Name: measuring bone remodeling and recreating the tumor-bone microenvironment using calvaria co-culture and histomorphometry
Uploaded: 2020-03-14T12:00:00
Description: Watch this Scientific Journal Video about Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry at JoVE.com

The authors thank Mario Nomura, M.D. and Rodolfo D&#237;az for their help with the histology, and Pierrick Fournier, Ph.D. for his valuable comments to improve the quality of the paper.

All mice used in these assays were obtained from BALB/c mice strains, using male and female mice indiscriminately. Previous culture experiments have also been performed using other strains, such as FVB, Swiss mice, CD-1, and CsA mice11,12,14. All mice were housed according to National Institutes of Health (NIH) guidelines, Appendix Q. Procedures involving animal subjects have been approved by the Institutional Animals Care and U...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+bone+metastasis:+Opportunities+for+the+study+of+cancer+dormancy.">Experimental models of bone metastasis: Opportunities for the study of cancer dormancy.</a> Advanced Drug Delivery Reviews. 94 (1), 141-150 (2015).</li><li>Deguchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+model+of+bone+to+facilitate+measurement+of+adhesion+forces+and+super-resolution+imaging+of+osteoclasts.">In vitro model of bone to facilitate measurement of adhesion forces and super-resolution imaging of osteoclasts.</a> Scientific Reports. 6 (22585), 1-13 (2016).</li><li>Marino, S., Staines, K. A., Brown, G., Howard-Jones, R. A., Adamczyk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+ex+vivo+explant+cultures:+applications+in+bone+research.">Models of ex vivo explant cultures: applications in bone research.</a> BoneKEY Reports. 5, 818 (2016).</li><li>Nordstrand, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+validation+of+an+in+vitro+coculture+model+to+study+the+interactions+between+bone+and+prostate+cancer+cells.">Establishment and validation of an in vitro coculture model to study the interactions between bone and prostate cancer cells.</a> Clinical &amp; Experimental Metastasis. 26 (8), 945-953 (2009).</li><li>Curtin, P., Youm, H., Salih, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cancer-bone+metastasis+model+using+ex+vivo+cocultures+of+live+calvaria+bones+and+cancer+cells.">Three-dimensional cancer-bone metastasis model using ex vivo cocultures of live calvaria bones and cancer cells.</a> Biomaterials. 33 (4), 1065-1078 (2012).</li><li>Garret, R., Helfrich, M. 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Here, we describe the protocol for a calvarial ex vivo model to evaluate bone formation or resorption and to study the interactions of cancer cells with calvarial mouse bone. The critical steps of this technique are the dissection, culture, embedding, and histomorphometrical analysis of the calvarias. During the dissection of the calvarias, it is crucial to cut the hemi-calvarias into a trapezoid, as it will strongly facilitate the orientation during the paraffin inclusion. When studying cancer cell interactions...

