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Ewing's sarcoma of bone tumor cells produces MCSF that stimulates monocyte proliferation in a novel mouse model of Ewing's sarcoma of bone
Bone 79 (2015) 121–130
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Original Full Length Article
Ewing's sarcoma of bone tumor cells produces MCSF that stimulates monocyte proliferation in a novel mouse model of Ewing's sarcoma of bone B.S. Margulies a,⁎, S.D. DeBoyace a, T.A. Damron a, M.J. Allen b a b
SUNY Upstate Medical University, Department of Orthopedic Surgery, USA The Ohio State University, College of Veterinary Medicine, Department of Veterinary Clinical Medicine, USA
a r t i c l e
i n f o
Article history: Received 4 June 2014 Revised 14 May 2015 Accepted 28 May 2015 Available online 5 June 2015 Edited by: Robert Recker Keywords: Ewing's sarcoma Osteoblasts Monocytes Osteoclasts Bone growth MCSF
a b s t r a c t Ewing's sarcoma of bone is a primary childhood malignancy of bone that is treated with X-radiation therapy in combination with surgical excision and chemotherapy. To better study Ewing's sarcoma of bone we developed a novel model of primary Ewing's sarcoma of bone and then treated animals with X-radiation therapy. We identified that uncontrolled tumor resulted in lytic bone destruction while X-radiation therapy decreased lytic bone destruction and increased limb-length asymmetry, a common, crippling complication of X-radiation therapy. Osteoclasts were indentified adjacent to the tumor, however, we were unable to detect RANK-ligand in the Ewing's tumor cells in vitro, which lead us to investigate alternate mechanisms for osteoclast formation. Ewing's sarcoma tumor cells and archival Ewing's sarcoma of bone tumor biopsy samples were shown to express MCSF, which could promote osteoclast formation. Increased monocyte numbers were detected in peripheral blood and spleen in animals with untreated Ewing's sarcoma tumor while monocyte number in animals treated with x-radiation had normal numbers of monocytes. Our data suggest that our Ewing's sarcoma of bone model will be useful in the study Ewing's sarcoma tumor progression in parallel with the effects of chemotherapy and X-radiation therapy. © 2015 Elsevier Inc. All rights reserved.
Introduction Ewing's sarcoma of bone (ESB) is a malignant, primary bone tumor of childhood; accounting for approximately 7% of pediatric bone tumors [1]. ESB is first treated with neo-adjuvant chemotherapy followed by Xradiation therapy (XRT), surgical excision or XRT in combination with surgical excision. Recent work by Choi et al. highlighted the controversy surrounding the efficacy of XRT alone, surgical excision alone or XRT in combination with surgical excision [21]. Unfortunately, the delivery of XRT to the growing skeleton is associated with a significant risk of crippling limb-length asymmetry and pathologic fracture [2]. In previous work, we sought to mitigate the negative effects of XRT administered to the growing bone through the use of chemoradioprotection drug therapies [3–5]. Nevertheless, the mechanism through which XRT has proven so effective as a treatment in ESB patients remains poorly understood. Further, the bone marrow signalingenvironment that facilitates ESB tumor progression remains unknown. Bone degradation (lysis) that occurs during ESB tumor expansion in bone is most likely mediated through a myeloid lineage cell, such as macrophages or osteoclasts. We have proposed that ESB tumor cells ⁎ Corresponding author at: SUNY Upstate Medical University, 750 E. Adams Street, 3113 Institute for Human Performance, Syracuse, NY, USA 13210. Fax: +1 315 464 6638. E-mail address: [email protected] (B.S. Margulies).
http://dx.doi.org/10.1016/j.bone.2015.05.041 8756-3282/© 2015 Elsevier Inc. All rights reserved.
alter the myeloid progenitor cell niche in the bone marrow as a means of creating a more favorable bone environment that favors sarcoma tumor growth. However, it remains unknown what factors mediate this process or how these tumor-produced factors may direct the fate of myeloid progenitors. Monocytes are hematopoietic progenitors of the myeloid-lineage that differentiate into macrophages, osteoclasts and dendritic cells. Monocyte differentiation is directed by number of growth factors that include MCSF, GCSF and GMCSF; with MCSF required for proliferation and GMCSF required hematopoietic stem cell differentiation. In addition, osteoclast differentiation requires interaction with mesenchymal lineage cells that express the ligand for the receptor-activator of NFκB (RANK-ligand). Monocytes and ESB are highly sensitive to XRT; with our previous in vitro work identifying the specific XRT-dose sensitivity, or D0-value, in both monocytes (0.94 Gy) and ESB (1.14 Gy) [16]. Further, the loss of monocytes after 17.5 Gy XRT in vivo resulted in decreased numbers of osteoclasts and increased bone mass [7]. To study ESB tumor progression we developed a novel mouse model of primary ESB tumor by injecting ESB tumor cells directly into the femoral bone marrow of a mouse. Our initial hypothesis was that ESB tumor associated bone lysis would be due to increased numbers of osteoclasts derived from an increase in RANK-ligand produced by the ESB tumor. In preliminary experiments using this novel mouse model we were unable to identify RANK-ligand gene expression in
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TC71 ESB tumor cells, however, we were able to identify MCSF gene expression. This lead us to hypothesize that ESB mediated osteolysis is due to an increase in the numbers of monocytes, which would in turn lead to increased numbers of osteoclasts and lytic bone destruction. We also hypothesized that the efficacy of XRT to treat ESB may be due the radio-sensitivity of monocytes in parallel with the radio-sensitivity of ESB, consistent with previous data [16]. Here we report that ESB tumor expansion in bone is driven by increased osteoclastic bone re-absorption due to increased numbers of monocytes, using a novel in vivo mouse model of ESB tumor progression. We also identify that the increase in monocyte numbers is due to the expression of MCSF produced by ESB tumor cells; with the application of XRT resulting in decreased tumor burden and corresponding monocyte numbers. Additionally, we found that ESB tumor cells do not express the most potent regulator of osteoclast differentiation from monocytes, RANK-ligand. We confirmed these observations in primary Ewing's sarcoma of bone biopsy samples. Thus, our current work suggests a mechanism through which the depletion of monocytes coupled with diminished MCSF expression from XRT results in decreased tumor burden. Methods Mouse model of primary Ewing's sarcoma of bone Skeletally immature female NCr nude mice (n = 66) were anesthetized with Telazol (45-mg/kg IM) and xylazine (7.5-mg/kg IM). Using a medial para-patellar approach, a sterile 26 G needle was inserted through the intra-patellar notch to a depth of 5 mm in the distal femur. A suspension of ESB TC71 tumor cells (1 × 105 cells in 20 μL) was injected into the right femoral medullary canal. Left femurs served as controls that were not implanted with tumor and were not treated. Tumor progression was observed with serial X-radiographs taken 2, 3 and 6 weeks after tumor inoculation. Animals were anesthetized using Telezol (30-mg/kg IP) prior to XRT with both limbs extended and placed under a therapeutic X-ray machine (Phillips MGC-30, operating at 300 kV and 10 mA and an effective dose rate 0.256 Gy/min for a 2 cm × 4 cm field, at a distance of 5 cm) and exposed to 0 Gy (n = 44), 5 Gy (n = 10) and 20 Gy (n = 10). Using beam collimation and lead shielding, only the right knee was exposed (distal half of the femur and proximal half of the tibia) [17]. At the completion of the experimental period or when the animals showed signs of distress/lameness, they were euthanized using carbon dioxide. The SUNY Upstate Medical University Institutional Animal Care and Use Committee approved all of the animal studies described in this paper. Tissue collection and histology Tibia were split and fixed in 2% glutaraldehyde + 2% paraformaldehyde + 0.7% hexa-amine ruthenium (III) chloride (RHT) in a 0.1 M cacodylate buffer (n = 4 for 0 Gy; n = 4 for 5 Gy; n = 4 for 20 Gy). Tibial halves were embedded in methyl methacrylate (PMMA) and sagitally sectioned on a rotary microtome at 5 μm [19]. Tibial sections were stained for bright-field observation and analysis with a combination of 2% periodic acid, 1% methylene blue (in 1% Borax), 0.15% basic fuchsin (in 10% ETOH), and azure II (with azure II and basic fuchsin mixed in equal parts). Alternatively, tibial sections were stained with 2% methyl green following immuno-histochemistry. Antibodies obtained from Santa Cruz Biotechnologies that recognized CD14 (1:250), CD99 (1:250) and cathepsin K (1:100) were detected using biotin conjugated secondary antibody (1:500) visualized by reacting streptavidin-horseradish peroxidase (VectaStain ABC kit, Vector Labs) with the chromogen 3, 3′-diaminobenzadine tetrahydrochloride (DAB, Biogenix). Alternatively, an anti-mouse-rabbit secondary antibody that is conjugated to a very active peroxidase micro-polymer (ImmPress kit, Vector Labs) was reacted with the chromogen Vecta NovaRed (Vector Labs). Tartrate resistant acid phosphatase (TRAP)
staining (Leukocyte Acid Phosphatase Kit 387-A, Sigma) was used to identify osteoclasts (n = 4 for 0 Gy; n = 4 for 20 Gy). DEXA analysis Animals were evaluated with a dual energy X-ray absorptometry (DEXA) 2, 3 and 6 weeks after tumor inoculation (n = 39 for 0 Gy; n = 10 for 5 Gy; n = 10 for 20 Gy) using a Lunar PixiMus2 (General Electric) scanner. A region of interest (ROI) was manually drawn around the right and lefts femur. Bone mineral density (BMD) was calculated by dividing the bone mineral content (BMC) by the cross-sectional area (A) of the mineralized tissue within the ROI for the right and left femurs using the proprietary software provided with the scanner. Whole body %Fat mass was calculated using the provided software. Limb length measurements of growth Radiographs of all the limbs were taken at baseline and following dissection (n = 15 for 0 Gy; n = 4 for 5 Gy; n = 14 for 20 Gy). Radiographs were subsequently scanned and measured using ImageJ analysis software (National Institutes of Health Research Services Branch http:// rsbweb.nih.gov/ij/) to determine femoral, tibial, and overall limb-length discrepancy. Limb-length discrepancy represents a measure of lost growth function by comparing the irradiated limb to the contralateral control limbs and represents the loss in cumulative growth (discrepancy = [Δ(left length) − Δ(right length)/Δ(left length)]) [3–5,17]. Measurement of total plate heights and terminal hypertrophic chondrocyte height Total growth plate heights were measured by selecting a measurement region bisected by the long axis of the tibia (n = 4 for 0 Gy; n = 4 for 5 Gy; n = 4 for 20 Gy). Lines were drawn along the contours of both the epiphysis and the chondro-osseous junction. The mean distance between the epiphyseal contour line and the chondro-osseous junction contour line was measured from three sections using ImageJ. Terminal, hypertrophic-chondrocyte heights were measured by following individual hypertrophic chondrocytes through serial section and measuring their greatest height. In both right and left limbs, counts of 300 terminal hypertrophic chondrocytes were obtained from each animal [10]. Measurement of bone volumes and 3D micro-architecture Femora were placed in PBS for μCT and stored at −20 °C. The distal 20% of the femur was scanned using a μCT (μCT40, Scanco) to ascertain 3D micro-architecture and tumor associated bone osteolysis using an isotrophic voxel resolution of 12 μm3 (n = 4 for 0 Gy; n = 5 for 5 Gy; n = 6 for 20 Gy). The distal 20% distance was determined as a percentage of the total length of bone. Thereafter, bone volumes were calculated as a percentage of the total tissue volume (Bv/Tv) using the proprietary analysis software supplies with the scanner. Spleen weights and differential blood smears Spleens were removed during routine necropsy, weighed and subsequently placed into 10% neutral buffered formalin (n = 10 for 0 Gy; n = 10 for 20 Gy). Differential blood smears were prepared from blood drawn from the tail-vein after euthanasia (n = 10 for 0 Gy; n = 10 for 20 Gy). A drop of blood was placed on a glass slide and smeared with cover glass. The blood smear was allowed to dry and the fixed with 70% ethanol and stained with a commercially prepared Giemsa stain (Sigma). Differential-cell counts were made by counting 100 cells in total from each of three slides produced for each animal in each experimental group.
