apoptotic rate， the expression of apoptosis-related proteins [B-cell lymphoma-2 （Bcl-2）， Bcl-2 related X protein （Bax）]， the proportion of decrease in mitochondrial membrane potential， the level of mitophagy as well as the protein expressions of PINK1， Parkin， and microtubule-associated protein 1 light chain-3Ⅱ （LC3-Ⅱ） were detected. PINK1 small interfering RNA （siRNA） was adopted to intervene in the expression of PINK1， the cells were divided into control group， PINK1 siRNA group， oxymatrine group， and PINK1 siRNA+oxymatrine group； the protein expressions of PINK1， Parkin， and LC3-Ⅱ， the proportion of decrease in mitochondrial membrane potential （MMP） as well as apoptotic rate were detected. The lentivirus infection technique was used to overexpress COX-2， the cells were divided into control group， oxymatrine group， COX-2 group， and COX-2+oxymatrine group. The protein expressions of COX-2， PINK1， and Parkin， as well as the proportion of decrease in MMP were detected. RESULTS After being treated with oxymatrine， the apoptotic rate， the protein expressions of Bax， PINK1， Parkin， and LC3-Ⅱ， the level of mitophagy as well as the proportion of decrease in MMP were significantly increased （P＜0.05）， while the protein expression of Bcl-2 was significantly decreased （P＜0.05）. Compared with the oxymatrine group， the protein expressions of PINK1， Parkin， and LC3-Ⅱ， apoptotic rate and the proportion of decrease in MMP were significantly decreased in PINK1 siRNA+oxymatrine group （P＜0.05）. Compared with the oxymatrine group， the protein expression of COX-2 in the COX-2+oxymatrine group was increased significantly （P＜0.05）， while the protein expressions of PINK1 and Parkin as well as the proportion of 526087266@qq.com decrease in MMP were decreased significantly （P＜0.05）. CONCLUSIONS Oxymatrine can mediate the overactivity of mitophagy based on the PINK1/Parkin signaling pathway by inhibiting COX-2 expression， thus promoting the apoptosis of the MG63 osteosarcoma cell line.

Research progress on autocrine and paracrine regulation of osteosarcoma cell growth / 中国生物制品学杂志

@#Osteosarcoma(OS)is one of the most common malignant tumors in bone tissue,and its specific mechanism is not yet fully clear. Studies have shown that OS cells express different cytokines(CKs)and their receptors in the development stage of tumors,and enable them to grow autonomously and confer metastatic ability through autocrine and paracrine effects. In this regard,the most important CKs mainly includes insulin-like growth factor-1,transforming growth factor-β,chemokine 5 and interleukin-8,which can regulate the tumor microenvironment that is conducive to tumor growth,invasion and metastasis. This review summarizes the role and mode of action of CKs and their biological relevance to OS cells,hoping to provide effective new markers and therapeutic targets for clinical treatment of OS.

Synergistic effects of methotrexate and piperine on human osteosarcoma cell lines – An in-vitro study

Background: Osteosarcoma is a malignant cancer that effect bone and metastasizing to many vital organs such as lungs. There are many available drugs to treat the disease including tamoxifen, methotrexate (MTX), and cisplatin which have their own side effects and hurdles to become drugs of choice for the disease. On the other hand, introduction of herbal drugs as chemotherapeutic agents opened up new arena to potentiate the existing treatment by exhibiting synergy. Piperine (PPN) is widely used drug as anti-cancer agent as well as it has anti-inflammatory, analgesic properties, and also used in the treatment of abdominal pains, tuberculosis, arthritis, and respiratory illness. Aims and Objective: Thus, this study was designed to investigate the synergistic inhibitory potential of PPN and MTX on the MG63 osteosarcoma cell lines in vitro. Materials and Methods: The cell lines were cultured on DMEM medium and investigated for cytotoxicity of the drugs using MTT assay at 540 nm in UV. Three groups of cell lines administered with PPN, MTX, and PPN+MTX (1:1) in various concentrations and IC50 values were calculated based on the % cell viability graphs. Results: Results showed that the IC50 of PPN was 38.65, MTX was 123.98, and PPN+MTX was 15.13 proving the significant synergistic cytotoxic effect of PPN and MTX in inhibiting the proliferation of MG63 cell lines. Conclusion: Further research needs to be conducted in this field to elucidate the synergistic pathways in which PPN has shown a better anti-osteosarcoma effect when combined with MTX.

Mesenchymal tumours of the head and neck: a 10-year institutional archival study / Tumores mesenquimales de cabeza y cuello: estudio de archivo institucional de 10 años de duración

Introduction: Histopathological characterisation of benign and malignant lesions of the head and neck in a systematic and coherent way is an essential part of Oral Pathology and Oral Medicine. Objective: To describe the frequency and histopathological profile of connective tissue tumours in the head and neck region reported in an Indian institute. Methods: A retrospective analysis was made of the 10-year records of reports of biopsy samples of patients maintained by the department of oral pathology showing histopathological diagnosis of connective tissue neoplasms. The data obtained was compiled for age, gender, site and histopathology of the lesions. Results: Majority of the tumours were benign and patients were found to be in the 2nd or 4th decade of life with female preponderance. The most common benign tumour was fibroma where buccal mucosa was the commonest location and malignant tumour was osteosarcoma where mandible was the commonest site. While fibromas were seen among general adult population, osteosarcomas were more in the males (7.2 percent) and in the younger population (< 20 years). The uncommon tumours among benign variety were leiomyoma and teratoma while in malignant category 1 case of undifferentiated sarcoma was reported. Conclusion: The findings in this study may be of help to oral and maxillofacial surgeons and general dentists in formulating diagnosis and rendering patient care in the existing local population(AU)

