The authors value the support of Liuying Chen for her administrative assistance and Long Zhao for his excellent technical assistance during the construction of bone extracellular matrix scaffolds. This study is supported by grants from the National Natural Science Foundation of China (31871413).

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
<ol>
	<li>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.</li>
	<li>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 &#176;C for months until required. 
	NOTE: PBS used in all the following steps has 10 mM PO43&#8722;.</li>
</ol>
2. Bone demineralization and decellularization
<ol>
	<li>Thaw the frozen bones at room temperature, and then freeze again at -80 &#176;C for 1 h. Subject the bones to more than 2&#8211;3 freeze-thaw cycles for cell lysis and tissue breakdown.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.&#160;Add 40 mL sterile 0.05% trypsin-EDTA (TE) into the tube and incubate bones for 23&#160;h in a CO2 incubator at 37&#160;&#176;C18.</li>
	<li>Discard the TE solution and rinse twice with sterile PBS supplemented with 90 &#956;g/mL ampicillin and 90 &#956;g/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 &#956;g/mL ampicillin and 90 &#956;g/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.</li>
	<li>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 &#176;C for 2 months until required.</li>
</ol>
3. Cell seeding and culture
<ol>
	<li>Take out the BEM from 4 &#176;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&#8217;s modified Eagle&#8217;s medium/F12 (DF12) containing 5% fetal bovine serum, 90 &#956;g/mL ampicillin and 90 &#956;g/mL kanamycin). Incubate the BEM overnight in a CO2 incubator at 37 &#176;C.</li>
	<li>Obtain human OS cell lines (MNNG/HOS and MG-63). Suspend approximately 1.0 x 105 OS cells with 100 &#956;L 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).</li>
	<li>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 &#176;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 &#176;C.</li>
	<li>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 &#176;C.</li>
	<li>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 &#176;C and refresh the culture medium according to the proliferation situation of OS cells.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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The authors declare that they have no competing financial interests.

Generally, OS can be classified as osteoblastic, chondroblastic, and &#64257;broblastic 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...

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 origin2,3,4,5. These cells can accumulate&#160;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 phenotypes6,7. 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 proliferation10,11. Synthesized bone-like ECM can regulate the pool size, fate decisions and lineage progression of MSCs12,13,14. Moreover, results evidence its clinical significance to provide osteogenic activity by stimulating cellular processes during bone formation and regeneration15,16,17.
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.

<table><tbody><tr><td>15 mL centrifuge tube</td><td>Greiner</td><td>188271</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Greiner</td><td>227270</td><td></td></tr><tr><td>6 cm cell culture dish</td><td>Greiner</td><td>628160</td><td></td></tr><tr><td>6-well plate</td><td>Greiner</td><td>657160</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9393</td><td></td></tr><tr><td>C57-BL/6J mouse</td><td>Sun Yat-sen University Laboratory Animal Center</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>SHEL LAB</td><td>SCO5A</td><td></td></tr><tr><td>Dibasic sodium phosphate</td><td>Guangzhou Chemical Reagent Factory</td><td>BE14-GR-500G</td><td></td></tr><tr><td>DMEM/F12</td><td>Sigma-Aldrich</td><td>D0547</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30084.03</td><td></td></tr><tr><td>Hemocytometer</td><td>BLAU</td><td>717805</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>PHR1487</td><td></td></tr><tr><td>MG-63</td><td>Chinese Academy of Science, Shanghai Cell Bank</td><td></td><td>Human osteosarcoma cell line</td></tr><tr><td>MNNG/HOS</td><td>Chinese Academy of Science, Shanghai Cell Bank</td><td></td><td>Human osteosarcoma cell line</td></tr><tr><td>Phenol red</td><td>Sigma-Aldrich</td><td>P4633</td><td>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.</td></tr><tr><td>Potassium chloride</td><td>Sangon Biotech</td><td>A100395</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Sangon Biotech</td><td>A501211</td><td></td></tr><tr><td>Sodium chloride</td><td>Sangon Biotech</td><td>A501218</td><td></td></tr></tbody></table>

three-dimensional bone extracellular matrix model for osteosarcoma

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.

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&#8211;5 &#956;m sections for hematoxylin-eosin (H&#38;E) staining. The thorough removal of cell nuclei is shown by b...

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Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

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.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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Research

JoVE Journal

Cancer Research

7.1K Views.  Sun Yat-sen University. Deze methode biedt een efficiënte strategie voor het voorbereiden van functionele steigers voor bottumoronderzoek. Decellularized het bot extracelullar matrix, toonde variabele serocompatibility voor de overleving en activiteiten van osteosarcoom cellen. Het belangrijkste voordeel van deze techniek is dat osteosarcoom cellen cultuur in bot-afgeleide matrix tonen zeer heterogene morfologie vergelijkbaar met een klinische osteosarcoom histopathologie.De methode biedt ideaal model voor h...

Video: Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Medicine

Protocol for Osteosarcoma PDX Mouse Models: Studying Growth and Drug Resistance

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular matrix (osteoid) express the Pdpn gene to form cellular dendrites and transform into preosteocytes. Later, preosteocytes express the Dmp1 gene to promote matrix mineralization and thereby transform into mature osteocytes.This process is called osteocytogenesis. IDG-SW3 is a well-known cell line for in vitro studies of osteocytogenesis. Many previous methods have used collagen I as the main or the only component of the culturing matrix. However, in addition to collagen I, the osteoid also contains a ground substance, which is an important component in promoting cellular growth, adhesion, and migration. In addition, the matrix substance is transparent, which increases the transparency of the collagen I-formed gel and, thus, aids the exploration of dendrite formation through imaging techniques. Thus, this paper details a protocol to establish a 3D gel using an extracellular matrix along with collagen I for IDG-SW3 survival. In this work, dendrite formation and gene expression were analyzed during osteocytogenesis. After 7 days of osteogenic culture, an extensive dendrite network was clearly observed under a fluorescence confocal microscope. Real-time PCR showed that the mRNA levels of Pdpn and Dmp1 continually increased for 3 weeks. At week 4, the stereomicroscope revealed an opaque gel filled with mineral particles, consistent with the X-ray fluorescence (XRF) assay. These results indicate that this culture matrix successfully facilitates the transition from osteoblasts to mature osteocytes.

Here, we present a protocol for culturing IDG-SW3 cells in a three-dimensional (3D) extracellular matrix.

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

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