WO2017112919A1 - Tissue-engineered three-dimensional model for tumor analysis - Google Patents

Abstract

A tissue engineered three-dimensional model is provided. The three-dimensional includes Ewing's sarcoma (ES) tumor cells; and an engineered human bone scaffold. The engineered human bone scaffold further includes osteoblasts that secrete substance of the human bone, and osteoclasts that absorb bone tissue during growth and healing. The engineered human bone scaffold includes the tissue engineered three dimensional model which recapitulates the osteolytic process. The engineered human bone scaffold is engineered by co-culturing of osteoblasts and osteoclasts. The osteoblast is produced by cell differentiation process from mesenchymal stem cells. The osteoclast is produced by cell differentiation from human monocytes, wherein the human monocytes are isolated from buffy coats.

Description

TISSUE-ENGINEERED THREE-DIMENSIONAL MODEL FOR TUMOR

ANALYSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

62/387, 121 filed December 23, 2015.

BACKGROUND

Technical Field

[0002] The embodiments herein generally relate to a three-dimensional tissue engineered model of tumor, Ewing's Sarcoma within human bone niche, recapitulating the osteolytic process observed in patients, and, more particularly, three-dimensional bone scaffold or bone tissue engineered by co-culturing osteoblasts and osteoclasts, that provides a controlled biomimetic environment for Ewing's Sarcoma growth.

Description of the Related Art [0003] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment and circulate in the blood stream. They contain cell-specific cargo molecules (i.e. proteins, mRNA, miRNA, DNA), membrane proteins, and lipids. Consequently, exosomes are finding application as diagnostic biomarkers in a number of cancers. Also, tumor-derived exosomes were shown to transfer a variety of bioactive molecules to other cells, inducing modifications of their environment and facilitating tumor growth and invasion. [0004] Our knowledge about the putative roles of the microenvironment on tumor exosomes is limited, due to a lack of experimental models that efficiently mimic the human in vivo situation. Animal models used to study the effects of exosomes on cancer development often fail in representing the context of human disease. In vitro, cancer cells are typically cultured under conditions not recapitulating the 3D tumor environment. The absence of physiological cell-cell and cell-matrix-interactions and the currently used non-physiological substrates cause disparity from the in vivo situation and lead to changes in cell morphology, proliferation and cellular processes, such as endo and exocytosis. Despite the growing notion of the importance of cell microenvironment for cancer signaling, supernatants from monolayer cultures still represent the main source of tumor- derived exosomes such that their micro-environmental regulation remains largely unknown. Bioengineering methods are just about starting to bridge the gap between studies in cell monolayers and experimental animals, providing the models of human tumors that enable studies of how the microenvironment modulates cancer biology.

[0005] Historically, evaluation of therapeutic targets and anti-cancer drugs has been done mostly in simple cultures of cell monolayers and animal models. Although many drugs showed promise in these systems, most failed to translate into human patients and only -5% showed anti-tumor activity in clinical trials. This discrepancy is caused by the lack of ability to sufficiently replicate the human microenvironment in these models. Cells in monolayers are known to rapidly lose their native features, while animal models do not recapitulate human tumors. Therefore, there is a real need for more effective cancer therapy, which requires better experimental models. [0006] Ewing's sarcoma (ES) is the second most frequent bone tumor affecting children and young adults that generally arises and metastasizes in bone. It is characterized by fast growth and progressive bone destruction by osteolysis. Notably, ES cells are incapable to directly degrade bone matrix. Instead, they orchestrate the process of bone resorption through a vicious cycle of recruitment and activation of osteoclasts that is mediated by osteoblasts. Bone destruction by osteoclasts releases calcium and growth factors from the bone matrix that favor acidosis and tumor growth and thereby the osteoclasts activation and increased bone resorption.

[0007] Under physiological conditions, bone is remodeled in a fine-tuned process by which osteoblasts produce new extracellular matrix of the bone and osteoclasts resorb old bone. During this process, minerals (i.e. calcium and phosphorus), growth factors and cytokines are released from the bone matrix to maintain mineral homeostasis and acid-base balance in the body. However, the crosstalk between tumor cells, osteoblasts and osteoclasts disrupts the bone remodeling and initiates either bone destruction (osteolytic tumors) or abnormal bone formation (osteoblastic tumors).

[0008] The lack of ability to replicate in vitro the bone osteolysis associated with the ES represents a critical barrier to understanding of the mechanisms underlying tumor progression and evaluating the new therapeutics. Bioengineered tumor models are becoming invaluable tools for cancer research. However, modeling the bone invasion by cancer remains a challenge. Due to the intrinsic biology of osteolytic tumors, it is of paramount importance to include both osteoblasts and osteoclasts into the bone that will be populated by cancer cells, within the mineralized bone matrix. [0009] In the last few decades, a number of 2-dimensional (2D) cultures and animal models of Ewing's sarcoma (ES) have contributed critical information about cancer biology and served as preclinical systems for therapeutic screens. Unfortunately, the existing ES models have failed to faithfully predict human physiology and support the development of effective treatment modalities. In spite to large investments, the use of these models have delayed drug discovery and exposed children to unnecessary chemicals, suggesting that modeling of the tumor progression requires interactions between tumor cells and their surrounding microenvironment.

[0010] Recently, tissue-engineered models have started to bridge the gap between 2D in vitro cultures (used for discovery and screening) and in vivo animal models (used for efficacy and safety assessment before proceeding to clinical trials) providing a predictive, inexpensive and low time-consuming alternative. However, recapitulating tumor features in vitro is still a major challenge in the field. Therefore, there is a real need for better bioengineered experimental model to which can biomimetic human microenvironment.

SUMMARY

[0011] In view of the foregoing, an embodiment herein provides a tissue engineered three- dimensional model. The three-dimensional model includes Ewing's sarcoma (ES) tumor cells; and an engineered human bone scaffold. The engineered human bone scaffold further includes osteoblasts that secrete substance of the human bone, and osteoclasts that absorb bone tissue during growth and healing. In one embodiment, the engineered human bone scaffold includes the tissue engineered three dimensional model which recapitulates the osteolytic process. In another embodiment, the engineered human bone scaffold is engineered by co-culturing of osteoblasts and osteoclasts. In alternate embodiment, the osteoblast is produced by cell differentiation process from mesenchymal stem cells. In another embodiment, the osteoclast is produced by cell differentiation from human monocytes. The human monocytes are isolated from buffy coats.

[0012] In one embodiment, the mesenchymal stem cells are human mesenchymal stem cells. In another embodiment, the three-dimensional model recapitulates using an osteolytic process. The osteoblasts and osteoclasts are cell differentiated for 12 days. The Ewing's sarcoma aggregates were infused in the engineered human bone scaffold. The infused Ewing's sarcoma aggregates are cultured for 7 days. In one embodiment, the Ewing's sarcoma aggregates are cultured in the engineered human bone scaffold to form a tumor model. In one embodiment, the three-dimensional model comprises a biomimetic environment for the Ewing's sarcoma tumor cells growth. The three-dimensional model mimics tumor microenvironment.