Introducción: La caracterización histopatológica de las lesiones benignas y malignas de cabeza y cuello de forma sistemática y coherente es una parte esencial de la Patología Oral y la Medicina Oral. Objetivo: Describir la frecuencia y el perfil histopatológico de los tumores del tejido conjuntivo de la región de cabeza y cuello notificados en un instituto indio. Métodos: Se realizó un análisis retrospectivo de los registros de 10 años de informes de muestras de biopsia de pacientes mantenidos por el departamento de patología oral que mostraban diagnóstico histopatológico de neoplasias del tejido conectivo. Se recopilaron los datos obtenidos en cuanto a edad, sexo, localización e histopatología de las lesiones. Resultados: La mayoría de los tumores eran benignos y los pacientes se encontraban en la 2ª o 4ª década de la vida, con preponderancia del sexo femenino. El tumor benigno más frecuente fue el fibroma, cuya localización más frecuente fue la mucosa bucal, y el tumor maligno fue el osteosarcoma, cuya localización más frecuente fue la mandíbula. Mientras que los fibromas se observaron entre la población adulta general, los osteosarcomas fueron más frecuentes en los varones (7,2 por ciento) y en la población más joven (< 20 años). Los tumores menos frecuentes en la variedad benigna fueron el leiomioma y el teratoma, mientras que en la categoría maligna se registró un caso de sarcoma indiferenciado. Conclusiones: Los hallazgos de este estudio pueden ser de ayuda para los cirujanos orales y maxilofaciales y los odontólogos generales en la formulación de diagnósticos y la prestación de atención al paciente en la población local existente (AU)

Role of incisional vacuum therapy in endoprosthetic bone reconstruction surgery / Papel da terapia a vácuo incisional em cirurgia de reconstrução óssea com endoprótese

ABSTRACT Reconstructive surgery with endoprostheses is the chosen method for treating bone malignancies. Postoperative infections are frequent complications, and their treatment involves prolonged hospital stays and antibiotic therapy. Among the advancements aimed at reducing the rate of postoperative infection, the use of incisional negative pressure therapy (iNPT) has shown promising results, with no reports in the literature regarding its use in patients with such conditions. Objective: To evaluate the effectiveness of iNPT in reducing postoperative complications in surgeries for resection of bone tumors associated with modular endoprosthesis reconstruction. Methods: Retrospective case series of 16 patients diagnosed with osteosarcoma, who underwent resection and reconstruction with endoprosthesis associated with iNPT during the postoperative period. Follow-up was performed for a period of six months, and the evaluated outcomes were the incidence of postoperative infection and complications of the surgical wound. Results: The use of iNPT for a postoperative period of seven days resulted in only three (18.7%) cases of postoperative infection. No cases of wound dehiscence, seroma formation, or hematoma at the surgical site were observed. Conclusion: The rate of surgical wound complications in our case series is lower than that reported in most of the literature, and iNPT appears to be an efficient way to reduce the rate of local complications in reconstructive surgeries with endoprosthesis after resection of bone malignancies. Level of Evidence III, Retrospective Study.

RESUMO A cirurgia reconstrutiva com endopróteses é o método escolhido no tratamento de malignidades ósseas. As infecções pós-operatórias são complicações frequentes, e seu tratamento envolve internações e antibioticoterapia prolongadas. Entre os avanços que visam reduzir a taxa de infecção pós-operatória, o uso da terapia com pressão negativa incisional (TPNi) vem mostrando resultados promissores, não havendo relatos na literatura de seu emprego em pacientes com tal quadro. Objetivo: Avaliar a eficácia da TPNi em reduzir complicações pós-operatórias em cirurgias de ressecção de tumores ósseos associadas à reconstrução com endopróteses modulares. Métodos: Série de casos retrospectiva de 16 pacientes diagnosticados com osteossarcoma, submetidos à ressecção e reconstrução com endoprótese associada à TPNi durante o pós-operatório. Foi realizado seguimento por um período de seis meses e os desfechos avaliados foram incidência de infecção pós-operatória e complicações da ferida operatória. Resultados: O uso da TPNi por um período pós-operatório de sete dias resultou em apenas três (18,7%) casos de infecção pós-operatória. Não foram observados casos em que ocorreu deiscência da ferida operatória, formação de seromas ou hematomas no sítio cirúrgico. Conclusão: A taxa de complicações de ferida operatória em nossa série de casos é menor que a da maior parte da literatura, e a TPNi parece ser uma forma eficiente de reduzir a taxa de complicações locais em cirurgias reconstrutivas com endoprótese após ressecção de malignidades ósseas. Nível de Evidência III, Estudo Retrospectivo.

