Osteosarcoma in a ceRNET perspective.

Osteosarcoma (OS) is the most prevalent and fatal type of bone tumor. It is characterized by great heterogeneity of genomic aberrations, mutated genes, and cell types contribution, making therapy and patients management particularly challenging. A unifying picture of molecular mechanisms underlying the disease could help to transform those challenges into opportunities.This review deeply explores the occurrence in OS of large-scale RNA regulatory networks, denominated "competing endogenous RNA network" (ceRNET), wherein different RNA biotypes, such as long non-coding RNAs, circular RNAs and mRNAs can functionally interact each other by competitively binding to shared microRNAs. Here, we discuss how the unbalancing of any network component can derail the entire circuit, driving OS onset and progression by impacting on cell proliferation, migration, invasion, tumor growth and metastasis, and even chemotherapeutic resistance, as distilled from many studies. Intriguingly, the aberrant expression of the networks components in OS cells can be triggered also by the surroundings, through cytokines and vesicles, with their bioactive cargo of proteins and non-coding RNAs, highlighting the relevance of tumor microenvironment. A comprehensive picture of RNA regulatory networks underlying OS could pave the way for the development of innovative RNA-targeted and RNA-based therapies and new diagnostic tools, also in the perspective of precision oncology.

System analysis based on Anoikis-related genes identifies MAPK1 as a novel therapy target for osteosarcoma with neoadjuvant chemotherapy.

BACKGROUND: Osteosarcoma (OS) is the most common bone malignant tumor in children, and its prognosis is often poor. Anoikis is a unique mode of cell death.However, the effects of Anoikis in OS remain unexplored. METHOD: Differential analysis of Anoikis-related genes was performed based on the metastatic and non-metastatic groups. Then LASSO logistic regression and SVM-RFE algorithms were applied to screen out the characteristic genes. Later, Univariate and multivariate Cox regression was conducted to identify prognostic genes and further develop the Anoikis-based risk score. In addition, correlation analysis was performed to analyze the relationship between tumor microenvironment, drug sensitivity, and prognostic models. RESULTS: We established novel Anoikis-related subgroups and developed a prognostic model based on three Anoikis-related genes (MAPK1, MYC, and EDIL3). The survival and ROC analysis results showed that the prognostic model was reliable. Besides, the results of single-cell sequencing analysis suggested that the three prognostic genes were closely related to immune cell infiltration. Subsequently, aberrant expression of two prognostic genes was identified in osteosarcoma cells. Nilotinib can promote the apoptosis of osteosarcoma cells and down-regulate the expression of MAPK1. CONCLUSIONS: We developed a novel Anoikis-related risk score model, which can assist clinicians in evaluating the prognosis of osteosarcoma patients in clinical practice. Analysis of the tumor immune microenvironment and chemotherapeutic drug sensitivity can provide necessary insights into subsequent mechanisms. MAPK1 may be a valuable therapeutic target for neoadjuvant chemotherapy in osteosarcoma.

Machine learning survival prediction using tumor lipid metabolism genes for osteosarcoma.

Osteosarcoma is a primary malignant tumor that commonly affects children and adolescents, with a poor prognosis. The existence of tumor heterogeneity leads to different molecular subtypes and survival outcomes. Recently, lipid metabolism has been identified as a critical characteristic of cancer. Therefore, our study aims to identify osteosarcoma's lipid metabolism molecular subtype and develop a signature for survival outcome prediction. Four multicenter cohorts-TARGET-OS, GSE21257, GSE39058, and GSE16091-were amalgamated into a unified Meta-Cohort. Through consensus clustering, novel molecular subtypes within Meta-Cohort patients were delineated. Subsequent feature selection processes, encompassing analyses of differentially expressed genes between subtypes, univariate Cox analysis, and StepAIC, were employed to pinpoint biomarkers related to lipid metabolism in TARGET-OS. We selected the most effective algorithm for constructing a Lipid Metabolism-Related Signature (LMRS) by utilizing four machine-learning algorithms reconfigured into ten unique combinations. This selection was based on achieving the highest concordance index (C-index) in the test cohort of GSE21257, GSE39058, and GSE16091. We identified two distinct lipid metabolism molecular subtypes in osteosarcoma patients, C1 and C2, with significantly different survival rates. C1 is characterized by increased cholesterol, fatty acid synthesis, and ketone metabolism. In contrast, C2 focuses on steroid hormone biosynthesis, arachidonic acid, and glycerolipid and linoleic acid metabolism. Feature selection in the TARGET-OS identified 12 lipid metabolism genes, leading to a model predicting osteosarcoma patient survival. The LMRS, based on the 12 identified genes, consistently accurately predicted prognosis across TARGET-OS, testing cohorts, and Meta-Cohort. Incorporating 12 published signatures, LMRS showed robust and significantly superior predictive capability. Our results offer a promising tool to enhance the clinical management of osteosarcoma, potentially leading to improved clinical outcomes.