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Tissue culture The Ewing's sarcoma of bone tumor cell line TC71 (Dr. Tim Triche, USC/CHLA) were maintained in RPMI 1640 media while the Ewing's sarcoma of bone tumor cell lines RD-ES, HS822.t and HS863.t (ATCC) were maintained in DMEM and then supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin–streptomycin–glutamine (PSG). The MB231 breast carcinoma and HBL100 breast epithelium cell-lines were maintained in DMEM supplemented with 10% FCS and 1% PSG as controls that express RANK-ligand for conditioned media experiments. In order to produce conditioned media (CM); cells were seeded at 1 × 104 cells per well (5 × 103 cells/cm2), which results in an actively proliferating culture. CM or stale media were collected 72 h after the start of culture from each tumor cell (ESBCM, MB231CM and HBL100CM), filtered and then 0%, 0.1%, 0.5%, 1%, 5% and 10% (v/v) aliquots were added to fresh media. To test an interaction of between ESB cells and osteoprogenitors we administered ESB conditioned media (ESBCM) to osteoblasts (OB) cultured from human trabecular fragments and monocytes derived from corresponding bone marrow (BM) aspirates, both retrieved during total hip arthroplasty from consenting adult patients as part of an IRB approved study (n = 6). In parallel, we also cultured the adherent fraction of cells derived from bone marrow, which are pluripotent mesenchymal stem cells (MSC). MSC allowed grown to confluence, at which point they begin to express a primitive osteoblastic phenotype. Primary human OBs were dissociated from metaphyseal trabecular bone fragments removed during total hip-arthroplasty using a mixture of 1% collagenase (Worthington Biochemical Corp.) 0.25% trypsin-EDTA (Cellgro) mixture. Primary human bone marrow cells were collected using gradient centrifugation (lymphocyte separation media; Lonza) from bone marrow aspirates. Bone marrow cells were seeded at 1 × 107 into T-25, 25 mm2 flasks and maintained in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) PSG and 100 ng/mL of recombinant human MCSF for three days. Non-adherent MCSF-dependent bone-marrow cells (monocytes) were collected and then seeded at 1 × 106 cells per well supplemented with 25 ng/mL MCSF and treated with CM and then assayed for osteoclast formation by staining for TRAP expression. Positive controls cultures were administered 25-ng/mL of MSCF with and without the addition of 25 ng/mL recombinant human RANK-ligand (R&D Systems). OB and monocytes were also seeded into culture plates at 1 × 104 cells per well (5 × 103 cells/cm2) and assayed for CM effects on proliferation using the MTT assay (5 mg/ml (w/v), Sigma) (n = 3 with n = 4 replicates per condition) [16]. Gene expression analysis Cells were assayed for changes in MCSF and RANK-ligand gene expression. mRNA was purified using RNeasy Plus Mini columns (Qiagen) and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). Gene expression was analyzed using quantitative PCR (qPCR) using 100 ng of cDNA mixed with Fast Plus EvaGreen Master Mix (Biotium). In each experiment GAPDH served as a control, negative controls contained no-template and a standard curve was generated using serial dilutions of a chemically synthesized sequence for GAPDH (0, 1, 5, 10 and 100 fg; Integrated DNA Technologies). Gene expression was evaluated using Pfaffl's method, in which the efficiency of each primer (E) and the starting concentration of gene product (N0) are calculated from the linear region of the fluorescence-crossing threshold curve using the software LinRegPCR (v2013.0) [18]. Experiments were considered valid when the control gene GAPDH fell within the standard curve and the primer efficiencies (E) were calculated to be E N 1.8. Protein expression through western blot analysis Cells were lysed with a cold buffer composed of the following: 1% sodium dodecyl sulfate (SDS), 20 mM TRIS (pH 8.0), 1 mM
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β-glycerophosphate, 150 mM sodium chloride, 2 mM iodoacetamide, 2 mM benzamidine hydrochloride, 0.1 mM ethylmaleimide, 1% PMSF, 1% β mercaptoethanol and the Halt Protease Inhibitor Cocktail (Pierce Thermo Scientific) [16]. Total protein was assayed using the BCA Protein Assay Kit (Pierce Thermo Scientific). Samples were loaded (20 μg/well) onto a 10–20% Mini-Protean Tris-Tricine Precast Gel (Bio-Rad) with the Page Ruler Pre-stained NIR Protein Ladder (Bio-Rad) and transferred to an Immun-Blot Low Fluorescence PVDF Membrane (Bio-Rad). Membranes were blocked using Protein Free T20 (TBS) Blocking Buffer (Pierce Thermo Scientific) and incubated with primary antibodies (Santa Cruz Biotechnologies) directed against RANK-ligand (1:500) and MCSF (1:500). Archival sarcoma biopsy samples Archival primary sarcoma biopsy samples that were collected from patients prior to therapy (n = 5 Ewing's sarcoma of bone and n = 5 osteosarcoma) were sectioned and stained for hematoxylin and eosin (H&E), the ESB specific marker CD99 (1:250), MCSF (1:100) and RANK-ligand (1:200). Sections were counter-stained using 2% methyl green and imaged at 20 ×. Archival primary sarcoma biopsy samples were analyzed as part of an IRB approved study. Statistics Prism (v5.0d, Graphpad Software Inc.) was used for all statistical calculations. Radio-graphically determined limb-lengths, DEXA and μCT for each femur or tibia from each of the 10 animals per treatment group were used to calculate means and standard deviations. Histomorphometric analyses (growth plate heights, terminal hypertrophic chondrocyte height) on each limb (left and right) were done using a minimum of 2 sections separated by 30 μm from 4 animals randomly chosen per group, from which means and standard deviations were calculated. In vitro experiments were done in triplicate (TRAP/MTT and Gene expression). Means and standard deviations were calculated and ANOVA with Bonferroni's post-hoc was used to determine significance (p b 0.05). Results ESB cells injected into the distal femur of mice resulted in bone destruction Previous work by Scotlandi et al. demonstrated that athymic mice inoculated with ESB tumor cells subcutaneously develop metastases to bone and lung [6]. We developed a primary ESB tumor model by injecting TC71 tumor cells into the distal femur of the right-limbs of skeletally immature athymic Ncr mice. Two-weeks after tumor implantation 86% of the animals inoculated with ESB presented with a lytic lesion. At 6-weeks all of the animals that did not undergo XRT had a lytic lesion in bone, of which 75% had significant cortical discontinuity (Fig. 1a). None of the animals receiving 5 or 20 Gy XRT presented with cortical discontinuity (Fig. 1a). Mice implanted with ESB that were not administered XRT were observed to have increased total body fat (%Fat) of 3.85% relative to mice exposed to 20 Gy XRT (p b 0.001) (Fig. 1b). We also compared the right-limb femoral bone mineral density (BMD) to the left-limb femoral BMD 6 weeks after tumor implantation and found that BMD in mice implanted with ESB decreased 6.11% relative to the left-limb controls (Fig. 1c). BMD increased 8.45% for the 5 Gy and 6.93% for the 20 Gy treatment groups 6 weeks after tumor implantation relative to the left-limb controls (p b 0.001); with these XRT-associated increases in BMD being consistent with our previous work in growing bone [7] (Fig. 1c). Significant bone destruction was observed using μCT imaging in limbs implanted with ESB but not in limbs implanted with ESB and exposed to 20 Gy XRT (Fig. 1d). Fractional metaphyseal femoral bone volume (Bv/Tv) was decreased in the limbs implanted with ESB relative to left-limb controls while Bv/Tv was
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Fig. 1. ESB cells injected into the distal femur of mice resulted in bone destruction. We developed a primary ESB tumor model by inoculating the distal femur within the right-limb of growing mice with TC71 ESB tumor cells. The knees of the right-limbs were then exposed to 0, 5 and 20 Gy XRT, with the left-limbs serving as internal controls (n = 44 for 0 Gy; n = 10 for 5 Gy; n = 20 for 20 Gy). Radiographs (a) showed bone destruction in the tumor bearing limbs not treated with XRT (arrow). %Fat (b) and BMD (c) were quantified using DEXA in mice after tumor implantation (n = 39 for 0 Gy; n = 10 for 5 Gy; n = 10 for 20 Gy). %Fat was measured to be 3.85% greater in the tumor-bearing limbs not treated with XRT (* = p b 0.001). BMD was diminished 6.11% in tumor-bearing limbs not treated with XRT relative to control limbs while BMD increased 8.45% after 5 Gy and 6.93% after 20 Gy XRT. ANCOVA was used to differentiate between the linear regression lines for each group versus control limbs (p b 0.001). Saggittal images (d) and Bv/Tv measurements (e) were performed using μCT (n = 4 for 0 Gy; n = 5 for 5 Gy; n = 6 for 20 Gy). Tumor-bearing limb Bv/Tv values were found to be 55.4% less than control limbs while after 20 Gy XRT Bv/Tv values increased 49.4% (* = p b 0.04).
increased in the 5 and 20 Gy XRT groups relative to non-irradiated controls (p b 0.04) (Fig. 1e). ESB tumor cells were observed to be in direct contact with osteoclasts Femurs implanted with ESB and not treated with XRT possessed numerous cortical discontinuities (Fig. 2a). Strong CD99 expression was observed in the bone marrow of mice implanted with ESB that did not receive XRT (Fig. 2b). CD99-positive ESB cells were closely associated with TRAP-staining multinucleate cells that express the enzyme cathepsin K, which is required for bone reabsorption (Fig. 2b).