[0013] In another aspect, a tissue engineered three-dimensional model is provided. The tissue engineered three-dimensional model includes a) tumor cells, and b) Engineered human bone scaffold. The three-dimensional model consists of tumor microenvironment. In one embodiment, the three-dimensional model comprises a Ewing's sarcoma tumor microenvironment. In another embodiment, the three dimensional model mimics physical and chemical properties of the tumor microenvironment by collagen 1 (coll) and hyaluronic acid (HA) proteins. In another embodiment, the tumor microenvironment releases tumor exosome. The tumor exosome matches shape, size and cargo of tumor patients. The tumor exosome signals the growth of tumor cells in healthy bone cells.

[0014] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] A detailed description of various aspects, features, and embodiments of the subject matter described herein is provided with reference to the accompanying drawings, which are briefly described below. The drawings are illustrative and are not necessarily drawn to scale, with some components and features being exaggerated for clarity. The drawings illustrate various aspects and features of the present subject matter and may illustrate one or more embodiment s) or example(s) of the present subject matter in whole or in part:

[0016] FIG. 1 illustrates human tissue engineered bone in vitro containing osteoblasts and osteoclasts according to an embodiment of invention;

[0017] FIG. 2 A-E illustrates characterization of osteoclasts within a tissue-engineered bone including osteoblasts according to an embodiment of the invention;

[0018] FIG. 3 A-C illustrates evaluation of bone microstructure in the tissue-engineered bone according to an embodiment of the invention; [0019] FIG. 4 A-C illustrates generation and characterization of the tissue-engineered model of Ewing's sarcoma according to an embodiment of the invention;

[0020] FIG. 5 A-C illustrates analysis of bone microstructure and zoledronic acid effects in the tissue-engineered model of Ewing's sarcoma according to an embodiment of the invention;

[0021] FIG. 6 A-C illustrates differentiation of human mesenchymal stem cells according to the embodiment of the invention.

[0022] FIG. 7 A-C illustrates differentiation of human mesenchymal stem cells to osteoblast in scaffold according to an embodiment of the invention;

[0023] FIG. 8 A-F illustrates differentiation of human monocytes to osteoclast in monolayer according to an embodiment of the invention;

[0024] FIG. 9 A-C illustrates differentiation of human monocytes to osteoclasts in co-culture with human osteoblasts in bone scaffold according to an embodiment of the invention;

[0025] FIG. 10 A-F illustrates Ewing's sarcoma type 1 model in a 3-dimensional Collagen 1- Hyaluronic acid scaffold;

[0026] FIG. 11 A-F illustrates recapitulation of exosomes' size in the bioengineered tumors; and

[0027] FIG. 12 A-E illustrates effects of engineered microenvironment on exosome cargo.

[0028] FIG. 13 A-F illustrates exosome-mediated transfer of EZH2 _mRNA.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0029] Reference will now be made in detail to exemplary embodiments of the disclosed subject matter, an example of which is illustrated in the accompanying drawings. Methods and corresponding steps of the disclosed subject matter will be described in conjunction with the detailed description of the system.

[0030] As mentioned, there remains a need for a new tool to better bioengineered experimental model to which can biomimetic human microenvironment, embodiment herein achieve this by providing a three-dimensional bone scaffold or bone tissue engineered by co- culturing osteoblasts and osteoclasts that provides a controlled biomimetic environment for Ewing's Sarcoma growth.

[0031] A three dimensional tumor model was built, with generated TE-bone containing mature osteoblasts and mature osteoclasts, differentiated for 12 days. Ewing's sarcoma aggregates were infused (cultured for 1 week to allow aggregate formation) into the construct and the tumor model was cultured for an additional 1 week (as shown in FIG. 1). Two different Ewing's sarcoma models were generated: type 1 (using SK-N-MC cell line) and type 2 (using RD-ES cell line). Confirmation of the presence of cancer cells in the tumor model was done by morphological studies (as shown in FIG. 4A) and by evaluating the expression levels of EWS/FLI and KX2.2 genes that are specifically expressed at high levels in Ewing's sarcoma (as shown in FIG. 4B). Additionally, decreases in BSP levels in the tumor model relatively to the corresponding bone constructs were observed (as shown in FIG. 4C).

[0032] Interactions between cancer cells and bone cells orchestrate a vicious cycle in which tumor cells induce osteoclast activation and osteoblasts inhibition, resulting in bone resorption and osteolysis. To evaluate the possible effect of Ewing's sarcoma cells on bone resorption, CT scans of the tumor models with RD-ES and SK-N-MC cells were performed (as shown in FIG. 5A). Consistent with the previous studies in animal models of bone osteolytic tumors, a marked decrease in Bone Volume Density per unit Tissue Volume (BV/TV) in the constructs with Ewing's sarcoma cells was observed (as shown in FIG. 5B). The same tendency was observed for the Connectivity Density (Conn. D) parameter (as shown in FIG. 5B). These results suggest that Ewing' sarcoma cells induce bone resorption and osteoclast activation. Calcium release in tumor model supernatants was quantified, but did not observe any difference compared to the bone construct with osteoclasts.

[0033] For further characterization, bone sialoprotein (BSP) distribution in the tumor model was examined by immunohistochemistry. Bone extracellular matrix lacking BSP was observed, while BSP co-localized with the CD99 Ewing's sarcoma marker (as shown in FIG.5C). The capability of Ewing's sarcoma cells to produce BSP in a 3D environment was reported. Thus, BSP observed (as shown in FIG. 5C) could be secreted by Ewing's sarcoma cells, and not by the osteoblasts. This result reinforces the idea of Ewing's sarcoma cell- mediated bone matrix degradation. Zoledronic acid (ZA) has been shown to target both osteoclasts and Ewing's sarcoma cells. To determine whether cancer cells inhibit the ability of osteoblasts to produce BSP, tumor model was treated with ZA (20μΜ or 2 days). BSP was detectable in the whole construct after treatment that suggests re-activation of osteoblasts (as shown in FIG.5C), recapitulating the effects observed in mice models.

[0034] At least three weeks of cultivation is necessary for hMSC to differentiate into osteoblasts has been demonstrated. Differentiation protocol showed expression of high levels of osteoblast markers. CD 14+ monocytes were co-cultured with the bone engineered from osteoblasts only for 1, 2 or 3 weeks in osteoclastogenic differentiation medium. Osteoclasts at week 2 by morphological analysis, expression of osteoclasts markers and activity assays that confirmed physiological bone remodeling in vitro was identified. The average lifespan of human osteoclasts is about 2-4 weeks, at week 2 the maximum peak of activity and after that, a slightly decreased activity in all the readouts was observed.

[0035] After 12 days of osteoclast differentiation, ES aggregates were infused into the tissue- engineered bone, and the three-dimensional tumor model was maintained for one week in order to secure the activity of the osteoclasts. Living tissue-engineered bone niche provided a biomimetic and controlled environment for recapitulating ES growth and development was observed. ES cells cultured in this niche recapitulated lytic lesions found in patient's tumors (i.e. loss of BSP, decreased Bone Volume Density and Connectivity Density). Additionally, ZA that modulates bone metabolism and has demonstrated some efficacy in Ewing's sarcoma patients, had effects in the tissue-engineered model that was comparable to those observed in animal studies.

