CT-derived Radiomics Predicts the Efficacy of Tyrosine Kinase Inhibitors in Osteosarcoma Patients with Pulmonary Metastasis.

BACKGROUND: To construct and validate the CT-based radiomics model for predicting the tyrosine kinase inhibitors (TKIs) effects in osteosarcoma (OS) patients with pulmonary metastasis. METHODS: OS patients with pulmonary metastasis treated with TKIs were randomly separated into training and testing cohorts (2:1 ratio). Radiomic features were extracted from the baseline unenhanced chest CT images. The random survival forest (RSF) and Kaplan-Meier survival analyses were performed to construct and evaluate radiomics signatures (R-model-derived). The univariant and multivariant Cox regression analyses were conducted to establish clinical (C-model) and combined models (RC-model). The discrimination abilities, goodness of fit and clinical benefits of the three models were assessed and validated in both training and testing cohorts. RESULTS: A total of 90 patients, 57 men and 33 women, with a mean age of 18 years and median progression-free survival (PFS) of 7.2 months, were enrolled. The R-model was developed with nine radiomic features and demonstrated significant predictive and prognostic values. In both training and testing cohorts, the time-dependent area under the receiver operating characteristic curves (AUC) of the R-model and RC-model exhibited obvious superiority over C-model. The calibration and decision curve analysis (DCA) curves indicated that the accuracy of the R-model was comparable to RC-model, which exhibited significantly better performance than C-model. CONCLUSIONS: The R-model showed promising potential as a predictor for TKI responses in OS patients with pulmonary metastasis. It can potentially identify pulmonary metastatic OS patients most likely to benefit from TKIs treatment and help guide optimized clinical decisions.

Lamin A/C and Vimentin as a Coordinated Regulator during Amoeboid Migration in Microscale Confined Microenvironments.

Cell migration occurs in confined microenvironments, which plays a vital role in the process of tumor metastasis. However, it is challenging to study their behaviors in vivo. Here we developed a cell squeeze system that can be scaled down to micrometers to mimic native physical confined microenvironments, wherein degrees of surface adhesion and mechanical constraints could be manipulated in order to investigate cell-migrating behaviors. Based on the microscale cell squeeze system, we found the synergistic role of lamin A/C and vimentin in cell transition and migration under strong confinement. The dynamic variations in lamin A/C and vimentin expression establish a positive feedback loop in response to confinement, effectively promoting amoeboid migration by modulating nuclear deformability while ensuring cell viability. This work shed light on modulating cell response to microenvironments by altering the expression of lamin A/C and/or vimentin, which may be a more efficient way of inhibiting cancer metastasis.

Sorafenib inhibits doxorubicin-induced PD-L1 upregulation to improve immunosuppressive microenvironment in Osteosarcoma.

PURPOSE: Although undergoing conventional chemotherapy significantly improves the prognosis of Osteosarcoma, chemoresistance and failure of therapy is still a significant challenge. Furthermore, conventional chemotherapy, like doxorubicin, would upregulate the expression of programmed death-ligand 1 (PD-L1) which caused an immunosuppressive microenvironment and unsatisfied treatment result in Osteosarcoma. Thus, it is urgent to explore a strategy to overcome this disadvantage. METHODS: Human Osteosarcoma cell line MG63 and mouse Osteosarcoma cell line K7 were included in this study. Subcutaneous tumor model was used by injection of K7 cells in BALB/C mice to test the effect of doxorubicin and sorafenib on tumor growth. PD-L1 expression was tested in vitro (flow cytometry, western blot and PCR) and in vivo (flow cytometry and immunohistochemistry). Proportion of immune cells (CD4, CD8, Tregs, and cytotoxic T lymphocytes) in vivo was analyzed with flow cytometry. RESULTS: Combination of sorafenib and doxorubicin inhibited tumor growth significantly in vivo. Doxorubicin increased PD-L1 expression in vitro and in vivo, while sorafenib inhibited doxorubicin-induced PD-L1 upregulation in vitro and in vivo. Proportion of interferon-γ-secreting CD8 + T lymphocytes in tumor tissue was increased significantly when sorafenib was combined with doxorubicin, while proportion of CD4, CD8, and Tregs was not significantly changed. Extracellular signal-regulated kinases (ERK) pathway could be one of the key mechanisms by which doxorubicin induced upregulation of PD-L1 in Osteosarcoma cells. CONCLUSION: Combination of sorafenib and conventional chemotherapeutic reagents is a potent strategy to improve treatment effectiveness by modulating tumor microenvironment in Osteosarcoma through increasing proportion of cytotoxic T lymphocytes.

