A short-term in vivo model for giant cell tumor of bone

1471-2407-11-2411471-2407 Research article A short-term in vivo model for giant cell tumor of bone BalkeMauricemaurice.balke@gmail.com NeumannAnnaanna.neumann@ukmuenster.de SzuhaiKárolyk.szuhai@lumc.nl AgelopoulosKonstantinagelopoulos@uni-muenster.de AugustChristianaugustc@uni-muenster.de GoshegerGeorggeorg.gosheger@ukmuenster.de HogendoornCWPancrasp.c.w.hogendoorn@lumc.nl AthanasouNicknick.Athanasou@noc.nhs.uk BuergerHorstbuerger@histopatho.eu HagedornMartinm.hagedorn@angio.u-bordeaux1.fr

Department of Trauma and Orthopedic Surgery, University of Witten-Herdecke, Cologne-Merheim Medical Center, Ostmerheimer Str., 200, 51109 Cologne, Germany

Department of Orthopedic Surgery, University of Muenster, Albert-Schweitzer-Str. 33, 48149 Muenster, Germany

Gerhard-Domagk-Institute of Pathology, University of Muenster, Domagkstr. 17, 48149 Muenster, Germany

Department of Pathology, Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands

Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2300 RC, Leiden, The Netherlands

Department of Medicine, Hematology and Oncology, University of Muenster Domagkstrasse 3, 48149 Muenster, Germany

Institute of Pathology, Klinikum Hanau GmbH, Leimenstr. 20, 63450 Hanau am Main, Germany

Department of Pathology, Nuffield Orthopaedic Centre, University of Oxford, Oxford OX3 7LD, UK

Institute of Pathology, Husener Str. 46 a 33098 Paderborn, Germany

INSERM U1029, Avenue des Facultés, Bâtiment B2, 33405 Talence cedex, France

University Bordeaux 1, Avenue des Facultés, Bâtiment B2, 33405 Talence cedex, France

BMC Cancer 1471-2407 2011 11 1 241 http://www.biomedcentral.com/1471-2407/11/241 10.1186/1471-2407-11-24121668953 1612201013620111362011 2011Balke et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Because of the lack of suitable in vivo models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus in vitro testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose.

Methods

Fresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using in vivo biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence in situ hybridization (FISH).

Results

The suspension of all 10 patients formed solid tumors when grafted on the CAM. In vivo microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells whichwere generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries.

Conclusions

A reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first in vivo model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.

Background

Giant cell tumor of bone (GCT) is an aggressive skeletal lesion typically located in the epiphyseal end of a long bone 1 2 3 . The tumor predominantly occurs in the third and fourth decade of life with a slight predilection for females 3 4 5 6 7 8 .

GCT is characterized by locally aggressive growth usually leading to extensive bone destruction 9 . The biological behavior of the tumor is, however unpredictable, and attempts to histologically grade the tumors have failed 10 11 12 . At the genomic level however recurrent cases are characterized by random individual cell aneusomy, while malignant cases show abnormalities at array CGH level 13 .

GCT is characterized by the presence of numerous Cathepsin-K producing, CD33 +, CD14 - multinucleated osteoclast-like giant cells and plump spindle-shaped stromal cells that represent the main proliferating cell population 14 15 16 17 . The spindle-shaped mononuclear cells are believed to represent the neoplastic population and are characterized at the cytogenetic level by telomeric associations and a peculiar telomere-protecting capping mechanism 18 . Areas of regressive change such as necrosis or fibrosis as well as extensive hemorrhage are frequently present.

The treatment of choice is intralesional curettage and bone cement packing leading to a local recurrence rate of 10 to 40% 1 19 20 ; treatment options are limited and recurrence rates are higher when GCT arises at a surgical inaccessible location (e.g. spine and sacrum). In addition, some GCT may rarely arise at multiple sites or undergo sarcomatous transformation. In about 2% of cases, patients develop lung metastases, which are thought to represent benign pulmonary implants that arise following vascular invasion 21 22 23 24 25 .

The underlying pathobiology of GCT growth and development of these complications is unknown. There is no successful adjuvant treatment option, although there are reports of a limited effect on tumor growth following treatment with bisphosphonates 26 27 and anti-RANKL antibodies 28 , agents that inhibit the formation and activity of the osteoclastic giant cells in the tumor.

