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.

High expression of SRSF1 facilitates osteosarcoma progression and unveils its potential mechanisms.

BACKGROUND: SRSF1, a member of Serine/Arginine-Rich Splicing Factors (SRSFs), has been observed to significantly influence cancer progression. However, the precise role of SRSF1 in osteosarcoma (OS) remains unclear. This study aims to investigate the functions of SRSF1 and its underlying mechanism in OS. METHODS: SRSF1 expression level in OS was evaluated on the TCGA dataset, TAGET-OS database. qRT-PCR and Western blotting were employed to assess SRSF1 expression in human OS cell lines as well as the interfered ectopic expression states. The effect of SRSF1 on cell migration, invasion, proliferation, and apoptosis of OS cells were measured by transwell assay and flow cytometry. RNA sequence and bioinformatic analyses were conducted to elucidate the targeted genes, relevant biological pathways, and alternative splicing (AS) events regulated by SRSF1. RESULTS: SRSF1 expression was consistently upregulated in both OS samples and OS cell lines. Diminishing SRSF1 resulted in reduced proliferation, migration, and invasion and increased apoptosis in OS cells while overexpressing SRSF1 led to enhanced growth, migration, invasion, and decreased apoptosis. Mechanistically, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) revealed that the biological functions of SRSF1 were closely associated with the dysregulation of the protein targeting processes, location of the cytosolic ribosome, extracellular matrix (ECM), and proteinaceous extracellular matrix, along with the PI3K-AKT pathway, Wnt pathway, and HIPPO pathway. Transcriptome analysis identified AS events modulated by SRSF1, especially (Skipped Exon) SE events and (Mutually exclusive Exons) MXE events, revealing potential roles of targeted molecules in mRNA surveillance, RNA degradation, and RNA transport during OS development. qRT-PCR confirmed that SRSF1 knockdown resulted in the occurrence of alternative splicing of SRRM2, DMKN, and SCAT1 in OS. CONCLUSIONS: Our results highlight the oncogenic role of high SRSF1 expression in promoting OS progression, and further explore the potential mechanisms of action. The significant involvement of SRSF1 in OS development suggests its potential utility as a therapeutic target in OS.

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.

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.

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

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

Deprivation of methionine inhibits osteosarcoma growth and metastasis via C1orf112-mediated regulation of mitochondrial functions.

Osteosarcoma is a malignant bone tumor that primarily inflicts the youth. It often metastasizes to the lungs after chemotherapy failure, which eventually shortens patients' lives. Thus, there is a dire clinical need to develop a novel therapy to tackle osteosarcoma metastasis. Methionine dependence is a special metabolic characteristic of most malignant tumor cells that may offer a target pathway for such therapy. Herein, we demonstrated that methionine deficiency restricted the growth and metastasis of cultured human osteosarcoma cells. A genetically engineered Salmonella, SGN1, capable of overexpressing an L-methioninase and hydrolyzing methionine led to significant reduction of methionine and S-adenosyl-methionine (SAM) specifically in tumor tissues, drastically restricted the growth and metastasis in subcutaneous xenograft, orthotopic, and tail vein-injected metastatic models, and prolonged the survival of the model animals. SGN1 also sharply suppressed the growth of patient-derived organoid and xenograft. Methionine restriction in the osteosarcoma cells initiated severe mitochondrial dysfunction, as evident in the dysregulated gene expression of respiratory chains, increased mitochondrial ROS generation, reduced ATP production, decreased basal and maximum respiration, and damaged mitochondrial membrane potential. Transcriptomic and molecular analysis revealed the reduction of C1orf112 expression as a primary mechanism underlies methionine deprivation-initiated suppression on the growth and metastasis as well as mitochondrial functions. Collectively, our findings unraveled a molecular linkage between methionine restriction, mitochondrial function, and osteosarcoma growth and metastasis. A pharmacological agent, such as SGN1, that can achieve tumor specific deprivation of methionine may represent a promising modality against the metastasis of osteosarcoma and potentially other types of sarcomas as well.

Slipped capital femoral epiphysis after tumor prosthesis implantation in a patient receiving chemotherapy.