[0036] Tissue-engineered models of human tumors are now designed to conform to the three

R's: Reduction, Refinement and Replacement. The three-dimensional models of ES can faithfully recapitulate the osteolytic process observed in the patients' bones. While animal models have limitations, and display a range of complexity associated with systemic factors that tissue-engineered systems still lack. A challenging and desirable goal is to engineer a bone niche that can maintain osteoclast and osteoblast precursors in undifferentiated state, in order to maintain active osteolysis and self-renewal over long periods of time. A less biomimetic but perhaps more feasible option is to introduce medium perfusion into the system, and to infuse bone precursors at timed intervals. The described three-dimensional model has high transformative potential, as the three-dimensional model enables critical advances in tumor modeling under conditions predictive of human physiology.

[0037] In alternate embodiment, the three-dimensional tissue-engineered model described above is for studying tumor exosomes, designed to mimic the native tumor microenvironment. As a clinically relevant example, Ewing's sarcoma (ES) is selected, a solid tumor with aggressive biologic behavior, that affects children and young adults, and is associated with frequent metastases and poor prognosis. ES is characterized by chromosomal rearrangements of the EWSRJ (22ql2) gene with one of the members of the ETS family of transcription factors: the FLU gene (Hq24) in 85% of cases. Expression of EWSRI-FLI1 fusion protein has been the main approach to study the development of ES. Recent studies also demonstrated the presence of EWSR1-FLI1 mRNA in ES-derived exosomes.

[0038] Human mesenchymal stem cells (hMSC) were the only cell type found to provide an appropriate cellular context for EWSRI-FLI1 expression, supporting the notion that Ewing's sarcoma is derived from hMSCs. Surprisingly, hMSCs were unable to form tumors in immunocompromised mice. The studies show that EWSRI-FLI1 is necessary to activate the oncogenic program, but not sufficient for oncogenic transformation of hMSCs. Therefore, recent research has focused on downstream transcriptional targets such as EZH2. EWSRI- FLU was shown to bind to the EZH2 promoter and to induce EZH2 expression in Ewing's sarcoma in vivo and hMSCs in vitro. The EZH2 methyltransferase is a major component of the polycomb repressive complex 2 (PRC2) that is related to transcriptional repression of tumor suppressors such as pl4ARF and pl6INK4a. EZH2 is involved in the maintenance of cell pluripotency and oncogenic transformation of Ewing's sarcoma cells. Additionally, expression of EZH2 correlates with poor prognosis in several tumor types including ES. Thus far, the presence of EZH2 in ES-derived exosomes has not been documented.

[0039] The effects of the microenvironment on tumor-derived exosomes, and the effects of exosomes on cell populations in the bone niche are studied and ES cells are cultured in 3- dimensional biomaterial scaffolds designed to mimic the biological and mechanical properties of ES. The size distributions and EZH2 mRNA cargo are analyzed and compared in exosomes from the plasma of patients and culture medium from monolayers (in culture dishes with different matrix coatings), cell aggregates (in polypropylene), and 3D tissue- engineered tumors (in scaffolds resembling native tumor matrix) as shown in FIG.1 OA, the transfer of EZH2 mRNA from tumor-secreted exosomes to the mesenchymal stem cells is investigated, osteoblasts and osteoclasts of the ES bone niche.

[0040] Cancer cell lines: Ewing's sarcoma cell lines SK-N-MC (HTB-10) and RD-ES (HTB- 166) were purchased from the American Type Culture Collection (ATCC) and cultured according to the manufacturer's specifications. RD-ES cells were cultured in ATCC282 formulated RPMI-1640 Medium (RPMI) and SK-N-MC cells were cultured in ATCC283 formulated Eagle's Minimum Essential Medium (EMEM). Both media were supplemented with 10% (v/v) Hyclone FBS and 1% penicillin/streptomycin. Cells were cultured at 37°C in a humidified incubator at 5% C02.

[0041] Tumor aggregates: Tumor aggregates were prepared by using aliquots of 300,000 Ewing's sarcoma cells, which were centrifuged in 15ml Falcon tubes (5 min at 12,000 rpm), and cultured in 4 mL of osteoclast differentiation medium without cytokines: Minimum Essential Medium Eagle Alpha modification, consisting of a-MEM (Sigma, M4526) supplemented with 10% (v/v) heat inactivated Hy clone FBS, 1% penicillin/streptomycin and L-Glutamine (Gibco #25030-081) for 1 week.

[0042] Human mesenchymal stem cells (hMSCs): unprocessed human bone marrow aspirates were purchased from Lonza. Aspirates from two different donors: donor 1 (code 26737) and donor 2 (code 26798). Human mesenchymal stem cells (hMSCs) were isolated from these aspirates, characterized and prepared as in our previous studies.

[0043] Derivation of osteoblasts from hMSCs: Cell culture and differentiation into osteoblasts were carried out as per protocol. Briefly, hMSC were cultured in expansion medium (DMEM supplemented with 10% (v/v) Hy clone FBS, 1% penicillin/streptomycin and 1 ng/mL of basic fibroblast growth factorb, bFGF). Differentiation into osteoblasts was performed by culturing hMSC in osteoblast differentiation medium (DMEM supplemented with 10%) v/v Hy clone FBS and 1%> penicillin/streptomycin, 1 NM dexamethasone, 10 mM β-glycerophosphate, 50 NM ascorbic acid-2-phosphate) for 3 weeks. Due to the highly osteogenic nature of the mineralized bone scaffolds used to culture the cells, the supplementation of BMP-2 was not necessary. hMSC and osteoblasts were cultured at 37°C in a humidified incubator at 5% C0₂.

[0044] Isolation of monocytes: Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of human blood (fully de-identified samples obtained from the New York Blood Center) by density gradient centrifugation with Ficoll-paque PLUS (17-1440-02, GE Healthcare). Monocytes were derived from the PBMC preparations by immunomagnetic isolation (The big easy EasySep Magnet, #180001, Stem Cell Technologies) using a negative selection (EasySep Human Monocyte Isolation Kit #19359, Stem Cell Technologies), following the manufacturer's protocol. Then, 8x106 monocytes were cultured on 25cm2 ultra-low attachment flasks (Corning #3815) with 10 mL of maintenance medium: RPMI 1640 (ATCC, 30-2001) supplemented with 10% heat inactivated human serum (Corning #35- 060), 1% penicillin/streptomycin, 20ng/ml Recombinant Human M-CSF (Prepotech #300- 25) during 2 days at 37°C in a humidified incubator at 5% C0₂.