Multinucleate, TRAP-positive and cathepsin K-positive cells are likely osteoclasts, which are the myeloid-lineage cells that regulate normal bone reabsorption [8]. Following the administration of 20 Gy XRT we were unable to detect CD99-positive ESB cells in the bone marrow (Fig. 2c). XRT adversely affected longitudinal bone growth XRT resulted in an increase in limb-length discrepancy, which results when one limb is growing at a slower rate than the contralateral limb (Fig. 3a). Limb-length discrepancy increased 91.1% and
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Fig. 2. ESB tumor cells were observed to be in direct contact with osteoclasts. Femurs implanted (a) with ESB and not treated with XRT possessed numerous cortical discontinuities (arrows) (n = 4 for 0 Gy; n = 4 for 5 Gy; n = 4 for 20 Gy). CD99 is a surface marker that is highly expressed in ESB. Numerous CD99-positive cells were observed in the bone marrow of mice implanted with ESB that did not receive XRT (b). CD99-positive ESB cells were closely associated with TRAP staining multinucleate cells that express the enzyme cathepsin K, which is required for bone reabsorption (arrows). Following the administration of 20 Gy XRT we were unable to detect CD99-positive ESB cells in the bone marrow (c).
96.4% after 5 and 20 Gy XRT (p b 0.001) (Fig. 3b). Growth plate chondrocyte organization was disorganized after 5 and 20 Gy XRT, consistent with impaired longitudinal growth (Fig. 3c). An analysis of growth plate height demonstrated that 5 Gy XRT reduced height by 30.6% (p b 0.015) whereas after 20 Gy XRT the growth plate heights were apparently larger (40.1%) than controls (p b 0.025). The relatively non-cellular appearance of the 20 Gy growth plates coupled with the growth plate heights that we measured suggest that longitudinal growth ceased in the proximal tibial growth plates following 20 Gy XRT and was sufficient to induce tissue level necrosis, a phenomenon that has been observed previously when the XRT dose exceeds a poorly understood dose threshold [9] (Fig. 3d). Mean terminal hypertrophic chondrocyte heights are strongly correlated with the growth rate [10]. We found that mean terminal hypertrophic chondrocyte heights decreased for the right-limb tumor bearing limbs (0 Gy) and the 5 and 20 Gy XRT treatment groups relative to the left-limb controls (Fig. 3e). The unexpected decrease observed in the right-limb tumor bearing limbs that did not receive XRT (0 Gy) is likely due to an increase in IGF produced by the tumor [11]. Otherwise, the decrease in terminal hypertrophic chondrocyte height following 5 and 20 Gy reflect the decreased limb-length observed for these two treatment groups. ESB tumor bearing mice were observed to have splenomegaly and increased numbers of osteoclasts in tumor-inoculated limbs The spleen weights from tumor-bearing mice that were not treated with XRT were 41% greater than control mice and the 20 Gy treatment group (p b 0.01) (Fig. 4a). Spleens from tumor-bearing mice that were not administered XRT were observed to have increased CD14 staining (monocytes) in the presence of CD99 staining cells (ESB); whereas following 20 Gy XRT CD14 staining was diminished and CD99 was not
observed (Fig. 4b). Differential blood smears were prepared to examine whether a particular white blood cell component was causing the increased spleen weight (Figs. 4c, d). The numbers of monocytes were significantly increased 78.4% over controls and the 20 Gy treatment group (p b 0.002). TRAP staining was observed to be highly increased in the tumor-bearing mice that did not receive XRT while limbs exposed to 20 Gy XRT were observed to possess significantly fewer TRAPpositive multinucleate cells (Fig. 4e). TRAP-positive osteoclasts were quantified and found to be 54.6% increased in the tumor-bearing mice that were not exposed to XRT relative to control left-limbs. After 20 Gy XRT osteoclast numbers decreased 60% relative to left-limb controls (p b 0.001); consistent with previous work that demonstrated a similar 71.9% decrease in osteoclast number after 17.5 Gy XRT [3]. ESB tumor cells express MSCF and do not express RANK-ligand To test a potential interaction of between ESB cells and osteoprogenitors we administered ESB conditioned media (ESBCM) to osteoblasts (OB) cultured from human trabecular fragments and monocytes derived from corresponding bone marrow (BM) aspirates, both retrieved during total hip arthroplasty. RANK-ligand represents the most common and potent route for osteoclast formation and lytic tumor progression in osteotropic tumors [12]. The addition of conditioned media (CM) to monocyte cultures demonstrated that ESB was unable to directly cause monocyte differentiation into osteoclasts; with cultures treated with ESB CM or monocytes and ESB cells were cocultured (Fig. 5a). TRAP-positive staining was only observed in RANKligand + MCSF-treated positive controls (Fig. 5a). The addition of ESBCM significantly increased osteoblast numbers 8.0%, 16.7% and 17.1% for the 0.1%, 0.5% and 1% conditioned media concentrations, relative to the control conditioned media derived from the MB231 breast
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Fig. 3. The adverse effects of XRT on longitudinal bone growth. Radiographs (a) showed limb-length discrepancy (n = 15 for 0 Gy; n = 4 for 5 Gy; n = 14 for 20 Gy). When compared to control limbs, limb length discrepancy (b) was increased 91.1% and 96.4% after 5 Gy and 20 Gy XRT, respectively (* = p b 0.001). Histology (c) revealed significant cellular disorganization in growth plates that have been exposed to XRT. An analysis of growth plate heights (d) showed a 30.6% (p b 0.015) decrease after 5 Gy XRT and a 40.1% increase after 20 Gy XRT (* = p b 0.025) relative to controls. Hypertrophic chondrocyte heights (e) decreased 25.3%, 38.6% and 91.1% after 0, 5 and 20 Gy XRT (* = p b 0.01) relative to control terminal hypertrophic chondrocytes heights.
carcinoma and HBL100 breast epithelium cell-lines (p b 0.001) (Fig. 5b). Control cell-lines were chosen because both express PTHrP; and, metastatic breast carcinoma, in particular, is known to promote osteoprogenitor proliferation through a mechanism called the ‘viscous cycle’ that we had originally hypothesized would be the mechanism for ESB tumor progression [13]. Surprisingly, experiments that tested the ESBCM, MB321CM or HBL100CM effects on bone marrow derived monocytes produced significant increases of 43.4%, 35.9% and 28.3% in monocyte numbers for the 0.1%, 0.5% and 1% ESBCM concentrations relative to control conditioned media (p b 0.02) (Fig. 5c). An analysis of gene expression demonstrated that the ESB cell-lines (RDES, HS822.t, HS863.t) express MCSF and not RANK-ligand while, in parallel,
MCSF and RANK-ligand were expressed in SaOS2 osteosarcoma tumor cells and human MSC (Fig. 5d). Protein expression was confirmed using Western blots and immunocytochemistry and ESB tumor cell lines were observed to only express MCSF and not RANK-ligand, while the SaOS2 and MSC expressed both MCSF and RANK-ligand (Figs. 5e and f). Archival Ewing's sarcoma of bone tumor biopsy samples did not stain with RANK-ligand but did stain with MCSF Ewing's sarcoma of bone and osteosarcoma tumor biopsy samples were stained H&E, which showed tumor associated with bone tissue
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Fig. 4. ESB tumor bearing mice were observed to have splenomegaly and increased numbers of osteoclasts in tumor-inoculated limbs. Spleens (n = 10 for 0 Gy; n = 10 for 20 Gy) were weighed (a) and were found to be larger in mice inoculated with tumor but not treated with XRT (* = p b 0.01) and stained for CD14 (monocytes, green) and CD99 (ESB; red) expression (b). Monocyte numbers were quantified (c) (* = p b 0.002) from differential blood smears prepared from whole blood (d). TRAP stained (red staining) femurs (e) were used to quantify osteoclast numbers (f). Tumor-bearing limbs had 54.6% more osteoclasts than controls, while osteoclasts numbers were decreased 60% after 20 Gy XRT (* = p b 0.001).
(Figs. 6a and e). Ewing's sarcoma of bone tumor samples strongly stained with the ESB specific tumor marker CD99 (Fig. 6b). Ewing's sarcoma of bone tumor samples expressed MCSF but not RANK-ligand (Figs. 6c and d). In contrast, osteosarcoma tumor samples, which served as positive controls, stained with MCSF and RANK-ligand (Figs. 6f and g). Discussion Prior to the work presented herein, there were no mouse models of primary sarcoma and this work represents the first mouse model of primary Ewing's sarcoma of bone. One of the goals of this work was to characterize our Ewing's sarcoma animal model and then determine if this model would be useful for testing potential therapies in the future. In the context of a therapeutic challenge to our model, we chose to expose mice implanted with tumor to X-radiation therapy, in part, due to the ESB sensitivity to XRT. Treating Ewing's sarcoma of bone with XRT often results in limb-length asymmetry, which is a crippling complication of therapy that we observed in our mouse model. The implanted ESB tumor cells also produced significant bone lysis that was effectively treated with XRT. Both the limb-length asymmetry, the ESB tumor associated bone lysis, efficacy of XRT at treating the ESB that we observed are consistent with clinical experience [2,9,13–15,
21–27]. One of our initial hypotheses was that ESB would express a growth factor that would mediate significant bone lysis via increased numbers of osteoclasts or accelerated osteoclastic bone reabsorption. Examination of the ESB cell lines revealed that they expressed high levels of MCSF, but did not express RANK-ligand, the latter of which is one of the most potent stimulators of osteoclast activity. Increased expression of MCSF in the ESB tumor cells suggested that there would be an increase in the numbers of monocytes associated with ESB tumor cells, which we observed in peripheral blood and in the spleens of mice implanted with ESB tumor. Further, an examination of archival ESB biopsy samples corroborated our findings by demonstrating that ESB tumor samples expressed MCSF and not RANK-ligand. In this regard, we believe that our mouse model ESB tumor represents a more promising means for assessing therapeutic interventions in future work. Our initial hypothesis was that Ewing's sarcoma of bone tumor cells would stimulate osteoclast bone reabsorption, which would promote bone lysis. However, we were unable to detect RANK-ligand in ESB tumor cells or in archival biopsy samples of Ewing's sarcoma of bone. This is in contrast to the work of Taylor et al., who identified RANKligand expression and MCSF expression in ESB tumor cells and tumor needle biopsy samples [20]. Recent work by Fujiwara et al. has identified
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Fig. 5. ESB tumor cells express MSCF and do not express RANK-ligand. The addition of ESB CM to cultures (a) did not result in the formation of osteoclasts. CM from HBL100 and MB231 control cells were also unable to stimulate osteoclast formation. In control cultures, in which both MCSF and RANK-ligand were added, we observed robust osteoclastogenesis. We also tested the effects of CM on osteoblast proliferation (b) and found no significant effect from ESB, HBL100 or MB2321 cells. The addition of ESB CM to primary human monocytes (c) resulted in a significant increased in monocyte cell proliferation 43.4%, 35.9% and 28.3% after the addition of 0.1%, 0.5% or 1% ESB CM relative to the CM controls cultures treated with HBL100 or MB231 CM (* = p b 0.02). We tested different ESB tumor cell lines for MCSF gene expression (d) in parallel with control cells SaOS2 cells (osteosarcoma) and human MSC. We were unable to observed RANK-ligand gene expression. We used immunochemistry (e) to test if ESB tumor cells, SaOS2 cell or human MSC expressed MCSF and RANK-ligand in parallel. While the ESB lines expressed MCSF, we were unable to detect RANK-ligand. Whereas, in both SaOS2 and human MSC MCSF and RANK-ligand were detected. We tested protein lysates for the expression of MCSF (f). We observed robust MCSF (green bands) expression in all of the ESB lines tested. Actin served as a loading control (green bands). Molecular weight markers are shown as red bands.
