Deposition of onco-histone H3.3-G34W leads to DNA repair deficiency and activates cGAS/STING-mediated immune responses.

Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.

[Corrigendum] Dexamethasone-induced inhibition of miR-132 via methylation promotes TGF-ß-driven progression of pancreatic cancer.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that two pairs of the culture plate images in Fig. 4A-C on p. 60 appeared to be the same, although the images were shown in different orientations; moreover, the 'NC/0 and DEX+miR132' and 'DEX and miR132' pairings of images in the scratch-wound assay experiments shown in Fig. 4B also appeared to be overlapping, such that these were apparently derived from the same original source where the results of differently performed experiments were intended to have been portrayed. After re­examining their original data, the authors have realized that some of the data in Fig. 4A and B were inadvertently assembled incorrectly. The revised version of Fig. 4, showing all the correct data for the culture plate images in Fig. 4A-C (specifically, the images fifth along on the right for Fig. 4B and C have been revised) and the correct images for 'NC/0' and 'DEX/0' in Fig. 4D is shown on on the next page. The authors are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a Corrigendum, and all the authors agree with its publication. Furthermore, the authors apologize to the readership for any inconvenience caused. [International Journal of Oncology 54: 53­64, 2019; DOI: 10.3892/ijo.2018.4616].

Selective and caspase-independent cytotoxicity of bioactive glasses towards giant cell tumor of bone derived neoplastic stromal cells but not to bone marrow derived stromal cells.

Semi-malignant giant cell tumors of bone (GCTB) are associated with large osteolytic defects and significant bone destructions. Surgical resection remains the standard therapy that is, however, associated with very high recurrence rates. Bioactive glasses (BGs) that are osteogenic but under certain conditions also cytotoxic might be suitable to achieve biological reconstruction with simultaneous reduction of tumor recurrence in GCTB. In this study, a concentration and time dependent cytotoxic effect of five different BG compositions towards neoplastic GCTB cells was identified while bone marrow derived mesenchymal stromal cells were mostly unaffected. Time course and extent of the cytotoxic effect were dependent on the BG composition and were not associated with caspases activation, indicating that apoptotic mechanisms are not involved. Rather, detection of BG-induced disruption of the cell membranes and a rapid drop of intracellular HMG1 (High Mobility Group Box 1 protein) levels suggest a necrotic cell death. Notably, the cytotoxic effects were dependent on a direct contact of cells and BGs and could not be observed using indirect cultivation settings. Our data suggest that BGs might represent promising materials for the treatment of GCTB in order to reduce tumor recurrence with simultaneous enhancement of bone regeneration.

Effect of manganese, zinc, and copper on the biological and osteogenic properties of mesoporous bioactive glass nanoparticles.

Mesoporous bioactive glass nanoparticles (MBGNs) have demonstrated promising properties for the local delivery of therapeutically active ions with the aim to improve their osteogenic properties. Manganese (Mn), zinc (Zn), and copper (Cu) ions have already shown promising pro-osteogenic properties. Therefore, the concentration-dependent impact of MBGNs (composition in mol%: 70 SiO2 , 30 CaO) and MBGNs containing 5 mol% of either Mn, Zn, or Cu (composition in mol%: 70 SiO2 , 25 CaO, 5 MnO/ZnO/CuO) on the viability and osteogenic differentiation of human marrow-derived mesenchymal stromal cells (BMSCs) was assessed in this study. Mn-doped MBGNs (5Mn-MBGNs) showed a small "therapeutic window" with a dose-dependent negative impact on cell viability but increasing pro-osteogenic features alongside increasing Mn concentrations. Due to a constant release of Zn, 5Zn-MBGNs showed good cytocompatibility and upregulated the expression of genes encoding for relevant members of the osseous extracellular matrix during the later stages of cultivation. In contrast to all other groups, BMSC viability increased with increasing concentration of Cu-doped MBGNs (5Cu-MBGNs). Furthermore, 5Cu-MBGNs induced an increase in alkaline phosphatase activity. In conclusion, doping with Mn, Zn, or Cu can enhance the biological properties of MBGNs in different ways for their potential use in bone regeneration approaches.

Globally altered epigenetic landscape and delayed osteogenic differentiation in H3.3-G34W-mutant giant cell tumor of bone.

The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.