First-in-Maintenance Therapy for Localized High-Grade Osteosarcoma: An Open-Label Phase I/II Trial of the Anti-PD-L1 Antibody ZKAB001.

PURPOSE: We investigated the safety and preliminary efficacy of anti-PD-L1 antibody (ZKAB001) as maintenance therapy for localized patients with high-grade osteosarcoma to reduce the risk of recurrence and metastasis. PATIENTS AND METHODS: This open-label Phase I/II study was divided into dose-escalation Phase I and expansion Phase II. Phase I used a 3+3 design with ZKAB001 at three escalating doses ranging: 5, 10, 15 mg/kg every 2 weeks in 9 patients with localized high-grade osteosarcoma and Phase II tested 10 mg/kg in 12 patients for up to 24 cycles. Primary endpoints were safety and tolerability assessed using CTCAE4.0.3. RESULTS: Between October 2018 and 2019, 21 eligible patients were enrolled and accepted ZKAB001 treatment: 9 in the dose-escalation phase, and 12 in expansion phase. Six patients with disease progression withdrew from this study and follow-up is ongoing. The MTD was not defined in Phase I. All doses had a manageable safety profile. The recommended dose in Phase II was set at 10 mg/kg. Most frequent immune-related adverse events were thyroiditis (76.2%) and dermatitis (42.9%). Only 1 (4.8%) of 21 patients had a Grade 3 skin rash. The median 3-year event-free survival (EFS) and overall survival (OS) were not established; however, 24-month EFS was 71.4% (95% confidence interval, 47.2-86.0) and 2-year OS was 100%. Preliminary efficacy data showed EFS benefits in patients with PD-L1 positive or an MSI-H sub-population. CONCLUSIONS: Switching to maintenance using ZKAB001 showed an acceptable safety profile and provided preliminary evidence of clinical activity in localized patients with osteosarcoma.

Superenhancers activate the autophagy-related genes Beclin1 and LC3B to drive metastasis and drug resistance in osteosarcoma.

Metastasis and drug resistance are the leading causes of poor prognosis in patients with osteosarcoma. Identifying the relevant factors that drive metastasis and drug resistance is the key to improving the therapeutic outcome of osteosarcoma. Here, we reported that autophagy was highly activated in metastatic osteosarcoma. We found increased autophagolysosomes in metastatic osteosarcoma cell lines by using electron microscopy, Western blot, and immunofluorescence experiments. We further examined the expression of the autophagy-related genes Beclin1 and LC3B in 82 patients through immunohistochemistry and found that Beclin1 and LC3B were highly related to unfavorable prognosis of osteosarcoma. Knockdown of Beclin1 and LC3B reduced invasion, metastasis, and proliferation in metastatic osteosarcoma cells. In vitro and in vivo studies also demonstrated that inhibiting by 3-MA inhibited cell growth and metastasis. Moreover, we demonstrated that autophagy-related genes were activated by SEs and that the inhibition of SEs by JQ-1 decreased the metastasis of osteosarcoma. Overall, our findings highlighted the association of autophagy with osteosarcoma progression and shed new light on autophagy-targeting therapy for osteosarcoma.

Integrated microfluidic-SERS for exosome biomarker profiling and osteosarcoma diagnosis.

Osteosarcoma is one of the most frequent primary sarcoma of bone among adolescents. Early diagnosis of osteosarcoma is the key factor to achieve high survival rate of patients. Nevertheless, traditional histological biopsy is highly invasive and associated with the risk of arousing tumor spread. Herein, we develop a method integrating microfluidics and surface-enhanced Raman spectroscopy (SERS) to isolate plasma-derived exosomes and profile multiple exosomal biomarkers for the diagnosis of osteosarcoma. The method showed highly efficient isolation of exosomes directly from human plasma and can profile exosomes based on protein biomarkers, with the detection limit down to 2 exosomes per µL. The whole assay can be performed in 5 h and only consumed 50 µL of plasma for one analysis. With the method, we analyzed the level of three protein biomarkers, i.e., CD63, vimentin (VIM) and epithelial cell adhesion molecule (EpCAM), on plasma-derived exosomes from 20 osteosarcoma patients and 20 heathy controls. Significantly higher levels of CD63, VIM and EpCAM were observed on plasma exosomes from the osteosarcoma patients compared to the healthy controls. Based on the level of the exosomal biomarkers, a classification model was built for the rapid diagnosis of osteosarcoma, with the sensitivity, specificity and accuracy of 100%, 90% and 95%, respectively. The proposed method does not require complex operations nor expensive equipment, and has great promise in clinical diagnosis of cancer as a liquid biopsy technique.