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Fig. 6. Archival Ewing's sarcoma of bone tumor biopsy samples did not stain with RANK-ligand but did stain with MCSF. Ewing's sarcoma of bone (a) and osteosarcoma (e) tumor biopsy samples were stained H&E, which showed tumor associated with bone tissue. (b) Ewing's sarcoma of bone tumor samples strongly stained with the ESB specific tumor marker CD99 (brown/red staining). Ewing's sarcoma of bone tumor samples expressed MCSF (c) (MCSF = brown/red staining) but not RANK-ligand (c). Osteosarcoma tumor samples served as positive controls that stained with MCSF (f) and RANK-ligand (g).
TRAP-positive tumor-associated-macrophages (TAM) located near subcutaneously implanted Ewing's tumor in a Balb/c mouse model [14]. Further, these investigators identified that MCSF and RANK-ligand gene expression were observed in several Ewing's sarcoma tumor cell lines. Our work also disagrees with the expression of RANK-ligand observed in the Fujiwara et al. study. Both Taylor et al. and Fujiwara et al. detected RANK-ligand in ESB tumor cells; however, only Taylor et al. identified RANK-ligand in needle biopsy samples. One of the strengths of our study was that we employed an osteosarcoma cell line positive control and archival osteosarcoma biopsy sample positive control, both of which expressed MCSF and RANK-ligand. This study, the work of Taylor et al., and Fujiwara et al. all agree that MCSF is expressed by ESB. Nevertheless, future work will need to be completed to determine what role RANK-ligand may or may not play in mediating ESB driven bone lysis. The observation of elevated MCSF expression lead us to examine monocyte number since monocytes are acutely sensitive to the MCSF growth factor, which stimulates proliferation and promotes survival. These observation lead to the hypothesis that the increased MCSF produced by the ESB tumor cells would increase the numbers of monocytes. We observed that TRAP-positive, cathepsin K-positive multinucleate osteoclasts were observed in the bone marrow niche occupied by the tumor in the tumor-bearing mice. Further, we hypothesized that the application of XRT would result in decreased tumor burden and corresponding monocyte numbers, due to the well-known radiosensitivity of both monocytes and ESB tumor cells [16]. Additionally, we observed that the numbers of osteoclasts were significantly diminished after the application of XRT; an observation that is consistent
with our previous work [7]. In the latter regard, our work also agrees with Fujiwara et al., in which they identified TRAP-positive multinucleate cells could reduced in number by chemically depleting the monocyte fraction. XRT has proven to be effective at treating ESB, with XRT being applied before or in some cases in lieu of surgical excision [1,2, 15]. Thus, the efficacy of XRT in treating ESB may derive in part from the extreme sensitivity of ESB (D0 = 1.14 Gy) tumor cells and monocytes (D0 = 0.94 Gy) to XRT; as was demonstrated in our previous work [16]. Therefore, we propose a mechanism through which XRT is effective as a treatment for ESB by concurrently decreasing the circulating concentration of MCSF via diminished numbers of ESB tumor cells and the available pool of progenitors (monocytes) able to differentiate into osteoclasts. Conclusions Using a novel Ewing's sarcoma of bone mouse model, we demonstrated that ESB tumor growth in bone is driven by increased osteoclastic bone reabsorption that we associated with increased numbers of monocytes. We further identify that the increase in monocyte numbers is due to the expression of MCSF produced by ESB tumor cells; with the application of XRT resulting in decreased tumor burden and corresponding monocyte numbers. Additionally, we found that ESB tumor cells do not express the most potent regulator of osteoclast differentiation from monocytes, RANK-ligand. Thus, our current work suggests a mechanism through which the depletion of monocytes coupled with diminished MCSF expression from XRT results in decreased tumor burden.
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Acknowledgments Margulies: Department of Orthopedic Surgery Funding and NIH NCCAM (F31AT001994) F31 Kirschstein NRSA Individual Pre-doctoral Fellowship; Damron: NIH NCI (R01CA083892) and the David G. Murray Endowed Professorship. Judith A. Strauss for tissue processing and Gustavo de la Roza, MD for providing access to the human sarcoma tissue samples. References [1] Gurney JG, Swensen AR, Bulterys M. Malignant bone tumors. In: Ries LAG, Smith MA, Gurney JG, Linet M, Tamra T, Young JL, Bunin GR, editors. Cancer Incidence and Survival among Children and Adolescents: United States SEER Program 19751995, NCI, SEER Program. Bethesda, MD: Cancer Statistics Branch; 1999. p. 99–110 [NIH Pub. No. 99-4649.]. [2] Butler MS, Robertson Jr WW, Rate W, D'Angio GJ, Drummond DS. Skeletal sequelae of radiation therapy for malignant childhood tumors. Clin Orthop 1990;251:235–40. [3] Damron TA, Spadaro JA, Horton JA, Margulies BS, Strauss JA, Farnum CE. Novel radioprotectant drugs for sparing radiation-induced damage to the physis. Int J Radiat Biol 2004;80(3):217–28. [4] Damron T, Margulies B, Strauss J, Spadaro J, Farnum C. Sequential histomorphometric changes following irradiation of the growth plate with and without radioprotectant. J Bone Joint Surg Am 2003;85-A:1302–13. [5] Damron TA, Horton JA, Naqvi A, Loomis RM, Margulies BS, Strauss JA, et al. Combination radioprotectors maintain proliferation better than single agents by decreasing early parathyroid hormone-related protein changes after growth plate irradiation. Radiat Res 2006;165(3):350–8. [6] Scotlandi K, Benini S, Manara MC, Serra M, Nanni P, Lollini PL, et al. Murine model for skeletal metastases of Ewing's sarcoma. J Orthop Res 2000;18(6):959–66. [7] Margulies B, Morgan H, Allen M, Strauss J, Spadaro J, Damron T. Transiently increased bone density after irradiation and the radioprotectant drug amifostine in a rat model. Am J Clin Oncol 2003;26(4):e106–14. [8] Henriksen K, Tanko LB, Qvist P, Delmas PD, Christiansen C, Karsdal MA. Assessment of osteoclast number and function: application in the development of new and improved treatment modalities for bone diseases. Osteoporos Int 2007;18(5):681–5. [9] Barr JS, Lingley JR, Gall EA. The effect of roentgen irradiation on epiphyseal growth: I. Experimental studies on the albino rat. Am J Roentgenol 1943;49(1):104–15. [10] Damron TA, Mathur S, Horton JA, Strauss J, Margulies B, Grant W, et al. Temporal changes in PTHrP, Bcl-2, Bax, caspase, TGF-beta, and FGF-2 expression following growth plate irradiation with or without radioprotectant. J Histochem Cytochem 2004;52(2):157–67.
[11] Fisher MC, Meyer C, Garber G, Dealy CN. Role of IGFBP2, IGF-I and IGF-II in regulating long bone growth. Bone 2005;37(6):741–50. [12] Clohisy DR, Ogilvie CM, Ramnaraine ML. Tumor osteolysis in osteopetrotic mice. J Orthop Res 1995;13(6):892–7. [13] Guise TA, Mohammad KS, Clines G, Stebbins EG, Wong DH, Higgins LS, et al. Basic mechanisms responsible for osteolytic and osteoblastic bone metastases. Clin Cancer Res 2006;12(20 Pt 2):6213s–6s. [14] Fujiwara T, Fukushi J, Yamamoto S, Matsumoto Y, Setsu N, Oda Y, et al. Macrophage infiltration predicts a poor prognosis for human Ewing sarcoma. Am J Pathol 2011; 179(3):1157–70. [15] Bölling T, Hardes J, Dirksen U. Management of bone tumours in paediatric oncology. Clin Oncol (R Coll Radiol) 2013;25(1):19–26. [16] Margulies BS, Damron TA, Allen MJ. The differential effects of the radioprotectant drugs amifostine and sodium selenite treatment in combination with radiation therapy on constituent bone cells, Ewing's sarcoma of bone tumor cells, and rhabdomyosarcoma tumor cells in vitro. J Orthop Res 2008;26(11):1512–9. [17] Tamurian RM, Damron TA, Spadaro JA. Sparing radiation-induced damage to the physis by radioprotectant drugs: laboratory analysis in a rat model. J Orthop Res 1999;17(2):286–92. [18] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29(9):2002–7. [19] Erben RG. Embedding of bone samples in methylmethacrylate: an improved method suitable for bone histomorphometry, histochemistry, and immunohistochemistry. J Histochem Cytochem 1997;45(2):307–13. [20] Taylor R, Knowles HJ, Athanasou NA. Ewing sarcoma cells express RANKL and support osteoclastogenesis. J Pathol 2011;225(2):195–202. [21] Choi Y, Lim DH, Lee SH, Lyu CJ, Im JH, Lee YH, et al. Role of radiotherapy in the multimodal treatment of Ewing sarcoma family tumors. Cancer Res Treat Feb 16 2015 [Epub Ahead of Print]. [22] Burchill SA. Ewing's sarcoma: diagnostic, prognostic, and therapeutic implications of molecular abnormalities. J Clin Pathol 2003;56:96–102. [23] Costantino PD, Friedman CD, Steinberg MJ. Irradiated bone and its management. Otolaryngol Clin North Am 1995;28(5):1021–38. [24] Koscielniak E, Morgan M, Treuner J. Soft tissue sarcoma in children: prognosis and management. Paediatr Drugs 2002;4(1):21–8. [25] Merchant TE, Kushner BH, Sheldon JM, LaQuaglia M, Healey JH. Effect of low-dose radiation therapy when combined with surgical resection for Ewing sarcoma. Med Pediatr Oncol 1999;33(2):65–70. [26] Paulussen M, Frolich B, Jurgens H. Ewing tumor: incidence, prognosis and treatment options. Paediatr Drugs 2001;3(12):899–913. [27] Wall JE, Kaste SC, Greenwald CA, Jenkins JJ, Douglass EC, Pratt CB. Fractures in children treated with radiotherapy for soft tissue sarcoma. Orthopedics 1996; 19(8):657–64.