Thus far, attempts to grow GCT in animal models as well as to derive suitable cell lines from primary tumors have failed. This has limited the study of pathobiology of GCT and the development of specific anti-GCT agents. To address this problem we have examined whether it is possible to establish the growth of GCT short-term in vivo in a chick chorio-allantoic membrane (CAM) assay.

The CAM is characterized by an extremely dense vascular network with large vessels situated within the somatic mesoderm and capillaries located within or directly under the splanchnic mesoderm. This double-layer membrane develops by fusion of the chorion with the allantoic vesicle on embryonic day 4 - 5 29 . Until hatching the CAM physiologically absorbs calcium from the shell, stores waste products and serves as a respiratory organ 30 .

The CAM assay has been utilized as a model system for more than a century to demonstrate development of embryonic blood vessels, and to provide a host for the grafting of bacteria, viruses and embryonic tissue. In the last 25 years, the CAM assay has become established as a model for angiogenesis research; this has been used to provide highly reproducible models for aggressive and malignant tumors including glioblastoma and pancreatic adenocarcinoma 31 32 .

The use of the CAM assay in bone tumor research has only been sporadically reported. We recently published the successful establishment of human osteosarcoma cell lines on a CAM assay and provided evidence that the MNNG-HOS cell line reproduces the key features of human osteosarcoma growth when grafted on the CAM 33 . This relatively simple experimental approach enables tumor growth and vascularization to be easily studied and permits the growth of tumors to be studied in an inexpensive way.

In this report, we present the results of successful establishment of human GCT in a CAM assay with emphasis on the morphological characteristics of the grafted tumors.

Methods

Patients

The patients included in this study had typical, histologically confirmed cases of giant cell tumors of bone (GCT). The mean age of the five male and five female patients was 29.8 years; eight of ten were localized in the extremities, one in the spine and one in the pelvis. Four were recurrent cases (see Additional file 1). All patients gave their written consent prior to tumor tissue isolation for research studies. All samples were handled in a coded fashion and the experiments were performed according to the local ethical guidelines.

Additional file 1

Table 1. Information on patients, anatomical localization of tumor, mortality/growth rate and tumor size. M = male, F = Female, prox = proximal, dist = distal, R = Recurrence, V mm³= mean tumor volume calculated by V = 4/3*p*r³(r = 1/2 * square root of diameter 1 * diameter 2), SD = standard deviation.

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Giant cell suspension

Cell suspensions isolated from GCT tissue of 10 patients were used in the experiment. Tissue samples were minced and incubated at 37°C in RPMI with 5-10 ml DNAse (2200 KU/100 ml - Sigma-Aldrich, Germany; cat. no. DN-25-10MG) and 5-10 ml collagenase Type 2 (500 U/ml - PAA; Austria; cat. no. K21-240) for 3-8 hours. DNAse and collagenase solutions were mixed in equal parts. The homogenized tissue solution was centrifuged at 1200 rpm for 5 min and the cell pellet was subsequently washed twice with RPMI 1640 (PAA Austria; cat. no. E15-840) supplemented with 10% Foetal Bovine Serum FBS Gold (PAA Austria; cat. no. A15-649) and 1% penicilline/streptomycine (PAA Austria; cat. no. P11-010). This procedure was repeated four times.

Freezing giant cell suspension

After the last washing step, the cell pellet was re-suspended in CryoMaxx S freezing medium (PAA, Austria cat. no. J05-013 - approximately 50 μl to 200 μl cells per ml freezing medium). One ml suspension was frozen per cryotube (Nunc; Germany; cat. no. 368632). Finished vials were frozen overnight at -70°C in a freezing container (NALGENE^®Labware, Hereford, United Kingdom Cat. No. 5100-0001) and stored in liquid nitrogen.

Thawing giant cell suspension

Cell culture medium RPMI 1640 (PAA Austria; cat. no. E15-840) supplemented with 10% Foetal Bovine Serum FBS Gold (PAA Austria; cat. no. A15-649) and 1% penicilline/streptomycine (PAA Austria; cat. no. P11-010) was preheated at 37°C and 50 ml were propounded in a conical centrifuge tube. The frozen vial of giant cells was thawed in a 37°C water bath to that point that it was possible to decant the cells into the RPMI (a rest of ice in the tube is necessary). The cells were decanted into the RPMI medium and centrifuged at 1200 rpm for 5 min. The resultant cell pellet was subsequently washed with RPMI 1640 four times. The yield of isolated cells was re-suspended and seeded on the day 10 CAM (20 μl each).

