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 ...

This method can help answer key questions in the intercellular communications field about reciprocal paracrine versus juxtacrine interactions between two types of cells. The main advantage of this method is that it is a very simple and reproducible technique that accurately captures paracrine signaling under typical culture conditions while at the same time preventing effective juxtacrine signaling. The implications of this technique extend to a better understanding of the mechanism of the crosstalk between the cancer cells and the tumor microenvironment.Though this method can provide insight into the interactions between the cancer cells and the tumor stroma, it can also be applied to other systems such as wound healing and angiogenesis. Begin by detaching the ovarian cancer cells from an 80%confluent mixture with one milliliter of 0.25%trypsin for 40 to 60 seconds, followed by neutralization of the reaction with six milliliters of complete growth medium. Collect the cells by centrifugation and resuspend the pellet in five milliliters of fresh complete growth medium.After counting, dilute the cells to a 100, 000 ovarian cancer cells per milliliter of complete growth medium concentration and place three inverted 0.4 micrometer pore tissue culture treated polycarbonate membrane cell culture inserts per experimental condition in an appropriately sized sterile tissue culture dish. Label the inserts according to the experimental condition and carefully pipette the cancer cells onto the bottom of each insert in concentric circles, starting from the middle of the membrane and moving outward to form an 800 microliter dome. Be extremely careful when seeding the cells onto the bottom surface of the insert so that the dome of the medium containing cells does not run off the edges of the insert during the incubation.When all of the inserts have been seeded, cover the dish and carefully place the inserts into the cell culture incubator. After about four hours, detach the HPMC with two milliliters of 0.25%trypsin for one to two minutes, followed by neutralization of the reaction with 12 milliliters of complete growth medium. Collect the HPMC by centrifugation and resuspend the pellet in 10 milliliters of complete growth medium for counting.Dilute the cells to a 300, 000 HPMC per milliliter of complete growth medium and add 2.5 milliliters of fresh complete growth medium to each well of a six-well culture plate. Using sterile forceps, place each insert right side up into each well of the six-well plate so that the lower ovarian cancer cell attached surfaces are immersed in the media. Then seed 1.5 milliliters of HPMC into the well of each insert.Then place the plate in the cell culture incubator for 72 hours. At the end of the incubation, carefully remove the inserts from each well and rinse both sides of the inserts with PBS. Place each insert into a new six-well plate and add 0.5 milliliters of trypsin to the insert wells and two milliliters of trypsin to the bottom wells.After two minutes at 37 degrees Celsius, add one milliliter of complete growth medium to each insert and carefully pipette the solution a few times without tearing the membrane or spilling the cell suspension into the well below. While pipetting, care should be taken to prevent the cross-contamination of the cells from one chamber with the others. Transfer the resuspended cells into a labeled collection tube and further neutralize the trypsin with an additional two milliliters of complete growth medium.To collect the cells on the bottom of the inserts, flush the bottoms of the membranes two to three times with the two milliliters of trypsin from the corresponding well of the six-well plate so that the detached cells are caught in the appropriate well bottoms. When all of the cells have been detached, neutralize the trypsin in each well with six milliliters of fresh growth medium and transfer the cell suspensions to labeled collection tubes. Then collect all of the cells by centrifugation and resuspend the pellets in 0.7 milliliters of phenol and guanidine thiocyanate-based lysis reagent per tube for RNA extraction and isolation according to standard protocols.Proximal culture of HPMC with ovarian cancer cells results in an increased expression of fibronectin and Transforming Growth Factor or TGF beta one compared to control HPMC seeded on inserts in the absence of cancer cells. The expression of both fibronectin and TGF beta one also significantly increases in ovarian cancer cells upon proximal culture with HPMCs compared to control ovarian cell cultured in the absence of HPMC. Conversely, ovarian cancer cells treated with HPMC conditioned medium demonstrate a significantly reduced fibronectin expression with no change in TGF beta one expression.Blocking of TGF beta one with a neutralizing antibody however results in a significant decrease in TGF beta one expression in ovarian cancer cells in proximal culture with HPMCs. Interestingly, a slight increase in TGF beta one expression is observed in control non-proximally cultured ovarian cancer cells likely as a compensatory response. Proximal culture with ovarian cancer cells slightly increases E-cadherin expression in HPMCs while a marked decrease in E-cadherin is observed in ovarian cancer cells grown in proximity with HPMCs compared to controls further demonstrating the effectiveness of the proximal culture system for studying paracrine signaling between ovarian cancer cells and normal cells at the site of metastasis.While attempting this procedure, it's important to optimize the number of cells seeded and the duration of the proximal culture. Following this procedure, other methods like immunoblotting can be performed to answer additional questions about the changes in protein expression and the activation of downstream processing signaling pathways during the tumor cell mesothelial cell co-culture. After its development, this technique paved the way for researchers in the field of tumor microenvironment, immunology, developmental biology to explore reciprocal paracrine interactions between cells through secreted factors.

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