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Bone journal homepage: www.elsevier.com/locate/bone
Original Full Length Article
Ewing's sarcoma of bone tumor cells produces MCSF that stimulates monocyte proliferation in a novel mouse model of Ewing's sarcoma of bone B.S. Margulies a,⁎, S.D. DeBoyace a, T.A. Damron a, M.J. Allen b a b
SUNY Upstate Medical University, Department of Orthopedic Surgery, USA The Ohio State University, College of Veterinary Medicine, Department of Veterinary Clinical Medicine, USA
a r t i c l e
i n f o
Article history: Received 4 June 2014 Revised 14 May 2015 Accepted 28 May 2015 Available online 5 June 2015 Edited by: Robert Recker Keywords: Ewing's sarcoma Osteoblasts Monocytes Osteoclasts Bone growth MCSF
a b s t r a c t Ewing's sarcoma of bone is a primary childhood malignancy of bone that is treated with X-radiation therapy in combination with surgical excision and chemotherapy. To better study Ewing's sarcoma of bone we developed a novel model of primary Ewing's sarcoma of bone and then treated animals with X-radiation therapy. We identified that uncontrolled tumor resulted in lytic bone destruction while X-radiation therapy decreased lytic bone destruction and increased limb-length asymmetry, a common, crippling complication of X-radiation therapy. Osteoclasts were indentified adjacent to the tumor, however, we were unable to detect RANK-ligand in the Ewing's tumor cells in vitro, which lead us to investigate alternate mechanisms for osteoclast formation. Ewing's sarcoma tumor cells and archival Ewing's sarcoma of bone tumor biopsy samples were shown to express MCSF, which could promote osteoclast formation. Increased monocyte numbers were detected in peripheral blood and spleen in animals with untreated Ewing's sarcoma tumor while monocyte number in animals treated with x-radiation had normal numbers of monocytes. Our data suggest that our Ewing's sarcoma of bone model will be useful in the study Ewing's sarcoma tumor progression in parallel with the effects of chemotherapy and X-radiation therapy. © 2015 Elsevier Inc. All rights reserved.
Introduction Ewing's sarcoma of bone (ESB) is a malignant, primary bone tumor of childhood; accounting for approximately 7% of pediatric bone tumors [1]. ESB is first treated with neo-adjuvant chemotherapy followed by Xradiation therapy (XRT), surgical excision or XRT in combination with surgical excision. Recent work by Choi et al. highlighted the controversy surrounding the efficacy of XRT alone, surgical excision alone or XRT in combination with surgical excision [21]. Unfortunately, the delivery of XRT to the growing skeleton is associated with a significant risk of crippling limb-length asymmetry and pathologic fracture [2]. In previous work, we sought to mitigate the negative effects of XRT administered to the growing bone through the use of chemoradioprotection drug therapies [3–5]. Nevertheless, the mechanism through which XRT has proven so effective as a treatment in ESB patients remains poorly understood. Further, the bone marrow signalingenvironment that facilitates ESB tumor progression remains unknown. Bone degradation (lysis) that occurs during ESB tumor expansion in bone is most likely mediated through a myeloid lineage cell, such as macrophages or osteoclasts. We have proposed that ESB tumor cells ⁎ Corresponding author at: SUNY Upstate Medical University, 750 E. Adams Street, 3113 Institute for Human Performance, Syracuse, NY, USA 13210. Fax: +1 315 464 6638. E-mail address: [email protected] (B.S. Margulies).
http://dx.doi.org/10.1016/j.bone.2015.05.041 8756-3282/© 2015 Elsevier Inc. All rights reserved.
alter the myeloid progenitor cell niche in the bone marrow as a means of creating a more favorable bone environment that favors sarcoma tumor growth. However, it remains unknown what factors mediate this process or how these tumor-produced factors may direct the fate of myeloid progenitors. Monocytes are hematopoietic progenitors of the myeloid-lineage that differentiate into macrophages, osteoclasts and dendritic cells. Monocyte differentiation is directed by number of growth factors that include MCSF, GCSF and GMCSF; with MCSF required for proliferation and GMCSF required hematopoietic stem cell differentiation. In addition, osteoclast differentiation requires interaction with mesenchymal lineage cells that express the ligand for the receptor-activator of NFκB (RANK-ligand). Monocytes and ESB are highly sensitive to XRT; with our previous in vitro work identifying the specific XRT-dose sensitivity, or D0-value, in both monocytes (0.94 Gy) and ESB (1.14 Gy) [16]. Further, the loss of monocytes after 17.5 Gy XRT in vivo resulted in decreased numbers of osteoclasts and increased bone mass [7]. To study ESB tumor progression we developed a novel mouse model of primary ESB tumor by injecting ESB tumor cells directly into the femoral bone marrow of a mouse. Our initial hypothesis was that ESB tumor associated bone lysis would be due to increased numbers of osteoclasts derived from an increase in RANK-ligand produced by the ESB tumor. In preliminary experiments using this novel mouse model we were unable to identify RANK-ligand gene expression in
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TC71 ESB tumor cells, however, we were able to identify MCSF gene expression. This lead us to hypothesize that ESB mediated osteolysis is due to an increase in the numbers of monocytes, which would in turn lead to increased numbers of osteoclasts and lytic bone destruction. We also hypothesized that the efficacy of XRT to treat ESB may be due the radio-sensitivity of monocytes in parallel with the radio-sensitivity of ESB, consistent with previous data [16]. Here we report that ESB tumor expansion in bone is driven by increased osteoclastic bone re-absorption due to increased numbers of monocytes, using a novel in vivo mouse model of ESB tumor progression. We also identify that the increase in monocyte numbers is due to the expression of MCSF produced by ESB tumor cells; with the application of XRT resulting in decreased tumor burden and corresponding monocyte numbers. Additionally, we found that ESB tumor cells do not express the most potent regulator of osteoclast differentiation from monocytes, RANK-ligand. We confirmed these observations in primary Ewing's sarcoma of bone biopsy samples. Thus, our current work suggests a mechanism through which the depletion of monocytes coupled with diminished MCSF expression from XRT results in decreased tumor burden. Methods Mouse model of primary Ewing's sarcoma of bone Skeletally immature female NCr nude mice (n = 66) were anesthetized with Telazol (45-mg/kg IM) and xylazine (7.5-mg/kg IM). Using a medial para-patellar approach, a sterile 26 G needle was inserted through the intra-patellar notch to a depth of 5 mm in the distal femur. A suspension of ESB TC71 tumor cells (1 × 105 cells in 20 μL) was injected into the right femoral medullary canal. Left femurs served as controls that were not implanted with tumor and were not treated. Tumor progression was observed with serial X-radiographs taken 2, 3 and 6 weeks after tumor inoculation. Animals were anesthetized using Telezol (30-mg/kg IP) prior to XRT with both limbs extended and placed under a therapeutic X-ray machine (Phillips MGC-30, operating at 300 kV and 10 mA and an effective dose rate 0.256 Gy/min for a 2 cm × 4 cm field, at a distance of 5 cm) and exposed to 0 Gy (n = 44), 5 Gy (n = 10) and 20 Gy (n = 10). Using beam collimation and lead shielding, only the right knee was exposed (distal half of the femur and proximal half of the tibia) [17]. At the completion of the experimental period or when the animals showed signs of distress/lameness, they were euthanized using carbon dioxide. The SUNY Upstate Medical University Institutional Animal Care and Use Committee approved all of the animal studies described in this paper. Tissue collection and histology Tibia were split and fixed in 2% glutaraldehyde + 2% paraformaldehyde + 0.7% hexa-amine ruthenium (III) chloride (RHT) in a 0.1 M cacodylate buffer (n = 4 for 0 Gy; n = 4 for 5 Gy; n = 4 for 20 Gy). Tibial halves were embedded in methyl methacrylate (PMMA) and sagitally sectioned on a rotary microtome at 5 μm [19]. Tibial sections were stained for bright-field observation and analysis with a combination of 2% periodic acid, 1% methylene blue (in 1% Borax), 0.15% basic fuchsin (in 10% ETOH), and azure II (with azure II and basic fuchsin mixed in equal parts). Alternatively, tibial sections were stained with 2% methyl green following immuno-histochemistry. Antibodies obtained from Santa Cruz Biotechnologies that recognized CD14 (1:250), CD99 (1:250) and cathepsin K (1:100) were detected using biotin conjugated secondary antibody (1:500) visualized by reacting streptavidin-horseradish peroxidase (VectaStain ABC kit, Vector Labs) with the chromogen 3, 3′-diaminobenzadine tetrahydrochloride (DAB, Biogenix). Alternatively, an anti-mouse-rabbit secondary antibody that is conjugated to a very active peroxidase micro-polymer (ImmPress kit, Vector Labs) was reacted with the chromogen Vecta NovaRed (Vector Labs). Tartrate resistant acid phosphatase (TRAP)
staining (Leukocyte Acid Phosphatase Kit 387-A, Sigma) was used to identify osteoclasts (n = 4 for 0 Gy; n = 4 for 20 Gy). DEXA analysis Animals were evaluated with a dual energy X-ray absorptometry (DEXA) 2, 3 and 6 weeks after tumor inoculation (n = 39 for 0 Gy; n = 10 for 5 Gy; n = 10 for 20 Gy) using a Lunar PixiMus2 (General Electric) scanner. A region of interest (ROI) was manually drawn around the right and lefts femur. Bone mineral density (BMD) was calculated by dividing the bone mineral content (BMC) by the cross-sectional area (A) of the mineralized tissue within the ROI for the right and left femurs using the proprietary software provided with the scanner. Whole body %Fat mass was calculated using the provided software. Limb length measurements of growth Radiographs of all the limbs were taken at baseline and following dissection (n = 15 for 0 Gy; n = 4 for 5 Gy; n = 14 for 20 Gy). Radiographs were subsequently scanned and measured using ImageJ analysis software (National Institutes of Health Research Services Branch http:// rsbweb.nih.gov/ij/) to determine femoral, tibial, and overall limb-length discrepancy. Limb-length discrepancy represents a measure of lost growth function by comparing the irradiated limb to the contralateral control limbs and represents the loss in cumulative growth (discrepancy = [Δ(left length) − Δ(right length)/Δ(left length)]) [3–5,17]. Measurement of total plate heights and terminal hypertrophic chondrocyte height Total growth plate heights were measured by selecting a measurement region bisected by the long axis of the tibia (n = 4 for 0 Gy; n = 4 for 5 Gy; n = 4 for 20 Gy). Lines were drawn along the contours of both the epiphysis and the chondro-osseous junction. The mean distance between the epiphyseal contour line and the chondro-osseous junction contour line was measured from three sections using ImageJ. Terminal, hypertrophic-chondrocyte heights were measured by following individual hypertrophic chondrocytes through serial section and measuring their greatest height. In both right and left limbs, counts of 300 terminal hypertrophic chondrocytes were obtained from each animal [10]. Measurement of bone volumes and 3D micro-architecture Femora were placed in PBS for μCT and stored at −20 °C. The distal 20% of the femur was scanned using a μCT (μCT40, Scanco) to ascertain 3D micro-architecture and tumor associated bone osteolysis using an isotrophic voxel resolution of 12 μm3 (n = 4 for 0 Gy; n = 5 for 5 Gy; n = 6 for 20 Gy). The distal 20% distance was determined as a percentage of the total length of bone. Thereafter, bone volumes were calculated as a percentage of the total tissue volume (Bv/Tv) using the proprietary analysis software supplies with the scanner. Spleen weights and differential blood smears Spleens were removed during routine necropsy, weighed and subsequently placed into 10% neutral buffered formalin (n = 10 for 0 Gy; n = 10 for 20 Gy). Differential blood smears were prepared from blood drawn from the tail-vein after euthanasia (n = 10 for 0 Gy; n = 10 for 20 Gy). A drop of blood was placed on a glass slide and smeared with cover glass. The blood smear was allowed to dry and the fixed with 70% ethanol and stained with a commercially prepared Giemsa stain (Sigma). Differential-cell counts were made by counting 100 cells in total from each of three slides produced for each animal in each experimental group.