Curcumin in Treatment of Osteosarcoma： A Review / 中国实验方剂学杂志

Osteosarcoma （OS） is the most common primary malignant bone tumor originating from mesenchymal stem cells， which features high degree of malignancy， strong invasiveness， easy early metastasis， and high recurrence rate. The clinical manifestations of OS are pain， local mass， limited movement， and pathological fracture. OS mainly occurs in children， adolescents， and the elderly， seriously threatening physical and mental health of patients， as well as their quality of life. The currently available therapies for OS are surgery， chemoradiotherapy， and the combination of the two. Although the therapeutic effect has been improved， tumor recurrence and metastasis and multidrug resistance still occur. Thus， the therapeutic effect is not satisfactory， especially in improving the overall survival rate of patients with metastatic OS. As a result， clinicians and researchers have been making efforts to find an effective therapy. In recent years， the mechanism of curcumin （CUR） against OS has attracted wide attention. CUR， a pigment extracted from the rhizomes or tubers of many plants， such as Curcuma longa， C. rcenyujin， and C. phaeocaulis， has a variety of pharmacological effects. Scholars have found that CUR has the effects of inhibiting proliferation， inducing apoptosis， and reversing multidrug resistance （MDR） of tumor cells， but also it has poor water solubility and low bioavailability， which limit the clinical application. This paper mainly discusses the mechanism of CUR against OS， the existing problems， new treatment methods， and future research directions， which is expected to provide new ideas for scientific researchers and provide a reference for the development and utilization of CUR in the future.

Prognostic value and mechanism of long non-coding RNA DLEU1 in osteosarcoma / 中国骨伤

OBJECTIVE@#To investigate the prognostic value and mechanism of long non-coding RNA DLEU1(LncRNA DLEU1) in osteosarcoma.@*METHODS@#The tissue samples and clinical data of 86 patients with osteosarcoma treated by orthopaedic surgery in our hospital from January 2012 to December 2014 were retrospectively collected. The expression of LncRNA DLEU1 in pathological tissues was detected by qRT-PCR, then the patients were divided into high and low expression of LncRNA DLEU1 groups. Osteosarcoma cell line HOS was divided into two groups, down-regulated expression group (si-DLEU1 group) and negative control group (si-NC group). LncRNA DLEU1 siRNA and negative control sequence were transfected by Lipofectamine 3000. Chi-square test was used to analyze the relationship between the expression of LncRNA DLEU1 and the clinicopathological factors of osteosarcoma. Kaplan-Meier method was used to compare the difference of the overall survival rate of osteosarcoma patients between the high and low expression groups of LncRNA DLEU1. The risk factors affecting the overall survival rate of osteosarcoma were analyzed by single factor and multifactor analysis. The number of invasive cells in the two groups was determined and compared by Transwell assay.@*RESULTS@#The expression of LncRNA DLEU1 in osteosarcoma tissue was higher than that in adjacent tissues (P<0.001). The expression of LncRNA DLEU1 in human osteosarcoma cell lines (MG-63, U-2 OS, and HOS) was significantly higher than that in human osteoblast line hFOB 1.19 (P<0.001). The expression of LncRNA DLEU1 was significantly correlated with Enneking stage (P<0.001), distant metastasis (P=0.016), and histological grade (P=0.028). The 1-year overall survival rate of the LncRNA DLEU1 high expression group was significantly higher than that of the low expression group (90.7% vs 60.5%, P<0.001). The 5-year overall survival rate of the LncRNA DLEU1 high expression group was significantly higher than that of the low expression group (32.6% vs 11.6%, P<0.001). Univariate analysis showed that Enneking stage (P<0.001), tumor size (P=0.043), distant metastasis (P<0.001), histological grade (P<0.001), and expression of LncRNA DLEU1 (P<0.001) were risk factors for overall survival of osteosarcoma patients. Multivariate analysis showed that high expression of LncRNA DLEU1 [HR=1.948, 95% CI(1.141, 3.641), P=0.012] and distant metastasis[HR=4.108, 95% CI(2.169, 7.780), P<0.001] were independent risk factors for overall survival of osteosarcoma patients. The number of invasive cells in si-DLEU1 group was significantly lesser than that in si-NC group(139±13 vs 357±31, P<0.001).@*CONCLUSION@#High expression of LncRNA DLEU1 is a molecular marker affecting the prognosis of osteosarcoma patients. Downregulation of LncRNA DLEU1 can inhibit the invasion of osteosarcoma cells.

Study on the relationship between long non-coding RNA DHRS4-AS1 and disease-free survival in patients with osteosarcoma and its effect on osteosarcoma cell proliferation and migration in vitro / 肿瘤研究与临床