Primary osteoblasts, osteoblast precursor cells or osteoblast-like cell lines: Which human cell types are (most) suitable for characterizing 45S5-bioactive glass?

The question how bioactive glasses (BGs) influence the viability and osteogenic differentiation of human osteogenic cells has already been addressed by several studies. However, a literature review revealed great differences in the type of cells used for these experiments. Primary human osteoblasts (hOBs) represent the desired standard, but possess the limitation of patient variability and time-consuming isolation protocols. Therefore, several alternative cell types have been used including primary mesenchymal stromal cells (BMSCs) and the "osteoblast-like" cell lines MG-63, Saos-2, HOS, and U2OS. The aim of our study was the identification of the cell type most suitable for tissue engineering projects involving BGs by comparative analysis of cell viability and osteogenic differentiation in response to crystallized 45S5-BG. We observed that hOBs, BMSCs, and MG-63 cells were resistant to 45S5-BG induced cytotoxicity, while the viability of Saos-2, HOS, and U2OS cells was significantly reduced. In addition, we detected alkaline phosphatase activity, except in U2OS cells, that increased upon 45S5-BG cocultivation, demonstrating the induction of osteogenic differentiation. Our data and the fact that the donor-dependent variations can be avoided when using MG-63 cells suggest that these are a promising alternative to primary cells and remain an important cell line for future BG related studies.

Optimization of the chicken chorioallantoic membrane assay as reliable in vivo model for the analysis of osteosarcoma.

Survival rates of osteosarcoma patients could not be significantly improved by conventional chemotherapeutic treatment regimens since the introduction of high-dose chemotherapy 35 years ago. Therefore, there is a strong clinical need for new therapeutic targets and personalized treatment strategies, requiring reliable in vivo model systems for the identification and testing of potential new treatment approaches. Conventional in vivo rodent experiments face ethical issues, are time consuming and costly, being of particular relevance in orphan diseases like osteosarcoma. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. The CAM is a highly vascularized, non-innervated extra-embryonic membrane that is perfectly suited for the engraftment of tumor cells. However, only few reports are available for osteosarcoma and reported data are inconsistent. Therefore, the aim of this study was the adaptation and optimization of the CAM assay for its application in osteosarcoma research. Tumor take rates and volumes of osteosarcoma that developed on the CAM were analyzed after modification of several experimental parameters, including egg windowing, CAM pretreatment, inoculation technique and many more. Eight osteosarcoma cell lines were investigated. Our optimized OS-CAM-assay was finally validated against a rat animal xenograft model. Using the cell line MNNG HOS as reference we could improve the tumor take rates from 51% to 94%, the viability of the embryos from initially 40% to >80% and achieved a threefold increase of the tumor volumes. We were able to generate solid tumors from all eight osteosarcoma cell lines used in this study and could reproduce results that were obtained using an osteosarcoma rat animal model. The CAM assay can bridge the gap between in vitro cell culture and in vivo animal experiments. As reliable in vivo model for osteosarcoma research the optimized CAM assay may speed up preclinical data collection and simplifies research on potential new agents towards personalized treatment strategies. Further, in accordance with Russell's and Burch's "Principles of Humane Experimental Technique" the reasonable use of this model provides a refinement by minimizing pain and suffering of animals and supports a considerable reduction and/or replacement of animal experiments.

Retrospective analysis of 51 intralesionally treated cases with progressed giant cell tumor of the bone: local adjuvant use of hydrogen peroxide reduces the risk for tumor recurrence.