[0045] Derivation of osteoclasts from human monocytes: Human CD14+ monocytes were incubated with differentiation medium consisting of Minimum Essential Medium Eagle Alpha modification (a-MEM, Sigma, M4526) supplemented with 10% (v/v) heat inactivated Hyclone FBS, 1% penicillin/streptomycin, L-Glutamine (Gibco #25030-081), 20ng/ml Recombinant Human M-CSF (Prepotech #300-25) and 40ng/ml Recombinant Human sRA K Ligand (Prepotech #310-01). Cytokines were replenished every 3 days. Cells were maintained at 37°C in a humidified incubator at 5% C0₂.

[0046] Resorption pit assay: Human CD 14+ monocytes were plated into 24-well osteo assay plate (100,000 cells per well) (Corning, #3987) and cultured either in complete osteoclast differentiation medium, or without sRA KL as a control for cell differentiation. At different time points, 10% bleach solution was added to each well and cells were incubated for 10 minutes at room temperature. Then, wells were washed 3 times with distilled water and air dried overnight. Resorption pits were visualized at lOx magnification and, for improving the quality of the image, a blue filter was used.

[0047] Engineered bone tissue containing osteoblasts and osteoclasts: Scaffolds (4 mm diameter x 4 mm high plugs) were prepared from decellularized bovine bone. hMSC (1.5x106 per scaffold) were seeded into each scaffold and cultured with osteoblasts differentiation medium for 3 weeks, with a complete medium change twice a week. The scaffolds were then incubated in osteoclasts differentiation medium without cytokines (M- CSF and sRA K Lingand) for 1 hour, and bisected. One half of the tissue construct was placed into a 4 mm x 4 mm (inner diameter x height) PDMS ring and cultured with the addition of 500,000 osteoclasts in ΙΟμΙ of osteoclast differentiation medium for 30min at 37°C in a humidified incubator at 5% C0₂. The scaffolds were flipped and seeded again with 500,000 osteoclasts in ΙΟμΙ of osteoclast differentiation medium for 30min at 37°C in a humidified incubator at 5% C0₂.

[0048] The resulting scaffolds were placed into low attachment six well plates (1 construct per well) containing 5 ml of osteoclast differentiation medium. Medium was changed twice a week. This group was termed hOB + hOC. The other half of each tissue scaffolds that contained only osteoblasts was termed the hOB group, and cultured with osteoclast differentiation medium without cytokines.

[0049] Tissue engineered tumor model: Tumor cells were introduced into the osteoblast- osteoclast bone niche using methods from our previous studies. Aggregates of Ewing's sarcoma cells (RD-ES or SK- MC cell lines) containing 0.3x106 cells were injected into the tissue constructs (3 aggregates per construct) and the resulting cancer cell-bone constructs were cultured for 1 week in osteoclast differentiation medium without supplemental cytokines. This group was termed hOB + hOC + RD-ES or hOB + hOC + SK-N-MC, depending on the Ewing's sarcoma cell line used for model generation. Bone tissue constructs (hOB + hOC) without cancer cells were used as a control.

[0050] Quantitative real-time PCR (qRT-PCR): qRT-PCR was carried out using DNA Master SYBR Green I mix (Applied Biosystems), mRNA expression levels were quantified applying the ACt method, ACt = (Ct of gene of interest - Ct of GAPDH). qRT-PCR primer sequences that were obtained from the PrimerBank data base (http://pga.mgh.harvard.edu/primerbank/) are listed below:

[0051] Histology and immunohistochemistry: Tumor tissue constructs and all controls were fixed in 10% formalin for 24 h and then decalcified with Immunocal (StatLab Corp., McKinney, TX) for 2 days. Samples were dehydrated in graded ethanol washes, and embedded in paraffin. Serial sections (3 μπι thick) were prepared for histology and stained with hematoxylin and eosin (H/E).

[0052] Immunohistochemistry stainings were performed using primary antibodies specific to CD99 (dilution 1 :500; Signet antibodies, SIG-3620) and bone sialoprotein (dilution 1 :500, Abeam, ab33022), and developed using the Vector Elite ABC kit (Vector Laboratories), following manufacturer instructions. Briefly, sections were blocked with serum for 30 min and incubated with the primary antibody overnight at 4 °C. After washing with PBS, samples were incubated with secondary antibodies and developed (Vector Laboratories). Negative controls were prepared by omitting the primary antibody step. Alkaline phosphatase and von Kossa stainings were performed as previously described (48). Tartrate- resistant Acidic Phosphatase (TRAP) staining was performed using the K-assay (Kamiya Biomedical Company #KY-008).

[0053] Monocytes (300,000 per well in 6-well plates) were cultured in complete osteoclast differentiation medium, or without sRANKL as a control for differentiation. At timed intervals (1, 2 and 3 weeks), culture medium was removed and cells were fixed and stained for TRAP, by following the manufacturer's protocol.

[0054] Tissue-engineered bone constructs were fixed in 10% formalin, decalcified in 12.5% EDTA, embedded in paraffin, sectioned to 4 μπι, stained for TRAP according to the manufacturer's instructions, and counterstained with Hematoxylin QS (Vector Labs).

[0055] Calcium release analysis: Supernatants of culture medium were sampled (1 mL per sample), snap frozen in liquid nitrogen and stored at -80 °C. The Ca²⁺ concentrations were analyzed using the Ca²⁺ Detection Kit (Abeam, ab 102505) following the manufacturer protocol. Briefly, supernatants were centrifuged for 2-5 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material. Supernatants were collected and transferred to clean tubes. 90 μΙ_, of the chromogenic reagent were added to each sample. The chromogenic complex formed between calcium ions and o-cresolphthalein was measured using a microplate reader at OD = 575 nm. The measured absorbance values for each standard were plotted as a function of the final concentration of calcium. Finally, the calcium concentrations in the samples were calculated from the standard curve.

[0056] Micro-Computed Tomography (NCT): Samples were scanned and analyzed using a Scanco VivaCT 40 micro-computed tomography system (Scanco Medical, Basserdorf, Switzerland). Scans were performed using 55 kVp, 109 μΑ, and 200 ms integration time, and resulted in images with 21 pm isotropic voxel size. Reconstructed images were smoothed using a Gaussian filter (sigma 0.8, support 1), segmented using a global threshold of 30% maximum gray-scale value, and processed using the standard trabecular morphometry evaluation.

[0057] Collection of the tissue samples from patients: Fully de-identified Ewing's sarcoma tumors were obtained from the Columbia University Tissue Bank, on an IRB-approved protocol. Frozen tissue samples from three different patients were cut into sets of contiguous sections for mechanical, histological, and immunohistochemical studies.

[0058] Fully de-identified blood plasma samples from Ewing's sarcoma patients for exosome isolation and characterization were collected in Dr. Moore's laboratory on an IRB-approved protocol at Memorial Sloan-Kettering Cancer Centre (New York, USA).