DNA Base Pairing-Inspired Supramolecular Nanodrug Camouflaged by Cancer-Cell Membrane for Osteosarcoma Treatment.

Osteosarcoma (OS) is one of the most common bone malignant tumors which mainly develops in adolescents. Although neoadjuvant chemotherapy has improved the prognosis of patients, numerous chemotherapeutic challenges still limit their use. Here, inspired by the Watson-Crick base pairing in nucleic acids, hydrophobic (methotrexate) and hydrophilic (floxuridine) chemo-drugs are mixed and self-assembled into M:F nanoparticles (M:F NPs) through molecular recognition. Then, the obtained NPs are co-extruded with membranes derived from OS cells to form cancer-cell membrane-coated NPs (CCNPs). With protected membranes at the outer layer, CCNPs are highly stable in both physiological and weak acid tumor conditions and possess homologous tumor targeted capability. Furthermore, the proteomic analysis first identifies over 400 proteins reserved in CCNPs, most of them participating in tumor cell targeting and adhesion processes. In vitro studies reveal that CCNPs significantly inhibit the PI3K/AKT/mTOR pathway, which promotes cell apoptosis and cell cycle arrest. More importantly, cell membrane camouflage significantly prolongs the circulation half-life of CCNPs, elevates the drug accumulation at tumor sites, and promotes anti-tumor efficacy in vivo. As a convenient and effective strategy to construct a biomimetic NP with high drug loading ratio, the CCNPs provide new potentials for precise and synergistic antitumor treatment.

Patterns and Trends of the Mortality From Bone Cancer in Pudong, Shanghai: A Population-Based Study.

Introduction: The burden of cancer-related mortality of common malignancies has been reported worldwide. However, whether bone cancer (BC), as a highly aggressive and heterogeneous group of rare cancers, followed a similar or distinct epidemiological pattern during such process remains largely unknown. We aimed to analyze the mortality and the temporal trends of BC in relation to gender, age, and premature death in Shanghai, China. Methods: We conducted a population-based analysis of the mortality data of BC in Shanghai Pudong New Area (PNA) from 2005 to 2020. The epidemiological characteristics and long-term trends in crude mortality rates (CMRs), age-standardized mortality rates worldwide (ASMRWs), and rate of years of life lost (YLL) was analyzed using the Joinpoint regression program. The demographic and non-demographic factors affecting the mortality rate were evaluated by the decomposition method. Results: There are 519 BC-specific deaths accounting for 0.15% of all 336,823 deaths and 0.49% of cancer-specific death in PNA. The CMR and ASMRW of BC were 1.15/105 person-year and 0.61/105 person-year, respectively. The YLL due to premature death from BC was 6,539.39 years, with the age group of 60-69 years having the highest YLL of 1,440.79 years. The long-term trend of CMR, ASMRW, and YLL rate significantly decreased by -5.14%, -7.64%, and -7.27%, respectively, per year (all p < 0.05) in the past 16 years. However, the proportion of BC-specific death within the total cancer-specific death dropped to a plateau without further improvement since 2016, and a remarkable gender and age disparity was noticed in the observed reduction in mortality. Specifically, the elderly benefited less but accounted for a larger percentage of BC population in the last decades. Although the overall mortality of BC decreased, there was still a significant upward trend toward an increased mortality rate caused by the aging of the BC patients. Conclusion: Our study provides novel insights on the epidemiological characteristics and longitudinal dynamics of BC in a fast urbanization and transitioning city. As a rare disease affecting all ages, the burden of BC among the elderly emerged to form an understudied and unmet medical need in an aging society.

Construction and validation of a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis and immune infiltration.

