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 Use Committee (IACUC) at the Center for Scientific Research and Higher Education at Ensenada (CICESE).
1. Calvarial dissection
<ol>
	<li>Sterilize dissection instruments (e.g., 1 x 2 teeth tissue forceps, straight surgical scissors, dissecting scissors, fine-tipped tweezers, fine curved tip dissecting forceps, dissecting forceps, scalpel). Sterile distilled water can be used to clean the surgical tools between each dissection, and sterile 1x PBS can be used for cleaning each hemi-calvaria.</li>
	<li>Place the sterile dissection instruments, water, 1x PBS, and required materials to carry out the procedure (e.g., micropipettes, precipitate glasses, sterile Petri dishes) in a laminar flow hood. 
	NOTE: Carry out the entire procedure under sterile conditions.</li>
	<li>Add 12 mL of sterile 1x PBS into two 10 cm Petri dishes.</li>
	<li>Select the pups and place them near the hood. 
	NOTE: Dissect the calvarias from 5-7 day old pups.</li>
	<li>Pick and hold the mouse carefully using dissecting forceps.</li>
	<li>Decapitate the mouse using dissecting scissors and place the head in a Petri dish with PBS. 
	NOTE: Decapitation is not suitable for older mice. If the experiment must be done with older mice, the method of euthanasia should be modified according to the animal experimentation rules.</li>
	<li>Hold the head firmly by the nose area with 1 x 2 teeth tissue forceps and remove the skin over the skull until the calvaria is visible.</li>
	<li>Identify the sutures (i.e., sagittal, coronal, and lambdoid) of the skull on the exposed calvaria.</li>
	<li>Penetrate with the tip of the microscissors approximately 2 mm behind the lambdoid suture and make a straight cut alongside it.</li>
	<li>Insert the tip of the microscissors on the rear side of the skull. Make a straight cut along the distal side of the lambdoid suture towards the coronal suture. Proceed similarly with the other lambdoid suture.</li>
	<li>Make another cut (at a 45&#176; angle) to connect the end of the cut made with the cut of the anterior fontanel.</li>
	<li>Repeat steps 1.10 and 1.11 with the other lambdoid suture. 
	NOTE: It is important to identify the sutures to make appropriate cuts and to be able to perform adequate embedding and data analysis.</li>
	<li>Use fine-tip forceps to remove the calvaria and place it in a Petri dish. With a scalpel, make a straight cut from the posterior fontanel along the sagittal suture through the coronal suture and ending in the anterior fontanel to obtain two hemi-calvarias.</li>
	<li>Pick up each of the hemi-calvarias with the fine-tipped tweezers and place them in a new Petri dish containing PBS.</li>
</ol>
2. Calvaria culture
<ol>
	<li>Add 1 mL of high-glucose DMEM medium supplemented with 0.1% bovine serum albumin (BSA) and 1% antibiotic/antimycotic (i.e., penicillin and streptomycin) in the wells of a 24 well plate. With fine-tip forceps, pick up each hemi-calvaria and place it in a well. 
	NOTE: Put the hemi-calvarias concave side down.</li>
	<li>Incubate the hemi-calvarias for 24 h at 37 &#176;C with 5% CO2.</li>
	<li>Remove the culture medium from each well and add 1 mL of fresh media containing the compound to test or conditioned media from other cells. 
	NOTE: Use at least three hemi-calvarias for each treatment group and use negative and positive controls.</li>
	<li>Incubate the hemi-calvarias for 7 days, changing the media every 2-3 days.</li>
</ol>
3. Culture with cancer cells
<ol>
	<li>Select a Petri dish with cancer cells at 80-90% confluence and trypsinize them. To trypsinize, wash the cancer cells 1x using PBS (5 mL per 75 cm2 flask) followed by incubation in an HBSS solution containing 0.05% trypsin and 0.53 mM EDTA (2 mL per 75 cm2 flask).</li>
	<li>Transfer the cell suspension into a 15 mL conical tube and centrifuge at 800 x g for 5 min at room temperature (RT).</li>
	<li>Remove and discard the supernatant.</li>
	<li>Resuspend the pellet in 2 mL of DMEM containing 2% fetal bovine serum (FBS) and 1% antibiotic/antimycotic. Count live cells.</li>
	<li>Assign hemi-calvarias to each study group (i.e., the negative control and coculture group for RNA or histology analysis).</li>
	<li>Remove the culture media from the hemi-calvarias and add 1 mL of DMEM containing 2% FBS and 1% antibiotic/antimycotic supplemented or not supplemented with cancer cells. 
	NOTE: The amount of cells to add can change depending on the cell line tested. With cancer cells like MDA-MB-231 or PC-3, 500,000 cells per well work appropriately.</li>
	<li>Incubate for 24 h at 37 &#176;C with 5% CO2. Pass each hemi-calvaria to a new well to retain only cancer cells that adhered to the bone tissue.</li>
	<li>Incubate for 7 days at 37 &#176;C with 5% CO2 and change the media every 2-3 days.</li>
</ol>
4. Fixation
<ol>
	<li>Cut squares of tissue paper to wrap the hemi-calvarias. 
	NOTE: Biopsy foam pads or steel capsules for small biopsies can also be used.</li>
	<li>Use straight fine-tip forceps to pick up each hemi-calvaria from the sagittal suture, place on a tissue paper, and wrap.</li>
	<li>Place the wrapped hemi-calvaria inside a labeled embedding cassette.</li>
	<li>Place the cassette into a container with 10% phosphate-buffered formalin.</li>
	<li>Fix for 24 h at 4 &#176;C.</li>
</ol>
5. Decalcification
<ol>
	<li>Remove the formalin, add 1x PBS, and agitate the tissues for 30 min to rinse the hemi-calvarias.</li>
	<li>Remove the PBS and add 10% EDTA (pH = 8, 0.34 M). 
	NOTE: Verify that the solution covers the tissues completely.</li>
	<li>Decalcify the tissues for 48 h at 4 &#176;C.</li>
	<li>Discard the EDTA solution and add 1x PBS to rinse the tissue.</li>
	<li>Store the hemi-calvarias in 70% ethanol until the histology processing.</li>
</ol>
6. Tissue processing
<ol>
	<li>Dehydrate the tissues with rounds of 96% ethanol, 60 min (3x), followed by 100% ethanol, 60 min (3x).</li>
	<li>Replace the ethanol by 100% xylene, 60 min (3x).</li>
	<li>Incubate the cassettes in paraffin wax, 60 min (2x).</li>
</ol>
7. Embedding
<ol>
	<li>Open the cassettes, carefully remove the tissue paper, and unwrap the calvaria.</li>
	<li>Pick up each calvaria carefully and stack all the hemi-calvarias from the same group in the same orientation.</li>
	<li>Put some paraffin in a mold.</li>
	<li>Pick up all hemi-calvarias with the forceps and place them inside the mold with the sagittal suture down towards the base of the mold. 
	NOTE: Orientation of the hemi-calvarias in the mold during inclusion is crucial to obtain consistent histological sections and reproducible results because this orientation will allow posterior identification of the front and parietal bones from the calvarias and the coronal suture facilitating the quantitative assessment.</li>
	<li>Release the forceps and verify that the hemi-calvarias stay in place.</li>
	<li>Place the labeled cassette on top of the mold and fill it with more paraffin to cover the hemi-calvarias.</li>
	<li>Move the molds to the cold surface.</li>
</ol>
8. Sectioning
<ol>
	<li>Trim 500-600 &#181;m of the sagittal suture with the microtome.</li>
	<li>Cut 4 &#181;m thick sections, and collect the sections needed.</li>
	<li>Trim another 300 &#181;m.</li>
	<li>Cut and collect another six 4 &#181;m thick sections.</li>
	<li>Trim the block 300 &#181;m further and collect more sections.</li>
	<li>Mount the sections onto glass microscope slides.</li>
	<li>Dry the slides at RT.</li>
</ol>
9. Staining
<ol>
	<li>Immerse the sections in 100% xylene for 3 min.</li>
	<li>Submerge in 100% ethanol for 1 min.</li>
	<li>Merge in 96% ethanol for 1 min.</li>
	<li>Immerse in 80% ethanol for 1 min.</li>
	<li>Submerge in 70% ethanol for 1 min.</li>
	<li>Rinse in water for 3 min.</li>
	<li>Submerge in hematoxylin for 3 min.</li>
	<li>Rinse in water until the section is clear.</li>
	<li>Submerge in a saturated lithium carbonate solution for 10 sec.</li>
	<li>Rinse in water for 3 min.</li>
	<li>Submerge in 96% ethanol for 1 min.</li>
	<li>Submerge in eosin for 3 min.</li>
	<li>Immerse in 96% ethanol for 1 min.</li>
	<li>Submerge in 100% ethanol for 1 min.</li>
	<li>Submerge in 100% xylene for 3 min.</li>
	<li>Add some per mount-mounting medium to the slide and protect it with a coverslip. 
	NOTE: You can complement the hematoxyline and eosin (H&#38;E) staining with tartrate-resistant acid phosphatase (TRAP) staining to evaluate osteoclast and osteolysis.</li>
</ol>
10. Quantitative assessment: defining area for analysis
<ol>
	<li>Examine the sections under low power (i.e., 4x) to identify the orientation and the sutures.</li>
	<li>Define the coronal suture and identify the long bone surface on one side and the short surface on the other.</li>
	<li>Identify the coronal suture under 40x magnification, and move two or three optical fields away from the suture along the long surface. Capture images of this area for analysis.</li>
	<li>Define the old bone area and the new bone area. The eosin Y with orange G stains the old bone darker and the new bone lighter.</li>
	<li>Measure the total bone area of the old and new bone with Image J software using the color threshold tool. Express the results as &#181;m2.</li>
</ol>
11. Data analysis
<ol>
	<li>Analyze the results using statistical software. Significant differences between groups can be determined using appropriate tests (e.g., nonparametric Mann-Whitney U test as is done herel).</li>
</ol>