While the usual etiology of slipped capital femoral epiphysis (SCFE) is idiopathic, there are many other factors that increase the predisposition to slippage. Chemotherapy can be one of them. In this article, we report a rare case of acute SCFE after tumor prosthesis implantation in a patient who received chemotherapy. A 10-year-old girl with osteosarcoma of the right distal femur underwent (neo-) adjuvant chemotherapy, wide tumor resection, and reconstruction using a growing tumor prosthesis and a short non-cemented femoral stem. Half a year after implantation, she developed aseptic loosening. Revision surgery was performed using a hydroxyapatite (HA)-coated cementless femoral stem. Postoperative plain radiographs revealed SCFE that was treated by closed reduction and screw fixation. The patient recovered without complications, and unaffected hip showed no radiographic signs of slippage on follow-up. The forces of implanting a tumor prosthesis, particularly with a non-cemented stem, can increase the risk of an acute SCFE. The controversy over prophylactic pinning of the uninvolved hip in chemotherapy-associated SCFE is unresolved. Pinning can be considered only in the presence of abnormal prodromal radiological findings.

[Osteosarcoma Secondary to Polyostotoic Fibrous Dysplasia of the Ribs].

Sarcomatous transformation of fibrous dysplasia is extremely rare. We present the case of a 54-yearold man with multiple rib masses, multiple enlarged lymph nodes throughout the body, and multiple osteolytic lesions on computed tomography( CT). A positron emission tomography( PET) scan showed abnormal enhancement in each. A needle biopsy of the right supraclavicular fossa lymph node revealed sarcoidosis. Considering the possibility of malignancy associated with sarcoidosis, a rib tumor resection and mediastinal lymph node biopsy were performed to confirm the diagnosis of the rib lesion. The pathology results showed that the rib mass was a low-grade central osteosarcoma and the mediastinal lymph node was sarcoidosis. The distribution of the lesions was consistent with osteosarcoma secondary to multiple fibrous bone dysplasia. As the osteosarcoma was low grade, the patient was followed up. Three years after surgery, there was no increase in residual disease.

A Successful Clinical Outcome 29 Months Following Improper Placement of the Compress Implant: A Case Report.

CASE: A 24-year-old woman presented with dedifferentiated parosteal osteosarcoma of the proximal femur and was treated with limb salvage surgery using the Compress implant. It was implanted with a technical error, was not revised, and has demonstrated no negative outcomes 29 months postoperatively. CONCLUSION: An instance of incorrect pin placement during the implantation of a Zimmer Compress implant is presented with good survivorship. This report acts as information for other surgeons who might inadvertently obtain unicortical fixation. In this patient, revision surgery was avoided and an excellent, short-term outcome was achieved while avoiding the potentially devastating complications associated with revision implantation.

Analysis of the effects of M2 macrophage-derived PDE4C on the prognosis, metastasis and immunotherapy benefit of osteosarcoma.

Tumour-associated macrophages (TAMs), encompassing M1 and M2 subtypes, exert significant effects on osteosarcoma (OS) progression and immunosuppression. However, the impacts of TAM-derived biomarkers on the progression of OS remains limited. The GSE162454 profile was subjected to single-cell RNA (scRNA) sequencing analysis to identify crucial mediators between TAMs and OS cells. The clinical features, effects and mechanisms of these mediators on OS cells and tumour microenvironment were evaluated via biological function experiments and molecular biology experiments. Phosphodiesterase 4C (PDE4C) was identified as a pivotal mediator in the communication between M2 macrophages and OS cells. Elevated levels of PDE4C were detected in OS tissues, concomitant with M2 macrophage level, unfavourable prognosis and metastasis. The expression of PDE4C was observed to increase during the conversion process of THP-1 cells to M2 macrophages, which transferred the PDE4C mRNA to OS cells through exosome approach. PDE4C increased OS cell proliferation and mobility via upregulating the expression of collagens. Furthermore, a positive correlation was observed between elevated levels of PDE4C and increased TIDE score, decreased response rate following immune checkpoint therapy, reduced TMB and diminished PDL1 expression. Collectively, PDE4C derived from M2 macrophages has the potential to enhance the proliferation and mobility of OS cells by augmenting collagen expression. PDE4C may serve as a valuable biomarker for prognosticating patient outcomes and response rates following immunotherapy.

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.

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.

Biomimetic bone-periosteum scaffold for spatiotemporal regulated innervated bone regeneration and therapy of osteosarcoma.