BACKGROUND: Apoptosis played vital roles in the formation and progression of osteosarcoma. However, no studies elucidated the prognostic relationships between apoptosis-associated genes (AAGs) and osteosarcoma. METHODS: The differentially expressed genes associated with osteosarcoma metastasis and apoptosis were identified from GEO and MSigDB databases. The apoptosis-associated prognostic signature was established through univariate and multivariate cox regression analyses. The Kaplan-Meier (KM) survival curve, ROC curve and nomogram were constructed to investigate the predictive value of this signature. CIBERSORT algorithm and ssGSEA were used to explore the relationships between immune infiltration and AAG signature. The above results were validated in another GEO dataset and the expression of AAGs was also validated in osteosarcoma patient samples by immunohistochemistry. RESULTS: HSPB1 and IER3 were involved in AAG signature. In training and validation datasets, apoptosis-associated risk scores were negatively related to patient survival rates and the AAG signature was regarded as the independent prognostic factor. ROC and calibration curves demonstrated the signature and nomogram were reliable. GSEA revealed the signature related to immune-associated pathways. ssGSEA indicated that one immune cell and three immune functions were significantly dysregulated. The immunohistochemistry analyses of patients' samples revealed that AAGs were significantly differently expressed between metastasis and non-metastasis osteosarcomas. CONCLUSIONS: The present study identified and validated a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis. It could serve as the potential biomarker and therapeutic targets for osteosarcoma in the future.

Reversal of neuropathic pain is associated with corticostriatal functional reorganization after nerve repair in the spared nerve injury model.

ABSTRACT: Following surgical repair after peripheral nerve injury, neuropathic pain diminishes in most patients but can persist in a small proportion of cases, the mechanism of which remains poorly understood. Based on the spared nerve injury (SNI), we developed a rat nerve repair (NR) model, where a delayed reconstruction of the SNI-injured nerves resulted in alleviating chronic pain-like behavior only in a subpopulation of rats. Multiple behavioral measures were assayed over 11-week presurgery and postsurgery periods (tactile allodynia, pain prick responses, sucrose preference, motor coordination, and cold allodynia) in SNI (n = 10), sham (n = 8), and NR (n = 12) rats. All rats also underwent resting-state functional magnetic resonance imaging under anesthesia at multiple time points postsurgery, and at 10 weeks, histology and retrograde labeling were used to calculate peripheral reinnervation. Behavioral measures indicated that at approximately 5 weeks postsurgery, the NR group separated to pain persisting (NR persisting, n = 5) and recovering (NR recovering, n = 7) groups. Counts of afferent nerves and dorsal root ganglion cells were not different between NR groups. Therefore, NR group differences could not be explained by peripheral reorganization. By contrast, large brain functional connectivity differences were observed between NR groups, where corticolimbic reorganization paralleled with pain recovery (repeated-measures analysis of variance, false discovery rate, P < 0.05), and functional connectivity between accumbens and medial frontal cortex was related both to tactile allodynia (nociception) and to sucrose preference (anhedonia) in the NR group. Our study highlights the importance of brain circuitry in the reversal of neuropathic pain as a natural pain-relieving mechanism. Further studies regarding the therapeutic potentials of these processes are warranted.

The Outcome of Discontinuing Tyrosine Kinase Inhibitors in Advanced Sarcoma Following a Favorable Tumor Response to Antiangiogenics Therapy.

(1) Background: The use of antiangiogenic TKIs (AA-TKIs) has recently emerged as a major paradigm shift in the treatment of advanced sarcoma. However, the feasibility of drug holidays for patients demonstrating a very favorable response remains unknown. (2) Methods: We aim to explore the outcomes of patients with advanced sarcoma who discontinued AA-TKIs after a (near-) complete remission or were long-term responders. Patients with advanced disease were included if they had bilateral or multiple lung metastases, extrapulmonary recurrence, a short disease-free interval, etc., at the initiation of AA-TKIs. (3) Results: A total of 22 patients with AA-TKI discontinuation were analyzed, with a median follow-up of 22.3 months post-discontinuation. Prior to discontinuation, there were four drug-induced complete remissions (CRs), twelve surgical CRs, and six long-term responders. Disease progression was observed in 17/22 (77.3%) patients, with a median of 4.2 months. However, since the majority were still sensitive to the original AA-TKIs and amenable to a second surgical remission, 7 out of these 17 patients achieved a second CR after disease progression and were thus considered as relapse-free post-discontinuation (pd-RFS). Therefore, the pd-RFS and post-discontinuation overall survival (pd-OS) in the last follow-up were 12/22 (54.5%) and 16/22 (72.7%), respectively. Remarkably, surgical CR and drug tapering off (versus abrupt stopping) were associated with a greater pd-RFS and pd-OS (p < 0.05). Furthermore, higher necrosis rates (p = 0.040) and lower neutrophil-to-lymphocyte ratios (NLR) (p = 0.060) before discontinuation tend to have a better pd-RFS. (4) Conclusions: Our results suggest that AA-TKI discontinuation with a taper-off strategy might be safe and feasible in highly selected patients with advanced sarcoma. Surgical CR, NLR, and tumor necrosis rates before discontinuation were potential biomarkers for AA-TKI withdrawal.