Use of a haired angularis oris axial pattern flap in a dog to correct a large oronasal fistula secondary to resection of a hard palate multilobular osteochondrosarcoma.

A 6-year-old neutered male mixed-breed dog underwent curative-intent surgical resection of a hard palatal multilobular osteochondrosarcoma and closure of the defect using bilateral buccal mucosal flaps. However, failure of the flaps resulted in a massive hard palatal defect that was subsequently repaired using a haired skin angularis oris axial pattern flap. This report describes the clinical outcome using this surgical approach and novel complications encountered. Key clinical message: The haired skin angularis oris axial pattern flap appears to be a suitable and robust option for reconstruction of large palatal defects.

Utilisation d'un lambeau cutanée poilus avec rotation axiale au niveau de l'artère angularis oris chez un chien pour corriger une fistule oronasale volumineuse secondaire à la résection d'un ostéochondrosarcome multilobulaire du palais dur. Un chien croisé mâle castré de 6 ans a subi une résection chirurgicale à visée curative d'un ostéochondrosarcome multilobulaire du palais dur et une fermeture de l'anomalie par des lambeaux de la muqueuse buccale. Cependant, la défaillance des lambeaux a entraîné un défaut important du palais dur qui a ensuite été réparé à l'aide d'un lambeau de peau avec poils avec rotation axiale au niveau de l'artère angularis oris. Ce rapport décrit les résultats cliniques de cette approche chirurgicale et les nouvelles complications rencontrées.Message clinique clé :L'utilisation d'un lambeau de peau avec poils avec rotation axiale au niveau de l'artère angularis oris semble être une option appropriée et robuste pour la reconstruction des défauts importants du palais.(Traduit par Dr Serge Messier).

Predictors of pulmonary metastases on chest computed tomography in children and adolescents with osteosarcoma-tips for qualifying patients for thoracotomy.

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumour in children and adolescents. Lungs are the most frequent and often the only site of metastatic disease. The presence of pulmonary metastases is a significant unfavourable prognostic factor. Thoracotomy is strongly recommended in these patients, while computed tomography (CT) remains the gold imaging standard. The purpose of our study was to create tools for the CT-based qualification for thoracotomy in osteosarcoma patients in order to reduce the rate of useless thoracotomies. METHODS: Sixty-four osteosarcoma paediatric patients suspected of lung metastases on CT and their first-time thoracotomies (n = 100) were included in this retrospective analysis. All CT scans were analysed using a compartmental evaluation method based on the number and size of nodules. Calcification and location of lung lesions were also analysed. Inter-observer reliability between two experienced radiologists was assessed. The CT findings were then correlated with the histopathological results of thoracotomies. Various multivariate predictive models (logistic regression, classification tree and random forest) were built and predictors of lung metastases were identified. RESULTS: All applied models proved that calcified nodules on the preoperative CT scan best predict the presence of pulmonary metastases. The rating of the operated lung on the preoperative CT scan, dependent on the number and size of nodules, and the total number of nodules on this scan were also found to be important predictors. All three models achieved a relatively high sensitivity (72-92%), positive predictive value (81-90%) and accuracy (74-79%). The positive predictive value of each model was higher than of the qualification for thoracotomy performed at the time of treatment. Inter-observer reliability was at least substantial for qualitative variables and excellent for quantitative variables. CONCLUSIONS: The multivariate models built and tested in our study may be useful in the qualification of osteosarcoma patients for metastasectomy through thoracotomy and may contribute to reducing the rate of unnecessary invasive procedures in the future.