[0059] Scaffold preparation: Highly porous scaffolds were produced from Coll-HA solutions by freeze-drying. A 1% (wt/v) solution was prepared from low molecular weight (10-20 kDa) or high molecular weight (500 kDa) Sodium Hyaluronate (HA, Lifecore, US) in distilled water. Four parts of Collagen 1 solution (8-11 mg/ml in 0.02 N acetic acid, Corning, US) were mixed with one part of HA solution (4: 1). After mixing, 200 μΐ of the solution was spread over a 8 mm x 5.5 mrn mold, frozen at -40°C for 4 hours, and sublimed at -40°C overnight under a vacuum of < 100 mTorr. Lyophilized collagen-HA scaffolds were cross- linked with a water-soluble carbodiimide using a previously described method. The scaffolds were immersed in 95% ethanol solution containing 33 mM EDC (Sigma-Aldrich Co. Ltd., UK) and 6 mM NHS (Sigma-Aldrich Co. Ltd., UK) for 4 h at 25°C. After crosslinking, the scaffolds were washed thoroughly in distilled water (5 min x 5 times), refrozen and re- lyophilized at the same freeze-drying cycle as specified above.

[0060] Preparation of matrix-coated plates: Three different types of solutions were prepared for coating culture plates. For collagen-coated plates, a solution of collagen 1 (8-10mg/mL, BD™) was diluted in distilled water (4: 1 dilution ratio). For HA coated plates, a suspension of HA (1% weight) was prepared from the low molecular weight sodium hyaluronate (10-20 lilla, Lifecore biomedical) in distilled water. For Coll/HA coated plates, the above solutions of collagen 1 and HA were mixed in the 4: 1 ratio of Coll : HA. 2mL of each of the three above solutions were added into each well of a 6-well plate, and left for lh at room temperature in a sterile hood. The remaining unattached solutions were carefully aspirated. Each well was plated with 0.3 ^χ 106 SK-N-MC cells.

[0061] Culture of cells in aggregates and in 3D scaffolds. Ewing's sarcoma cell line SK-N- MC (HTB-IO) was purchased from the American Type Culture Collection (ATCC) and cultured according to the manufacturer's specifications, in ATCC-formulated Eagle's Minimum Essential Medium (EM EM) supplemented with 10% (v/v) Hy clone FBS and 1% penicillin/streptomycin. To form tumor cell aggregates, 0.3 x 10⁶ SK-N-MC cells were centrifuged in 15 mL Falcon tubes, 5 minutes at 1200 rpm, with 4 mL of medium and cultured for 7 days at 37°C in a humidified incubator at 5% C02.

[0062] To seed 3D Collagen 1-HA scaffolds, single-cell suspension of SK-N-MC cells was adjusted to the cell concentration of 1 x 10⁶ cells/mL in a 50 ml Falcon tube. A total of 15 scaffolds were added to 30 mL of cell suspension, and the Falcon was set onto a rotary platform for 3h at 37°C/5% C02. Cell seeded scaffolds were then transferred to non-treated wells in 12-multiwell plates (Nunc) and cultured in 2 mL of medium at 37°C / 5% C02. Cell numbers and were determined by Quant-iT PicoGreen dsDNA Assay Kit (Life technologies) according to the manufacturer's instructions.

[0063] Mechanical testing: The mechanical properties of native Ewing's sarcoma tumors collected from the patients at the Memorial Sloan-Kettering Cancer Centre (New York, USA) were measured using a previously established protocol. Briefly, the Young's modulus was determined under unconfined compression in phosphate-buffered saline (PBS) at room temperature. An initial tare load of 0.2 N was applied, and followed by a series of stress- relaxation steps, where specimens were compressed at a ramp velocity of 1% per second up to the 10%) strain, and maintained at each position for 1,800 s. The Young's modulus was calculated from the equilibrium force measured at the 10%> strain.

[0064] Scanning Electron Microscopy (SEM): The morphology of the bioengineered tumors was examined by SEM. Samples were washed twice in PBS and fixed in 4% paraformaldheyde in PBS (Santa Cruz, US) for 1 hour. Fixed specimens underwent a graded dehydration series of ethanol (70, 85, 95, 100% for 5 min each) and hexamethyldisilazane drying for 15 min (HMDS, Sigma). Samples were dried overnight in the fume hood, sputter- coated with gold and palladium, and imaged using SEM (Hitachi S-4700).

[0065] Fluid uptake by the Scaffolds: Dried samples were weighed (Wd) and immersed in distilled water at 37°C for different periods of time (2 hours, 3, 7 and 10 days). At each time point, specimens were removed from distilled water and the ability of the scaffold structure to absorb water was measured using a previously described method. At each time point, the samples were removed from water and weighed (Ww). The water uptake was calculated as: Fluid uptake (%) = (Ww-Wd)AVdx lOO. Each sample was measured in triplicate.

[0066] Scaffold degradation: Dried samples were weighed (Wd) and immersed in distilled water at 37°C in a humid atmosphere for timed intervals (2 hours, 3, 7 and 10 days). At each time point, specimens were removed from distilled water, air-dried for 24 hand weighed (Wa). The weight loss was calculated as: Weight loss (%) = (Wd-Wa)AVd xlOO. Each sample was measured in triplicate.

[0067] Histology and immunohistochemistry (IHC): Frozen sections of the native Ewing's sarcoma tumors and bioengineered tumors were fixed in pre-cooled acetone (-20°C) for 10 min. Sections were washed with PBS and treated with 0.3% H202 solution in PBS at room temperature for 10 min to block endogenous peroxidase activity, and incubated with a blocking buffer from Vectastain Elite ABC Kit (Vector Labs), according to the manufacturer's instructions. Then, sections were stained for CD99 (dilution 1 :500; Signet antibodies, SIG-3620) and Collagen 1 (dilution 1 :500; Abeam, ab34710). Slides were counterstained with Hematoxylin QS (Vector Labs). For the hyaluronan acid binding protein (HABP) staining, the sections were blocked using 1% BSA in HBSS at room temperature for 30 min, and incubated with a biotinylated HABP antibody (dilution 1 : 100; Millipore #385911). A Streptavidin Alexa fluor 488 conjugate (dilution 1 :500; Molecular Probes) was used as the secondary antibody.

[0068] Live-Dead assay: At timed intervals (day 3 and day 7), Bioengineered tumor models were incubated in EMEM medium containing 2 μΜ Calcein and 4 μΜ of ethidium homodimer-I for 30 min at 37°C, 5% C02, as indicated by the manufacturer's protocol (UVEIDEAD^® Viability/Cytotoxicity Kit, Molecular Probes). Samples were imaged with a fluorescence microscope (Olympus 1X81 light microscope, Center Valley PA).

[0069] Exosome isolation and size analysis: Cells cultured in monolayers, aggregates and 3D scaffolds were washed with PBS twice and cultured in EMEM supplemented with 10% (v/v) Exosome-depleted FBS (SBI) and 1% penicillin/streptomycin for 12h. The supematants were collected and exosomes were isolated from cell culture media using the total exosome isolation kit (Invitrogen), according to the manufacturer's protocol. Exosomes from plasma samples were also isolated using the total exosome isolation kit (Invitrogen). The size distributions of exosomes were determined by Nanoparticle Tracking Analysis (NTA) using the Nanosight machine.

