WO2017112919A1 - Modèle tridimensionnel obtenu par ingénierie tissulaire pour l'analyse de tumeurs - Google Patents

Modèle tridimensionnel obtenu par ingénierie tissulaire pour l'analyse de tumeurs Download PDF

Info

Publication number
WO2017112919A1
WO2017112919A1 PCT/US2016/068478 US2016068478W WO2017112919A1 WO 2017112919 A1 WO2017112919 A1 WO 2017112919A1 US 2016068478 W US2016068478 W US 2016068478W WO 2017112919 A1 WO2017112919 A1 WO 2017112919A1
Authority
WO
WIPO (PCT)
Prior art keywords
dimensional model
tumor
engineered
cells
ewing
Prior art date
Application number
PCT/US2016/068478
Other languages
English (en)
Inventor
Gordana Vunjak-Novakovic
Original Assignee
The Trustees Of Columbia University In The City Of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of Columbia University In The City Of New York filed Critical The Trustees Of Columbia University In The City Of New York
Publication of WO2017112919A1 publication Critical patent/WO2017112919A1/fr
Priority to US16/016,101 priority Critical patent/US10883083B2/en
Priority to US17/121,817 priority patent/US20210102170A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1311—Osteocytes, osteoblasts, odontoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1317—Chondrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • 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.
  • 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.
  • Ewing's sarcoma 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.
  • 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.
  • bone 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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. The human monocytes are isolated from buffy coats.
  • the mesenchymal stem cells are human mesenchymal stem cells.
  • 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.
  • the Ewing's sarcoma aggregates are cultured in the engineered human bone scaffold to form a tumor model.
  • the three-dimensional model comprises a biomimetic environment for the Ewing's sarcoma tumor cells growth.
  • the three-dimensional model mimics tumor microenvironment.
  • a tissue engineered three-dimensional model includes a) tumor cells, and b) Engineered human bone scaffold.
  • the three-dimensional model consists of tumor microenvironment.
  • the three-dimensional model comprises a Ewing's sarcoma tumor microenvironment.
  • the three dimensional model mimics physical and chemical properties of the tumor microenvironment by collagen 1 (coll) and hyaluronic acid (HA) proteins.
  • 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.
  • FIG. 1 illustrates human tissue engineered bone in vitro containing osteoblasts and osteoclasts according to an embodiment of invention
  • FIG. 2 A-E illustrates characterization of osteoclasts within a tissue-engineered bone including osteoblasts according to an embodiment of the invention
  • FIG. 3 A-C illustrates evaluation of bone microstructure in the tissue-engineered bone according to an embodiment of the invention
  • FIG. 4 A-C illustrates generation and characterization of the tissue-engineered model of Ewing's sarcoma according to an embodiment of the invention
  • 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
  • FIG. 6 A-C illustrates differentiation of human mesenchymal stem cells according to the embodiment of the invention.
  • FIG. 7 A-C illustrates differentiation of human mesenchymal stem cells to osteoblast in scaffold according to an embodiment of the invention
  • FIG. 8 A-F illustrates differentiation of human monocytes to osteoclast in monolayer according to an embodiment of the invention
  • 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
  • FIG. 10 A-F illustrates Ewing's sarcoma type 1 model in a 3-dimensional Collagen 1- Hyaluronic acid scaffold
  • FIG. 11 A-F illustrates recapitulation of exosomes' size in the bioengineered tumors
  • FIG. 12 A-E illustrates effects of engineered microenvironment on exosome cargo.
  • FIG. 13 A-F illustrates exosome-mediated transfer of EZH2 m RNA.
  • 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.
  • BSP bone sialoprotein
  • Zoledronic acid has been shown to target both osteoclasts and Ewing's sarcoma cells.
  • tumor model was treated with ZA (20 ⁇ 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.
  • 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).
  • 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.
  • 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.
  • the three-dimensional tissue-engineered model described above is for studying tumor exosomes, designed to mimic the native tumor microenvironment.
  • Ewing's sarcoma ES
  • ES Ewing's sarcoma
  • 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.
  • 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.
  • hMSC Human mesenchymal stem cells
  • 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.
  • PRC2 polycomb repressive complex 2
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • hMSCs Human mesenchymal stem cells
  • hMSC differentiation medium DMEM supplemented with 10%
  • PBMC Peripheral blood mononuclear cells
  • 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.
  • 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% C0 2 .
  • 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% C0 2 .
  • 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.
  • 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.
  • cytokines M- CSF and sRA K Lingand
  • 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 ⁇ of osteoclast differentiation medium for 30min at 37°C in a humidified incubator at 5% C0 2 .
  • the scaffolds were flipped and seeded again with 500,000 osteoclasts in ⁇ of osteoclast differentiation medium for 30min at 37°C in a humidified incubator at 5% C0 2 .
  • 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.
  • 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.
  • qRT-PCR Quantitative real-time PCR
  • 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).
  • TRIP Tartrate- resistant Acidic Phosphatase
  • 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.
  • Tissue-engineered bone constructs were fixed in 10% formalin, decalcified in 12.5% EDTA, embedded in paraffin, sectioned to 4 ⁇ , stained for TRAP according to the manufacturer's instructions, and counterstained with Hematoxylin QS (Vector Labs).
  • Micro-Computed Tomography 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 ⁇ , 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.
  • a Gaussian filter Sigma 0.8, support 1
  • 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.
  • 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.
  • ATCC American Type Culture Collection
  • EM EM Eagle's Minimum Essential Medium
  • Hy clone FBS 1% penicillin/streptomycin.
  • 0.3 x 10 6 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.
  • SEM Scanning Electron Microscopy
  • IHC immunohistochemistry
  • HABP hyaluronan acid binding protein
  • Live-Dead assay At timed intervals (day 3 and day 7), Bioengineered tumor models were incubated in EMEM medium containing 2 ⁇ Calcein and 4 ⁇ 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).
  • 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.
  • NTA Nanoparticle Tracking Analysis
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • EMEM ATCC-formulated Eagle's Minimum Essential Medium
  • SBI Exosome-depleted FBS
  • penicillin/streptomycin for 12h.
  • Supernatants were harvested and exosomes were isolated.
  • protein concentration by Bradford assay, the concentration of protein was adjusted to ⁇ 0.1 ⁇ g/ ⁇ L in PBS, and the samples were diluted 1 :50 (20 ⁇ in 1ml of PBS) for NTA analysis.
  • 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.
  • hMSC human mesenchymal stem cells
  • 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.
  • 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).
  • TE-hOB bone containing only osteoblasts
  • Fig 8A bone containing only osteoblasts
  • osteoclast precursors CD 14+ monocytes
  • FIG. 8A The capability of osteoclast precursors (CD 14+ monocytes) to differentiate into mature osteoclasts 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.
  • acidic hydrolases such as cathepsin K and the tartrate-resistant acid phosphatase (TRAP)
  • 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).
  • 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).
  • ES Native Ewing's sarcoma
  • HA hyaluronic acid
  • FIG.10B soft tissue matrix characterized by an equilibrium modulus of ⁇ 2 kPa
  • purified preparations of natural col 1 and HA low molecular weight, LMW; high molecular weight, HMW
  • Coll -HA LMW 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 ).
  • 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).
  • 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).
  • FIG.10D 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • hOB human osteoblasts
  • hOC human osteoclasts