The complexity of repairing large segment defects and eradicating residual tumor cell puts the osteosarcoma clinical management challenging. Current biomaterial design often overlooks the crucial role of precisely regulating innervation in bone regeneration. Here, we develop a Germanium Selenium (GeSe) co-doped polylactic acid (PLA) nanofiber membrane-coated tricalcium phosphate bioceramic scaffold (TCP-PLA/GeSe) that mimics the bone-periosteum structure. This biomimetic scaffold offers a dual functionality, combining piezoelectric and photothermal conversion capabilities while remaining biodegradable. When subjected to ultrasound irradiation, the US-electric stimulation of TCP-PLA/GeSe enables spatiotemporal control of neurogenic differentiation. This feature supports early innervation during bone formation, promoting early neurogenic differentiation of Schwann cells (SCs) by increasing intracellular Ca2+ and subsequently activating the PI3K-Akt and Ras signaling pathways. The biomimetic scaffold also demonstrates exceptional osteogenic differentiation potential under ultrasound irradiation. In rabbit model of large segment bone defects, the TCP-PLA/GeSe demonstrates promoted osteogenesis and nerve fibre ingrowth. The combined attributes of high photothermal conversion capacity and the sustained release of anti-tumor selenium from the TCP-PLA/GeSe enable the synergistic eradication of osteosarcoma both in vitro and in vivo. This strategy provides new insights on designing advanced biomaterials of repairing large segment bone defect and osteosarcoma.

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.

An ALP-responsive, anionic iridium complex for specific recognition of osteosarcoma cells.

Poor cellular permeability greatly hampers the utilization of anionic Ir(III) complexes, though efficiently emissive and remarkably stable, in cell-based diagnosis. To overcome this barrier, we present the development of an alkaline phosphatase (ALP)-responsive, anionic, and aggregation-induced emission (AIE)-active Ir(III) complex (Ir1) for specific recognition of osteosarcoma cells. Containing phosphate moieties, Ir1 exhibits a net -1 charge, enabling charge repulsion from the cell membrane and resulting in low cellular uptake and good biocompatibility in normal osteoblast cells. Upon ALP-mediated hydrolysis of phosphate groups, the resulting dephosphorylated product, Ir2, demonstrates a positive charge and increased lipophilicity, promoting cellular uptake and activating its AIE properties for specific recognition of osteosarcoma cells that express elevated levels of ALP. This study elucidates the role of ALP as an ideal trigger for enhancing the cellular permeability of phosphate ester-containing Ir(III) complexes, thus expanding the potential of anionic Ir(III) complexes for biomedical applications.

Bioinformatics analysis and validation of mesenchymal stem cells related gene MT1G in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is a primary malignant bone tumor arising from mesenchymal cells. The standard clinical treatment for OS involves extensive tumor resection combined with neoadjuvant chemotherapy or radiotherapy. OS's invasiveness, lung metastasis, and drug resistance contribute to a low cure rate and poor prognosis with this treatment. Metallothionein 1G (MT1G), observed in various cancers, may serve as a potential therapeutic target for OS. METHODS: OS samples in GSE33382 and TARGET datasets were selected as the test cohorts. As the external validation cohort, 13 OS tissues and 13 adjacent cancerous tissues from The Second Affiliated Hospital of Nanchang University were collected. Patients with OS were divided into high and low MT1G mRNA-expression groups; differentially expressed genes (DEGs) were identified as MT1G-related genes. The biological function of MT1G was annotated using Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and gene set enrichment analysis (GSEA). Gene expression correlation analysis and competing endogenous RNA (ceRNA) regulatory network construction were used to determine potential biological regulatory relationships of DEGs. Survival analysis assessed the prognostic value of MT1G. RESULTS: MT1G expression increased in OS samples and presented higher in metastatic OS compared with non-metastatic OS. Functional analyses indicated that MT1G was mainly associated with spliceosome. A ceRNA network with DEGs was constructed. MT1G is an effective biomarker predicting survival and correlated with increased recurrence rates and poorer survival. CONCLUSIONS: This research identified MT1G as a potential biomarker for OS prognosis, highlighting its potential as a therapy target.

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.

WNT5B drives osteosarcoma stemness, chemoresistance and metastasis.

