Nestin expression in osteosarcomas and derivation of nestin/CD133 positive osteosarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>Nestin was originally identified as a class VI intermediate filament protein that is expressed in stem cells and progenitor cells in the mammalian CNS during development. This protein is replaced in the adult organism by other intermediate filament proteins; however, nestin may be re-expressed under certain pathological conditions such as ischemia, inflammation, brain injury, and neoplastic transformation. Nestin has been detected in many kinds of tumors, especially in tumors derived from the CNS. Co-expression of nestin and the CD133 surface molecule is considered to be a marker for cancer stem cells in neurogenic tumors. Our work was aimed at a detailed study of nestin expression in osteosarcomas and osteosarcoma-derived cell lines.</p> <p>Methods</p> <p>Using immunodetection methods, we examined nestin in tumor tissue samples from 18 patients with osteosarcomas. We also successfully established permanent cell lines from the tumor tissue of 4 patients and immunodetection of nestin and CD133 was performed on these cell lines.</p> <p>Results</p> <p>Nestin-positive tumor cells were immunohistochemically detected in all of the examined osteosarcomas, but the proportion of these cells that were positively stained as well as the intensity of staining varied. Nestin-positive cells were rarely observed in 2 tumor samples, and the remaining 16 tumor samples showed various nestin expression patterns ranging from very sporadic occurrence to an overwhelming proportion of cells with strong positive staining. Three of the established osteosarcoma cell lines were demonstrated to be nestin-positive, and only one cell line showed no expression of nestin; this finding corresponds with the rare occurrence of nestin-positive cells in the respective tumor sample. Moreover, three of these osteosarcoma cell lines were undoubtedly proven to be Nes+/CD133+.</p> <p>Conclusion</p> <p>Our results represent the first evidence of nestin expression in osteosarcomas and suggest the possible occurrence of cells with a stem-like phenotype in these tumors.</p

Outcomes of Intercalary Endoprostheses as a Treatment for Metastases in the Femoral and Humeral Diaphysis

Background: The purpose of this study was to evaluate the implant survival, functional score and complications of intercalary endoprostheses implanted for metastatic involvement of the femoral and humeral diaphysis. Methods: The selected group covered patients with bone metastasis who were surgically treated with an intercalary endoprosthesis between 2012 and 2021. The functional outcome was evaluated with the Musculoskeletal Tumor Society (MSTS) scoring system, and complications were evaluated by using the failure classification for prosthetics designed by Henderson. Results: The mean follow-up was 29.8 months. In our group of 25 patients with 27 intercalary endoprostheses (18 femurs, 9 humeri), there were 7 implant-related complications (25.9%), which were more common on the humerus (4 cases, 44.4%) than on the femur (3 cases, 16.7%). Only type II failure—aseptic loosening (5 cases, 18.5%)—and type III failure—structural failure (2 cases, 7.4%)—occurred. There was a significantly higher risk of aseptic loosening of the endoprosthesis in the humerus compared with that in the femur (odds ratio 13.79, 95% confidence interval 1.22–151.05, p = 0.0297). The overall cumulative implant survival was 92% 1 year after surgery and 72% 5 years after surgery. The average MSTS score was 82%. The MSTS score was significantly lower (p = 0.008) in the humerus (75.9%) than in the femur (84.8%). Conclusions: The resection of bone metastases and replacement with intercalary endoprosthesis has excellent immediate functional results with an acceptable level of complications in prognostically favourable patients

Proximal Tibia Tumour Location and Curettage Are Major Risk Factors of Local Recurrence in Giant Cell Tumour of Bone