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Tissue culture The Ewing's sarcoma of bone tumor cell line TC71 (Dr. Tim Triche, USC/CHLA) were maintained in RPMI 1640 media while the Ewing's sarcoma of bone tumor cell lines RD-ES, HS822.t and HS863.t (ATCC) were maintained in DMEM and then supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin–streptomycin–glutamine (PSG). The MB231 breast carcinoma and HBL100 breast epithelium cell-lines were maintained in DMEM supplemented with 10% FCS and 1% PSG as controls that express RANK-ligand for conditioned media experiments. In order to produce conditioned media (CM); cells were seeded at 1 × 104 cells per well (5 × 103 cells/cm2), which results in an actively proliferating culture. CM or stale media were collected 72 h after the start of culture from each tumor cell (ESBCM, MB231CM and HBL100CM), filtered and then 0%, 0.1%, 0.5%, 1%, 5% and 10% (v/v) aliquots were added to fresh media. To test an interaction of between ESB cells and osteoprogenitors we administered ESB conditioned media (ESBCM) to osteoblasts (OB) cultured from human trabecular fragments and monocytes derived from corresponding bone marrow (BM) aspirates, both retrieved during total hip arthroplasty from consenting adult patients as part of an IRB approved study (n = 6). In parallel, we also cultured the adherent fraction of cells derived from bone marrow, which are pluripotent mesenchymal stem cells (MSC). MSC allowed grown to confluence, at which point they begin to express a primitive osteoblastic phenotype. Primary human OBs were dissociated from metaphyseal trabecular bone fragments removed during total hip-arthroplasty using a mixture of 1% collagenase (Worthington Biochemical Corp.) 0.25% trypsin-EDTA (Cellgro) mixture. Primary human bone marrow cells were collected using gradient centrifugation (lymphocyte separation media; Lonza) from bone marrow aspirates. Bone marrow cells were seeded at 1 × 107 into T-25, 25 mm2 flasks and maintained in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) PSG and 100 ng/mL of recombinant human MCSF for three days. Non-adherent MCSF-dependent bone-marrow cells (monocytes) were collected and then seeded at 1 × 106 cells per well supplemented with 25 ng/mL MCSF and treated with CM and then assayed for osteoclast formation by staining for TRAP expression. Positive controls cultures were administered 25-ng/mL of MSCF with and without the addition of 25 ng/mL recombinant human RANK-ligand (R&D Systems). OB and monocytes were also seeded into culture plates at 1 × 104 cells per well (5 × 103 cells/cm2) and assayed for CM effects on proliferation using the MTT assay (5 mg/ml (w/v), Sigma) (n = 3 with n = 4 replicates per condition) [16]. Gene expression analysis Cells were assayed for changes in MCSF and RANK-ligand gene expression. mRNA was purified using RNeasy Plus Mini columns (Qiagen) and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). Gene expression was analyzed using quantitative PCR (qPCR) using 100 ng of cDNA mixed with Fast Plus EvaGreen Master Mix (Biotium). In each experiment GAPDH served as a control, negative controls contained no-template and a standard curve was generated using serial dilutions of a chemically synthesized sequence for GAPDH (0, 1, 5, 10 and 100 fg; Integrated DNA Technologies). Gene expression was evaluated using Pfaffl's method, in which the efficiency of each primer (E) and the starting concentration of gene product (N0) are calculated from the linear region of the fluorescence-crossing threshold curve using the software LinRegPCR (v2013.0) [18]. Experiments were considered valid when the control gene GAPDH fell within the standard curve and the primer efficiencies (E) were calculated to be E N 1.8. Protein expression through western blot analysis Cells were lysed with a cold buffer composed of the following: 1% sodium dodecyl sulfate (SDS), 20 mM TRIS (pH 8.0), 1 mM
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β-glycerophosphate, 150 mM sodium chloride, 2 mM iodoacetamide, 2 mM benzamidine hydrochloride, 0.1 mM ethylmaleimide, 1% PMSF, 1% β mercaptoethanol and the Halt Protease Inhibitor Cocktail (Pierce Thermo Scientific) [16]. Total protein was assayed using the BCA Protein Assay Kit (Pierce Thermo Scientific). Samples were loaded (20 μg/well) onto a 10–20% Mini-Protean Tris-Tricine Precast Gel (Bio-Rad) with the Page Ruler Pre-stained NIR Protein Ladder (Bio-Rad) and transferred to an Immun-Blot Low Fluorescence PVDF Membrane (Bio-Rad). Membranes were blocked using Protein Free T20 (TBS) Blocking Buffer (Pierce Thermo Scientific) and incubated with primary antibodies (Santa Cruz Biotechnologies) directed against RANK-ligand (1:500) and MCSF (1:500). Archival sarcoma biopsy samples Archival primary sarcoma biopsy samples that were collected from patients prior to therapy (n = 5 Ewing's sarcoma of bone and n = 5 osteosarcoma) were sectioned and stained for hematoxylin and eosin (H&E), the ESB specific marker CD99 (1:250), MCSF (1:100) and RANK-ligand (1:200). Sections were counter-stained using 2% methyl green and imaged at 20 ×. Archival primary sarcoma biopsy samples were analyzed as part of an IRB approved study. Statistics Prism (v5.0d, Graphpad Software Inc.) was used for all statistical calculations. Radio-graphically determined limb-lengths, DEXA and μCT for each femur or tibia from each of the 10 animals per treatment group were used to calculate means and standard deviations. Histomorphometric analyses (growth plate heights, terminal hypertrophic chondrocyte height) on each limb (left and right) were done using a minimum of 2 sections separated by 30 μm from 4 animals randomly chosen per group, from which means and standard deviations were calculated. In vitro experiments were done in triplicate (TRAP/MTT and Gene expression). Means and standard deviations were calculated and ANOVA with Bonferroni's post-hoc was used to determine significance (p b 0.05). Results ESB cells injected into the distal femur of mice resulted in bone destruction Previous work by Scotlandi et al. demonstrated that athymic mice inoculated with ESB tumor cells subcutaneously develop metastases to bone and lung [6]. We developed a primary ESB tumor model by injecting TC71 tumor cells into the distal femur of the right-limbs of skeletally immature athymic Ncr mice. Two-weeks after tumor implantation 86% of the animals inoculated with ESB presented with a lytic lesion. At 6-weeks all of the animals that did not undergo XRT had a lytic lesion in bone, of which 75% had significant cortical discontinuity (Fig. 1a). None of the animals receiving 5 or 20 Gy XRT presented with cortical discontinuity (Fig. 1a). Mice implanted with ESB that were not administered XRT were observed to have increased total body fat (%Fat) of 3.85% relative to mice exposed to 20 Gy XRT (p b 0.001) (Fig. 1b). We also compared the right-limb femoral bone mineral density (BMD) to the left-limb femoral BMD 6 weeks after tumor implantation and found that BMD in mice implanted with ESB decreased 6.11% relative to the left-limb controls (Fig. 1c). BMD increased 8.45% for the 5 Gy and 6.93% for the 20 Gy treatment groups 6 weeks after tumor implantation relative to the left-limb controls (p b 0.001); with these XRT-associated increases in BMD being consistent with our previous work in growing bone [7] (Fig. 1c). Significant bone destruction was observed using μCT imaging in limbs implanted with ESB but not in limbs implanted with ESB and exposed to 20 Gy XRT (Fig. 1d). Fractional metaphyseal femoral bone volume (Bv/Tv) was decreased in the limbs implanted with ESB relative to left-limb controls while Bv/Tv was
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Fig. 1. ESB cells injected into the distal femur of mice resulted in bone destruction. We developed a primary ESB tumor model by inoculating the distal femur within the right-limb of growing mice with TC71 ESB tumor cells. The knees of the right-limbs were then exposed to 0, 5 and 20 Gy XRT, with the left-limbs serving as internal controls (n = 44 for 0 Gy; n = 10 for 5 Gy; n = 20 for 20 Gy). Radiographs (a) showed bone destruction in the tumor bearing limbs not treated with XRT (arrow). %Fat (b) and BMD (c) were quantified using DEXA in mice after tumor implantation (n = 39 for 0 Gy; n = 10 for 5 Gy; n = 10 for 20 Gy). %Fat was measured to be 3.85% greater in the tumor-bearing limbs not treated with XRT (* = p b 0.001). BMD was diminished 6.11% in tumor-bearing limbs not treated with XRT relative to control limbs while BMD increased 8.45% after 5 Gy and 6.93% after 20 Gy XRT. ANCOVA was used to differentiate between the linear regression lines for each group versus control limbs (p b 0.001). Saggittal images (d) and Bv/Tv measurements (e) were performed using μCT (n = 4 for 0 Gy; n = 5 for 5 Gy; n = 6 for 20 Gy). Tumor-bearing limb Bv/Tv values were found to be 55.4% less than control limbs while after 20 Gy XRT Bv/Tv values increased 49.4% (* = p b 0.04).
increased in the 5 and 20 Gy XRT groups relative to non-irradiated controls (p b 0.04) (Fig. 1e). ESB tumor cells were observed to be in direct contact with osteoclasts Femurs implanted with ESB and not treated with XRT possessed numerous cortical discontinuities (Fig. 2a). Strong CD99 expression was observed in the bone marrow of mice implanted with ESB that did not receive XRT (Fig. 2b). CD99-positive ESB cells were closely associated with TRAP-staining multinucleate cells that express the enzyme cathepsin K, which is required for bone reabsorption (Fig. 2b).