Exploration and Validation of a Novel Inflammatory Response-Associated Gene Signature to Predict Osteosarcoma Prognosis and Immune Infiltration.

BACKGROUND: Inflammatory response took part in the progression of tumor and was regarded as the hallmark of cancer. However, the prognostic relationship between osteosarcoma and inflammatory response-associated genes (IRGs) was unclear. This research aimed to explore the correlations between osteosarcoma prognosis and IRG signature. METHODS: The inflammatory response-associated differentially expressed messenger RNAs (DEmRNAs) were screened out through Gene Expression Omnibus (GEO) and Molecular Signature Database (MSigDB) databases. Univariate and multivariate cox regression analyses were utilized to construct the IRG signature. The prognostic value of signature was investigated through Kaplan-Meier (KM) survival curve and nomogram. DEmRNAs among high and low inflammatory response-associated risks were identified and functional enrichment analyses were conducted. ESTIMATE, CIBERSORT and single-sample gene set enrichment analyses (ssGSEA) were implied to reveal the alterations in immune infiltration. All the above results were validated in Target database. The expression of IRGs was also validated in different cell lines by quantitative real-time PCR (qRT-PCR) and osteosarcoma patient samples by immunohistochemistry. RESULTS: The IRG signature that consisted of two genes (MYC, CLEC5A) was established. In training and validation datasets, patients with lower risk scores survived longer and the IRG signature was confirmed as the independent prognostic factor in osteosarcoma. The nomogram was constructed and the calibration curves demonstrated the reliability of this model. Functional analysis of risk score-associated DEmRNAs indicated that immune-related pathways and functions were significantly enriched. ssGSEA revealed that 14 immune cells and 11 immune functions were significantly dysregulated. The qRT-PCR results indicated IRGs were significantly differently expressed in osteosarcoma and osteoblast cell lines. The immunohistochemistry analyses of patients' samples revealed the same result. CONCLUSION: The novel osteosarcoma inflammatory response-associated prognostic signature was established and validated in this study. This model could serve as the biomarker and therapeutic target for osteosarcoma in the future.

A Novel Microfluidic Chip for Fast, Sensitive Quantification of Plasma Extracellular Vesicles as Biomarkers in Patients With Osteosarcoma.

Plasma circulating extracellular vesicle (EV) has emerged as a promising biomarker for diagnosis and prognosis of various epithelial tumors. However, fast and efficient capture of EVs with microfluidic chip in sarcoma remains to be established. Herein, we reported a ZnO-nanorods integrated (ZNI) microfluidic chip, where EV capture antibody was uniformly grafted to the surface of the ZnO-nanorods of the chip to enhance the plasma turbulence formation and the capture efficiency at the micro-scale. Based on osteosarcoma (OS) cell line, we demonstrated that a combination of CD81 and CD63 antibody on ZNI chip yielded the greatest amount of total EVs, with an extra sensitive limit of detection (LOD) of ~104 particles mL-1. Furthermore, the addition of fluorescent labeling of Vimentin (VIM), a previously reported sarcoma cell surface biomarker, could enabled the dual visualization of total plasma EVs and VIM-positive EVs from OS patients' plasma. Based on our ZNI chip, we found that the amount of plasma total EVs was significantly different between OS and healthy donors (1562 a.u. versus 639 a.u., p< 0.05), but not between metastatic and nonmetastatic OS (p> 0.05). Interestingly, patients with metastatic disease had a significantly greater amount of VIM-positive EVs (1411 a.u. versus 231 a.u.., p< 0.05) and increased VIM-positive/total EVs ratio (0.943 versus 0.211, p< 0.05) in comparison with the nonmetastatic counterpart. Therefore, our ZNI microfluidic chip has great potential for the fast quantification of plasma EVs, and the microfluidic-based quantification of total and VIM-positive EVs might serve as a promising biomarker for the diagnosis and surveillance in OS patients.