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Rheumatology (AREA)
  • Oncology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un modèle tridimensionnel obtenu par ingénierie tissulaire. Le modèle tridimensionnel comprend des cellules tumorales de sarcome d'Ewing (ES) ; et une matrice osseuse humaine obtenue par ingénierie tissulaire. La matrice osseuse humaine obtenue par ingénierie tissulaire comprend en outre des ostéoblastes sécrétant la substance de l'os humain, et des ostéoclastes qui résorbent le tissu osseux pendant la croissance et la cicatrisation. La matrice osseuse humaine obtenue par ingénierie tissulaire comprend le modèle tridimensionnel obtenu par ingénierie tissulaire, récapitulant le processus ostéolytique. La matrice osseuse humaine obtenue par ingénierie tissulaire est construite par la coculture d'ostéoblastes et d'ostéoclastes. Les ostéoblastes sont produits par un processus de différentiation cellulaire à partir de cellules souches mésenchymateuses. Les ostéoclastes sont produits par différenciation cellulaire de monocytes humains, les monocytes humains étant isolés à partir de la couche leucoplaquettaire de prélèvements sanguins.
PCT/US2016/068478 2013-08-02 2016-12-23 Modèle tridimensionnel obtenu par ingénierie tissulaire pour l'analyse de tumeurs WO2017112919A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/016,101 US10883083B2 (en) 2013-08-02 2018-06-22 Tissue-engineered three-dimensional model for tumor analysis
US17/121,817 US20210102170A1 (en) 2013-08-02 2020-12-15 Tissue-engineered three-dimensional model for tumor analysis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562387121P 2015-12-23 2015-12-23
US62/387,121 2015-12-23

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2014/049416 Continuation-In-Part WO2015017784A1 (fr) 2013-08-02 2014-08-01 Modèles de cancers obtenus par génie tissulaire
US14/908,870 Continuation-In-Part US20160168542A1 (en) 2013-08-02 2014-08-01 Tissue engineered models of cancers

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/016,101 Continuation US10883083B2 (en) 2013-08-02 2018-06-22 Tissue-engineered three-dimensional model for tumor analysis

Publications (1)

Publication Number Publication Date
WO2017112919A1 true WO2017112919A1 (fr) 2017-06-29