[0070] Genomics Analysis: Overexpression of EZH2 in Ewing's sarcoma tumors at mRNA levels were compared using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl.) The R2 platform is an online genomics analysis tool that can analyze a large collection of public data. We selected EZH2 as gene of interest to generate a MegaSampler using the following dataset: [0071] Tumor Ewing Sarcoma-Francesconi (37 samples). Source: GEO 10: gse34620 Dataset Date: 2000-01-01. Pubmed link: 22327514. A genome-wide association study of at least 401 French ES patients compared to either 684 French or 3668 US self-described Caucasian controls consistently revealed candidate loci at chromosomes 1 and 10 (p<10-6) [0072] Tumor Ewing Sarcoma-Delattre (117 samples). Source: GEO 10: gse 12102 Dataset Date: 2008-06-15. Pubmed link: 22327514. Available tracks in R2: group (CAT) [ ews metastasis tumor (metastasis) ews primary tumor (no evidence of disease) ews primary tumor (relapse)]

[0073] Healthy: Normal Various -Roth- (353 samples). Source: GEO 10: GSE3526 Dataset Date: 2006-03-30. Pubmed link: 16572319. Normal human tissue samples from ten postmortem donors were processed to generate total RNA, which was subsequently analyzed for gene expression using Affymetrix U133 plus 2.0 arrays. Donor information: Donor} 25 year old male; donor 2 - 38 year old male; donor 3 - 39 year old female; donor 4 - 30 year old male; donor 5 - 35 year old male; donor 6 - 52 year old male; donor 7 - 50 year old female; donor 8 - 48 year old female; donor 9 - 53 year old female; donor 10 - 23 year old female.

[0074] Quantitative real-time PCR (qRT-PCR): Total RNA from cells was obtained using Trizol (Life Technologies) and total RNA from exosomes was obtained using the Total Exosome RNA & Protein Isolation Kit (ThennoFisher scientific) following the manufacturer's instructions. RNA preparations were treated with "Ready-to-go youprime first-strand beads" (GE Healthcare) to generate cDNA. Quantitative real-time PCR was performed using DNA Master SYBR Green I mix (Applied Biosystems). mRNA expression levels were quantified applying the ACt method, Δ Ct = (Ct of gene of interest- Ct of Actin). EZH2 primers were obtained from the PrimerBank database (http : //pga. mgh . harvard . edu/ primerb ankL) .

[0075] RNA quality: Total RNA quality and size distribution from cells and exosomes were determined by electropherograms from the Bioanalyzer 2100 using the RNA Pico Chip kit (Agilent Technologies).

[0076] Western blot: Cells were lysed in R1PA buffer containing protease inhibitors (Sigma-Aldrich, P8340) and exosomes extracts were obtained using the total Exosome RNA & Protein Isolation Kit (ThermoFisher scientific) following the manufacturer's instructions. Cell preparations were centrifugated at 12,000 g for 10 min and supernatants containing soluble proteins were collected for analysis. 20μg of cells and exosomes extracts were loaded on 4-12% gradient Bis-Tris gels (BioRad), transferred to a nitrocellulose membrane and incubated with antibodies against EZH2 (1:500; Millipore 07-689), Calnexin (1 :500; Santa Cruz, sc- 11397, CD8} (1 :500; Santa Cruz, sc-7637) at 4 degrees over night and GAPDH (1 :5000; Invitrogen 437000) at room temperature for one hour.

[0077] For detection, membranes were incubated with a secondary antibody anti-rabbit or anti-mouse conjugated with Alexa Fluor 680 dye (1 :5000; ThermoFisher Scientific) at room temperature for one hour and imaged on Licor Odyssey scanner.

[0078] Exosome-mediated transfer of RNA: SKNMC cells were cultured on Col 1-HA scaffolds for 7 days in ATCC-formulated Eagle's Minimum Essential Medium (EMEM) supplemented with 10% (v/v) Hy clone FBS and 1% penicillin/streptomycin. For exosome isolation, cells were cultured with 10% Exosome-depleted FBS (SBI) and 1% penicillin/streptomycin for 12h. Supernatants were harvested and exosomes were isolated. To measure protein concentration (by Bradford assay), the concentration of protein was adjusted to ~ 0.1 μg/μL in PBS, and the samples were diluted 1 :50 (20 μΐ in 1ml of PBS) for NTA analysis. The same volumes, dilutions and the same camera shutter were used to obtain similar concentrations of particles for measuring size distributions in cell monolayer and TE- Tumors. 10μg of exosomes protein were labeled with SYTO RNA Select green fluorescent (Invitrogen) during 30 min at 37°C/5% C0₂ at a final dye concentration of 10 μΜ. Exosome Spin Columns (MW 3000) were used to remove unincorporated dye from exosome labeling. The same volume of PBS without exosomes was also treated with SYTO RNA and exosome spin columns to serve as a control. Cells (5,000cells/well) were seeded in an 8-wells chamber slide the day before the exosome- mediated transferring assay. 10 μg of labeled exosomes in PBS, or same volume of PBS control, were incubated with hMSC passage 3, human osteoblasts or human osteoclasts during 2h at 37°C/5% C0₂. Cells were fixed for 20 min with 4% PFA in PBS and mounting with Vectashield-DAPI.

[0079] RESULTS - Derivation of bone cell precursors: According to the embodiment for derivation of bone precursor human mesenchymal stem cells (hMSC) are used to differentiate into osteoblasts, hMSCs from various sources have been used to engineer bone.

The decellularized bone scaffold preserves not only the structural and mechanical features of the original bone, but also maintains its inorganic mineral phase and many of the growth factors. Notably, owing to the highly osteogenic properties of these scaffolds, the supplementation of BMP-2 during bone tissue engineering is not necessary.

[0080] hMSC are used as a source of osteoblasts for engineering bone in vitro. hMSC (from two different donors) ability to differentiate into osteoblasts, both in cell monolayers and in decellularized bone scaffolds was confirmed (as shown in FIGS. 7A& 8A). hMSC from both donors were positive for Alkaline phosphatase and Von Kossa after 3 weeks of differentiation in monolayer culture (as shown in FIG. 7B). Increased expression of bone markers (BGLAP, OPN and BSP) was observed by qRT-PCR, relatively to the hMSC cultured in expansion medium (Fig 7C). Then bone containing only osteoblasts (TE-hOB) was engineered, by culturing hMSC in decellularized bone scaffolds using osteogenic medium, for 3 weeks (Fig 8A). The ability to generate hMSC-derived osteoblasts and form new bone matrix (by histology, Fig 8B) and expression of bone markers (by qRT-PCR, Fig 8C) was confirmed.

[0081] The capability of osteoclast precursors (CD 14+ monocytes) to differentiate into mature osteoclasts (as shown in FIG. 8A) was assessed and identified based on their unique morphology and function. Osteoclasts are large, multinucleated and polarized cells with the nuclei localized toward the apical membrane and a ruffled border membrane. These cells are specialized in bone resorption that proceeds with degradation of organic matrix and demineralization of the mineral matrix in specific regions known as "resorption lacunae", and inducing increases in local concentrations of calcium and phosphate. Activated osteoclasts resorb bone by lowering the pH in the resorption lacunae, following secretion of acidic hydrolases such as cathepsin K and the tartrate-resistant acid phosphatase (TRAP), and express considerable levels of calcitonin receptor.