MCM4 Is a Novel Biomarker Associated With Genomic Instability, BRCAness Phenotype, and Therapeutic Potentials in Soft-Tissue Sarcoma.

Soft-tissue sarcoma (STS) is represented by a heterogeneous group of rare malignancies with various molecular oncogenesis. Therapies targeting DNA repair pathways in STS have achieved minimal progress, potentially due to the lack of molecular biomarker(s) beyond the histology subtype. In this report, we comprehensively analyzed the expression profiles of 100 liposarcomas (LPSs), the most common STS subtype, in comparison with 21 adipose tissues from multiple GEO datasets to identify the potential prognostic and therapeutic biomarker for LPS. Furthermore, we investigated TCGA database, our archived tumor samples, and patient-derived tumor cell cultures (PTCCs) as a validation. We identified a total of 69 common differentially expressed genes (DEGs) among public datasets, with mini-chromosome maintenance protein 4 (MCM4) identified as a novel biomarker correlated with patients' clinical staging and survival outcome. MCM4-high expression LPS was characterized by MCM4 copy number increase, genomic instability, and BRCAness phenotype compared with the MCM4-low expression counterpart. In contrast, the mutational and the immune landscape were minimally different between the two groups. Interestingly, the association of MCM4-high expression with genomic instability and BRCAness were not only validated in LPS samples from our institution (n = 66) but also could be expanded to the pan-sarcoma cohort from TCGA database (n = 263). Surprisingly, based on four sarcoma cell lines and eight PTCCs (three LPS and five other sarcoma), we demonstrated that MCM4 overexpression tumors were therapeutically sensitive to PARP inhibitor (PARPi) and platinum chemotherapy, independent of the histology subtypes. Our study, for the first time, suggested that MCM4 might be a novel prognostic biomarker, associated with dysregulated DNA repair pathways and potential therapeutic vulnerability in STS.

Development and Validation of a Hypoxia-Associated Prognostic Signature Related to Osteosarcoma Metastasis and Immune Infiltration.

BACKGROUND: Increasing evidence has shown that hypoxia microenvironment relates to tumor initiation and progression. However, no studies focus on the application of hypoxia-associated genes in predicting osteosarcoma patients' prognosis. This research aims to identify the hypoxia-associated genes related to osteosarcoma metastasis and construct a gene signature to predict osteosarcoma prognosis. METHODS: The differentially expressed messenger RNAs (DEmRNAs) related to osteosarcoma metastasis were identified from Therapeutically Applicable Research to Generate Effective Treatments (Target) database. Univariate and multivariate cox regression analyses were performed to develop the hypoxia-associated prognostic signature. The Kaplan-Meier (KM) survival analyses of patients with high and low hypoxia risk scores were conducted. The nomogram was constructed and the gene signature was validated in the external Gene Expression Omnibus (GEO) cohort. Single-sample gene set enrichment analysis (ssGSEA) was conducted to investigate the relationships between immune infiltration and gene signature. RESULTS: Two genes, including decorin (DCN) and prolyl 4-hydroxylase subunit alpha 1 (P4HA1), were involved in the hypoxia-associated gene signature. In training and testing datasets, patients with high-risk scores showed lower survival rates and the gene signature was identified as the independent prognostic factor. Receiver operating characteristic (ROC) curves demonstrated the robustness of signature. Functional analyses of DEmRNAs among high- and low-risk groups revealed that immune-associated functions and pathways were significantly enriched. Furthermore, ssGSEA showed that five immune cells (DCs, macrophages, neutrophils, pDCs, and TIL) and three immune features (CCR, APC co inhibition, and Check-point) were down-regulated in the high-risk group. CONCLUSION: The current study established and validated a novel hypoxia-associated gene signature in osteosarcoma. It could act as a prognostic biomarker and serve as therapeutic guidance in clinical applications.

Impaired Limb Functional Outcome of Peripheral Nerve Regeneration Is Marked by Incomplete Recovery of Paw Muscle Atrophy and Brain Functional Connectivity in a Rat Forearm Nerve Repair Model.