The chick chorio-allantoic membrane assay

Fertilized white leghorn chicken eggs (Valo-SPF eggs, Lohmann Tierzucht GmbH, Cuxhaven, Germany) were incubated at a humidity of 70% and 37°C. At embryonic day 3, 2 - 3 ml of albumen were removed with a syringe, thus allowing detachment of the embryo and a small window was cut into the eggshell. After verification of normal development of the embryo the window was sealed with tape. After 10 days of incubation small plastic rings made out of Thermanox™ cover discs were placed on the CAM. After gentle laceration of the CAM surface 20 μl of re-suspended tumor suspension were deposited into the rings. For the controls only 20 μl of RPMI was used.

Until day 16 CAMs were examined and photographed in ovo with a digital camera (Olympus E330) attached to a stereomicroscope. All embryos that died before day 16 were excluded from further analyses. Tumor volumes were estimated by the following formula: V = 4/3*p*r³(r = 1/2 * square root of diameter 1 * diameter 2) 31 .

For further information of the technique of the CAM assay see instructional videos in the 'additional files' section (Additional files 2, 3, 4, 5, 6 and 7).

Additional file 2

Opening of the eggs. Video showing the process of opening of the egg.

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Additional file 3

Preparation of plastic rings. Video demonstrating the preparation of the plastic rings.

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Additional file 4

Placement of the plastic ring on the CAM. Video showing the placement of the plastic ring on the CAM.

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Additional file 5

Gentle laceration of the CAM surface. Video showing the process of gentle laceration of the CAM surface.

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Additional file 6

Grafting of the tumor cells. Video demonstrating the technique of tumor cell grafting.

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Additional file 7

Fixation and further processing of the tumor tissue. Video demonstration the method of fixation and preparation of the tissue for further processing.

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Histology and Immunohistochemistry

At embryonic day 16, (6 days of tumor growth), tumors were fixed in vivo using 4% paraformaldehyde for 20 min. Tumors were removed and transferred into culture dishes and samples were observed and photographed. Relevant samples were embedded in paraffin and cut into 10 μm sections. Tissue sections were stained with hematoxylin-eosin and by immunohistochemistry using an indirect immunoperoxidase technique, with mouse monoclonal antibodies MIB-1, and KPI (both obtained from DAKO-UK) and NCL-CD14 and NCL-CD51 (Novocastra, UK) directed against the proliferation marker Ki67, the macrophage/osteoclast marker CD68, the monocyte/macrophage marker CD14, and the osteoclast marker CD51 (vitronectin receptor) respectively. Results were analyzed by standard light microscopy (Leica DM2500 with Leica EC3 camera).

Interphase fluorescence in situ hybridization (FISH)

for the positive identification of cells with human origin we performed an interphase FISH using human haploid repeat sequence containing probe sets 34 . These alpha-satellite probes specifically recognize (peri)centromeric sequences of human chromosomes. Based on the size and specificity of these alpha-satellite probes we selected human chromosome 1 (PUC 1.77) and 15 (D15Z1) 35 .

Interphase FISH was performed according to previously described protocols on formalin-fixed paraffin-embedded tissue slides 36 . Chromosome 1 (detected by FITC, green) and chromosome 15 (detected by Cy3, red) specific alpha satellite probes were labeled by using standard nick translation procedure, hybridized and analyzed as previously described 37 . All slides were embedded in Citifluor anti-fading solution containing DAPI for visualization of DNA of the interphase nuclei.

Results

In vivo observation

All of the ten GCT samples were able to form solid vascularized tumors when grafted to the CAM (Additional file 1, Figure 1 and 2). No significant differences in the growth rate were observed according to the primary lesion. The percentage of tumors after 6 days of growth in living embryos was 86.9% (60 of 69). The overall death rate after grafting of the tumor tissue was 55% (69 of 125) and was significantly higher (P = 0.001, Fisher's exact test - Figure 3) than the death rate of the controls, which was 19% (5 of 26).