High expression of DEC2 distinguishes chondroblastic osteosarcoma and promotes tumour growth by activating the VEGFC/VEGFR2 signalling pathway.

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and young adults. Account for 80% of all OS cases, conventional OS are characterized by the presence of osteoblastic, chondroblastic and fibroblastic cell types. Despite this heterogeneity, therapeutic treatment and prognosis of OS are essentially the same for all OS subtypes. Here, we report that DEC2, a transcriptional repressor, is expressed at higher levels in chondroblastic OS compared with osteoblastic OS. This difference suggests that DEC2 is disproportionately involved in the progression of chondroblastic OS, and thus, DEC2 may represent a possible molecular target for treating this type of OS. In the human chondroblastic-like OS cell line MNNG/HOS, we found that overexpression of DEC2 affects the proliferation of the cells by activating the VEGFC/VEGFR2 signalling pathway. Enhanced expression of DEC2 increased VEGFR2 expression, as well as increased the phosphorylation levels at sites Y951 and Y1175 of VEGFR2. On the one hand, activation of VEGFR2Y1175 enhanced cell proliferation through VEGFR2Y1175-PLCγ1-PKC-SPHK-MEK-ERK signalling. On the other hand, activation of VEGFR2Y951 decreased mitochondria-dependent apoptosis rate through VEGFR2Y951-VARP-PI3K-AKT signalling. Activation of these two signalling pathways resulted in enhanced progression of chondroblastic OS. In conclusion, DEC2 plays a pivotal role in cell proliferation and apoptosis-resistance in chondroblastic OS via the VEGFC/VEGFR2 signalling pathway. These findings lay the groundwork for developing focused treatments that target specific types of OS.

Identification of hub genes related to metastasis and prognosis of osteosarcoma and establishment of a prognostic model with bioinformatic methods.

Osteosarcoma (OS) is the most common primary malignant bone tumor occurring in children and adolescents. Improvements in our understanding of the OS pathogenesis and metastatic mechanism on the molecular level might lead to notable advances in the treatment and prognosis of OS. Biomarkers related to OS metastasis and prognosis were analyzed and identified, and a prognostic model was established through the integration of bioinformatics tools and datasets in multiple databases. 2 OS datasets were downloaded from the Gene Expression Omnibus database for data consolidation, standardization, batch effect correction, and identification of differentially expressed genes (DEGs); following that, gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the DEGs; the STRING database was subsequently used for protein-protein interaction (PPI) network construction and identification of hub genes; hub gene expression was validated, and survival analysis was conducted through the employment of the TARGET database; finally, a prognostic model was established and evaluated subsequent to the screening of survival-related genes. A total of 701 DEGs were identified; by gene ontology and KEGG pathway enrichment analyses, the overlapping DEGs were enriched for 249 biological process terms, 13 cellular component terms, 35 molecular function terms, and 4 KEGG pathways; 13 hub genes were selected from the PPI network; 6 survival-related genes were identified by the survival analysis; the prognostic model suggested that 4 genes were strongly associated with the prognosis of OS. DEGs related to OS metastasis and survival were identified through bioinformatics analysis, and hub genes were further selected to establish an ideal prognostic model for OS patients. On this basis, 4 protective genes including TPM1, TPM2, TPM3, and TPM4 were yielded by the prognostic model.

Neoadjuvant chemotherapy-induced hemoglobin decline as a prognostic factor in osteosarcoma around the knee joint: a single-center retrospective analysis of 242 patients.

