Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Summary

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Abstract

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

Introduction

Osteosarcoma (OS) usually occurs in actively growing areas, the metaphysis of long bones, during adolescence. More than 80% of the OS-affected sites have preference for the metaphysis of proximal tibia and proximal humerus as well as both distal and proximal femur, corresponding to the location of the growth plate¹. OS comprises multiple cell subtypes with mesenchymal properties and considerable diversity in histologic features and grade. Evidences support mesenchymal stem cells (MSCs), osteoblasts committed precursors and pericytes as the cells of origin^2,3,4,5. These cells can accumulate genetic or epigenetic alterations and give rise to OS under the influence of certain bone microenvironmental signals. Both intrinsic and extrinsic mechanisms result in the genomic instability and heterogeneity of OS, with multiple morphological and clinical phenotypes^6,7. For individualized therapies or screening of new drugs, novel models need to be generated to against heterogeneity or other clinical disorders.

OS is an intra-osseous malignant solid tumor. The complexity and activity of surrounding microenvironment elements confer phenotypic and functional differences upon OS cells in different locations of a tumor. Bone extracellular matrix (BEM) provides a structural and biochemical scaffold for mineral deposition and bone remodeling. The organic portion of extracellular matrix (ECM) mainly consists of type I collagen secreted by osteoblastic lineage cells, while its mineralized portion is composed of calcium phosphate in the form of hydroxyapatite⁸. The dynamic role of ECM networks is to regulate cell adhesion, differentiation, cross-talk and tissue function maintenance⁹.

Demineralized BEM and ECM hydrogels have been successfully used in cell culture and can enhance cell proliferation^10,11. Synthesized bone-like ECM can regulate the pool size, fate decisions and lineage progression of MSCs^12,13,14. Moreover, results evidence its clinical significance to provide osteogenic activity by stimulating cellular processes during bone formation and regeneration^15,16,17.

In this article, our group establishes a modified model and favorable alternative for three-dimensional long-term culture. OS cells injected into the tissue-derived BEM present a heterogeneously mesenchymal phenotype readily as compared to plastic two-dimensional cultures. BEM derived from site-specific homologous tissue show its dramatic advantage as being a native niche for OS cells in vitro and has great potential in OS theoretical and clinical research. This characterized BEM platform is simple but efficient for in vitro research and may be extended in modeling multiple cancers.

Protocol

Animal care and use are conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication NO.80-23, revised in 1996) after approval from the Animal Ethics Committee of Sun Yat-sen University.

1. Bone preparation

1. Obtain 4 to 6-week-old BALB/c mice (without sex-specific requirement). Euthanize a mouse aseptically by cervical dislocation and cut off fresh fibula, tibia and femur from a hindlimb with sterile surgical scissors. Peel off the epithelial tissue and then remove as much of the soft tissue as possible using scissors and tweezers.
2. Rinse the leg bones with sterile 10 mM phosphate buffered saline (PBS) twice to remove blood in a 6 cm dish. Immerse the bones in 75% ethanol for 3 min, then rinse with PBS twice. The clean bones can be stored in a sterile 50 mL centrifuge tube with sterile PBS at -80 °C for months until required.
   NOTE: PBS used in all the following steps has 10 mM PO₄³⁻.

2. Bone demineralization and decellularization

1. Thaw the frozen bones at room temperature, and then freeze again at -80 °C for 1 h. Subject the bones to more than 2–3 freeze-thaw cycles for cell lysis and tissue breakdown.
2. Incubate the bones in a sterile 50 mL centrifuge tube with 0.5 N HCl overnight at room temperature on a rocking platform or orbital shaker with gentle rocking/shaking to ensure complete and even coverage of bones.
   NOTE: Make sure the bones are entirely immersed during motion in the acid and do not settle during the process. The volume of HCl solution should be more than ten times as that of the bones.
3. After decalcification, decant the HCl solution completely and rinse under running water for 1 h. Then, wash the bones twice for 15 min per wash with distilled water on a rocking platform or orbital shaker.
   NOTE: Make sure to completely remove the solution or water between washes and after the final wash with distilled water.
4. Extract the lipids in the demineralized bones with a 1:1 mixture of methanol and chloroform in a 50 mL centrifuge tube wrapped with tin foil for 1 h at room temperature¹⁰.
5. Then, transfer the bones into another tube of methanol wrapped with tin foil for 30 min. Remove the methanol completely and rinse with distilled water twice for 15 min with gently shaking. Decant final wash water and proceed with the following steps under sterile condition.
   NOTE: During the extraction step, light must be avoided to prevent chloroform decomposition. The mixture can be stored in light-resistant container or a centrifuge tube wrapped with tin foil. Perform all treatment and wash steps under modest rotation or rocking motion.
6. Rinse the bones in a 6 cm dish with sterile PBS for 3 min, and then transfer the bones into a new 50 mL centrifuge tube. Add 40 mL sterile 0.05% trypsin-EDTA (TE) into the tube and incubate bones for 23 h in a CO₂ incubator at 37 °C¹⁸.
7. Discard the TE solution and rinse twice with sterile PBS supplemented with 90 μg/mL ampicillin and 90 μg/mL kanamycin. After decanting the final wash PBS completely, replenish with 40 mL sterile PBS. Wash thoroughly for 24 h at room temperature with gentle rocking or shaking for antibiotic soak.
   NOTE: All the sterile PBS used in this and the following steps contains 90 μg/mL ampicillin and 90 μg/mL kanamycin. Overnight wash under rotation or rocking motion are performed for long periods thorough immersion with antibiotics to achieve effective sterilization of pore spaces.
8. Remove the PBS and transfer the bones into a fresh 50 mL centrifuge tube filled with sterile PBS. The prepared demineralized and decellularized bones are called bone extracellular matrix (BEM) and can be stored at 4 °C for 2 months until required.