Figure 1

In vivo observations of tumor growth

In vivo observations of tumor growth. The tumor solute is seeded into the plastic ring on the CAM (0h). After 24 h a solid tumor develops which gets further vascularized (24 to 144 h). The typical red/yellow-brownish color as well as areas of haemorrhage are visible. Upper row magnification 10 ×, scale bar 1 mm; lower row magnification 20×, scale bar 500 μm.

Figure 2

Photographs of day 6 tumor

Photographs of day 6 tumor. Another example of a GCT grown on the CAM. Note the typical yellow-brownish color in A and the strong vascularization of the tumor when the CAM is turned upside down after fixation in B. Magnification 40 ×, scale bar 250 μm.

Figure 3

Survival of embryos after tumor grafting

Survival of embryos after tumor grafting. The overall death rate after grafting of the tumor tissue is siginficantly higher than the death rate of the controls. P = 0.001, Fisher's exact test.

24h after grafting of the suspension, a solid tumor became apparent which then progressively further vascularized without significantly increasing in size (Figure 1). With the typical yellow-brownish color and the strong vascularization the tumors resembled the macroscopical aspect of GCT during surgery (Figure 2). The overall mean estimated tumor volume was 12.3 mm³(4.3 - 35.6 mm³, Additional file 1).

Histological and Immunohistochemical findings

The tumor samples cultured on the CAM contained both (osteoclast-like) giant cell and mononuclear components of GCT (Figure 4). The giant cells reacted for CD68, which is expressed by both macrophages and osteoclasts, and exhibited the typical immunophenotypic profile of osteoclasts, being CD14- and CD51+ (Figure 5); giant cells in GCT exhibit a similar antigenic phenotype 38 39 . The mononuclear component contained cells expressing CD68, CD14 and CD51. Giant cells were numerous and widely scattered throughout the original tumors but fewer were noted in tumors cultured on the CAM. Tumor giant cells frequently contained more than five nuclei in the original tumors but were smaller and contained fewer nuclei in the cultured samples. The tumors appear to grow on the membrane rather than invade it, producing an implant-like rather than infiltrative growth pattern. Vessels were recruited from the CAM to vascularize the tumor. Ki-67 revealed a very low proliferating fraction (less than 1%) of cells. The tumors contained a background chronic inflammatory cell infiltrate including lymphocytes and plasma cells.

Figure 4

Histology and Ki67 staining

Histology and Ki67 staining. Hematoxylin-eosin stain of a day 6 tumor (A and B) shows all cell components of a GCT and closely resembles original tumor (C). Note the fewer giant cells (arrowheads) in A and B containing fewer nuclei compared to original tumor in C. Note the very low proliferation activity (arrow) in the nuclear staining of MIB-1 in D. Magnification in A+C 20 × (scale bar 50 μm), B: 40 × (scale bar 25 μm), D: 10 × (scale bar 100 μm).

Figure 5

Immunophenotypic profile of giant cells

Immunophenotypic profile of giant cells. Typical immunophenotypic profile of osteoclasts, being CD51 + (A) and CD14 - (B). Giant cells reacted for CD68 (C), which is expressed by both macrophages and osteoclasts. Magnification 400 ×.

Interphase fluorescence in situ hybridization (FISH)

For the discrimination between the human and chicken cells, we performed interphase FISH using human alpha-satellite probes specific to the heterochromatic region of chromosome 1q12 and the (peri)centromeric region of chromosomes 15. The two color labeling of these two probes allows the identification of human cells with FISH signals while chicken cells would be stained with DAPI only. The use of a similar approach to discriminate between human and mouse cells have been shown by us earlier 34 . Despite the very strong auto-fluorescence coming from extracellular matrix material of the CAM, a clear recognition of the FISH positive human cells were possible (Figure 6). FISH image using two human centromeric probes (red, green) showed that there was no cross reactivity between human and chicken centromeres. There was no signal in the CAM nor in the remaining chicken erythrocytes in the tumor nor in the vascular endothelium (Figure 6B). The giant cells were positive for FISH indicating that they were of human origin.

Figure 6

Fluorescence in situ hybridization

Fluorescence in situ hybridization. Interphase FISH overview using probes specific to human chromosome 1 (green) and chromosome 15 (red) alpha-satellite sequences. Sections were counterstained by DAPI (blue) showing nuclei of both human and chicken cells. A: FISH signals were detected in human cells only, chicken erythrocytes showed no signals (indicated by white arrows). B: Human cells are well demarcated from CAM cells (dashed white line) and attracted numerous blood vessels and erythrocytes (white arrows).

