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 border="0" cellpadding="0" cellspacing="0" style="width:1049px;" width="1049">
	<tbody>
		<tr height="21">
			<td height="21">15 mL centrifuge tube</td>
			<td>Greiner</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">50 mL centrifuge tube</td>
			<td>Greiner</td>
			<td>227270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">6 cm cell culture dish</td>
			<td>Greiner</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">6-well plate</td>
			<td>Greiner</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>A9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42">C57-BL/6J mouse</td>
			<td>Sun Yat-sen University Laboratory Animal Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25">CO2 incubator</td>
			<td>SHEL LAB</td>
			<td>SCO5A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42">Dibasic sodium phosphate</td>
			<td>Guangzhou Chemical Reagent Factory</td>
			<td>BE14-GR-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">DMEM/F12</td>
			<td>Sigma-Aldrich</td>
			<td>D0547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Hemocytometer</td>
			<td>BLAU</td>
			<td>717805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1487</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42">MG-63</td>
			<td>Chinese Academy of Science, Shanghai Cell Bank</td>
			<td></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42">MNNG/HOS</td>
			<td>Chinese Academy of Science, Shanghai Cell Bank</td>
			<td></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42">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 height="21">
			<td height="21">Potassium chloride</td>
			<td>Sangon Biotech</td>
			<td>A100395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">Potassium Phosphate Monobasic</td>
			<td>Sangon Biotech</td>
			<td>A501211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21">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...

Watch this Scientific Journal Video about Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma at JoVE.com

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 ...

three-dimensional-bone-extracellular-matrix-model-for-osteosarcoma

Research

JoVE Journal

Cancer Research

7.1K Views.  Sun Yat-sen University. 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.

Video: Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

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 ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

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 ...