PURPOSE: Anemia is relatively common in cancer patients, and is associated with poor survival in patients with various malignancies. However, how anemia would affect prognosis and response to neoadjuvant chemotherapy (NAC) in osteosarcoma (OS) is still without substantial evidence. METHODS: We retrospectively analysed 242 patients with stage II OS around the knee joint in our institute. Changed hemoglobin (Hb) levels (before and after NAC) were recorded to assess the prognostic value in DFS (disease-free survival) and tumor response to NAC. Univariate and multivariate analyses were conducted to identify prognostic factors related with outcome in OS patients. RESULTS: The mean Hb level significantly decreased after NAC (134.5 ± 15.3 g/L vs. 117.4 ± 16.3 g/L). The percentage of mild (21%), moderate (4.2%) and severe (0%) anemia patients markedly increased after NAC: 41%, 24% and 4.1% respectively. There was higher percentage of ≥ 5% Hb decline in patients with tumor necrosis rate < 90% (141 out of 161), compared with those with tumor necrosis rate ≥ 90% (59 out of 81). Further univariate and survival analysis demonstrated that Hb decline had a significant role in prediction survival in OS patients. Patients with ≥ 5% Hb decline after NAC had an inferior DFS compared with those with < 5% Hb decline. CONCLUSION: In osteosarcoma, patients with greater Hb decrease during neoadjuvant treatment were shown to have worse DFS and a poorer response to NAC than those without. Attempts to correct anemia and their effects on outcomes for osteosarcoma patients should be explored in future studies.

Osteosarcoma After Total Knee Arthroplasty: A Case Report.

CASE: A 79-year-old woman presented with a periprosthetic fracture 8 years after a total knee arthroplasty (TKA). Radiographs demonstrated tibial implant loosening with severe osteolysis. A high-grade osteosarcoma around the prosthesis was diagnosed, and a supracondylar femoral amputation was performed. After 2 years, no complications have occurred. CONCLUSIONS: A malignant tumor around a TKA is extremely rare. Surgeons should remain vigilant with patients who present with rapidly progressive or very aggressive implant loosening with osteolysis. Owing to its complexity and potentially devastating prognosis, treatment should be guided by a specialist multidisciplinary team. Complex limb salvage procedures or amputation is usually required.

Noscapine modulates hypoxia-induced angiogenesis and hemodynamics: Insights from a zebrafish model investigation.

We investigated the angiogenesis-modulating ability of noscapine in vitro using osteosarcoma cell line (MG-63) and in vivo using a zebrafish model. MTT assay and the scratch wound healing assay were performed on the osteosarcoma cell line (MG-63) to analyze the cytotoxic effect and antimigrative ability of noscapine, respectively. We also observed the antiangiogenic ability of noscapine on zebrafish embryos by analyzing the blood vessels namely the dorsal aorta, and intersegmental vessels development at 24, 48, and 72 h postfertilization. Real-time polymerase chain reaction was used to analyze the hypoxia signaling molecules' gene expression in MG-63 cells and zebrafish embryos. The findings from the scratch wound healing demonstrated that noscapine stopped MG-63 cancer cells from migrating under both hypoxia and normoxia. Blood vessel development and the heart rate in zebrafish embryos were significantly reduced by noscapine under both hypoxia and normoxia which showed the hemodynamics impact of noscapine. Noscapine also downregulated the cobalt chloride (CoCl2) induced hypoxic signaling molecules' gene expression in MG-63 cells and zebrafish embryos. Therefore, noscapine may prevent MG-63 cancer cells from proliferating and migrating, as well as decrease the formation of new vessels and the production of growth factors linked to angiogenesis in vivo under both normoxic and hypoxic conditions.

The role of programmed cell death in osteosarcoma: From pathogenesis to therapy.

Osteosarcoma (OS) is a prevalent bone solid malignancy that primarily affects adolescents, particularly boys aged 14-19. This aggressive form of cancer often leads to deadly lung cancer due to its high migration ability. Experimental evidence suggests that programmed cell death (PCD) plays a crucial role in the development of osteosarcoma. Various forms of PCD, including apoptosis, ferroptosis, autophagy, necroptosis, and pyroptosis, contribute significantly to the progression of osteosarcoma. Additionally, different signaling pathways such as STAT3/c-Myc signal pathway, JNK signl pathway, PI3k/AKT/mTOR signal pathway, WNT/ß-catenin signal pathway, and RhoA signal pathway can influence the development of osteosarcoma by regulating PCD in osteosarcoma cell. Therefore, targeting PCD and the associated signaling pathways could offer a promising therapeutic approach for treating osteosarcoma.