Giant cell tumour of bone (GCTB) is one of the most common local aggressive tumourous lesions with a wide variety of biological behaviour. However, there are no clear indicative criteria when choosing the type of procedure and the complication rates remain high, especially in terms of local recurrence. The purpose of the study was to (1) identify the main risk factors for local recurrence, (2) evaluate the recurrence-free survival in dependence on neoadjuvant denosumab use and the type of procedure, and (3) compare the functional outcomes after curettage and en bloc resection. The group included 102 patients with GCTB treated between 2006 and 2020. The mean age of patients was 34.4 years (15–79). The follow-up period was 8.32 years (2–16) on average. Local recurrence occurred in 14 patients (29.8%) who underwent curettage and in 5 patients (10.6%) after en bloc resection. Curettage was shown to be a factor in increasing recurrence rates (OR = 3.64 [95% CI: 1.19–11.15]; p = 0.023). Tibial location was an independent risk factor for local recurrence regardless of the type of surgery (OR = 3.22 [95% CI: 1.09–9.48]; p = 0.026). The recurrence-free survival rate of patients treated with resection and denosumab was higher compared to other treatments at five years postoperatively (p = 0.0307). Functional ability and pain as reported by patients at the latest follow-up were superior after curettage compared to resection for upper and lower extremity (mean difference: −4.00 [95% CI: –6.81 to −1.18]; p p < 0.001, respectively). Proximal tibia tumour location and curettage were shown to be major risk factors for local recurrence in GCTB regardless of neoadjuvant denosumab treatment. The recurrence-free survival rate of patients treated with resection and denosumab was higher compared to other treatments. The functional outcome of patients after curettage was better compared to en bloc resection

Improved osteosarcoma survival with addition of mifamurtide to conventional chemotherapy – Observational prospective single institution analysis

Purpose: Conventional osteosarcoma is an orphan disease. Current treatment approaches include combining a three drug chemotherapy schedule and surgery. The 3- and 5-year event-free survival (EFS) in localized disease is roughly 65 and 60%, respectively. The registration study of mifamurtide reported survival benefit, but some methodological controversies have been insufficient for FDA market authorization in contrast to EMA. Methods: prospective single centre survival analysis of a mifamurtide addition to conventional therapy in 23 patients over a 5.5 year enrolment period is reported and compared to a historical control of 26 patient with localized disease. Bias arising from observational methodology was addressed using Landmark analysis and time-dependent Cox models. Blood count dynamics were analysed during the treatment. Results: The adverse event profile was as expected with no dose limiting toxicities. There were no local relapses observed, one patient died in the first complete remission due to doxorubicin cardiotoxicity, one patient had pulmonary metastatic relapse. The observed 3- and 5-year EFS was 87.4% (CI 72.4–100%) and 87.4% (CI 72.4–100%), progression free survival (PFS) was 92.9% (CI 80.3–100%) and 92.9% (CI 80.3–100%), overall survival was 94.1% (CI 83.6–100) and 80.7% (CI 58.3–100), respectively. Comparison to the historical control showed statistically significant better PFS for mifamurtide patients (Landmark analysis; p = 0.044). Risk of progression was 5-times lower for the mifamurtide group (Cox model; HR 0.21, p = 0.136). Only subtle differences in lymphocyte counts were observed across treatment. Conclusion: the PFS benefit of mifamurtide is reported herein. The addition of mifamurtide could be considered as a best treatment option for localized osteosarcoma

Serial Xenotransplantation in NSG Mice Promotes a Hybrid Epithelial/Mesenchymal Gene Expression Signature and Stemness in Rhabdomyosarcoma Cells

Serial xenotransplantation of sorted cancer cells in immunodeficient mice remains the most complex test of cancer stem cell (CSC) phenotype. However, we have demonstrated in various sarcomas that putative CSC surface markers fail to identify CSCs, thereby impeding the isolation of CSCs for subsequent analyses. Here, we utilized serial xenotransplantation of unsorted rhabdomyosarcoma cells in NOD/SCID gamma (NSG) mice as a proof-of-principle platform to investigate the molecular signature of CSCs. Indeed, serial xenotransplantation steadily enriched for rhabdomyosarcoma stem-like cells characterized by enhanced aldehyde dehydrogenase activity and increased colony and sphere formation capacity in vitro. Although the expression of core pluripotency factors (SOX2, OCT4, NANOG) and common CSC markers (CD133, ABCG2, nestin) was maintained over the passages in mice, gene expression profiling revealed gradual changes in several stemness regulators and genes linked with undifferentiated myogenic precursors, e.g., SOX4, PAX3, MIR145, and CDH15. Moreover, we identified the induction of a hybrid epithelial/mesenchymal gene expression signature that was associated with the increase in CSC number. In total, 60 genes related to epithelial or mesenchymal traits were significantly altered upon serial xenotransplantation. In silico survival analysis based on the identified potential stemness-associated genes demonstrated that serial xenotransplantation of unsorted rhabdomyosarcoma cells in NSG mice might be a useful tool for the unbiased enrichment of CSCs and the identification of novel CSC-specific targets. Using this approach, we provide evidence for a recently proposed link between the hybrid epithelial/mesenchymal phenotype and cancer stemness