Discussion

GCT is a locally aggressive primary tumor of bone that commonly recurs and in some cases can be life threatening 40 . Control of tumor growth and intervention to reduce complications such as the development of pulmonary metastases has hitherto not been possible 1 41 . The major problem in GCT research is the lack of animal models in which to study GCT growth and pathobiology. Although mononuclear stromal cells can be cultured from GCT, other significant cellular components, notably macrophages and giant cells, do not remain in culture after passaging 42 43 . Due to these difficulties, very little is known on the pathobiology of this particular tumor.

The CAM assay is an established in vivo model to study angiogenesis 44 . It is characterized by several advantages such as easy accessibility and relatively simple and cost effective experimental approach. Despite its natural immunodeficient environment (for review see 45 ) the CAM assay is still rarely used for tumor grafting. There are reports about the use of the CAM assay as a reliable model to study tumors such as glioblastoma 31 , prostatic cancer 46 , and melanoma 47 , but compared to murine models of tumor growth these are relatively rare. Reports of the use of the CAM assay for establishment of human bone and soft tissue tumors are largely anecdotal 48 49 .

Recently, we established the CAM assay for human osteosarcoma cell lines and were able to show that the MNNG-HOS, U2OS and SAOS cell lines consistently developed vascularized tumors that simulated key features of human osteosarcoma growth such as angiogenesis, necrosis and hemorrhage 33 . Compared to tumor formation with osteosarcoma cell lines, where the number of successful implanted tumors was around 50%, the number of tumors which resulted from suspensions of GCTs seeded on the CAM was nearly 90% (60 of 69). However, the overall embryonic mortality rate after tumor grafting was 55% (69 of 125); this was higher than in the previous osteosarcoma study where there was an embryonic mortality rate of 38% (58 of 152) 33 . The underlying reasons for this difference remain speculative, but it is possible that the mortality rate may correlate with tumor (graft) and host interactions; death might be caused by tumor cell dissemination directly or via secretion of specific factors causing blood coagulation. Metabolic stress such as hypoxia and/or serum deprivation 50 , may occur in the tumors grafted to the CAM and might induce tumor cells to secrete such molecules.

One theory of GCT lung metastasis formation is that tumor cells invade blood vessels and are disseminated via the blood stream, finally implanting in the lungs. Following implantation, GCTs become established by inducing angiogenesis and further vascularization. The cultured GCTs in our model appeared to grow on the CAM and showed some morphological resemblance to metastatic GCT lung nodules. Although the tumors which grew on the CAM contained all the cellular components of GCT, including stromal cells, macrophages and multinucleated osteoclast-like giant cells, the latter were generally less numerous than in the original tumor, a morphological finding which has been noted in GCT lung nodules 51 . Areas of necrosis and haemorrhage as well as extensive vascularization were present in the tumors grown on the CAM, all features which are typical for GCT. The Ki67 staining demonstrated a very low proliferation rate, in contrast to the original tumors. This might be due to the short time span (only 6 days) of tumor growth. Thus the model would appear to simulate the early phase of tumor seeding, one of the initial steps in the development of a metastasis or local recurrence.

The FISH analysis provided evidence that the experimental GCTs on the CAM are hybrid tumors composed of human graft and chicken host (vasculature) cells. Therefore the model might allow further studies clarifying these crucial steps of angiogenesis and tumor invasion and metastasis which are promising targets for new drugs against solid tumors 52 53 . As an example it might be possible to develop new antitumor drugs by simultaneous measuring of gene expression using Affymetrix chicken GeneChips and human GeneChips in the tumor cells as well as in newly formed blood vessels, as has been shown for pancreatic adenocarcinoma 32 . This model represents an excellent alternative to the commonly used animal models. It is cost effective and fulfills the recommendations of an ethically appreciable use of live animals in cancer research 54 . The fact that tumors were grown from a frozen cell suspension will also favor exchange of material between different centres, an important point given the rarity of GCT.