BACKGROUND: Giant cell tumor of the bone (GCT) has high local recurrence rates and the prognosis is hard to predict. We therefore retrospectively analyzed clinical outcome and recurrences of 51 GCT cases focusing on the effects of adjuvant local use of hydrogen peroxide. METHODS: The series enclosed 51 advanced GCT cases of the upper and lower extremities (n = 27 Campanacci grade III; n = 24 grade II; n = 39 surgery at our institution, n = 12 elsewhere). Mean follow-up was 88.3 (± 62.0) months. Surgical details, histology, metastases, recurrences, and interview-based data on satisfaction and function including the Musculoskeletal Tumor Society (MSTS) score were evaluated. It was investigated whether hydrogen peroxide was additionally used or not to clean the tumor cavity after curettage as we hypothesized influence on recurrences. To analyze the underlying mechanisms, GCT-derived stromal cell lines were cultured in vitro and tested for cell viability and apoptosis after treatment with hydrogen peroxide. Statistical analysis was performed with Student's t tests, analysis of variance (ANOVA) with post hoc testing, Mann-Whitney U tests, chi-square tests, Kaplan-Meier analysis, and multivariate Cox regression analysis. RESULTS: The whole series had 21 recurrences (41%). Eleven recurrences were found (28%) after surgery at our institution. Kaplan-Meier analysis of cumulative recurrence-free survival revealed at 2 years follow-up 69% (72%, only our institution) and at 10 years follow-up 54% (68%, only our institution). Intralesional resection was performed by vigorous curettage, burring, and defect filling with either polymethylmethacrylate bone cement (n = 45) or cancellous bone from the iliac crest (n = 6). Univariate chi-square analysis showed significantly lower recurrence rate after bone cement filling (2.3-fold, p = 0.024). Cleaning of the lesion cavity with hydrogen peroxide significantly reduced recurrence rate (whole collective 2.9-fold, p = 0.004; our institution 2.8-fold, p = 0.04) and significantly increased cumulative recurrence-free survival rate (whole collective at 10 years follow-up 74% versus 31%, p = 0.002; our institution 79% versus 48%, p = 0.02) compared to cases without hydrogen peroxide treatment. In multivariate analysis, significant risk factors for recurrence were pathological fracture (hazard ratio 3.7; p = 0.04), high mitosis rate (hazard ratio 15.6; p = 0.01), and lack of hydrogen peroxide use (hazard ratio 6.0; p = 0.02). In vitro cell culture analyses found apoptotic nature of hydrogen peroxide induced GCT cell death. CONCLUSIONS: The present series proved for the first time that additional cleaning of the tumor cavity with hydrogen peroxide before defect filling significantly reduced recurrence rate and significantly increased recurrence-free survival in advanced but intralesionally treated GCT cases.

Dexamethasone-induced inhibition of miR-132 via methylation promotes TGF-ß-driven progression of pancreatic cancer.

Glucocorticoids (GCs) such as dexamethasone (DEX) are administered as cancer co­treatment for palliative purposes due to their pro­apoptotic effects in lymphoid cancer and limited side effects associated with cancer growth and chemotherapy. However, there is emerging evidence that GCs induce therapy resistance in most epithelial tumors. Our recent data reveal that DEX promotes the progression of pancreatic ductal adenocarcinoma (PDA). In the present study, we examined 1 primary and 2 established PDA cell lines, and 35 PDA tissues from patients who had received (n=14) or not received (n=21) GCs prior to surgery. Through microRNA microarray analysis, in silico, and RT­qPCR analyses, we identified 268 microRNAs differentially expressed between DEX­treated and untreated cells. With a focus on cancer progression, we selected miR­132 and its target gene, transforming growth factor-ß2 (TGF­ß2), as top candidates. miR­132 mimics directly bound to the 3' untranslated region (3'UTR) of a TGF­ß2 luciferase construct and enhanced expression, as shown by increased luciferase activity. By contrast, DEX inhibited miR­132 expression via promoter methylation. miR­132 mimics also reduced DEX­induced clonogenicity, migration and expression of vimentin and E­cadherin in vitro and in tumor xenografts. In patients, GC intake prior to surgery enhanced global hypermethylation and expression of TGF­ß2 in tissues; expression of miR­132 was detected but could not be quantified. Our results demonstrate that DEX­mediated inhibition of miR­132 is a key mediator in the progression of pancreatic cancer, and the findings provide a foundation for miRNA­based therapies.

Transcriptome and protein interaction profiling in cancer cells with mutations in histone H3.3.

Mutations of histone variant H3.3 are highly recurrent in childhood glioblastoma and in young adults with Giant Cell Tumor of the Bone (GCTB). The heterozygotic representation of the mutations in the tumors, and with potential histone H3 and H3.3 redundancy, suggest that the mutations are gain-of-function by nature. To address common H3.3 point mutations, we have generated data from GCTB patient samples with H3.3 G34W substitutions and engineered human GFP-tagged H3.3-mutated isogenic cell lines for high throughput data comparisons. First, a total of thirty-six patient samples and cell lines were used to acquire gene expression transcriptome data using microarray and RNA-sequencing. The expression data were validated with the orthogonal nCounter assay. Second, to uncover the H3.3-GFP interaction proteomes from the isogenic cell lines, immunoprecipitation of unmutated wild type, K27M, G34R, and G34W substitutions were performed. The RNA-sequencing data and the H3.3 interaction proteome enable potentially important functional insight into the tumorigenic process and should spur further detailed analysis.

