Name: a proximal culture method to study paracrine signaling between cells
Uploaded: 2018-08-28T14:30:00
Description: Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

We are indebted to the patients for their participation in the tissue collection for these experiments. A DoD OCRP Ovarian Cancer Academy Award (W81XWH-15-0253) and a pilot award from Colleen&#39;s Dream Foundation to Anirban K. Mitra supported this research.

The protocol follows the guidelines of the Institutional Regulatory Board of Indiana University.
1. Cell Preparation
<ol>
	<li>Isolation and culture of human primary mesothelial cells
	<ol>
		<li>Isolate human primary mesothelial cells (HPMCs) from human omentum as described previously17,18 and grow them in complete growth medium [Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) containing 10% fetal bovine serum...

<ol>
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J., 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=EGFR+ligands+exhibit+functional+differences+in+models+of+paracrine+and+autocrine+signaling.">EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling.</a> Growth Factors. 30 (2), 107-116 (2012).</li><li>Kopan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling.">Notch signaling.</a> Cold Spring Harbor Perspectives in Biology. 4 (10), (2012).</li><li>Singh, A. B., Sugimoto, K., Harris, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxtacrine+activation+of+epidermal+growth+factor+(EGF)+receptor+by+membrane-anchored+heparin-binding+EGF-like+growth+factor+protects+epithelial+cells+from+anoikis+while+maintaining+an+epithelial+phenotype.">Juxtacrine activation of epidermal growth factor (EGF) receptor by membrane-anchored heparin-binding EGF-like growth factor protects epithelial cells from anoikis while maintaining an epithelial phenotype.</a> Journal of Biological Chemistry. 282 (45), 32890-32901 (2007).</li><li>Swartz, M. 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=Tumor+microenvironment+complexity:+emerging+roles+in+cancer+therapy.">Tumor microenvironment complexity: emerging roles in cancer therapy.</a> Cancer Research. 72 (10), 2473-2480 (2012).</li><li>Mitra, A. K., 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=MicroRNAs+reprogram+normal+fibroblasts+into+cancer-associated+fibroblasts+in+ovarian+cancer.">MicroRNAs reprogram normal fibroblasts into cancer-associated fibroblasts in ovarian cancer.</a> Cancer Discovery. 2 (12), 1100-1108 (2012).</li><li>Nieman, K. M., 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=Adipocytes+promote+ovarian+cancer+metastasis+and+provide+energy+for+rapid+tumor+growth.">Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.</a> Nature Medicine. 17 (11), 1498-1503 (2011).</li><li>Salimian Rizi, B., 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=Nitric+oxide+mediates+metabolic+coupling+of+omentum-derived+adipose+stroma+to+ovarian+and+endometrial+cancer+cells.">Nitric oxide mediates metabolic coupling of omentum-derived adipose stroma to ovarian and endometrial cancer cells.</a> Cancer Research. 75 (2), 456-471 (2015).</li><li>Mitra, A. K., 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=Microenvironment-induced+downregulation+of+miR-193b+drives+ovarian+cancer+metastasis.">Microenvironment-induced downregulation of miR-193b drives ovarian cancer metastasis.</a> Oncogene. 34 (48), 5923-5932 (2015).</li><li>Tomar, S., 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=ETS1+induction+by+the+microenvironment+promotes+ovarian+cancer+metastasis+through+focal+adhesion+kinase.">ETS1 induction by the microenvironment promotes ovarian cancer metastasis through focal adhesion kinase.</a> Cancer Letters. 414, 190-204 (2018).</li><li>Ovarian Cancer Metastasis: A Unique Mechanism of Dissemination. Tumor Metastasis Available from: <a target="_blank" href="https://www.intechopen.com/books/tumor-metastasis/ovarian-cancer-metastasis-a-unique-mechanism-of-dissemination">https://www.intechopen.com/books/tumor-metastasis/ovarian-cancer-metastasis-a-unique-mechanism-of-dissemination</a> (2016)</li><li>Iwanicki, M. P., 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=Ovarian+cancer+spheroids+use+myosin-generated+force+to+clear+the+mesothelium.">Ovarian cancer spheroids use myosin-generated force to clear the mesothelium.</a> Cancer Discovery. 1 (2), 144-157 (2011).</li><li>Kenny, H. 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=Mesothelial+cells+promote+early+ovarian+cancer+metastasis+through+fibronectin+secretion.">Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.</a> Journal of Clinical Investigation. 124 (10), 4614-4628 (2014).</li><li>Boelens, M. C., 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=Exosome+transfer+from+stromal+to+breast+cancer+cells+regulates+therapy+resistance+pathways.">Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways.</a> Cell. 159 (3), 499-513 (2014).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+function+of+exosomes+in+cancer.">The biology and function of exosomes in cancer.</a> Journal of Clinical Investigation. 126 (4), 1208-1215 (2016).</li><li>Kohlhapp, F. J., Mitra, A. K., Lengyel, E., Peter, M. 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Understanding the mechanism of paracrine and juxtacrine signaling between cells is essential for developing a better knowledge of normal tissue homeostasis and disease conditions7,8. Most paracrine signaling studies are conducted by collecting conditioned medium from one cell type and using it to treat the other cell type. This method has an advantage in its inherent simplicity. However, it does not accurately recapitulate the localized concentrations of the secr...