The typical GCT can usually be locally controlled by intralesional curettage or surgical removal. The treatment options for complicated cases, such as GCT with pulmonary metastases or GCT arising in a surgically inaccessible site, are limited. To date only a few promising therapies have been developed for adjuvant use in these cases. The few publications that exist on systemic treatment of GCT have focused on inhibition of osteoclastic bone resorption with bisphosphonates or disruption of the RANK/RANKL pathway of osteoclast formation with specific antibodies such as denosumab 28 55 . The limitation of this treatment is that only osteoclasts are inhibited, whereas the proliferating neoplastic stromal cells are mainly unaffected. Thus these treatments might only have a short term effect. Use of the CAM assay should permit the effect of therapeutic agents on both stromal and giant cell components of GCT to be studied in greater detail; this model should also facilitate further molecular characterization of the cellular components of this rare tumor.

Conclusions

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MB, AN, NA, KS, and MH designed experiments and wrote the manuscript. MB, AN, KA, KS, KA, and HB conducted the experiments. MB, and GG collected the patient data and performed the surgeries. CA, NA and HB performed the histology and immunohistochemistry. KS, and PH performed the FISH analysis. MB and MH developed the ideas, NA and PH corrected and edited the manuscript. All authors have read and approved the final manuscript.

Acknowledgements

This work was supported by EuroBoNet, a sixth framework Network of Excellence for studying pathology and genetics of bone tumors.

None of the authors has professional and financial affiliations that may be perceived to have biased the presentation. There is no conflict of interest.