[0082] Osteoclasts were derived from human monocytes isolated from buffy coats, and tested for purity. On average, the enrichment of CD14+ monocytes was 94%, as determined by flow cytometry analysis (as shown in FIG. 8B). The purified monocytes were cultured for up to 3 weeks in monolayer in the presence of RA KL to induce osteoclastic lineage differentiation. By week 1, the osteoclasts markers TRAP, calcitonin receptor and cathepsin K were expressed (Fig 8C), and this expression reached the maximum level at week 3. Morphology, differentiation and multi-nuclearity of osteoclasts by TRAP staining was evaluated (Fig 8D). Osteoclast activation and functionality were evaluated (as shown in FIG. 8E), and the calcium release over time was compared to the undifferentiated cell control (as shown in FIG. 8F).

[0083] Bioengineered tumor model: Native Ewing's sarcoma (ES) is a pediatric tumor rich in collagen 1 (col 1) and hyaluronic acid (HA) proteins (as shown in FIG.10 A), and soft tissue matrix characterized by an equilibrium modulus of ~2 kPa (as shown in FIG.10B). In order to mimic the composition and mechanical properties of the ES extracellular matrix, we used purified preparations of natural col 1 and HA (low molecular weight, LMW; high molecular weight, HMW) with a stiffness matching that of the native tumor (as shown in FIG. 10B). Two types of 3D porous scaffolds (Coll -HA LMW; Col l -HA HMW) were made by freeze- drying of Col 1/HA solutions, and cross-linking with l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride, EDC, in the presence of N-hydroxysuccinimide, NHS (as shown in FIG. IOC ).

[0084] The swelling behavior, measured by the liquid uptake, was similar for the two porous scaffolds, and in agreement with the previous studies. The rate of degradation was much slower for Col l -Ha LMW than Col l -Ha HMW scaffolds, presumably due to the higher density of chemical cross-links (as shown in FIG.10D). These results demonstrated that the Col l -Ha LMW scaffold was suitable for supporting the in vitro culture of tumor cells. In previous studies, LMW HA was shown to play a role in tumor progression in a number of cancers. Therefore, we selected the Coll -Ha LMW scaffold as an appropriate biomimetic environment for culturing ES cells.

[0085] To bioengineer the most common ES tumor type, SK-N-MC cell lines (type 1 rearrangement) were cultured in Coll -Ha LMW scaffolds. Mechanical properties of the TE- tumor did not change over time (as shown in FIG.1 OB), and the model was stable over one week of culture. The proliferation of ES cells cultured within the TE-tumor model was slower than when the same cells were cultured in monolayer (as shown in FIG. IOC), consistent with the known lower rates of cell proliferation in native tumors compared to cancer cells cultured in monolayers. Live dead analysis demonstrated uniform distribution of cells throughout the scaffolds at day 3 and day 7, and showed that most of the cells were viable after 7 days of culture (as shown in FIG. l 1).

[0086] Notably, the levels of expression of CD99 in the TE tumor model were comparable to those measured in the samples of patients' tumors (as shown in FIG.10D). These data show that cell culture on Coll/HA scaffolds does not modify the levels of this important membrane protein that is highly expressed in most cases of Ewing's sarcoma and maintains them at levels similar to those in tumors from patients. The cells cultured in the TE-tumor model formed small avascular aggregates that increased in size over time, mimicking the initiation of native tumor formation (as shown in FIGS. 10 E-F).

[0087] Evaluation of the purity of exosomes preparations: In order to check the purity of the exosome preparations, we performed two sets of analysis consisting in protein composition and total RNA profiles. Toward this end, first we analyzed the levels of the CD81 (exosomal marker) and calnexin (only detectable in cellular and apoptotic bodies extracts), in monolayer and the TE tumor model at day 3 and day 7 (as shown in FIG. 13 A). GAPDH levels were determined to address the possibility of using GAPDH as a loading control of the technique. Absence of calnexin was confirmed in the extracellular preparations. This suggests that there is not cellular or apoptotic bodies' contamination in the exosomes preparations. CD81 was detectable in exosomes preparations from cells in monolayer but not from TE-tumors preparations. GAPDH levels were similar between samples that points GAPDH as a good loading control.

[0088] Then, the quality of the exosomes isolation is analyzed by analyzing RNA profiles from cells and exosomes preparations from cells in monolayer and TE-tumor at day 7, using the Bioanalyzer 2100 (as shown in FIG. 12B). As expected, electropherograms showed different RNA size distributions between samples. The RNA profile from cells revealed two dominant peaks, corresponding to the ribosomal RNA (rRNA) subunits 18S and 28S. Both peaks are also observed in RNA profiles from preparations of apoptotic bodies. The RNA profile from extracellular vesicles lacked of both rRNA peaks and showed and enrichment in small RNAs, accordingly with the literature.

[0089] Exosome size: Using the Nanoparticle Tracking Analysis (NIA), the size distributions of exosomes released into the culture media from the bioengineered tumor and from cell monolayers, are determined and compared these to the size distributions of exosomes secreted into the blood plasma of ES patients. The sizes of exosomes isolated from human plasma (average mean ± SD: 88.7 ± 22 nm; average mode ± SD: 70.0 ± 20 nm, n=7 patients, as shown in FIG. 11 A) were consistent with the previously reported data, and significantly smaller than the exosomes from monolayer cultures of ES cells (average mean ± SD: 149.2 ± 19 nm; average mode ± SD = 103.3 ± 23 nm, n==3,**p < 0.01; as shown in FIG. 11 A). In addition, the numbers of particles per unit protein were not statistically different for cell monolayers and tissue engineered tumors (as shown in FIG. 13). Notably, the sizes of exosomes released from tumor models (average mean ± SD: 113.4 ± 10 nm, average mode ± SD: 76.7, ± 10.3 n = 6; as shown in FIG. 11 A) were indistinguishable from those in the patients' plasma. These data suggest that the 3 -dimensionality or composition of the scaffold (or both of these factors) regulate the exosomes to reach their native size. To distinguish the relative contributions of the matrix 3 -dimensionality and composition, we investigated the sizes of exosomes in multiple model systems.

[0090] To evaluate the role of 3 -dimensionality, we generated ES cell aggregates in a generic polypropylene context, in the range of sizes that we have observed for bioengineered tumors at day 7 (as shown in FIG. 1 IB). Neither the average mean nor the mode size of exosomes isolated from these aggregates recapitulated the values found in the patients' plasma (as shown in FIG. 1 IB). Mimicking the tumor size and morphology using 3D models without a biomimetic context was thus not sufficient to recapitulate the native exosome size. To evaluate the role of matrix composition, we cultured ES cells in monolayers formed on polystyrene dishes coated with different extracellular matrix proteins (HA LMW, Coll,

Coll -HA LMW, as shown in FIG. 11C). It was observed that there was no difference in the mean size or mode of exosomes secreted by the ES cells cultured on uncoated polystyrene dishes and on dishes coated with the proteins used for fabricating the scaffolds (as shown in

FIG. 11D). These results indicate that mimicking the native matrix composition without providing the native stiffness and 3D context was also not sufficient for reproducing the native size of exosomes. Providing both the 3 -dimensionality of cell culture and the composition or extracellular matrix found in ES was necessary for recapitulating the exosome size.