Objective:To investigate the relationship between long non-coding RNA (lncRNA) DHRS4-AS1 and disease-free survival in osteosarcoma patients and the mechanisms of its effect on proliferation and migration of osteosarcoma cells in vitro.Methods:The data of DHRS4-AS1 transcriptome levels and survival status of osteosarcoma patients in GEPIA database were collected since the database was established, and the patients were divided into high DHRS4-AS1 expression group and low DHRS4-AS1 expression group based on the median DHRS4-AS1 transcriptome level, with 59 cases in each group, and the Kaplan-Meier method was used to analyze the disease-free survival of the two groups. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of DHRS4-AS1 in osteosarcoma cell lines MG-63, HOS, 143B, U-2OS, Saos2 and normal osteoblast cell line hFOB1.19, and the osteosarcoma cell line with the lowest DHRS4-AS1 expression level was selected for subsequent experiments. The plasmid carrying DHRS4-AS1 sequence and the plasmid carrying negative control sequence were transfected into the selected osteosarcoma cells as DHRS4-AS1 group and control group. CCK-8 method was used to detect the proliferation of each group of cells, and the absorbance value was used as the cell proliferation ability; cell scratch assay was used to detect the migration of each group of cells. The bioinformatics website starBase V2.0 was used to predict the target genes of DHRS4-AS1, and the dual luciferase reporter gene assay was used to verify the targeting relationship between DHRS4-AS1 and the target genes. The expression levels of target genes and downstream genes of osteosarcoma cells in control group and DHRS4-AS1 group were detected by qRT-PCR and Western blotting.Results:Survival analysis showed that the disease-free survival of osteosarcoma patients in the high DHRS4-AS1 expression group in GEPIA database was superior to that of the low DHRS4-AS1 expression group ( P < 0.001). Compared with normal osteoblastic hFOB1.19 cells, the expression level of DHRS4-AS1 was low in all osteosarcoma cells (all P < 0.01), with the lowest expression level of DHRS4-AS1 in U-2OS cells ( P < 0.001). Cell proliferation ability was reduced in U-2OS cells of the DHRS4-AS1 group after 1, 2, 3 and 4 d of culture compared with the control group (all P < 0.05). The migration rate of U-2OS cells in the DHRS4-AS1 group was lower than that in the control group [(31±6)% vs. (63±4)%, t = 4.38, P = 0.005]. starBase V2.0 website predicted that DHRS4-AS1 complementarily bound to miRNA-411-3p (miR-411-3p); dual luciferase reporter gene assay showed that miR-411-3p overexpression reduced the luciferase activity of the wild-type DHRS4-AS1 reporter gene ( P < 0.001), but had no effect on the luciferase activity of the mutant DHRS4-AS1 reporter gene ( P > 0.05). qRT-PCR showed that the relative expression of miR-411-3p in U-2OS cells of the DHRS4-AS1 group was low (0.22±0.06 vs. 1.06±0.23, t = 3.55, P = 0.012) and the relative expression of metastasis suppressor MTSS1 mRNA was high (5.58±1.03 vs. 1.06±0.22, t = 4.28, P = 0.005) compared with the control group; Western blotting showed that MTSS1 expression was elevated, and the expression levels of cell proliferation phenotype proteins CDK3 and cyclin C and cell migration phenotype proteins ZEB2 and KLF8 were low. Conclusions:Osteosarcoma patients with high expression of lncRNA DHRS4-AS1 have better disease-free survival, and its expression is low in osteosarcoma cell lines. DHRS4-AS1 may promote MTSS1 gene expression and inhibit cell proliferation and migration by targeting and down-regulating miR-411-3p expression in osteosarcoma cells.

Research progress on the role of non-coding RNA in chemotherapy resistance of osteosarcoma / 中华骨科杂志

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. With the emergence of chemotherapy resistance in recent years, the survival rate of osteosarcoma patients has reached a bottleneck. Therefore, exploration of its chemoresistance mechanism is one of the popular research directions currently. Non-coding RNA (ncRNA) is a class of RNA without the ability to encode proteins, which is classified into microRNA, long non-coding RNA, and circular RNA based on length and shape. With the development of high-throughput sequencing technology, there is increasing evidence that some non-coding RNAs are abnormally upregulated or downregulated in osteosarcoma cells and affect the response of osteosarcoma to four commonly used chemotherapeutic drugs (methotrexate, doxorubicin, cisplatin and ifosfamide) through mechanisms such as regulation of apoptosis, cell cycle, signaling pathways, intracellular drug concentration, and cellular autophagy. Therefore, thesenon-coding RNAs are expected to be novel targets for osteosarcoma treatment. In this paper, the current studies were searched and reviewed on the above three non-coding RNAs mediating chemoresistance in osteosarcoma, aiming to provide a reference for breaking the bottleneck of survival rate of osteosarcoma patients.

A new 3D-printed ankle fusion prosthesis for the treatment of osteosarcoma in the distal tibia: a case report / 中华骨科杂志

To report the short-term clinical outcome of three cases of distal tibial osteosarcoma treated with a novel 3D-printed ankle fusion prosthesis for limb preservation. The patients were admitted to the Department of Bone Tumor, Shanghai General Hospital from January 2020 to June 2021, with one male and two female cases, aged 18, 12, and 14 years, respectively, all diagnosed with distal tibial osteosarcoma (Ennecking stage IIb). A new self-designed ankle fusion prosthesis was used to perform osteosarcoma resection and prosthetic reconstruction of the distal tibia. The operation time, blood loss, postoperative American Orthopedic Foot and Ankle Society Score (AOFAS) and ankle range of motion were recorded. All the 3 patients successfully completed the operation and were followed up for 22 months, 18 months and 12 months, respectively. The operation time was 140 min, 110 min and 200 min, and the blood loss was 200 ml, 200 ml and 350 ml, respectively. At the last follow-up, the AOFAS were 86, 90 and 95 points, and the range of motion of ankle flexion and extension were 30°, 15° and 30°. There was no local recurrence or lung metastasis at the last follow-up. The novel 3D-printed ankle fusion prosthesis in the distal tibia is safe and effective for the reconstruction of bone defects after resection of osteosarcoma in the distal tibia, and the early postoperative function is satisfactory.