Multinucleate, TRAP-positive and cathepsin K-positive cells are likely osteoclasts, which are the myeloid-lineage cells that regulate normal bone reabsorption [8]. Following the administration of 20 Gy XRT we were unable to detect CD99-positive ESB cells in the bone marrow (Fig. 2c). XRT adversely affected longitudinal bone growth XRT resulted in an increase in limb-length discrepancy, which results when one limb is growing at a slower rate than the contralateral limb (Fig. 3a). Limb-length discrepancy increased 91.1% and
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Fig. 2. ESB tumor cells were observed to be in direct contact with osteoclasts. Femurs implanted (a) with ESB and not treated with XRT possessed numerous cortical discontinuities (arrows) (n = 4 for 0 Gy; n = 4 for 5 Gy; n = 4 for 20 Gy). CD99 is a surface marker that is highly expressed in ESB. Numerous CD99-positive cells were observed in the bone marrow of mice implanted with ESB that did not receive XRT (b). CD99-positive ESB cells were closely associated with TRAP staining multinucleate cells that express the enzyme cathepsin K, which is required for bone reabsorption (arrows). Following the administration of 20 Gy XRT we were unable to detect CD99-positive ESB cells in the bone marrow (c).
96.4% after 5 and 20 Gy XRT (p b 0.001) (Fig. 3b). Growth plate chondrocyte organization was disorganized after 5 and 20 Gy XRT, consistent with impaired longitudinal growth (Fig. 3c). An analysis of growth plate height demonstrated that 5 Gy XRT reduced height by 30.6% (p b 0.015) whereas after 20 Gy XRT the growth plate heights were apparently larger (40.1%) than controls (p b 0.025). The relatively non-cellular appearance of the 20 Gy growth plates coupled with the growth plate heights that we measured suggest that longitudinal growth ceased in the proximal tibial growth plates following 20 Gy XRT and was sufficient to induce tissue level necrosis, a phenomenon that has been observed previously when the XRT dose exceeds a poorly understood dose threshold [9] (Fig. 3d). Mean terminal hypertrophic chondrocyte heights are strongly correlated with the growth rate [10]. We found that mean terminal hypertrophic chondrocyte heights decreased for the right-limb tumor bearing limbs (0 Gy) and the 5 and 20 Gy XRT treatment groups relative to the left-limb controls (Fig. 3e). The unexpected decrease observed in the right-limb tumor bearing limbs that did not receive XRT (0 Gy) is likely due to an increase in IGF produced by the tumor [11]. Otherwise, the decrease in terminal hypertrophic chondrocyte height following 5 and 20 Gy reflect the decreased limb-length observed for these two treatment groups. ESB tumor bearing mice were observed to have splenomegaly and increased numbers of osteoclasts in tumor-inoculated limbs The spleen weights from tumor-bearing mice that were not treated with XRT were 41% greater than control mice and the 20 Gy treatment group (p b 0.01) (Fig. 4a). Spleens from tumor-bearing mice that were not administered XRT were observed to have increased CD14 staining (monocytes) in the presence of CD99 staining cells (ESB); whereas following 20 Gy XRT CD14 staining was diminished and CD99 was not
observed (Fig. 4b). Differential blood smears were prepared to examine whether a particular white blood cell component was causing the increased spleen weight (Figs. 4c, d). The numbers of monocytes were significantly increased 78.4% over controls and the 20 Gy treatment group (p b 0.002). TRAP staining was observed to be highly increased in the tumor-bearing mice that did not receive XRT while limbs exposed to 20 Gy XRT were observed to possess significantly fewer TRAPpositive multinucleate cells (Fig. 4e). TRAP-positive osteoclasts were quantified and found to be 54.6% increased in the tumor-bearing mice that were not exposed to XRT relative to control left-limbs. After 20 Gy XRT osteoclast numbers decreased 60% relative to left-limb controls (p b 0.001); consistent with previous work that demonstrated a similar 71.9% decrease in osteoclast number after 17.5 Gy XRT [3]. ESB tumor cells express MSCF and do not express RANK-ligand To test a potential interaction of between ESB cells and osteoprogenitors we administered ESB conditioned media (ESBCM) to osteoblasts (OB) cultured from human trabecular fragments and monocytes derived from corresponding bone marrow (BM) aspirates, both retrieved during total hip arthroplasty. RANK-ligand represents the most common and potent route for osteoclast formation and lytic tumor progression in osteotropic tumors [12]. The addition of conditioned media (CM) to monocyte cultures demonstrated that ESB was unable to directly cause monocyte differentiation into osteoclasts; with cultures treated with ESB CM or monocytes and ESB cells were cocultured (Fig. 5a). TRAP-positive staining was only observed in RANKligand + MCSF-treated positive controls (Fig. 5a). The addition of ESBCM significantly increased osteoblast numbers 8.0%, 16.7% and 17.1% for the 0.1%, 0.5% and 1% conditioned media concentrations, relative to the control conditioned media derived from the MB231 breast
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Fig. 3. The adverse effects of XRT on longitudinal bone growth. Radiographs (a) showed limb-length discrepancy (n = 15 for 0 Gy; n = 4 for 5 Gy; n = 14 for 20 Gy). When compared to control limbs, limb length discrepancy (b) was increased 91.1% and 96.4% after 5 Gy and 20 Gy XRT, respectively (* = p b 0.001). Histology (c) revealed significant cellular disorganization in growth plates that have been exposed to XRT. An analysis of growth plate heights (d) showed a 30.6% (p b 0.015) decrease after 5 Gy XRT and a 40.1% increase after 20 Gy XRT (* = p b 0.025) relative to controls. Hypertrophic chondrocyte heights (e) decreased 25.3%, 38.6% and 91.1% after 0, 5 and 20 Gy XRT (* = p b 0.01) relative to control terminal hypertrophic chondrocytes heights.
carcinoma and HBL100 breast epithelium cell-lines (p b 0.001) (Fig. 5b). Control cell-lines were chosen because both express PTHrP; and, metastatic breast carcinoma, in particular, is known to promote osteoprogenitor proliferation through a mechanism called the ‘viscous cycle’ that we had originally hypothesized would be the mechanism for ESB tumor progression [13]. Surprisingly, experiments that tested the ESBCM, MB321CM or HBL100CM effects on bone marrow derived monocytes produced significant increases of 43.4%, 35.9% and 28.3% in monocyte numbers for the 0.1%, 0.5% and 1% ESBCM concentrations relative to control conditioned media (p b 0.02) (Fig. 5c). An analysis of gene expression demonstrated that the ESB cell-lines (RDES, HS822.t, HS863.t) express MCSF and not RANK-ligand while, in parallel,
MCSF and RANK-ligand were expressed in SaOS2 osteosarcoma tumor cells and human MSC (Fig. 5d). Protein expression was confirmed using Western blots and immunocytochemistry and ESB tumor cell lines were observed to only express MCSF and not RANK-ligand, while the SaOS2 and MSC expressed both MCSF and RANK-ligand (Figs. 5e and f). Archival Ewing's sarcoma of bone tumor biopsy samples did not stain with RANK-ligand but did stain with MCSF Ewing's sarcoma of bone and osteosarcoma tumor biopsy samples were stained H&E, which showed tumor associated with bone tissue
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Fig. 4. ESB tumor bearing mice were observed to have splenomegaly and increased numbers of osteoclasts in tumor-inoculated limbs. Spleens (n = 10 for 0 Gy; n = 10 for 20 Gy) were weighed (a) and were found to be larger in mice inoculated with tumor but not treated with XRT (* = p b 0.01) and stained for CD14 (monocytes, green) and CD99 (ESB; red) expression (b). Monocyte numbers were quantified (c) (* = p b 0.002) from differential blood smears prepared from whole blood (d). TRAP stained (red staining) femurs (e) were used to quantify osteoclast numbers (f). Tumor-bearing limbs had 54.6% more osteoclasts than controls, while osteoclasts numbers were decreased 60% after 20 Gy XRT (* = p b 0.001).
(Figs. 6a and e). Ewing's sarcoma of bone tumor samples strongly stained with the ESB specific tumor marker CD99 (Fig. 6b). Ewing's sarcoma of bone tumor samples expressed MCSF but not RANK-ligand (Figs. 6c and d). In contrast, osteosarcoma tumor samples, which served as positive controls, stained with MCSF and RANK-ligand (Figs. 6f and g). Discussion Prior to the work presented herein, there were no mouse models of primary sarcoma and this work represents the first mouse model of primary Ewing's sarcoma of bone. One of the goals of this work was to characterize our Ewing's sarcoma animal model and then determine if this model would be useful for testing potential therapies in the future. In the context of a therapeutic challenge to our model, we chose to expose mice implanted with tumor to X-radiation therapy, in part, due to the ESB sensitivity to XRT. Treating Ewing's sarcoma of bone with XRT often results in limb-length asymmetry, which is a crippling complication of therapy that we observed in our mouse model. The implanted ESB tumor cells also produced significant bone lysis that was effectively treated with XRT. Both the limb-length asymmetry, the ESB tumor associated bone lysis, efficacy of XRT at treating the ESB that we observed are consistent with clinical experience [2,9,13–15,
21–27]. One of our initial hypotheses was that ESB would express a growth factor that would mediate significant bone lysis via increased numbers of osteoclasts or accelerated osteoclastic bone reabsorption. Examination of the ESB cell lines revealed that they expressed high levels of MCSF, but did not express RANK-ligand, the latter of which is one of the most potent stimulators of osteoclast activity. Increased expression of MCSF in the ESB tumor cells suggested that there would be an increase in the numbers of monocytes associated with ESB tumor cells, which we observed in peripheral blood and in the spleens of mice implanted with ESB tumor. Further, an examination of archival ESB biopsy samples corroborated our findings by demonstrating that ESB tumor samples expressed MCSF and not RANK-ligand. In this regard, we believe that our mouse model ESB tumor represents a more promising means for assessing therapeutic interventions in future work. Our initial hypothesis was that Ewing's sarcoma of bone tumor cells would stimulate osteoclast bone reabsorption, which would promote bone lysis. However, we were unable to detect RANK-ligand in ESB tumor cells or in archival biopsy samples of Ewing's sarcoma of bone. This is in contrast to the work of Taylor et al., who identified RANKligand expression and MCSF expression in ESB tumor cells and tumor needle biopsy samples [20]. Recent work by Fujiwara et al. has identified
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Fig. 5. ESB tumor cells express MSCF and do not express RANK-ligand. The addition of ESB CM to cultures (a) did not result in the formation of osteoclasts. CM from HBL100 and MB231 control cells were also unable to stimulate osteoclast formation. In control cultures, in which both MCSF and RANK-ligand were added, we observed robust osteoclastogenesis. We also tested the effects of CM on osteoblast proliferation (b) and found no significant effect from ESB, HBL100 or MB2321 cells. The addition of ESB CM to primary human monocytes (c) resulted in a significant increased in monocyte cell proliferation 43.4%, 35.9% and 28.3% after the addition of 0.1%, 0.5% or 1% ESB CM relative to the CM controls cultures treated with HBL100 or MB231 CM (* = p b 0.02). We tested different ESB tumor cell lines for MCSF gene expression (d) in parallel with control cells SaOS2 cells (osteosarcoma) and human MSC. We were unable to observed RANK-ligand gene expression. We used immunochemistry (e) to test if ESB tumor cells, SaOS2 cell or human MSC expressed MCSF and RANK-ligand in parallel. While the ESB lines expressed MCSF, we were unable to detect RANK-ligand. Whereas, in both SaOS2 and human MSC MCSF and RANK-ligand were detected. We tested protein lysates for the expression of MCSF (f). We observed robust MCSF (green bands) expression in all of the ESB lines tested. Actin served as a loading control (green bands). Molecular weight markers are shown as red bands.