A Systemic Review of Primary Malignant Long Bone Tumors in Children and Adolescents.

PURPOSE OF THE STUDY: Managing bone tumours is complex, relying on limited evidence, expert opinions, and retrospective reviews. Multidisciplinary approaches and early diagnosis are crucial for better outcomes, especially in young patients with growing skeletons. The aim of this systemic review and meta-analysis is to give a comprehensive review of common malignant tumors affecting long bones in children and adolescents. MATERIAL AND METHODS: A PubMed/Medline search for "primary malignant long bone tumours in children" initially retrieved 1120 papers, which were subsequently narrowed down to 110 articles based on inclusion and exclusion criteria. These articles were reviewed, focusing on clinical presentation, diagnostic workup, treatment options, surgical planning, and variations in presentation, including rare tumours. The two most commonly reported tumours were osteosarcoma and Ewing sarcoma, leading to the division of studies into five groups. The inclusion criteria encompassed malignancies in patients aged 2-25 years, work-up, imaging, surgical treatment, rare tumour case reports, and surgical management principles, resulting in a heterogeneous group of articles. To enhance categorisation, it was clarified that studies with 10 or more cases were considered retrospective reviews. RESULTS: Reviewing of results thus demonstrate that the two likely tumours in children under consideration were osteosarcoma and Ewing sarcoma. Their presentation findings and clinical features were discussed in detail in the review. It is worth noting here that in case of differential diagnosis this should be the first on the list. DISCUSSION AND CONCLUSIONS: Although focus of literature is more on the two most common tumours. However, rare tumours should be considered as they can mimic these common tumors. KEY WORDS: primary, malignant, bone tumors, children, adolescent.

Transformation of IDH-wildtype glioblastoma to gliosarcoma with features of osteosarcoma.

Gliosarcoma (GS) is a rare variant of IDH-wildtype glioblastoma. It is classified as grade 4 in the latest WHO CNS classification of both glial and mesenchymal components. Gliosarcoma may arise de novo or secondary from glioblastoma. It occurs in up to 2% of patients diagnosed with glioblastoma. We present a case report of a 51-year-old patient who was initially diagnosed with glioblastoma multiforme, which transformed into secondary gliosarcoma with an osteosarcoma component 16 months after the initial diagnosis. We believe that increasing reporting of secondary gliosarcoma (sGS) will be helpful in understanding, diagnosing and providing more effective treatment for this cancer.

Exosome-transmitted circular RNA circ-LMO7 facilitates the progression of osteosarcoma by regulating miR-21-5p/ARHGAP24 axis.

The potential function and mechanism of circRNAs in regulating malignant performances of Osteosarcoma (OS) cells have not been well investigated. The expression level of CircLMO7, miR-21-5p and ARHGAP24 were detected by RT-qPCR. The relationship between miR-21-5p and circ-LMO7, as well as between miR-21-5p and ARHGAP24, was predicted and examined through bioinformatics analysis and luciferase reporter gene experiments. Moreover, OS cell growth, invasion, migration, and apoptosis were detected using the cell counting kit-8 (CCK-8), transwell and flow cytometry assays, respectively. ARHGAP24 protein level was measured using western blotting. In present study, we choose to investigate the role and mechanism of circ-LOM7 on OS cell proliferation, migration and invasion. circ-LOM7 was found to be down-regulated in OS tissues and cell lines. Enforced expression of circ-LOM7 suppressed the growth, invasion, and migration of OS cells. In contrast, decreasing circ-LMO7 expression had opposite effects. Furthermore, miR-21-5p was predicted to be sponged by circ-LMO7, and had an opposite role of circ-LMO7 in OS. Moreover, ARHGAP24 served as miR-21-5p's downstream target. Mechanistically, circ-LMO7 was packed in exosomes and acted as a cancer-suppresser on OS by sponging miR-21-5p and upregulating the expression of ARHGAP24. The exosomal circ-LMO7 expression was significantly decreased in OS cell exosomes, and co-culture experiments showed that exosomal circ-LMO7 suppressed the proliferation ability of OS cells. Circ-LMO7 exerts as a tumor suppressor in OS, and the circ-LMO7/miR-21-5P/ARHGAP24 axis is involved in OS progression.