Research on cell membrane-coated drug delivery nanovesicles in inducing the polarization of tumor-associated macrophages and treating osteosarcoma / 中华骨科杂志

Objective:To prepare cell membrane-coated nanovesicles with targeted delivery of toll-like receptor 4 (TLR4) agonist, and to explore the effect and mechanism of inducing the polarization of tumor-associated macrophages (TAMs) and treating osteosarcoma.Methods:TLR4 agonist loaded nanovesicles were prepared by polycarbonate membrane extruders. The morphology and size of nanovesicles were detected by transmission electron microscopy (TEM) and particle size analyzer, and the drug loading performance of the nanovesicles to TLR4 agonist was investigated. TLR4 agonist loaded nanovesicles were co-incubated with macrophages in vitro, and the targeting ability of nanovesicles to macrophages and its role in regulating the function of macrophages were detected by confocal fluorescence microscopy. In vitro experiments, a cell co-culture system was established. After the upper layer macrophages were treated by the control group, the TLR4 agonist group and the TLR4 agonist loaded nanovesicle group, the lower layer osteosarcoma cells were collected for CCK-8 and cloning formation experiments to evaluate their effects on the proliferation and migration of osteosarcoma cells. In vivo experiments, an osteosarcoma subcutaneous graft tumor model was established, and mice were randomly divided into the control group, the TLR4 agonist group, and the TLR4 agonist loaded nanovesicle group. After the treatment by caudal vein, the tumor targeting ability of nanovesicles in vivo was explored through the in vivo imaging system, and the volume of tumor tissue was continuously detected. The subcutaneous tumors were stained to detect macrophage-related markers, and their effect on the polarization of macrophages was evaluated. The TUNEL fluorescence of tumor tissues was further detected.Results:TEM showed the round shape of TLR4 agonist loaded nanovesicle and the size was about 200 nm. The co-incubation of 0.05 mg TLR4 agonist with 0.1 mg nanovesicles was the best condition for the preparation of drug-loaded nanovesicles. The drug loading efficiency was about 35% and the drug loading content was about 0.11 mg/mg. The membrane-coated nanovesicles could efficiently load and deliver TLR4 agonist. TLR4 agonist loaded nanovesicles were labeled with DiD red fluorescent dye, and then the labeled nanovesicles were co-incubated with macrophages. It was found by confocal fluorescence microscopy that DiD labeled TLR4 agonist loaded nanovesicles significantly accumulated in macrophages, and the fluorescence of M1-type macrophage marker (iNOS) was significantly enhanced, which could induce M1 polarization of macrophages. In vitro experiments, it was found that the number of osteosarcoma cells in the TLR4 agonist loaded nanovesicle group was significantly reduced under the light microscope, and the cell morphology was wrinkled and rounded. CCK-8 and cloning formation experiments showed that the proliferation and migration ability of osteosarcoma cells in the TLR4 agonist loaded nanovesicle group was significantly reduced compared with the control group and the TLR4 agonist group. A subcutaneous graft tumor model was established. In vivo imaging experiments showed that TLR4 agonist loaded nanovesicles locally accumulated in tumor tissues in vivo, but were not distributed in other organs. The growth of tumor tissue was significantly inhibited in the TLR4 agonist loaded nanovesicle group. Moreover, the fluorescence of M1-type macrophage marker (iNOS) was significantly enhanced (relative fluorescence intensity: 3.27±0.19), while the fluorescence of M2-type macrophage marker (CD163) was significantly decreased (relative fluorescence intensity: 0.14±0.04). TUNEL fluorescence staining showed that the apoptosis level of osteosarcoma cells was significantly increased (relative fluorescence intensity: 9.53±0.21).Conclusion:Membrane-coated nanovesicles could targeted deliver TLR4 agonist to osteosarcoma, induce TAMspolarization, remodel tumor immunosuppressive microenvironment, promote cell apoptosis, and effectively kill osteosarcoma.

The prognosis of patients with extremity osteosarcoma who have undergone misdiagnosis and mistreatment / 中华骨科杂志

Objective:To compare outcomes between standardized and misdiagnosis and mistreatment of osteosarcoma.Methods:A retrospective analysis of patients with high-grade osteosarcoma who received appropriate surgical treatment and chemotherapy (299 cases, control group) and those who were misdiagnosed (benign or infective) and received mistreatment (23 cases, study group) between January 2009 and December 2021. Gender, age, first operation mode, recurrence time, recurrence interval, metastasis time, metastasis interval, total survival time (months), survival status in the two group and tumor site reoperation mode in the study group were statistically analyzed. Further, chi-square test was performed for comparison of the clinical between two groups. The survival analysis was performed using Kaplan-Meier test and Log-rank test.Results:All the 322 patients were followed up. In the control group, the average follow-up time was 42 months (1-137 months), the average age was 24 years (3-80 years), male 184 cases, female 115 cases, and limb salvage rate was 85.3% (255/299). Seven patients underwent amputation, and the amputation rate was 17.7% (44/299). The recurrence rate was 8.4% (25/299), the average recurrence interval was 22.8 months (7-36 months), and the metastasis rate was 28.1% (85/299), the average metastasis time was 32.7 months (0-58 months). In the study group, the average of follow-up time was 30 months (9-117 months), the average age was 36 years (5-67 years), 17 males and 6 females. Among them, eleven patients were treated with limb salvage in the second stage, and the limb salvage rate was 47.8% (11/23). Seven patients underwent amputation, and the amputation rate was 30.4% (7/23). The recurrence rate was 26.1% (6/23), the average recurrence interval was 11 months (1-42 months), and the metastasis rate was 43.4% (10/23), the average metastasis time was 20.3 months (1-44 months). The 5-year survival rate was 50.7% in the study group and 56.1% in the control group. There was no significant difference between the two groups (χ 2=0.09, P=0.760). Conclusion:The overall prognosis of patients with high-grade osteosarcoma who receive active treatment after mistreatment is similar to that of patients with standardized treatment, but the recurrence and metastasis rate is higher, the recurrence time is earlier, and the amputation rate is higher.

