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 with the calvarias, it is important to use multiwell plates that are not treated for cell culture to prevent cancer cells from adhering and growing on the plastic instead of the bone. During the tissue inclusion, it is important to carefully orient each hemi-calvaria. They need to be placed in a vertical manner with the sagittal border on the outer side of the paraffin block. The orientation of the bone is essential to obtain consistent histological sections and reproducible results. Similarly, it is critical for consistency during the histomorphometric analysis to define a specific region in the calvaria to measure bone remodeling. Here, we identified first the coronal suture and then the long bone surface where the pictures were taken and the measurements made.
To achieve the standardization and develop the protocols described, we modified some established methodologies slightly. During the dissection of the hemi-calvarias, PBS was used to rinse the mouse heads instead of culture media. To determine the number of cells necessary to produce an effect on the calvarias, the tissue was incubated with different numbers of breast cancer cells. In general, 5 x 105 cancer cells work well for a 7 day assay. Also, during fixation, tissue paper was used instead of sponges to protect the calvarias. The tissue was well-preserved within the paper.
The model described has some limitations. The calvarias from neonatal mice lack some of the components of the bones that are the recipient of metastases, whether structural (i.e., trabecular bone) or cellular components such as immune cells or other cells of the bone marrow, including hematopoietic stem cells that cancer cells interact with. The culture of the calvarias in vitro cannot simulate the complete physiopathology. Also, when it comes to bone metastases, breast cancer cells seldom metastasize to the calvaria, and tend to favor bones with more active bone remodeling, such as the vertebrae, the hip, or the long bones of the arms and legs. Besides, the coculture of hemi-calvarias with cancer cells only represents the latest steps of the metastatic cascade: the colonization of the bone and the growth of the metastasis. Furthermore, the number of samples is limited by the number of pups per litter. It is recommended to use one litter per experiment and not mix litters to avoid variations within the results. We obtained similar results in bone remodeling when using calvarias from BALB/c or FVB mice, but whether the strain affects the time response of bone remodeling in this assay remains to be determined.
The main advantages of the calvarial ex vivo model compared to in vivo models include lower cost, simplicity of the assay, and shorter experimental time to obtain a bone remodeling response. The coculture of cancer cells and calvarias allows studying the cell-contact dependent interactions as well as the effect of secreted factors on bone remodeling, while the use of conditioned media allows focusing on the effect of soluble factors. In addition, the direct contact model can facilitate the characterization of intercellular interactions and molecular mechanisms of the bone metastasis microenvironment, as well as the study and evaluation of new drugs for the treatment of skeletal complications of malignancies.

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 to the control (Figure 2A). Quantitative histomorphometric analysis for the thickness and bone area of hemi-calvaria confirmed a significant increase for both parameters in the insulin-treated tissues compared to the control (Figure 2B). These data indicate the feasibility of the ex vivo model protocol to reproduce bone formation conditions.
Additionally, the calvarial ex vivo model can be used to reproduce cancer and bone microenvironment conditions. The protocol was used to evaluate the effect of the breast cancer cells MDA-MB-231 on murine calvarias. To perform this, the calvarial bones were directly cocultured with the cancer cells or cultured only with the conditioned media of the cancer cells. After 7 days of culture, the calvarias were embedded in paraffin, and H&#38;E staining was performed. The coculture with MDA-MB-231 breast cancer cells appeared to increase osteolysis, as shown by the decrease in bone and the damages to the calvarial structure when compared to the control calvarias cultivated only with media (Figure 2A). Bone area quantification of calvarias showed a significant decrease in the total bone area of the calvarias cultured with MDA-MB-231 cancer cells compared to the control (Figure 2B). These results demonstrated that the cocultures of cancer cells with calvarial tissue can recreate the cancer and bone microenvironment and could be used to investigate the mechanisms of osteolytic bone metastasis or to evaluate possible inhibitors of this process.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59028/59028fig1.jpg" /> 
Figure 1: Histological structure of the calvarial bone. Representative tissue section of hemi-calvaria after H&#38;E staining. <a href="https://www.jove.com/files/ftp_upload/59028/59028fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59028/59028fig2.jpg" /> 
Figure 2: Insulin increased the bone area and thickness of hemi-calvarias ex vivo while coculture of hemi-calvarias with MDA-MB-231 breast cancer cells induced osteolysis. For the bone formation model, the hemi-calvarias were cultured in the presence or absence of insulin (50 &#181;g/mL), while for the coculture with cancer cells, hemi-calvarias were cultured in the presence or absence of 5 x 105 MDA-MB-231 cells for 7 days and histomorphometrical analysis was performed on H&#38;E stained sections. (A) Representative tissue sections. (B) Histomorphometrical analysis of the bone area. Results are represented as the average &#177; SEM (n = 3). *P &#60; 0.05, nonparametric Mann-Whitney U test. <a href="https://www.jove.com/files/ftp_upload/59028/59028fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Universidad San Sebastian

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 ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

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 ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

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

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

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

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

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 ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...