Giant cell tumor of bone: treatment and outcome of 214 casesBalkeMSchremperLGebertCAhrensHStreitbuergerAKoehlerGHardesJGoshegerGJ Cancer Res Clin Oncol200813496997810.1007/s00432-008-0370-x18322700Giant-cell tumor of boneCampanacciMBaldiniNBorianiSSudaneseAJ Bone Joint Surg Am1987691061143805057Knochentumoren. 2. AuflageFreyschmidtJOstertagHJundtGBerlin, Heidelberg, New York, Tokio, Springer1998611649Giant cell tumorsCarrascoCHMurrayJAOrthop Clin North Am1989203954052662113[Differential diagnosis of giant cell tumor of bone]Salzer-KuntschikMVerh Dtsch Ges Pathol19988215415910095427Bone and Soft Tissue TumorsCampanacciMNew York: Springer1990117151Giant-cell tumor of bone. A demographic, clinical, and histopathological study of all cases recorded in the Swedish Cancer Registry for the years 1958 through 1968LarssonSELorentzonRBoquistLJ Bone Joint Surg Am1975571671731112843Update on giant cell tumor of boneSchwartzHSCompr Ther1998244884939801847A system of staging musculoskeletal neoplasmsEnnekingWFClin Orthop Relat Res1986924Giant cell tumor of bone: a clinicopathologic study of prognostic factorsMasuiFUshigomeSFujiiKPathol Int19984872372910.1111/j.1440-1827.1998.tb03973.x9778111Giant cell tumor of long bone: a Canadian Sarcoma Group studyTurcotteREWunderJSIslerMHBellRSSchacharNMasriBAMoreauGDavisAMClin Orthop Relat Res2002248258Local recurrences in giant cell tumour of bone. Long-term follow up of 31 casesLaustenGSJensenPKSchiodtTLundBInt Orthop19962017217610.1007/s0026400500578832321Genomic instability in giant cell tumor of bone. A study of 52 cases using DNA ploidy, relocalization FISH, and array-CGH analysisMoskovszkyLSzuhaiKKrenacsTHogendoornPCSzendroiMBenassiMSKopperLFuleTSapiZGenes Chromosomes Cancer20094846847910.1002/gcc.2065619242928Giant cell tumour of bone: morphological, biological and histogenetical aspectsWernerMInt Orthop20063048448910.1007/s00264-006-0215-717013643The nature of giant cell tumor of boneWullingMEngelsCJesseNWernerMDellingGKaiserEJ Cancer Res Clin Oncol200112746747410.1007/s00432010023411501745Cathepsin K is the principal protease in giant cell tumor of boneLindemanJHHanemaaijerRMulderADijkstraPDSzuhaiKBrommeDVerheijenJHHogendoornPCAm J Pathol200416559360010.1016/S0002-9440(10)63323-8161856515277232CD33+ CD14- phenotype is characteristic of multinuclear osteoclast-like cells in giant cell tumor of boneForsythRGDe BoeckGBaeldeJJTaminiauAHUyttendaeleDRoelsHPraetMMHogendoornPCJ Bone Miner Res200924707710.1359/jbmr.08090518767926Telomere biology in giant cell tumour of boneForsythRGDe BoeckGBekaertSDe MeyerTTaminiauAHUyttendaeleDRoelsHPraetMMHogendoornPCJ Pathol200821455556310.1002/path.230118278785Treatment of giant-cell tumors of long bones with curettage and bone-graftingBlackleyHRWunderJSDavisAMWhiteLMKandelRBellRSJ Bone Joint Surg Am19998181182010391546Local control of long bone giant cell tumour using curettage, burring and bone grafting without adjuvant therapyMalekFKruegerPHatmiZNMalayeriAAFaezipourHO'DonnellRJInt Orthop20063049549810.1007/s00264-006-0146-316896875Metastases from histologically benign giant-cell tumor of boneRockMGPritchardDJUnniKKJ Bone Joint Surg Am1984662692746693454Giant-cell tumour of bone metastasising to the lungs. A long-term follow-upSiebenrockKAUnniKKRockMGJ Bone Joint Surg Br199880434710.1302/0301-620X.80B1.78759460951Histologically verified lung metastases in benign giant cell tumours-14 cases from a single institutionDominkusMRuggieriPBertoniFBriccoliAPicciPRoccaMMercuriMInt Orthop20063049950410.1007/s00264-006-0204-x16909252Giant cell tumor of bone. Prognosis and treatment of pulmonary metastasesChengJCJohnstonJOClin Orthop Relat Res1997205214Late malignant transformation of a benign giant-cell tumor of bone. A case reportSakkersRJvan der HeulROKroonHMTaminiauAHHogendoornPCJ Bone Joint Surg Am1997792592629052550Bisphosphonates induce apoptosis of stromal tumor cells in giant cell tumor of boneChengYYHuangLLeeKMXuJKZhengMHKumtaSMCalcif Tissue Int200475717715037971Bisphosphonates reduce local recurrence in extremity giant cell tumor of bone: a case-control studyTseLFWongKCKumtaSMHuangLChowTCGriffithJFBone200842687310.1016/j.bone.2007.08.03817962092Denosumab in patients with giant-cell tumour of bone: an open-label, phase 2 studyThomasDHenshawRSkubitzKChawlaSStaddonABlayJYRoudierMSmithJYeZSohnWLancet Oncol2010Mapping of the microcirculation in the chick chorioallantoic membrane during normal angiogenesisDeFouwDORizzoVJSteinfeldRFeinbergRNMicrovasc Res19893813614710.1016/0026-2862(89)90022-82477666The avian egg: air-cell gas tension, metabolism and incubation timeRahnHPaganelliCVArARespir Physiol19742229730910.1016/0034-5687(74)90079-64475469Accessing key steps of human tumor progression in vivo by using an avian embryo modelHagedornMJaverzatSGilgesDMeyreAde