Physeal-sparing distal femur megaprothesis for skeletally immature patients with osteosarcoma / 中华骨科杂志

Objective:To evaluate the clinical outcome of a special physeal sparing knee prosthesis for pediatric distal femoral osteosarcoma regarding the functional outcome, retention of the growth potential of the proximal tibia, and postoperative complications.Methods:A retrospective study was conducted to review 37 pediatric patients with osteosarcoma of distal femur who were treated in a single musculuskeletal tumor center between August 2015 and January 2019. Among them, 21 were boys and 16 were girls, aged from 5 to 12 years at the time of operation, with an average age of 9.1±2.1 years and the height of 115 to 160 cm, with an average of 140±10 cm. Tumor resection of distal femur was performed and the bone defect was reconstructed by a special hinged knee prosthesis which can preserve the proximal tibial epiphyseal plate. Demographic data was recorded. Overall leg length and tibial length was assessed by full-length standing anteroposterior radiographs of bilateral lower extremity with the patella pointing anteriorly preoperativelly and postoperativelly at each follow up. And the growth potential of the affected proximal tibia was calculated by comparing with the preoperative length of tibia. Meanwhile, the functional outcome was assessed by using the Musculoskeletal Tumor Society (MSTS) system, and the postoperative complications were analysed.Results:All patients underwent the tumor resection and reconstruction operation successfully. The average operation duration was 143±41 minutes, ranging 90 to 250 minutes. The average intraoperative blood loss was 314±397 ml, ranging 30 to 2 200 ml. The patients were followed up for 24 to 64 months, averaging 42.3±12.1 months. The postoperative knee range of motion was 100-130 degrees, with an average of 115.6±7.2 degrees. The postoperative MSTS score was 23-30, with an average of 26.7±1.6. To the last follow-up, the limb length discrepancy of the lower limb was 1.3 to 10 cm, and the length of the tibia was shortened from 0 to 3.8 cm compared with the opposite side, with an average of 1.3±1.0 cm. The growth percentage of the proximal tibial epiphysis on the affected side was 30% to 100%, with an average of 70%±17%. Totally, 13 patients suffered postoperative complication, the overall incidence of complications was 35% (13/37), and prosthesis-related complications were 16% (6/37). Three patients with wound dehiscence were managed by debridement and antibiotics. Radiographs revealed femoral stem loosening in a single patient 3 years after the initial operation and then the prosthesis was converted to an adult tumor knee endoprosthesis. Two cases experienced breakage of the femoral stem at 30 and 33 months, respectively, due to an accidental injury. They received revision surgery, and a new femoral prosthesis component was replaced. One patient developed femoral stem breakage at 10 months after surgery due to fatigue fracture, which treated with revision surgery. Tumor recurrence occurred in 6 patients. Among them, tumor recurrence in soft tissue occurred in 4 patients, and treated with regional resection without further recurrence. The other 2 patients experienced tumor recurrence at the distal femoral site, and treated with resection and prosthetic revision.Conclusion:The physeal sparing pediatric knee prosthesis can preserve the growth potential of the proximal tibial epiphyseal plate with good postoperative function and low incidence of prosthesis complications. Therefore, it can be an alternativeespecially for skeletally immature patients with distal femur osteosarcoma.

Mid-term follow-up of reconstruction with liquid nitrogen-inactivated autologous bone graft in patients with diaphyseal malignant tumours / 中华骨科杂志