Surgical therapy of benign and low-grade malignant intramedullary chondroid lesions of the distal femur: intralesional resection and bone cement filling with or without osteosynthesis.

Surgical treatment of benign and low-grade malignant intramedullary chondroid lesions at the distal femur is not well analyzed compared to higher-grade chondrosarcomas. Localization at the distal femur offers high biomechanical risks requiring sophisticated treatment strategy, but scientific guidelines are missing. We therefore wanted to analyze a series of equally treated patients with intralesional resection and bone cement filling with and without additional osteosynthesis. Twenty-two consecutive patients could be included with intralesional excision and filling with polymethylmethacrylate bone cement alone (n = 10) or with compound bone cement osteosynthesis using a locking compression plate (n = 12). Clinical and radiological outcome was retrospectively evaluated including tumor recurrences, complications, satisfaction, pain, and function. Mean follow-up was 55 months (range 7-159 months). Complication rate was generally high with lesion-associated fractures both in the osteosynthesis group (n = 2) and in the non-osteosynthesis group (n = 2). All fractures occurred in lesions that reached the diaphysis. No fractures were found in meta-epiphyseal lesions. No tumor recurrence was found until final follow-up. Clinical outcome was good to excellent for both groups, but patients with additional osteosynthesis had significantly longer surgery time, more blood loss, longer postoperative stay in the hospital, more complications, more pain, less satisfaction, and worse functional outcome. Intralesional resection strategy was oncologically safe without local recurrences but revealed high risk of biomechanical complications if the lesion reached the diaphysis with an equal fracture rate no matter whether osteosynthesis was used or not. Additional osteosynthesis significantly worsened final clinical outcome and had more overall complications. This study may help guide surgeons to avoid overtreatment with additional osteosynthesis after curettage and bone cement filling of intramedullary lesions of the distal femur. Meta-epiphyseal lesions will need additional osteosynthesis rarely, contrary to diaphyseal lesions with considerable cortical thinning.

The histone variant H3.3 G34W substitution in giant cell tumor of the bone link chromatin and RNA processing.

While transcription as regulated by histones and their post-translational modifications has been well described, the function of histone variants in this process remains poorly characterized. Potentially important insight into this process pertain to the frequently occurring mutations of H3.3, leading to G34 substitutions in childhood glioblastoma and giant cell tumor of the bone (GCTB). In this study, we have established primary cell lines from GCTB patients and used them to uncover the influence of H3.3 G34W substitutions on cellular growth behavior, gene expression, and chromatin compaction. Primary cell lines with H3.3 G34W showed increased colony formation, infiltration and proliferation, known hallmarks of tumor development. Isogenic cell lines with H3.3 G34W recapitulated the increased proliferation observed in primary cells. Transcriptomic analysis of primary cells and tumor biopsies revealed slightly more downregulated gene expression, perhaps by increased chromatin compaction. We identified components related to splicing, most prominently hnRNPs, by immunoprecipitation and mass spectrometry that specifically interact with H3.3 G34W in the isogenic cell lines. RNA-sequencing analysis and hybridization-based validations further enforced splicing aberrations. Our data uncover a role for H3.3 in RNA processing and chromatin modulation that is blocked by the G34W substitution, potentially driving the tumorigenic process in GCTB.

MiR-127 and miR-376a act as tumor suppressors by in vivo targeting of COA1 and PDIA6 in giant cell tumor of bone.

Giant cell tumors of bone (GCTB) are generally benign bone tumors associated with expansive osteolytic defects, a high rate of recurrence and potential malignant transformation. We recently observed silencing of miR-127-3p and miR-376a-3p in GCTB and identified COA1 and PDIA6 as their target genes. Here, we investigate the impact of these microRNAs and their target genes on tumor engraftment and progression of giant cell tumor stromal cells (GCTSC) in vivo by xenotransplantation on the chorioallantoic membrane of chicken eggs. Prior to transplantation, the neoplastic GCTSCs were transfected with miRNA mimics or siRNAs directed against their target genes. Restoration of miR-127-3p and miR-376a-3p reduced the tumor take rate to 17% and 47% compared to 95% in the controls. The tumor volumes were significantly reduced to 29% by both miRNAs. Silencing of COA1 and PDIA6 significantly decreased the tumor volumes to 37.7% and 42.7%, while the tumor take rates remained stable. Our results indicate that re-expression of miR-127-3p and miR-376a-3p induces a strong tumor suppressor effect in GCTSC, which is partially mediated via COA1 and PDIA6.

Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma.

BACKGROUND: Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value. METHODS: All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities. RESULTS: Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006). CONCLUSION: In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma.

Restoration of miR-127-3p and miR-376a-3p counteracts the neoplastic phenotype of giant cell tumor of bone derived stromal cells by targeting COA1, GLE1 and PDIA6.

Although generally benign, giant cell tumors of bone (GCTB) display an aggressive behavior associated with significant bone destruction and lung metastasis in rare cases. This and the very high recurrence rate observed after surgical resection ranging from 20 to 55% necessitates the development of more effective treatment strategies. To identify valuable therapeutic targets, we screened a previously identified microRNA signature consisting of 23 microRNAs predominantly down-regulated in GCTB. We preselected eight candidate microRNAs and analyzed the impact of their restored expression on the neoplastic phenotype of GCTB stromal cells (GCTSC). A consistent and significant inhibition of cell proliferation, migration, colony formation and spheroid formation could be induced by transfection of primary GCTSC cell lines with miR-127-3p and miR-376a-3p, respectively. Genome wide expression analysis of miR-127-3p and miR-376a-3p transfected cells revealed four novel target genes for each microRNA. Luciferase reporter assays demonstrated direct interactions of miR-127-3p with COA1 and direct interaction of miR-376a-3p with GLE1 and PDIA6, suggesting a pivotal role of these genes in the molecular etiology of GTCB. Interestingly, both microRNAs are located within a chromosomal region frequently silenced in GCTB and many other types of cancers, indicating that these microRNAs and their target genes are valuable therapeutic targets for the treatment of GCTB and possibly other tumor entities.

Mesenchymal stroma cells trigger early attraction of M1 macrophages and endothelial cells into fibrin hydrogels, stimulating long bone healing without long-term engraftment.

Implantation of mesenchymal stroma cells (MSCs) is an attractive approach to stimulate closure of large bone defects but an optimal carrier has yet to be defined. MSCs may display trophic and/or immunomodulatory features or stimulate bone healing by their osteogenic activity. The aim of this study was to unravel whether fibrin hydrogel supports early actions of implanted MSCs, such as host cell recruitment, immunomodulation and tissue regeneration, in long bone defects. Female rats received cell-free fibrin or male MSCs embedded in a fibrin carrier into plate-stabilized femoral bone defects. Removed callus was analyzed for host cell invasion (day 6), local cytokine expression (days 3 and 6) and persistence of male MSCs (days 3, 6, 14 and 28). Fibrin-MSC composites triggered fast attraction of host cells into the hydrogel while cell-free fibrin implants were not invaded. A migration front dominated by M1 macrophages and endothelial progenitor cells formed while M2 macrophages remained sparse. Only MSC-seeded fibrin hydrogel stimulated early tissue maturation and primitive vessel formation at day 6 in line with significantly higher VEGF mRNA levels recorded at day 3. Local TNF-α, IL-1ß and IL-10 expression indicated a balanced immune cell activity independent of MSC implantation. Implanted MSCs persisted until day 14 but not day 28. Our results demonstrate that fibrin hydrogel is an attractive carrier for MSC implantation into long bone defects, supporting host cell attraction and pro-angiogenic activity. By this angiogenesis, implant integration and tissue maturation was stimulated in long bone healing independent of long-term engraftment of implanted MSCs.

Osteosarcoma microenvironment: whole-slide imaging and optimized antigen detection overcome major limitations in immunohistochemical quantification.

BACKGROUND: In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment. METHODS: Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis. RESULTS: More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35-65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method. CONCLUSION: Standard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations.

Rescue of silenced UCHL1 and IGFBP4 expression suppresses clonogenicity of giant cell tumor-derived stromal cells.