Bone is a dynamic connective tissue that has several functions, including supporting the muscles, protecting the internal organs and bone marrow, and storing and releasing calcium and growth factors1,2. To maintain its integrity and proper function, bone tissue is continuously under the process of remodeling. In general terms, a cycle of bone remodeling can be divided into bone resorption and bone formation1. An imbalance between these two phases of bone remodeling can lead to the development of bone pathologies. Also, diseases such as breast cancer often affect bone integrity; approximately more than 70% of patients in advanced stages have or will have bone metastases. When breast cancer cells enter the bones, they affect bone metabolism, resulting in excessive resorption (osteoclastic lesions) and/or formation (osteoblastic lesions)3.
To understand the biology of bone diseases and develop new treatments, it is necessary to understand the mechanisms involved in bone remodeling. In cancer research, it is essential to investigate the bone metastasis process and its relation to the metastatic microenvironment. In 1889, Stephen Paget hypothesized that metastases occur when there is compatibility between the tumor cells and the target tissue, and suggested that the metastatic site depends on the affinity of the tumor for the microenvironment4. In 1997, Mundy and Guise introduced the concept of the &#34;vicious cycle of bone metastases&#34; to explain how tumor cells modify the bone microenvironment to achieve their survival and growth, and how the bone microenvironment promotes their growth by providing calcium and growth factors5,6,7.
To characterize the mechanisms involved in bone remodeling and bone metastasis and to evaluate molecules with possible therapeutic potential, it has been necessary to develop in vitro and in vivo models. However, these models currently present many limitations, such as the simplified representation of the bone microenvironment, and their cost8,9. The culture of bone explants ex vivo has the advantage of maintaining the three-dimensional organization as well as the diversity of bone cells. In addition, experimental conditions can be controlled. The explant models include the culture of metatarsal bones, femoral heads, calvarias, and mandibular or trabecular cores10. The advantages of the ex vivo models have been demonstrated in diverse studies. In 2009, Nordstrand and collaborators reported the establishment of a coculture model based on the interactions between bone and prostate cancer cells11. Also, in 2012, Curtin and collaborators reported the development of a three-dimensional model using ex vivo cocultures12. The purpose of such ex vivo models is to recreate the conditions of the bone microenvironment as accurately as possible to be able to characterize the mechanisms involved in normal or pathological bone remodeling and evaluate the efficacy of new therapeutic agents.
The present protocol is based on the procedures published by Garrett13 and Mohammad et al.14. Neonatal mouse calvaria cultures have been used as an experimental model, as they retain the three-dimensional architecture of the bone under development and bone cells, including cells at all stages of differentiation (i.e., osteoblasts, osteoclasts, osteocytes, stromal cells) that lead to mature osteoclasts and osteoblasts, as well as the mineralized matrix14. The ex vivo model does not represent the pathological process of bone diseases totally. However, effects on bone remodeling or cancer-induced bone osteolysis can be accurately measured.
Briefly, this protocol consists of the following steps: the dissection of calvarias from 5-7 day old mice, calvaria preculture, calvaria culture applications (e.g., culture in the presence of insulin, cancer cells or conditioned medium, and even agents with therapeutic potential, according to the aim of the investigation), bone fixation and calvaria decalcification, tissue processing, histological analysis, and result interpretation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 well cell culture</td>
			<td>Corning</td>
			<td>CLS3524</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well non tissue culture</td>
			<td>Falcon</td>
			<td>15705-060</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL cryovial</td>
			<td>SSI</td>
			<td>2341-S0S</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics-Antimycotic</td>
			<td>Corning</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Biowest</td>
			<td>P6154-100GR</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugue</td>
			<td>Eppendorf</td>
			<td>22628188</td>
			<td>Centrifuge 5810R</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Corning</td>
			<td>2935-24X50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytoseal resin</td>
			<td>Richard Allen</td>
			<td>8310-10</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td></td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagles Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (10X)</td>
			<td>Corning</td>
			<td>20-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ebedding Cassettes</td>
			<td>Sigma</td>
			<td>Z672122-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Golden</td>
			<td>26400</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding Workstation</td>
			<td>Thermo Scientific</td>
			<td>A81000001</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Golden</td>
			<td>60600</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>JALMEK</td>
			<td>E5325-17P</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>BIO-S1650-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Corning</td>
			<td>CLS431229</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps and scissors</td>
			<td>LANCETA HG</td>
			<td>74165</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin buffered 10%</td>
			<td>Sigma</td>
			<td>HT501320</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides 25 x 75 mm</td>
			<td>Premiere</td>
			<td>9105</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris&#39;s Hematoxylin</td>
			<td>Jalmek</td>
			<td>SH025-13</td>
			<td></td>
		</tr>
		<tr>
			<td>High profile blades</td>
			<td>Thermo Scientific</td>
			<td>1001259</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoquinet</td>
			<td>Thermo Scientific</td>
			<td>813150</td>
			<td>STP 120</td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma</td>
			<td>16634</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>Axio Scope.A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Thermo Scientific</td>
			<td>905200</td>
			<td>MICROM HM 355S</td>
		</tr>
		<tr>
			<td>Mouse food, 18% prot, 2018S</td>
			<td>Harlan</td>
			<td>T.2018S.15</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer</td>
			<td>VWR</td>
			<td>631-0696</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Biobasic</td>
			<td>OB0674-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Paraplast</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Section Flotation Bath</td>
			<td>Electrothermal</td>
			<td>MH8517X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Corning</td>
			<td>CLS430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Phloxin B</td>
			<td>Probiotek</td>
			<td>166-02072</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Corning</td>
			<td>25-051-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Wax dispenser</td>
			<td>Electrothermal</td>
			<td>MH8523BX1</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Golden</td>
			<td>534056-500ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring bone remodeling and recreating the tumor-bone microenvironment using calvaria co-culture and histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

To evaluate bone formation in the calvarial model, we cultivated the hemi-calvarias in media with or without 50 &#181;g/mL of insulin. Tissue sections were prepared and stained with H&#38;E. In these conditions, the histology showed that the structural integrity of the calvarial bone was maintained, allowing the identification of its different components (Figure 1). The calvarias treated with insulin presented an increase in the amount of bone tissue compared...

Watch this Scientific Journal Video about Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry at JoVE.com

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

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