Objective:To explore the mid-term efficacy of liquid nitrogen-inactivated autologous tumor segment bone replantation for repairing bone defects after resection of malignant tumors in the long bone shaft.Methods:A retrospective analysis was performed on the clinical data of 16 patients treated with liquid nitrogen-inactivated autologous bone graft at Beijing Jishuitan Hospital from July 2015 to June 2017 to repair defects caused by malignant tumour resection of the diaphysis. There were 10 males and 6 females with a mean age of 23.4±11.6 years (range, 8-44 years), including 8 classic osteosarcoma, 2 high-grade surface osteosarcoma, 4 Ewing's sarcoma, 1 periosteal osteosarcoma, and 1 undifferentiated pleomorphic sarcoma. Tumors were located in the humerus in 2 cases, in the femur in 8 cases and in the tibia in 6 cases. The mean length of tumor was 12.4±4.8 cm (range, 5.5-26 cm). Postoperative imaging examination was performed every 6 months, and the healing status of the transplanted bone-host bone was evaluated based on the imaging assessment method of the International Society of Limb Salvage (ISOLS) imaging assessment after allogeneic bone transplantation, and the complications were assessed using the Henderson classification. The five-year survival rate for patients and grafted bone was calculated using the Kaplan-Meier survival curve.Results:The median follow-up was 64 (60.3, 69.8) months. At the end of follow-up, 13 patients were tumour free and 3 patients died of multiple metastases at 19, 20 and 33 months after surgery. There were 32 osteotomy ends in 16 patients, of which 30 healed, including 11 metaphyseal osteotomy ends, and the healing time was 9 (6, 12) months after replantation of the tumour segment with liquid nitrogen-inactivated autologous bone; 19 osteotomy ends in the diaphysis took 13 (9, 21) months to heal, with a statistically significant difference in healing time between different sites ( Z=-2.25, P=0.025). Sixteen patients had six complications, including two cases of non-union at the diaphyseal site, one case of failure of internal fixation due to non-union, three cases of recurrence, and no soft tissue complications or infections. One patient with failed internal fixation was treated with a vascularized tip iliac bone graft that healed 6 months after surgery. Another patient died of multiple metastases with 1 unhealed diaphysis left. Three cases of recurrence were all located in the extracranial soft tissue of the autologous tumor segment inactivated by liquid nitrogen. Among them, one case underwent reoperation and local radiotherapy, and there was still no tumor survival after 65 months of surgery, and two cases died due to multiple metastases. The five-year survival rate of patients was 81% as calculated using the Kaplan-Meier survival curve, and the graft survival rate was 100%. There was no amputation and the limb salvage rate was 100%. Conclusion:The use of liquid nitrogen-inactivated autologous tumor segment bone replantation for reconstruction of bone defects after resection of malignant tumors in the shaft has advantages of higher healing rate, shorter healing time at the metaphyseal end compared to the osteotomy end, fewer complications, and higher survival rate of the replanted bone.

Osteosarcoma with bone metastasis or pulmonary metastasis show distinct genomic manifestations / 中华骨科杂志

Objective:To investigate the genomic manifestation and pathogenesis of osteosarcoma with different relapse pattens, which were respectively initially presented with bone metastasis or pulmonary metastasis.Methods:From May 1, 2021 to October 1, 2021, 38 fresh tumor specimens and some paraffin-embedded specimens of high-grade osteosarcoma were collected in Peking University People's Hospital, including 29 males and 9 females, aged 19.6±2.2 years (range, 6-61 years). Among the 38 cases, 12 cases had initial bone metastasis (group A) and 26 cases had initial lung metastasis (group B), of which 15 cases (40%, 15/38) had paired specimens of primary and metastatic lesions. Based on Illumina NovaSeq 6000, we analyzed whole-exome sequencing (WES) as well as transcriptome for osteosarcoma with paired samples in different relapse patterns. During all their treatment courses, we also collected their paired samples to reveal these tumors' evolution. We sought to redefine disease subclassifications for osteosarcoma based on genetic alterations and correlate these genetic profiles with clinical treatment courses to elucidate potential evolving cladograms.Results:We found that osteosarcoma in group A mainly carried single-nucleotide variations (83%, 10/12), displaying higher tumor mutation burden [4.9 (2.8, 12.0) & 2.4 (1.4, 4.5), P=0.010] and neoantigen load [743.0 (316.5, 1,034.5) & 128.5 (49.0, 200.5), P=0.003], while those in group B mainly exhibit structural variants (58%, 15/26). The mutation spectrum showed that there was a significant difference in age-related gene imprinting 1 between the bone metastasis group and the lung metastasis group ( P=0.005). Samples were randomly selected from group A (3 patients) to investigate immunologic landscape by multiplex immunohistochemistry, from which we noticed tertiary lymphatic structure from one patient from group A. High conservation of reported genetic sequencing over time was found in their evolving cladograms. Conclusion:Osteosarcoma with mainly single-nucleotide variations other than structural variants might exhibit biological behavior predisposing toward bone metastases with older in age as well as better immunogenicity in tumor microenvironment.

Prognostic effect of peritumoral vascular invasion of tumor thrombus in osteosarcomas / 中华骨科杂志

Objective:To analyze the value of peritumoral vascular invasion (PVI) on the prognosis of patients with osteosarcoma.Methods:A total of 232 patients with primary osteosarcoma from 2007 to 2016 were retrospectively analyzed, including 142 males and 90 females. The average age was 17.9±8.2 years (range, 3-39 years). There were 22 positive and 210 negative cases of PVI, 94 deaths and 138 survivals. Univariate survival analysis (Log-rank test and univariate Cox regression) was used to evaluate the effects of age, gender, PVI status, tumor location, surgical method, sensitivity to chemotherapy, and chemotherapy regimen on the prognosis of osteosarcoma. The indicators with statistically significant differences were included in the multivariate Cox regression model to finally determine the risk factors affecting the prognosis of osteosarcoma. The relationship between PVI status and 5-year survival and the incidence of recurrence or metastasis was evaluated using the Kaplan-Meier method.Results:All patients were followed up for 7.6±4.5 years (range, 0.1-15 years). The differences in sensitivity to chemotherapy (χ 2=9.52, P=0.002), choice of chemotherapy regimen (χ 2=8.87, P=0.012), choice of surgical modality (χ 2=13.50, P<0.001), tumor metastasis rate (χ 2=8.51, P=0.004) and mortality rate (χ 2=5.39, P= 0.020) of PVI positive group and PVI negative group had statistically significant differences. Univariate survival analysis was performed on 232 patients with osteosarcoma (gender, age, PVI status, site of tumor development, surgical modality, sensitivity to chemotherapy, and chemotherapy regimen). Indicators with statistically significant differences were included in a multifactorial Cox regression model. The results showed PVI positive [5-year survival rate: HR=2.02, 95% CI (1.61, 2.79), P=0.010; 5-year recurrence or metastasis rate: HR=2.25, 95% CI (1.55, 3.14), P<0.001], surgical procedure as amputation [5-year survival rate: HR=1.22, 95% CI (0.94, 1.78), P=0.037; 5-year recurrence or metastasis rate: HR=1.58, 95% CI (1.11, 2.23), P=0.026] and poor sensitivity to chemotherapy [5-year survival rate: HR=2.71, 95% CI (1.84, 3.98), P=0.001; 5-year recurrence or metastasis rate: HR=2.52, 95% CI (1.88, 3.45), P<0.001] was associated with poor prognosis. Kaplan-Meier curve showed that the 5-year survival rate of PVI positive group was 34%, which was lower than 68% of PVI negative group. The 5-year recurrence or metastasis rate was 72% in the PVI negative group, which was significantly higher than 38% in the PVI negative group ( P<0.05). Conclusion:The 5-year survival rate of PVI positive group was lower than that of PVI negative group, and the 5-year recurrence or metastasis rate was higher than that of PVI negative group. The presence of microvascular angiosarcoma plugs infiltrating the peritumoral tissue in surgical specimens of osteosarcoma after neoadjuvant chemotherapy is a useful indicator to assess the prognosis of patients with osteosarcoma.