<ol>
	<li>Boyce, B., Coleman, R. E., Abrahamsson, P. A., Hadji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+biology+and+pathology.">Bone biology and pathology.</a> Handbook of Cancer-Related Bone Disease. , 3-21 (2012).</li><li>Clark, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy and Physiology: Understanding the Human Body. 474, (2005).</li><li>Fournier, P. G. J., Ju&#225;rez, P., Guise, T. A., Heymann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-bone+interactions:+there+is+no+place+like+bone.">Tumor-bone interactions: there is no place like bone.</a> Bone Cancer: Primary Bone Cancers and Bone Metastases. , 13-28 (2014).</li><li>Ribatti, D., Mangialardi, G., Vacca, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stephen+Paget+and+the+"seed+and+soil"+theory+of+metastatic+dissemination.">Stephen Paget and the "seed and soil" theory of metastatic dissemination.</a> Clinical and Experimental Medicine. 6 (4), 145-149 (2006).</li><li>Guise, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vicious+cycle+of+bone+metastases.">The vicious cycle of bone metastases.</a> Journal of Musculoskeletal &amp; Neuronal Interactions. 2 (6), 570-572 (2002).</li><li>Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+bone+metastasis.">Mechanisms of bone metastasis.</a> Cancer. 80 (8), 1546-1556 (1997).</li><li>Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+to+bone:+causes,+consequences+and+therapeutic+opportunities.">Metastasis to bone: causes, consequences and therapeutic opportunities.</a> Nature Reviews Cancer. 2 (8), 584-593 (2002).</li><li>Chong, S. K. 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. H., Ralston, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+bone+formation+using+mouse+calvarial+organ+cultures.">Assessing bone formation using mouse calvarial organ cultures.</a> Bone Research Protocols. , 183-198 (2003).</li><li>Mohammad, K. S., Chirgwin, J. M., Guise, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+new+bone+formation+in+neonatal+calvarial+organ+cultures.">Assessing new bone formation in neonatal calvarial organ cultures.</a> Methods in Molecular Biology. 455 (1), 37-50 (2008).</li></ol>