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Fig. 6. Archival Ewing's sarcoma of bone tumor biopsy samples did not stain with RANK-ligand but did stain with MCSF. Ewing's sarcoma of bone (a) and osteosarcoma (e) tumor biopsy samples were stained H&E, which showed tumor associated with bone tissue. (b) Ewing's sarcoma of bone tumor samples strongly stained with the ESB specific tumor marker CD99 (brown/red staining). Ewing's sarcoma of bone tumor samples expressed MCSF (c) (MCSF = brown/red staining) but not RANK-ligand (c). Osteosarcoma tumor samples served as positive controls that stained with MCSF (f) and RANK-ligand (g).
TRAP-positive tumor-associated-macrophages (TAM) located near subcutaneously implanted Ewing's tumor in a Balb/c mouse model [14]. Further, these investigators identified that MCSF and RANK-ligand gene expression were observed in several Ewing's sarcoma tumor cell lines. Our work also disagrees with the expression of RANK-ligand observed in the Fujiwara et al. study. Both Taylor et al. and Fujiwara et al. detected RANK-ligand in ESB tumor cells; however, only Taylor et al. identified RANK-ligand in needle biopsy samples. One of the strengths of our study was that we employed an osteosarcoma cell line positive control and archival osteosarcoma biopsy sample positive control, both of which expressed MCSF and RANK-ligand. This study, the work of Taylor et al., and Fujiwara et al. all agree that MCSF is expressed by ESB. Nevertheless, future work will need to be completed to determine what role RANK-ligand may or may not play in mediating ESB driven bone lysis. The observation of elevated MCSF expression lead us to examine monocyte number since monocytes are acutely sensitive to the MCSF growth factor, which stimulates proliferation and promotes survival. These observation lead to the hypothesis that the increased MCSF produced by the ESB tumor cells would increase the numbers of monocytes. We observed that TRAP-positive, cathepsin K-positive multinucleate osteoclasts were observed in the bone marrow niche occupied by the tumor in the tumor-bearing mice. Further, we hypothesized that the application of XRT would result in decreased tumor burden and corresponding monocyte numbers, due to the well-known radiosensitivity of both monocytes and ESB tumor cells [16]. Additionally, we observed that the numbers of osteoclasts were significantly diminished after the application of XRT; an observation that is consistent
with our previous work [7]. In the latter regard, our work also agrees with Fujiwara et al., in which they identified TRAP-positive multinucleate cells could reduced in number by chemically depleting the monocyte fraction. XRT has proven to be effective at treating ESB, with XRT being applied before or in some cases in lieu of surgical excision [1,2, 15]. Thus, the efficacy of XRT in treating ESB may derive in part from the extreme sensitivity of ESB (D0 = 1.14 Gy) tumor cells and monocytes (D0 = 0.94 Gy) to XRT; as was demonstrated in our previous work [16]. Therefore, we propose a mechanism through which XRT is effective as a treatment for ESB by concurrently decreasing the circulating concentration of MCSF via diminished numbers of ESB tumor cells and the available pool of progenitors (monocytes) able to differentiate into osteoclasts. Conclusions Using a novel Ewing's sarcoma of bone mouse model, we demonstrated that ESB tumor growth in bone is driven by increased osteoclastic bone reabsorption that we associated with increased numbers of monocytes. We further identify that the increase in monocyte numbers is due to the expression of MCSF produced by ESB tumor cells; with the application of XRT resulting in decreased tumor burden and corresponding monocyte numbers. Additionally, we found that ESB tumor cells do not express the most potent regulator of osteoclast differentiation from monocytes, RANK-ligand. Thus, our current work suggests a mechanism through which the depletion of monocytes coupled with diminished MCSF expression from XRT results in decreased tumor burden.
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Acknowledgments Margulies: Department of Orthopedic Surgery Funding and NIH NCCAM (F31AT001994) F31 Kirschstein NRSA Individual Pre-doctoral Fellowship; Damron: NIH NCI (R01CA083892) and the David G. Murray Endowed Professorship. Judith A. Strauss for tissue processing and Gustavo de la Roza, MD for providing access to the human sarcoma tissue samples. References [1] Gurney JG, Swensen AR, Bulterys M. Malignant bone tumors. In: Ries LAG, Smith MA, Gurney JG, Linet M, Tamra T, Young JL, Bunin GR, editors. Cancer Incidence and Survival among Children and Adolescents: United States SEER Program 19751995, NCI, SEER Program. Bethesda, MD: Cancer Statistics Branch; 1999. p. 99–110 [NIH Pub. No. 99-4649.]. [2] Butler MS, Robertson Jr WW, Rate W, D'Angio GJ, Drummond DS. Skeletal sequelae of radiation therapy for malignant childhood tumors. Clin Orthop 1990;251:235–40. [3] Damron TA, Spadaro JA, Horton JA, Margulies BS, Strauss JA, Farnum CE. Novel radioprotectant drugs for sparing radiation-induced damage to the physis. Int J Radiat Biol 2004;80(3):217–28. [4] Damron T, Margulies B, Strauss J, Spadaro J, Farnum C. Sequential histomorphometric changes following irradiation of the growth plate with and without radioprotectant. J Bone Joint Surg Am 2003;85-A:1302–13. [5] Damron TA, Horton JA, Naqvi A, Loomis RM, Margulies BS, Strauss JA, et al. Combination radioprotectors maintain proliferation better than single agents by decreasing early parathyroid hormone-related protein changes after growth plate irradiation. Radiat Res 2006;165(3):350–8. [6] Scotlandi K, Benini S, Manara MC, Serra M, Nanni P, Lollini PL, et al. Murine model for skeletal metastases of Ewing's sarcoma. J Orthop Res 2000;18(6):959–66. [7] Margulies B, Morgan H, Allen M, Strauss J, Spadaro J, Damron T. Transiently increased bone density after irradiation and the radioprotectant drug amifostine in a rat model. Am J Clin Oncol 2003;26(4):e106–14. [8] Henriksen K, Tanko LB, Qvist P, Delmas PD, Christiansen C, Karsdal MA. Assessment of osteoclast number and function: application in the development of new and improved treatment modalities for bone diseases. Osteoporos Int 2007;18(5):681–5. [9] Barr JS, Lingley JR, Gall EA. The effect of roentgen irradiation on epiphyseal growth: I. Experimental studies on the albino rat. Am J Roentgenol 1943;49(1):104–15. [10] Damron TA, Mathur S, Horton JA, Strauss J, Margulies B, Grant W, et al. Temporal changes in PTHrP, Bcl-2, Bax, caspase, TGF-beta, and FGF-2 expression following growth plate irradiation with or without radioprotectant. J Histochem Cytochem 2004;52(2):157–67.
[11] Fisher MC, Meyer C, Garber G, Dealy CN. Role of IGFBP2, IGF-I and IGF-II in regulating long bone growth. Bone 2005;37(6):741–50. [12] Clohisy DR, Ogilvie CM, Ramnaraine ML. Tumor osteolysis in osteopetrotic mice. J Orthop Res 1995;13(6):892–7. [13] Guise TA, Mohammad KS, Clines G, Stebbins EG, Wong DH, Higgins LS, et al. Basic mechanisms responsible for osteolytic and osteoblastic bone metastases. Clin Cancer Res 2006;12(20 Pt 2):6213s–6s. [14] Fujiwara T, Fukushi J, Yamamoto S, Matsumoto Y, Setsu N, Oda Y, et al. Macrophage infiltration predicts a poor prognosis for human Ewing sarcoma. Am J Pathol 2011; 179(3):1157–70. [15] Bölling T, Hardes J, Dirksen U. Management of bone tumours in paediatric oncology. Clin Oncol (R Coll Radiol) 2013;25(1):19–26. [16] Margulies BS, Damron TA, Allen MJ. The differential effects of the radioprotectant drugs amifostine and sodium selenite treatment in combination with radiation therapy on constituent bone cells, Ewing's sarcoma of bone tumor cells, and rhabdomyosarcoma tumor cells in vitro. J Orthop Res 2008;26(11):1512–9. [17] Tamurian RM, Damron TA, Spadaro JA. Sparing radiation-induced damage to the physis by radioprotectant drugs: laboratory analysis in a rat model. J Orthop Res 1999;17(2):286–92. [18] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29(9):2002–7. [19] Erben RG. Embedding of bone samples in methylmethacrylate: an improved method suitable for bone histomorphometry, histochemistry, and immunohistochemistry. J Histochem Cytochem 1997;45(2):307–13. [20] Taylor R, Knowles HJ, Athanasou NA. Ewing sarcoma cells express RANKL and support osteoclastogenesis. J Pathol 2011;225(2):195–202. [21] Choi Y, Lim DH, Lee SH, Lyu CJ, Im JH, Lee YH, et al. Role of radiotherapy in the multimodal treatment of Ewing sarcoma family tumors. Cancer Res Treat Feb 16 2015 [Epub Ahead of Print]. [22] Burchill SA. Ewing's sarcoma: diagnostic, prognostic, and therapeutic implications of molecular abnormalities. J Clin Pathol 2003;56:96–102. [23] Costantino PD, Friedman CD, Steinberg MJ. Irradiated bone and its management. Otolaryngol Clin North Am 1995;28(5):1021–38. [24] Koscielniak E, Morgan M, Treuner J. Soft tissue sarcoma in children: prognosis and management. Paediatr Drugs 2002;4(1):21–8. [25] Merchant TE, Kushner BH, Sheldon JM, LaQuaglia M, Healey JH. Effect of low-dose radiation therapy when combined with surgical resection for Ewing sarcoma. Med Pediatr Oncol 1999;33(2):65–70. [26] Paulussen M, Frolich B, Jurgens H. Ewing tumor: incidence, prognosis and treatment options. Paediatr Drugs 2001;3(12):899–913. [27] Wall JE, Kaste SC, Greenwald CA, Jenkins JJ, Douglass EC, Pratt CB. Fractures in children treated with radiotherapy for soft tissue sarcoma. Orthopedics 1996; 19(8):657–64.