BACKGROUND: Treatment for osteosarcoma, a paediatric bone cancer with no therapeutic advances in over three decades, is limited by a lack of targeted therapies. Osteosarcoma frequently metastasises to the lungs, and only 20% of patients survive 5 years after the diagnosis of metastatic disease. We found that WNT5B is the most abundant WNT expressed in osteosarcoma tumours and its expression correlates with metastasis, histologic subtype and reduced survival. METHODS: Using tumor-spheroids to model cancer stem-like cells, we performed qPCR, immunoblotting, and immunofluorescence to monitor changes in gene and protein expression. Additionally, we measured sphere size, migration and forming efficiency to monitor phenotypic changes. Therefore, we characterised WNT5B's relevance to cancer stem-like cells, metastasis, and chemoresistance and evaluated its potential as a therapeutic target. RESULTS: In osteosarcoma cell lines and patient-derived spheres, WNT5B is enriched in stem cells and induces the expression of the stemness gene SOX2. WNT5B promotes sphere size, sphere-forming efficiency, and cell proliferation, migration, and chemoresistance to methotrexate (but not cisplatin or doxorubicin) in spheres formed from conventional cell lines and patient-derived xenografts. In vivo, WNT5B increased osteosarcoma lung and liver metastasis and inhibited the glycosaminoglycan hyaluronic acid via upregulation of hyaluronidase 1 (HYAL1), leading to changes in the tumour microenvironment. Further, we identified that WNT5B mRNA and protein correlate with the receptor ROR1 in primary tumours. Targeting WNT5B through inhibition of WNT/ROR1 signalling with an antibody to ROR1 reduced stemness properties, including chemoresistance, sphere size and SOX2 expression. CONCLUSIONS: Together, these data define WNT5B's role in driving osteosarcoma cancer stem cell expansion and methotrexate resistance and provide evidence that the WNT5B pathway is a promising candidate for treating osteosarcoma patients. KEY POINTS: WNT5B expression is high in osteosarcoma stem cells leading to increased stem cell proliferation and migration through SOX2. WNT5B expression in stem cells increases rates of osteosarcoma metastasis to the lungs and liver in vivo. The hyaluronic acid degradation enzyme HYAL1 is regulated by WNT5B in osteosarcoma contributing to metastasis. Inhibition of WNT5B with a ROR1 antibody decreases osteosarcoma stemness.

[Knockdown mitochondrial transcription factor A (TFAM) inhibits autophagy, proliferation, invasion and migration in human cervical cancer and osteosarcoma cells].

Objective To investigate the effects of mitochondrial transcription factor A (TFAM) on mitochondrial function, autophagy, proliferation, invasion, and migration in cervical cancer HeLa cells and osteosarcoma U2OS cells. Methods TFAM small-interfering RNA (si-TFAM) was transfected to HeLa and U2OS cells for downregulating TFAM expression. Mito-Tracker Red CMXRos staining combined with laser confocal microscopy was used to detect mitochondrial membrane potential (MMP). MitoSOXTM Red labeling was used to test mitochondrial reactive oxygen species (mtROS) levels. The expression of mitochondrial DNA (mtDNA) was detected by real-time quantitative PCR. Changes in the number of autophagosomes were detected by immunofluorescence cytochemistry. Western blot analysis was used to detect the expressions of TFAM, autophagy microtubule associated protein 1 light chain 3A/B (LC3A/B), autophagy associated protein 2A (ATG2A), ATG2B, ATG9A, zinc finger transcription factor Snail, matrix metalloproteinase 2 (MMP2) and MMP9. CCK-8 assay and plate clony formation assay were used to detect cell proliferation, while TranswellTM assay and scratch healing assay were used to detect changes in cell invasion and migration. Results The downregulation of TFAM expression resulted in a decrease in MMP and mtDNA copy number, but an increase in mtROS production. The protein content of LC3A/B decreased significantly compared to the control group and the number of autophagosomes in the cytoplasm decreased significantly. The expressions of ATG2B and ATG9A in the early stage of autophagy were significantly reduced. The expressions of Snail, MMP2 and MMP9 proteins in HeLa and U2OS cells were also decreased. The proliferation, invasion and migration ability of HeLa and U2OS cells were inhibited after being interfered with TFAM expression. Conclusion Downregulation of TFAM expression inhibits mitochondrial function, delays autophagy process and reduces the proliferation, invasion and migration ability of cervical cancer cells and osteosarcoma cells.