[0091] To probe a possible mechanism underlying the observed effects of the tumor environment on exosome size, we modified the tension forces within the cells. To this end, we maintained the 3 -dimensionality, composition and stiffness of the microenvironment at levels comparable to the native tumor matrix, while eliminating tension-dependent changes in cell shape by using Blebbistatin, a well-known selective inhibitor of non-muscle myosin n. Cell morphology in blebbistatin-treated samples was different from untreated controls (as shown in FIG. 1 IE), with a partial disassembly of cell aggregates (as shown in FIG. 1 IE) and a shift of the exosome size distribution curve to higher values (as shown in FIG. 1 IF) when tensional forces within the cells were modified in a 3D setting.

[0092] Exosome cargo: Based on these findings, it was hypothesize that the exosome size is not the only property controlled by the microenvironment, and that their cargo is also a subject to regulation. To test this hypothesis, we analyzed the exosomal mRNA cargo and focused on EZH2, one of the most important mediators of Ewing's sarcoma tumor growth and progression. First, it was confirmed that over expression of EZH2 in ES tumors at mRNA levels using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl), by comparing the gene profiles for ES tumors (arrays from Francesconi n=37, and Delattre; n=117) and healthy tissues (array from Roth n=353) (as shown in FIG.12A). EZH2 overexpression in ES tumors by Immunohistochemistry was checked and EZH2 protein was almost undetectable by Western blot in ES cells cultured in monolayers (as shown in FIG. 12B), which also expressed low levels of EZH2 mRNA by qRT-PCR (as shown in FIG. 12C). However, EZH2 mRNA and EZH2 protein increased in TE-tumors, both at the protein level (as shown in FIG. 12B) and at the mRNA level (as shown in FIG. 12C). These data supported the notion that a native-like environment can modulate cancer biology and mimic, at least in part, the properties of real tumors. Exosomes released from the ES cells cultured in monolayers and bioengineered tumors were isolated and high levels of EZH2 mRNA in exosomes from TE-tumors, both at day 3 and day 7 was found, when compared to monolayers (as shown in FIG. 12D). Importantly, the measured levels of EZH2 in bioengineered tumors corresponded to those in the blood plasma of ES patients. EZH2 mRNA was detected in exosomes from Ewing's sarcoma type-I plasma (n=4), but not in plasma of healthy donors (n=4), non-type 1 patients (n=3) or an osteosarcoma patient (n=I) (as shown in FIG. 12E).

[0093] Transfer of exosome cargo: Because EZH2 induces an aberrant phenotype of Ewing's sarcoma in vivo and also affects the hMSCs cultured in vitro, Exosomes containing EZH2 mRNA can transfer their cargo to the cells hMSCs normally present in the bone niche were investigated. Labeled exosomes derived from the TE-tumor (Exo-TE-tumor) with the green RNA-selective nucleic acid stain SYTO RNA Select at day 7, the time point at which we observed high levels of EZH2 mRNA in these exosomes. The exosomes from the TE-tumors were taken up by bone marrow derived hMSCs, after 12 hours of incubation compared to the technical control (PBS treated with SYTO RNASelect) (as shown in FIG. 13 A). Significant increases in EZH2 mRNA levels were detected in hMSC treated with exosomes from TE- tumors, when compared with untreated hMSCs or hMSCs treated with hMSC-derived exosomes (as shown in FIG. 13B). Finally, we analyzed the effects of exosomes secreted by bioengineered tumors on human osteoblasts (hOB) and human osteoclasts (hOC). Labeled exosomes from TE-tumors were taken up by both hOB (4C) and hOC (as shown in FIG. 13E). However, this uptake had no effect on EZH2 mRNA levels in hOB (As shown in FIG. 13D), and resulted in down-regulation of EZH2 in hOC (As shown in FIG. 13F). These data confirm that EZH2 mRNA-loaded exosomes can be transferred in vitro from cancer cells to cell populations from the bone niche, with different effects on hMSC (upregulation of EZH2), hOC (downregulation of EZH2) and hOB.

[0094] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A tissue engineered three-dimensional model, comprising:

Ewing's sarcoma (ES) tumor cells; and

an engineered human bone scaffold,

wherein the engineered human bone scaffold further comprises:

osteoblasts that secretes substance of the human bone; and

osteoclasts that absorbs the human bone tissue during growth and healing.

2. The three-dimensional model of claim 1, wherein the engineered human bone scaffold is engineered by co-culturing of the osteoblasts and the osteoclasts.

3. The three-dimensional model of claim 1, wherein the osteoblast is produced by cell differentiation process from mesenchymal stem cells.

4. The three-dimensional model of claim 1, wherein the osteoclast is produced by cell differentiation from human monocytes, wherein the human monocytes are isolated from buffy coats.

5. The three-dimensional model of claim 3, wherein the mesenchymal stem cells are human mesenchymal stem cells.

6. The three-dimensional model of claim 1, wherein the three-dimensional model recapitulates using an osteolytic process.

7. The three-dimensional model of claim 1, wherein the osteoblasts and osteoclasts are cell differentiated for 12 days.

8. The three-dimensional model of claim 1, wherein the Ewing's sarcoma aggregates were infused in the engineered human bone scaffold.

9. The three-dimensional model of claim 8, wherein the infused Ewing's sarcoma aggregates are cultured for 7 days.

10. The three-dimensional model of claim 8, wherein the Ewing's sarcoma aggregates are cultured in the engineered human bone scaffold to form a tumor model.

11. The three-dimensional model of claim 1, wherein the three-dimensional model comprises a biomimetic environment for the Ewing's sarcoma tumor cells growth.

12. The three dimensional model of claim 1, wherein the three-dimensional model mimics tumor microenvironment.

13. A tissue engineered three-dimensional model, wherein the tissue engineered three- dimensional model comprises;

tumor cells; and

an engineered human bone scaffold;

wherein, the three-dimensional model consists of tumor microenvironment.

14. The tissue engineered three-dimensional model of claim 13, comprising Ewing's sarcoma tumor microenvironment.

15. The tissue engineered three dimensional model of claim 13, wherein the three dimensional model mimics physical and chemical properties of the tumor microenvironment by collagen 1 (coll) and hyaluronic acid (HA) proteins.

16. The tissue engineered three dimensional model of claim 13, wherein the tumor microenvironment releases tumor exosome.

17. The tissue engineered three dimensional model of claim 17, wherein the tumor exosome matches shape, size and cargo of tumor patients.

18. The tissue engineered three dimensional model of claim 17, wherein the tumor exosome signals the growth of tumor cells in healthy bone cells.