3. Cell seeding and culture

1. Take out the BEM from 4 °C refrigerator and immerse it in 75% ethanol for 30 s, then rinse with PBS twice. Transfer the BEM onto a clean 6-well cell culture plate. Add 2 mL complete culture medium (Dulbecco’s modified Eagle’s medium/F12 (DF12) containing 5% fetal bovine serum, 90 μg/mL ampicillin and 90 μg/mL kanamycin). Incubate the BEM overnight in a CO₂ incubator at 37 °C.
2. Obtain human OS cell lines (MNNG/HOS and MG-63). Suspend approximately 1.0 x 10⁵ OS cells with 100 μL PBS containing phenol red as indicator.
   NOTE: To better track and observe multi-layer cells within the three-dimensional BEM model, MNNG/HOS and MG-63 are infected with lentiviral vector expressing fluorescent mCherry and green fluorescent protein (GFP).
3. After the BEM is fully soaked in the medium, inject OS cells into BEM from proximal or distal epiphysis when the needle reaches the medullary cavity of BEM. Incubate the OS-BEM model for a minimum of 2 h in a humidified 5% CO₂ atmosphere at 37 °C to ensure the injected cells firmly adhere to BEM.
   NOTE: Pre-warm all the media used for cell culture. The incubator used for cell culture has a humidified 5% CO₂ atmosphere at 37 °C.
4. Add 1 mL complete culture medium into the plate to completely coat the surface of the BEM culture overnight in a CO₂ incubator at 37 °C.
5. Gently transfer the OS-BEM model into a new well of 6-well plate with a sterile tweezer and refeed 1 mL fresh culture medium. Culture the model for 14 days in a CO₂ incubator at 37 °C and refresh the culture medium according to the proliferation situation of OS cells.
6. Keep monitoring medium color and cell status under the inverted fluorescence microscope during the culture process. When OS cells expand to plate, gently transfer the OS-BEM model to another new well with sterile tweezer.
   NOTE: The culture medium is bright red at pH 7.4, which is the optimum pH value for most mammalian cell culture. If the medium turns into orange or even yellow, immediately refresh the medium to maintain a healthy environment for OS cells.
7. Transfer the OS-BEM model into a new well with tweezers and gently rinse with PBS to remove the culture medium. Then, transfer into a 15 mL centrifuge tube and fix with 10% buffered formalin for histological identification.

Results

After demineralization and decellularization, BEM appears to be translucent with stronger resilience and tenacity compared to native mouse bone. A little muscle residue and the space of medullary cavity can be clearly observed (Figure 1A, B). To determine the effective decellularization of BEM, BEM is embedded in paraffin after fixation, and then sliced into 3–5 μm sections for hematoxylin-eosin (H&E) staining. The thorough removal of cell nuclei is shown by bright-field...

Discussion

Generally, OS can be classified as osteoblastic, chondroblastic, and fibroblastic subtypes depending on its dominant histologic component. Its prognosis is dependent not only on histologic parameters but also on its anatomic site. It may occur inside the bones (in the intramedullary or intracortical compartment), on the surfaces of bones, and in extraosseous sites¹⁹. The emergence and heterogeneity of OS can be elucidated as a conjugation of oncogenic events and an adequate microenvironmenta...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL centrifuge tube	Greiner	188271
50 mL centrifuge tube	Greiner	227270
6 cm cell culture dish	Greiner	628160
6-well plate	Greiner	657160
Ampicillin	Sigma-Aldrich	A9393
C57-BL/6J mouse	Sun Yat-sen University Laboratory Animal Center
CO2 incubator	SHEL LAB	SCO5A
Dibasic sodium phosphate	Guangzhou Chemical Reagent Factory	BE14-GR-500G
DMEM/F12	Sigma-Aldrich	D0547
Fetal bovine serum	Hyclone	SH30084.03
Hemocytometer	BLAU	717805
Kanamycin	Sigma-Aldrich	PHR1487
MG-63	Chinese Academy of Science, Shanghai Cell Bank		Human osteosarcoma cell line
MNNG/HOS	Chinese Academy of Science, Shanghai Cell Bank		Human osteosarcoma cell line
Phenol red	Sigma-Aldrich	P4633	A solution of phenol red is used as a pH indicator: its color exhibits a gradual transition from yellow to red over the pH range 6.6 to 8.0.
Potassium chloride	Sangon Biotech	A100395
Potassium Phosphate Monobasic	Sangon Biotech	A501211
Sodium chloride	Sangon Biotech	A501218