The authors declare no competing interests.

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>
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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...

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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 ...

measuring-bone-remodeling-recreating-tumor-bone-microenvironment

Research

JoVE Journal

Cancer Research

6.7K Views.  Center for Scientific Research and Higher Education at Ensenada. 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.

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

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Establishing a 3D Ex Vivo Model for Ovarian Cancer-Omentum Interaction

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

Prognostic Significance of Immune Cell Infiltration in Hepatocellular Carcinoma

Mast Cells in the Microenvironment of Hepatocellular Carcinoma Confer Favorable Prognosis: A Retrospective Study using QuPath Image Analysis Software

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies investigating the expression and localization of immune cells within tumor tissues are associated with several challenges, including a lack of precise annotation for tumor regions and random selection of microscopic fields of view. QuPath is an open-source, user-friendly software that could meet the growing need for digital pathology in whole-slide image (WSI) analysis.
The infiltration of HCC and adjacent tissues by CD1a+ immature dendritic cells (iDCs), CD117+ mast cells, and NKp46+ natural killer cells (NKs) cells was assessed immunohistochemically in representative specimens of 67 patients with HCC who underwent curative resection. The area fraction (AF) of positively stained cells was assessed automatically in WSIs using QuPath in the tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells was evaluated for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS).
The AF of mast cells was significantly greater than the AF of NKs, and the AF of iDCs was significantly lower compared to NKs in each region of interest. High AFs of mast cells in the IM and PT areas were associated with longer DFS. In addition, high AF of mast cells in IM was associated with longer OS.
Computer-assisted analysis using this software is a suitable tool for obtaining prognostic information for tumor-infiltrating immune cells (iDCs, mast cells, and NKs) in different regions of HCC after resection. Mast cells displayed the greatest AF in all regions of interest (ROIs). Mast cells in the peritumor region and IM showed a positive prognostic significance.

Author Spotlight: Investigating Immune Cell Dynamics in the Tumor Microenvironment &#8212; Challenges and Innovations in Cancer Prognosis

The presence of mast cells in the inner margin and peritumor areas of hepatocellular carcinoma after resection confers a favorable prognosis. This study endorses QuPath image analysis software as a promising platform that could meet the need for reproducibility, consistency, and accuracy in digital pathology.

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies ...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...