Giant cell tumor (GCT) of bone is a generally benign tumor with a locally aggressive behavior. Histologically, GCTs consist of multinucleated giant cells, mononuclear histiocytes and the neoplastic fibroblast-like stromal cells (GCTSC). Growing evidence exists that GCTSCs develop from mesenchymal stem cells (MSCs), but little is known about the underlying molecular mechanisms. In previous studies we observed inactivation of the ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene in primary GCTSC due to strong DNA hypermethylation, indicating that epigenetic silencing might be involved in neoplastic transformation of MSCs. Here we investigated further candidate genes and identified strong hypermethylation of the insulin-like growth factor binding protein 4 (IGFBP4) promoter, resulting in IGFBP4 downregulation in GCTs compared to MSCs. Overexpression of UCHL1 and IGFBP4 by stable transfection of GCTSC did not influence cell viability, proliferation, migration and chemosensitivity compared to parental cells. However, colony-formation was significantly decreased suggesting that rescue of UCHL1 and IFGBP4 suppresses clonogenicity of GCT stromal cells. The observation of reduced expression of the stem-cell-specific transcription factors OCT4 and SOX2 in these cell lines further supported our findings. Epigenetic silencing of UCHL1 and IGFBP4 in GCTs might thus be a crucial event during the malignant transformation of MSCs in the context of GCT development and represent promising targets for the development of new diagnostic and therapeutic strategies.

Overexpression of inosine 5'-monophosphate dehydrogenase type II mediates chemoresistance to human osteosarcoma cells.

BACKGROUND: Chemoresistance is the principal reason for poor survival and disease recurrence in osteosarcoma patients. Inosine 5'-monophosphate dehydrogenase type II (IMPDH2) encodes the rate-limiting enzyme in the de novo guanine nucleotide biosynthesis and has been linked to cell growth, differentiation, and malignant transformation. In a previous study we identified IMPDH2 as an independent prognostic factor and observed frequent IMPDH2 overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance. METHODOLOGY/PRINCIPAL FINDINGS: Stable cell lines overexpressing IMPDH2 and IMPDH2 knock-down cells were generated using the osteosarcoma cell line Saos-2 as parental cell line. Chemosensitivity, proliferation, and the expression of apoptosis-related proteins were analyzed by flow cytometry, WST-1-assay, and western blot analysis. Overexpression of IMPDH2 in Saos-2 cells induced strong chemoresistance against cisplatin and methotrexate. The observed chemoresistance was mediated at least in part by increased expression of the anti-apoptotic proteins Bcl-2, Mcl-1, and XIAP, reduced activation of caspase-9, and, consequently, reduced cleavage of the caspase substrate PARP. Pharmacological inhibition of IMPDH induced a moderate reduction of cell viability and a strong decrease of cell proliferation, but no increase in chemosensitivity. However, chemoresistant IMPDH2-overexpressing cells could be resensitized by RNA interference-mediated downregulation of IMPDH2. CONCLUSIONS: IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression.

Silencing of the UCHL1 gene in giant cell tumors of bone.

Giant cell tumors are heterogeneous tumors consisting of multinucleated giant cells, fibroblast-like stromal cells and mononuclear histiocytes. The stromal cells have been identified as the neoplastic cell population, which promotes the recruitment of histiocytes and the formation of giant cells. Strong evidence exists that these cells develop from mesenchymal stem cells (MSCs) but little is known about the molecular mechanisms involved in GCT tumorigenesis. The aim of our study was the identification of cancer-related genes differentially expressed in GCTs compared to MSCs in order to identify possible targets for aberrant promoter methylation, which may contribute to MSC transformation and GCT development. Gene expression of 440 cancer-related genes was analyzed by DNA microarrays in GCT stromal cells and bone marrow-derived MSCs (BMSCs) isolated from the same patient (n = 3) to avoid interindividual variations. Differential expression was identified for 14 genes, which could be confirmed by quantitative PCR in further 21 GCT and 10 BMSC samples. The most pronounced difference in gene expression was detected for UCHL1, an important regulator of the ubiquitin proteasome pathway. Methylation-specific PCR and bisulfite sequencing revealed a strong methylation of the CpG island covering the UCHL1 promoter in GCT stromal cells, whereas methylation was completely absent in BMSCs. UCHL1 expression in stromal cells could be restored by the methylation inhibitor 5-aza-dC. These data demonstrate that the UCHL1 gene is inactivated in GCTs but not in MSCs, suggesting a possible role of UCHL1 in MSC transformation and GCT development.