Cell Cycle Checkpoints p16 and p21-Strong Predictors of Clinicopathologic Outcomes in High-Grade Osteosarcoma.

PURPOSE: In this study, we used a series of immunohistochemical measurements of 2 cell cycle regulators, p16 and p21, to evaluate their prognostic value, separately and in combination, for the disease outcomes. METHOD: A total of 101 patients with high-grade osteosarcoma were included in this study. Clinicopathologic data were collected, and immunohistochemistry for p16 and p21 was performed and interpreted by 3 independent pathologists. Statistical analysis was performed to assess the strength of each of these markers relative to disease outcome. RESULTS: Our results indicate that more than 90% expression (high) of p16 by immunohistochemistry on the initial biopsy has a strong predictive value for good histologic response to chemotherapy. The patients are also more likely to survive the past 5 years and less likely to develop metastasis than patients with less than 90% p16 (low) expression. The results for p21, on the other hand, show a unique pattern of relationship to the clinicopathologic outcomes of the disease. Patients with less than 1% (low) or more than 50% (high) expression of p21 by immunohistochemistry show a higher chance of metastasis, poor necrotic response to chemotherapy, and an overall decreased survival rate when compared with p21 expression between 1% and 50% (moderate). Our results also showed that the expression of p16 and combined p16 and p21 demonstrates a stronger predictive relationship to 5-year survival than tumor histologic necrosis and p21 alone. DISCUSSION: The results of this study, once proven to be reproducible by a larger number of patients, will be valuable in the initial assessment and risk stratification of the patients for treatment and possibly the clinical trials.

Methods to Enable Spatial Transcriptomics of Bone Tissues.

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with normal development and disease pathology. Spatial transcriptomics is a powerful method that enables the analysis of the whole transcriptome within tissue samples, thus providing information about the cellular gene expression and the histological context in which the cells reside. While this method has been extensively utilized for many soft tissues, its application for the analyses of hard tissues such as bone has been challenging. The major challenge resides in the inability to preserve good quality RNA and tissue morphology while processing the hard tissue samples for sectioning. Therefore, a method is described here to process freshly obtained bone tissue samples to effectively generate spatial transcriptomics data. The method allows for the decalcification of the samples, granting successful tissue sections with preserved morphological details while avoiding RNA degradation. In addition, detailed guidelines are provided for samples that were previously paraffin-embedded, without demineralization, such as samples collected from tissue banks. Using these guidelines, high-quality spatial transcriptomics data generated from tissue bank samples of primary tumor and lung metastasis of bone osteosarcoma are shown.

Inflammasome-related gene signatures as prognostic biomarkers in osteosarcoma.

Osteosarcoma, the primary bone cancer in adolescents and young adults, is notorious for its aggressive growth and metastatic potential. Our study delved into the prognostic impact of inflammasome-related gene signatures in osteosarcoma patients, employing comprehensive genetic profiling to uncover signatures linked with patient outcomes. We identified three patient subgroups through consensus clustering, with one showing worse survival rates correlated with high FGFR3 and RARB expressions. Immune profiling revealed significant immune cell infiltration differences among these subgroups, affecting survival. Utilising advanced machine learning, including StepCox and gradient boosting machine algorithms, we developed a prognostic model with a notable c-index of 0.706, highlighting CD36 and MYD88 as key genes. Higher inflammasome risk scores from our model were associated with poorer survival, corroborated across datasets. In vitro experiments validated CD36 and MYD88's roles in promoting osteosarcoma cell proliferation, invasion and migration, emphasising their therapeutic potential. This research offers new insights into inflammasomes' role in osteosarcoma, introducing novel biomarkers for risk assessment and potential therapeutic targets. Our findings suggest a pathway towards personalised treatment strategies, potentially improving patient outcomes in osteosarcoma.

Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Single-cell and spatial transcriptomics reveal metastasis mechanism and microenvironment remodeling of lymph node in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor and is highly prone to metastasis. OS can metastasize to the lymph node (LN) through the lymphatics, and the metastasis of tumor cells reestablishes the immune landscape of the LN, which is conducive to the growth of tumor cells. However, the mechanism of LN metastasis of osteosarcoma and remodeling of the metastatic lymph node (MLN) microenvironment is not clear. METHODS: Single-cell RNA sequencing of 18 samples from paracancerous, primary tumor, and lymph nodes was performed. Then, new signaling axes closely related to metastasis were identified using bioinformatics, in vitro experiments, and immunohistochemistry. The mechanism of remodeling of the LN microenvironment in tumor cells was investigated by integrating single-cell and spatial transcriptomics. RESULTS: From 18 single-cell sequencing samples, we obtained 117,964 cells. The pseudotime analysis revealed that osteoblast(OB) cells may follow a differentiation path from paracancerous tissue (PC) → primary tumor (PT) → MLN or from PC → PT, during the process of LN metastasis. Next, in combination of bioinformatics, in vitro and in vivo experiments, and immunohistochemistry, we determined that ETS2/IBSP, a new signal axis, might promote LN metastasis. Finally, single-cell and spatial dissection uncovered that OS cells could reshape the microenvironment of LN by interacting with various cell components, such as myeloid, cancer-associated fibroblasts (CAFs), and NK/T cells. CONCLUSIONS: Collectively, our research revealed a new molecular mechanism of LN metastasis and clarified how OS cells influenced the LN microenvironment, which might provide new insight for blocking LN metastasis.

Investigating the molecular mechanism of Mori Cortex against osteosarcoma by bioinformatics analysis and in vitro experimental.

OBJECTIVE: To explore the therapeutic mechanism of Mori Cortex against osteosarcoma (OS), we conducted bioinformatics prediction followed by in vitro experimental validation. METHODS: Gene expression data from normal and OS tissues were obtained from the GEO database and underwent differential analysis. Active Mori Cortex components and target genes were extracted from the Traditional Chinese Medicine System Pharmacology database. By intersecting these targets with differentially expressed genes in OS, we identified potential drug action targets. Using the STRING database, a protein-protein interaction network was constructed. Subsequent analyses of these intersected genes, including Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment, were performed using R software to elucidate biological processes, molecular functions, and cellular components, resulting in the simulation of signaling pathways. Molecular docking assessed the binding capacity of small molecules to signaling pathway targets. In vitro validations were conducted on U-2 OS cells. The CCK8 assay was used to determine drug-induced cytotoxicity in OS cells, and Western Blotting was employed to validate the expression of AKT, extracellular signal-regulated kinases (ERK), Survivin, and Cyclin D1 proteins. RESULTS: Through differential gene expression analysis between normal and OS tissues, we identified 12,364 differentially expressed genes. From the TCSMP database, 39 active components and 185 therapeutic targets related to OS were derived. The protein-protein interaction network indicated that AKT1, IL-6, JUN, VEGFA, and CASP3 might be central targets of Mori Cortex for OS. Molecular docking revealed that the active compound Morusin in Mori Cortex exhibits strong binding affinity to AKT and ERK. The CCK8 assay showed that Morusin significantly inhibits the viability of U-2 OS cells. Western Blot demonstrated a reduction in the p-AKT/AKT ratio, the p-ERK/ERK ratio, Survivin, and Cyclin D1. CONCLUSION: Mori Cortex may exert its therapeutic effects on OS through multiple cellular signaling pathways. Morusin, the active component of Mori Cortex, can inhibit cell cycle regulation and promote cell death in OS cells by targeting AKT/ERK pathway.