The role of productive reciprocal interactions between cancer cells and the tumor microenvironment in tumor progression has been well established and has become a major focus of research in cancer biology1. Similar instances of bidirectional signaling are crucial during wound healing, immune responses, angiogenesis, stem cell niches, and during development2,3,4,5,6,7,8. A common theme in all these biological processes is that cells respond in various ways to extracellular cues from their microenvironment which determine cell fate, tissue physiology, and disease progression. Therefore, the focus has increasingly turned toward developing a better understanding of the mechanisms involved in such cell-cell communications. A majority of such interactions involve paracrine or juxtacrine signaling between cells. Paracrine signaling involves the secretion of specific signaling factors by one cell which are perceived by corresponding receptors on another cell in the vicinity, triggering a response in it9,10, whereas juxtacrine signaling requires direct contact between cellular components of the two cells involved11,12.
Such signaling is a crucial component in tissue homeostasis, as well as in the tumor microenvironment. The cancer cells benefit from paracrine and juxtacrine factors from cells in the tumor stroma, including cancer-associated fibroblasts (CAFs), immune cells, and adipocytes13,14,15,16. The paracrine signaling can be mediated by growth factors, cytokines, chemokines, etc., while the juxtacrine signaling involves juxtaposed ligands and receptors as in Notch signaling, or interactions between integrins and their respective extracellular matrix proteins. We have demonstrated the importance of reciprocal interactions between ovarian cancer cells and CAFs in tumor progression and metastasis14. Similarly, the interactions of metastasizing ovarian cancer cells with the mesothelial cells covering the site of metastasis regulate key microRNAs and transcription factors in the cancer cells which promote metastatic colonization17,18.
Most studies on paracrine signaling involve the use of a conditioned medium collected from one cell type to treat the second cell type with. While this approach has been widely used, it does not effectively replicate the localized high-concentration levels of the secreted factor in the microenvironment of the receiving cell. It also fails to reproduce the kinetics of the continuous flow of the secreted factor being produced by one cell and received by the neighboring cell. Paracrine signaling is effective over short distances as the secreted factors are at the required concentrations only in the vicinity of the source cell and tend to diffuse and dilute out as the distance increases. This localized high concentration of the secreted factor is essential to trigger a response in the receptor cell. Moreover, the response in the recipient cells is also dependent on the balance of newly secreted factors and their continuous depletion through degradation, binding, and internalization in the recipient cells and diffusion away from the source cell. Conditioned medium can be concentrated to account for the higher localized concentrations present in the microenvironment, but that cannot accurately replicate the exact concentrations. Moreover, it cannot mimic the natural kinetics of production and depletion of the factor involved. To more accurately replicate paracrine signaling and separate it from juxtacrine signaling mechanisms, we have devised a novel proximal culture method, which involves growing the two cell types on either surface of a porous membrane. The pores are small enough to prevent juxtacrine interactions and yet allow the exchange of secreted factors at localized high concentrations. In that way, this system retains the kinetics of production and depletion of the paracrine factors.

<table>
	<tbody>
		<tr>
			<td>24 mm Transwell permeable support with 0.4 &#181;m Pore Polycarbonate Membrane Insert</td>
			<td>Corning (Costar)</td>
			<td>3412</td>
			<td>&#8226; 10 &#181;m thick translucent polycarbonate membrane 
			&#8226; Treated for optimal cell attachment 
			&#8226; Packaged 6 inserts in a 6 well plate, 4 plates per case 
			&#8226; Membrane must be stained for cell visibility 
			&#8226; Sterilized by gamma radiation</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning (Falcon)</td>
			<td>353046</td>
			<td>Flat Bottom, TC-treated, sterile, with Lid</td>
		</tr>
		<tr>
			<td>15 cm culture dish</td>
			<td>Corning (Falcon)</td>
			<td>353025</td>
			<td>Sterile, TC-treated Cell Culture Dish</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning (Cellgro)</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning (Cellgro)</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning (Cellgro)</td>
			<td>25-020-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin, 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipets</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>Biosafety level II cabinet</td>
			<td></td>
			<td></td>
			<td>Any make is fine</td>
		</tr>
		<tr>
			<td>FN1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01549976_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFB1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs00998133_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDH1 TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs01023895_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH TaqMan Gene Expression Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Hs99999905_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini RNA isolation Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>43-688-13</td>
			<td></td>
		</tr>
		<tr>
			<td>HeyA8 ovarian cancer cells</td>
			<td>Obtained from Ernst Lengyel Lab, University of Chicago</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946; Neutralizing Antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1835-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a proximal culture method to study paracrine signaling between cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Metastasizing ovarian cancer cells encounter mesothelial cells at the site of metastasis within the peritoneal cavity19. Productive paracrine and juxtacrine interactions with the mesothelial cells help in inducing adaptive responses in the ovarian cancer cells, which enable successful metastasis17,18,20,21. To test the effectiveness of th...

Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

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