Clinical outcome and prognosis of indeterminate pulmonary nodules in patients with osteosarcoma / 中华骨科杂志

Objective:To explore the incidence, clinical characteristics, imaging features, clinical outcome and prognosis of indeterminate pulmonary nodules (IPN) in patients with osteosarcoma.Methods:A total of 69 patients of osteosarcoma with IPN in lung treated in the Bone tumor Center of Eastern Theater General Hospital from January 2011 to January 2021 were collected retrospectively, there were 47 males and 22 females, with a median age of 19 years old (range 7-60 years old). The clinical characteristics including disease-free interval, the chemotherapy response, with recurrence/non-pulmonary, IPN presence before / during / after chemotherapy and imaging features of IPN including number of IPN, location of IPN, density of IPN, boundary clarity of IPN and outcome. The patients were divided into the metastasis pulmonary nodules group and the benign nodules group according to the final outcome of IPN. Further, χ 2 test was performed for comparison of the clinical and imaging characteristics between the two groups. The survival of patients was counted and the correlation between single factor and survival was compared by Kaplan-Meier test, and multivariate survival analyses were performed using Cox proportional hazards regression models. Results:Sixty-nine cases occurred IPN in 211 patients with osteosarcoma, with an incidence of 32.7%. Of the 69 patients, 45 patients (65.2%) with IPN were diagnosed as metastases, and 24 patients (34.8%) with IPN were diagnosed as benign nodules. Follow-up length ranged from 1 to 124 months, with the median follow up time 43 months. To the end of follow-up, 41 patients (59.4%) remained alive and 28 patients (40.6%) had died. The median survival time was 41.0 (20.0, 65.0) months and the median survival time after diagnosis of IPN was 25.0 (10.0, 43.0) months. There were significant differences in lung nodule density ( P<0.001), boundary ( P=0.002), history of recurrence/extra-pulmonary metastasis ( P=0.023) and chemotherapeutic effect ( P<0.001) between the metastasis pulmonary nodules group and the benign nodules group. Multivariate survival analysis showed that chemotherapeutic effect was an independent factor affecting the overall survival of patients [ HR=0.048, 95% CI (0.01, 0.26)]. Boundary definition [ HR=0.12, 95% CI (0.02, 0.93)] and chemotherapeutic effect [ HR=0.06, 95% CI (0.01, 0.29)] were independent factors influencing survival after diagnosis of IPN. Conclusion:Osteosarcoma patients with IPN have a poor prognosis. The poor effect of chemotherapy is an independent risk factor for the overall survival time of those patients and the survival time after diagnosis of IPN. The boundary definition of IPN is an independent risk factor for the survival time after diagnosis of IPN.

A meta-analysis of progressive pulmonary micro nodules in osteosarcoma / 中华骨科杂志

Objective:To identify the prevalence of pulmonary micro nodule (PMN) in osteosarcoma, investigate radiologic features of progressive PMN, and provide evidence for early diagnosis of pulmonary metastasis of osteosarcoma.Methods:Electronic articles published in PubMed, EMBASE, Web of Science and the Cochrane Library databases between January 1, 2000, and September 1, 2022, were searched and critically evaluated. The authors independently reviewed the abstracts and extracted data on the prevalence of PMN in osteosarcoma and radiologic features of progressive PMN. Seven high quality studies were finally included in the meta-analysis with evidence level III.Results:The pooled prevalence of PMN in osteosarcoma was 36.0%, 95% CI (14.6%, 57.3%). The pooled progressive rate of PMN was 52.5%, 95% CI (37.7%, 67.2%). As for a specific PMN, it was more likely to progress which had a larger Dmax, HR=2.40, 95% CI (1.06, 5.42), P=0.035. No significant difference was found in number, component, and border. Conclusion:PMN is quite common in patients with osteosarcoma. About half of the patients suffered the progression of PMN, and it is related to several risk factors.