LafargeBEichmannABikfalviAProc Natl Acad Sci USA20051021643164810.1073/pnas.040862210254784915665100Netrin-1 mediates early events in pancreatic adenocarcinoma progression, acting on tumor and endothelial cellsDumartinLQuemenerCLaklaiHHerbertJBicknellRBousquetCPyronnetSCastronovoVSchillingMKBikfalviAHagedornMGastroenterology138159516061606 e1591-1598Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membraneBalkeMNeumannAKerstingCAgelopoulosKGebertCGoshegerGBurgerHHagedornMBMC Res Notes201035810.1186/1756-0500-3-58283890620202196Human cardiomyocyte progenitor cell transplantation preserves long-term function of the infarcted mouse myocardiumSmitsAMvan LaakeLWden OudenKSchreursCSzuhaiKvan EchteldCJMummeryCLDoevendansPAGoumansMJCardiovasc Res20098352753510.1093/cvr/cvp14619429921Combined metaphase, interphase cytogenetic, and flow cytometric analysis of DNA content of pediatric acute lymphoblastic leukemiaPajorLSzuhaiKMehesGKosztolanyiGJaksoPLendvaiGSzanyiIKajtarPCytometry199834879410.1002/(SICI)1097-0320(19980415)34:2<87::AID-CYTO5>3.0.CO;2-99579606EWSR1-CREB1 and EWSR1-ATF1 fusion genes in angiomatoid fibrous histiocytomaRossiSSzuhaiKIjszengaMTankeHJZanattaLSciotRFletcherCDDei TosAPHogendoornPCClin Cancer Res2007137322732810.1158/1078-0432.CCR-07-174418094413Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISHSzuhaiKBezrookoveVWiegantJVrolijkJDirksRWRosenbergCRaapAKTankeHJGenes Chromosomes Cancer200028929710.1002/(SICI)1098-2264(200005)28:1<92::AID-GCC11>3.0.CO;2-210738307Immunophenotypic differences between osteoclasts and macrophage polykaryons: immunohistological distinction and implications for osteoclast ontogeny and functionAthanasouNAQuinnJJ Clin Pathol199043997100310.1136/jcp.43.12.9975029722266187Phenotypic and molecular studies of giant-cell tumors of bone and soft tissueLauYSSabokbarAGibbonsCLGieleHAthanasouNHum Pathol20053694595410.1016/j.humpath.2005.07.00516153456Treatment options for recurrent giant cell tumors of boneBalkeMAhrensHStreitbuergerAKoehlerGWinkelmannWGoshegerGHardesJJ Cancer Res Clin Oncol200813514915818521629Giant-cell tumor of the appendicular skeletonGhertMARizzoMHarrelsonJMScullySPClin Orthop Relat Res2002201210Establishment of a cell line from a human giant cell tumor of boneKitoMMoriyaHMikataAHarigayaKTakenouchiTTakadaNTatezakiSUmedaTClin Orthop Relat Res1993353360Characterization of a cell line derived from a human giant cell tumor that stimulates osteoclastic bone resorptionOreffoROMarshallGJKirchenMGarciaCGallwitzWEChavezJMundyGRBonewaldLFClin Orthop Relat Res1993229241VEGF coordinates interaction of pericytes and endothelial cells during vasculogenesis and experimental angiogenesisHagedornMBalkeMSchmidtABlochWKurzHJaverzatSRousseauBWiltingJBikfalviADev Dyn2004230233310.1002/dvdy.2002015108306HagedornMWiltingJChick chorioallantoic membrane assay: growth factor and tumor-induced angiogenesis and lymphangiogenesisBerlin, Heidelberg, Germany: Springer2004Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute modelKunzi-RappKGenzeFKuferRReichEHautmannREGschwendJEJ Urol20011661502150710.1016/S0022-5347(05)65820-X11547121Intravital videomicroscopy of the chorioallantoic microcirculation: a model system for studying metastasisMacDonaldICSchmidtEEMorrisVLChambersAFGroomACMicrovasc Res19924418519910.1016/0026-2862(92)90079-51474926Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of disseminationDeryuginaEIZijlstraAPartridgeJJKupriyanovaTAMadsenMAPapagiannakopoulosTQuigleyJPCancer Res200565109591096910.1158/0008-5472.CAN-05-222816322244Expression of type-IV collagenases in human tumor cell lines that can form liver colonies in chick embryosTsuchiyaYEndoYSatoHOkadaYMaiMSasakiTSeikiMInt J Cancer19945646518262676Tumor hypoxia, the physiological link between Trousseau's syndrome (carcinoma-induced coagulopathy) and metastasisDenkoNCGiacciaAJCancer Res20016179579811221857Morphological and immunophenotypic features of primary and metastatic giant cell tumour of boneAlberghiniMKliskeyKKrenacsTPicciPKindblomLForsythRAthanasouNAVirchows Arch20104569710310.1007/s00428-009-0863-220012988Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesisAlmogNHenkeVFloresLHlatkyLKungALWrightRDBergerRHutchinsonLNaumovGNBenderEFaseb J20062094794910.1096/fj.05-3946fje16638967Novel agents on the horizon for cancer therapyMaWWAdjeiAACA Cancer J Clin20095911113710.3322/caac.2000319278961RussellWMSBurchRLThe Principles of Humane Experimental TechniquePotters Bar, UK: Universities Federation for Animal Welfare (UFAW)New edition1992Bisphosphonate treatment of aggressive primary, recurrent and metastatic Giant Cell Tumour of BoneBalkeMCampanacciLGebertCPicciPGibbonsMTaylorRHogendoornPKroepJWassJAthanasouNBMC Cancer20101046210.1186/1471-2407-10-462294080220799989

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