CD133 Expression and Identification of CD133/nestin Positive Cells in Rhabdomyosarcomas and Rhabdomyosarcoma Cell Lines

Background: Co-expression of CD133, cell surface glycoprotein, and nestin, an intermediate filament protein, was determined to be a marker of neural stem cells and of cancer stem cells in neurogenic tumors. Methods: We examined the expression of CD133 and nestin in ten tumor tissue samples taken from patients with rhabdomyosarcomas and in five rhabdomyosarcoma cell lines. Immunohistochemistry and immunofluorescence were used to examine FFPE tumor tissue samples. Cell lines were analyzed by immunofluorescence, immunoblotting, flow cytometry, and RT-PCR. Functional assays (clonogenic in vitro assay and tumorigenic in vivo assay) were also performed using these cell lines. Results: CD133 and nestin were detected in all 10 tumor tissue samples and in all 5 cell lines; however, the frequency of CD133+, Nes+, and CD133+/Nes+ cells, as well as the intensity of fluorescence varied in individual samples or cell lines. The expression of CD133 and nestin was subsequently confirmed in all cell lines by immunoblotting. Furthermore, we observed an increasing expression of CD133 in relation to the cultivation. All cell lines were positive for Oct3/4 and nucleostemin; NSTS-11 cells were also able to form xenograft tumors in mice. Conclusion: Our results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and in rhabdomyosarcoma cell lines. In addition, the co-expression of CD133 and nestin as well as results of the functional assays suggest a possible presence of cancer cells with a stem-like phenotype in these tumors

Transcription factor c-Myb: novel prognostic factor in osteosarcoma

The transcription factor c-Myb is an oncoprotein promoting cell proliferation and survival when aberrantly activated/expressed, thus contributing to malignant transformation. Overexpression of c-Myb has been found in leukemias, breast, colon and adenoid cystic carcinoma. Recent studies revealed its expression also in osteosarcoma cell lines and suggested its functional importance during bone development. However, the relevance of c-Myb in control of osteosarcoma progression remains unknown. A retrospective clinical study was carried out to assess a relationship between c-Myb expression in archival osteosarcoma tissues and prognosis in a cohort of high-grade osteosarcoma patients. In addition, MYB was depleted in metastatic osteosarcoma cell lines SAOS-2 LM5 and 143B and their growth, chemosensitivity, migration and metastatic activity were determined. Immunohistochemical analysis revealed that high c-Myb expression was significantly associated with poor overall survival in the cohort and metastatic progression in young patients. Increased level of c-Myb was detected in metastatic osteosarcoma cell lines and its depletion suppressed their growth, colony-forming capacity, migration and chemoresistance in vitro in a cell line-dependent manner. MYB knock-out resulted in reduced metastatic activity of both SAOS-2 LM5 and 143B cell lines in immunodeficient mice. Transcriptomic analysis revealed the c-Myb-driven functional programs enriched for genes involved in the regulation of cell growth, stress response, cell adhesion and cell differentiation/morphogenesis. Wnt signaling pathway was identified as c-Myb target in osteosarcoma cells. Taken together, we identified c-Myb as a negative prognostic factor in osteosarcoma and showed its involvement in the regulation of osteosarcoma cell growth, chemosensitivity, migration and metastatic activity