Skilled sensorimotor deficit is an unsolved problem of peripheral nerve injury (PNI) led by limb trauma or malignancies, despite the improvements in surgical techniques of peripheral nerve anastomosis. It is now accepted that successful functional recovery of PNI relies tremendously on the multilevel neural plasticity from the muscle to the brain. However, animal models that recapitulate these processes are still lacking. In this report, we developed a rat model of PNI to longitudinally assess peripheral muscle reinnervation and brain functional reorganization using noninvasive imaging technology. Based on such model, we compared the longitudinal changes of the rat forepaw intrinsic muscle volume and the seed-based functional connectivity of the sensorimotor cortex after nerve repair. We found that the improvement of skilled limb function and the recovery of paw intrinsic muscle following nerve regeneration are incomplete, which correlated with the functional connectivity between the primary motor cortex and dorsal striatum. Our results were highly relevant to the clinical observations and provided a framework for future investigations that aim to study the peripheral central sensorimotor circuitry underlying skilled limb function recovery after PNI.

Development and analysis of long non-coding RNA-associated competing endogenous RNA network for osteosarcoma metastasis.

BACKGROUND: Osteosarcoma is the primary bone malignant neoplasm that often develops metastasis. Increasing evidences have shown that non-coding RNAs (ncRNAs) relate to the progression of osteosarcoma. However, the ncRNAs' roles in osteosarcoma metastasis are still unknown. METHODS: Differentially expressed (DE) RNAs were identified from Gene Expression Omnibus (GEO) database. Protein-protein interaction (PPI) of DE messenger RNAs (DEmRNAs) was built through STRING database. The target mRNAs and long ncRNAs (lncRNAs) of microRNAs (miRNA) were predicted through miRDB, Targetscan and Genecode databases, which then cross-checked with previously obtained DERNAs to construct competing endogenous RNA (ceRNA) network. All networks were visualized via Cytoscape and the hub RNAs were screened out through Cytoscape plug-in Cytohubba. The gene functional and pathway analyses were performed through DAVID and MirPath databases. The survival analyses of hub RNAs were obtained through Kaplan-Meier (KM) survival curves. RESULTS: Five hundred sixty-four DEmRNAs, 16 DElncRNAs and 22 DEmiRNAs were screened out. GO functional and KEGG pathway analyses showed that DERNAs were significantly associated with tumor metastasis. The ceRNA network including 6 lncRNAs, 55 mRNAs and 20 miRNAs were constructed and the top 10 hub RNAs were obtained. Above all, PI3K/AKT signaling pathway was identified as the most important osteosarcoma metastasis-associated pathway and its hub ceRNA module was constructed. The survival analyses showed that the RNAs in hub ceRNA module closely related to osteosarcoma patients' prognosis. CONCLUSIONS: The current study provided a new perspective on osteosarcoma metastasis. More importantly, the RNAs in hub ceRNA module might act as the novel therapeutic targets and prognostic factors for osteosarcoma patients.

Clinical factors affecting prognosis of limb osteosarcoma in China: a multicenter retrospective analysis.

OBJECTIVE: This study was performed to explore the relationship between various clinical factors and the prognosis of limb osteosarcoma. METHODS: We retrospectively analyzed the clinical data of 336 patients with limb osteosarcoma treated from June 2000 to August 2016 at 7 Chinese cancer centers. Data on the patients' clinical condition, treatment method, complications, recurrences, metastasis, and prognosis were collected and analyzed. Kaplan-Meier analysis and Cox regression models were used to analyze the data. RESULTS: The patients comprised 204 males and 132 females ranging in age from 6 to 74 years (average, 21.1 years). The overall 3- and 5-year survival rates were 65.0% and 55.0%, respectively. The 5-year overall survival rate was 64.0% with standard chemotherapy and 45.6% with non-standard chemotherapy. Cox regression analysis demonstrated that standard chemotherapy, surgery, recurrence, and metastasis were independent factors associated with the prognosis of limb osteosarcoma. CONCLUSION: The survival of patients with limb osteosarcoma can be significantly improved by combining standard chemotherapy and surgery. The overall survival rate can also be improved by adding methotrexate to doxorubicin-cisplatin-ifosfamide triple chemotherapy.

Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples.

BACKGROUND: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. METHODS: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. RESULTS: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. CONCLUSIONS: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research.