Family

ID=59091259

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/068478 WO2017112919A1 (fr) 2013-08-02 2016-12-23 Modèle tridimensionnel obtenu par ingénierie tissulaire pour l'analyse de tumeurs

Country Status (1)

Country Link
WO (1) WO2017112919A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114181904A (zh) * 2021-12-06 2022-03-15 华中科技大学 模拟骨物理特征的肿瘤细胞三维培养支架及其制备与应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100324677A1 (en) * 2007-10-24 2010-12-23 The University Of Sydney Biocompatible material and uses thereof
US20130202644A1 (en) * 2010-04-06 2013-08-08 John W. Holaday Methods of treating cancer
US8834928B1 (en) * 2011-05-16 2014-09-16 Musculoskeletal Transplant Foundation Tissue-derived tissugenic implants, and methods of fabricating and using same
WO2015017784A1 (fr) * 2013-08-02 2015-02-05 The Trustees Of Columbia University In The City Of New York Modèles de cancers obtenus par génie tissulaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100324677A1 (en) * 2007-10-24 2010-12-23 The University Of Sydney Biocompatible material and uses thereof
US20130202644A1 (en) * 2010-04-06 2013-08-08 John W. Holaday Methods of treating cancer
US8834928B1 (en) * 2011-05-16 2014-09-16 Musculoskeletal Transplant Foundation Tissue-derived tissugenic implants, and methods of fabricating and using same
WO2015017784A1 (fr) * 2013-08-02 2015-02-05 The Trustees Of Columbia University In The City Of New York Modèles de cancers obtenus par génie tissulaire

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114181904A (zh) * 2021-12-06 2022-03-15 华中科技大学 模拟骨物理特征的肿瘤细胞三维培养支架及其制备与应用
CN114181904B (zh) * 2021-12-06 2024-02-09 华中科技大学 模拟骨物理特征的肿瘤细胞三维培养支架及其制备与应用

Similar Documents

Publication Publication Date Title
JP7442852B2 (ja) 生理活性腎臓細胞
Duan et al. Unsaturated polyurethane films grafted with enantiomeric polylysine promotes macrophage polarization to a M2 phenotype through PI3K/Akt1/mTOR axis
Villasante et al. Recapitulating the size and cargo of tumor exosomes in a tissue-engineered model
Nayak et al. Tissue mimetic 3D scaffold for breast tumor-derived organoid culture toward personalized chemotherapy
JP6054299B2 (ja) 臓器強化のための注射可能な製剤
US20210102170A1 (en) Tissue-engineered three-dimensional model for tumor analysis
US20190201586A1 (en) Three-Dimensional Tissue Matrix Scaffold System
De Luca et al. Positively charged polymers modulate the fate of human mesenchymal stromal cells via ephrinB2/EphB4 signaling
Chaicharoenaudomrung et al. Fabrication of 3D calcium‐alginate scaffolds for human glioblastoma modeling and anticancer drug response evaluation
KR102215476B1 (ko) 신장 세포 모집단 및 이의 용도
Lacombe et al. Plant-based scaffolds modify cellular response to drug and radiation exposure compared to standard cell culture models
Amorim et al. Hyaluronic acid/poly-L-lysine bilayered silica nanoparticles enhance the osteogenic differentiation of human mesenchymal stem cells
Koistinen et al. EMT induced by EGF and wounding activates hyaluronan synthesis machinery and EV shedding in rat primary mesothelial cells
Han et al. In vitro bone metastasis dwelling in a 3D bioengineered niche
EP2834348A1 (fr) Construction d'os / cartilage bi-phasique in vitro en 3d
Recha‐Sancho et al. Heparin‐based self‐assembling peptide scaffold reestablish chondrogenic phenotype of expanded de‐differentiated human chondrocytes
Monteiro et al. Three‐dimensional osteosarcoma models for advancing drug Discovery and development
He et al. Honeycomb-like hydrogel microspheres for 3D bulk construction of tumor models
Zhang et al. Enhanced multi-lineage differentiation of human mesenchymal stem/stromal cells within poly (N-isopropylacrylamide-acrylic acid) microgel-formed three-dimensional constructs
Li et al. The PCK2-glycolysis axis assists three-dimensional-stiffness maintaining stem cell osteogenesis
Lim et al. Advanced 3D dynamic culture system with transforming growth factor-β3 enhances production of potent extracellular vesicles with modified protein cargoes via upregulation of TGF-β signaling
Abay et al. Bone formation from porcine dental germ stem cells on surface modified polybutylene succinate scaffolds
US20230174912A1 (en) System, device, and method for single-cell encapsulation and culture
Yoon et al. Microphysiological system recapitulating the pathophysiology of adipose tissue in obesity
Campbell et al. Isolating and characterizing lymphatic endothelial progenitor cells for potential therapeutic lymphangiogenic applications

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16880134

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16880134

Country of ref document: EP

Kind code of ref document: A1