A.P.S is supported by DebRA International and the British Skin Foundation. Y.Z.N is supported by A*STAR - University of Dundee Partnership PhD Programme.

This study was conducted according to the Declaration of Helsinki Principles and was approved by the appropriate Ethics Committees.
1. Preparation of Media and Reagents
<ol>
	<li>Preparation of 200x of L-ascorbic acid 2-phosphate stock
	<ol>
		<li>Dissolve 29 mg of L-ascorbic acid 2-phosphate per 5 ml of Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) solution and filter through 0.22 &#956;m membrane filter. Store as 0.25 ml sterile aliquots in -20 &#176;C.</li>
		<li>Add 0.25 ml aliquot of 200x L-ascorbic acid 2-phosphate stock to every 50 ml of fibroblast media (DMEM with 1% L-glutamine and 10% fetal bovine serum) on the day required, for a final concentration of 0.1 mM of L-ascorbic acid 2-phosphate.</li>
	</ol>
	</li>
	<li>Preparation of keratinocyte growth media
	<ol>
		<li>Isolation and culture of primary SCC keratinocytes have been described previously21. Preparation of keratinocyte growth media is described therein as well.</li>
		<li>Briefly, prepare the media as follows: 
		300 ml of DMEM and 100 ml of Ham&#8217;s F-12 supplemented with 10% FBS 
		0.4 mg/ml hydrocortisone 
		5 mg/ml insulin 
		10 ng/ml EGF 
		5 mg/ml transferrin 
		8.4 ng/ml cholera toxin 
		13 ng/ml liothyronine 
		1x penicillin-streptomycin solution</li>
	</ol>
	</li>
</ol>
2. In Vitro Construction of Fibroblast-derived Native Matrix in L-Ascorbic Acid 2-Phosphate Supplemented Media
<ol>
	<li>Seed 200,000 fibroblasts per well in 6-well plates (20,000 cells/cm2) in fibroblast media supplemented with of L-ascorbic acid 2-phosphate. Refeed every 2-3 days with 2-5 ml of media. (Note: Refeeding frequency and volume may be adjusted to suit the individual needs of different cells. See &#8220;Discussions&#8221;).</li>
	<li>A thick layer of cells embedded in extracellular matrix will form at the end of 6 weeks, visible to the naked eye (Figure 1). To release this layer from the tissue culture plate, gently scrape the circumference of matrix with a 1 ml micropipette tip and then push the edges of the matrix towards center of the well. This layer of cells and matrix (native matrix) should be easily detachable from the plate surface, and now be floating in the media. Spread the native matrix out in the media to avoid it folding up onto itself.</li>
	<li>Let the native matrix float and remodel for 5 days, changing media every 2-3 days. Due to tensile strength and intrinsic remodeling within matrix, the matrix will contract drastically and become reduced to a smaller, but thicker native matrix, and is ready to be utilized for invasion assay.</li>
</ol>
3. Invasion Assay with Tumor SCC Keratinocytes
<ol>
	<li>Pick up the native matrix gently with blunt forceps and transfer to Nylon net. Once on the Nylon net, spread the matrix gently to lie as flat as possible using a 1 ml micropipette tip and blunt forceps.</li>
	<li>Prepare the sterile clonal cylinders by smearing a small amount of sterile Vaseline on one end.</li>
	<li>Place the clonal cylinders on the native matrix, with the Vaseline side down. This is to ensure a tight seal between the native matrix and the clonal rings.</li>
	<li>Add cSCC cells to the clonal cylinders (250,000 cells in 100 &#181;l of keratinocyte growth media).</li>
	<li>Remove the clonal cylinders after 6 hr when the cSCC cells have settled down on the native matrix.</li>
	<li>Lift the Nylon net with the native matrix and cSCC cells to air-liquid interface onto bent stainless steel wire mesh support.</li>
	<li>Add keratinocyte growth media supplemented with ascorbic acid until the media level touches the bottom of the native matrix.</li>
	<li>Change media every 2-3 days and harvest at 7 and 14 days post-seeding of cSCC cells.</li>
</ol>
4. Harvesting of 3D Cultures and Preparation for Histology
<ol>
	<li>Fix in 4% paraformaldehyde overnight at room temperature.</li>
	<li>Bisect the samples and embed in wax for formalin-fixed paraffin embedded blocks, with the cut surface facing outwards.</li>
	<li>Alternatively, embed the bisected samples in OCT and snap-freeze immediately in liquid nitrogen for fresh frozen tissue blocks.</li>
	<li>Cut 4 &#956;m sections on microtome and dewax (if necessary). Stain using standard haematoxylin and eosin. To visualize the cSCC cells, mouse monoclonal antibody against keratin 14 (LL001, in-house) can be used in immunohistology staining.</li>
</ol>

<ol>
	<li>Bell, E., Ehrlich, H. P., Buttle, D. J., Nakatsuji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+tissue+formed+in+vitro+and+accepted+as+skin-equivalent+tissue+of+full+thickness.">Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness.</a> Science. 211 (4486), 1052-1054 (1981).</li><li>Asselineau, D., Bernhard, B., Bailly, C., Darmon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+morphogenesis+and+induction+of+the+67+kD+keratin+polypeptide+by+culture+of+human+keratinocytes+at+the+liquid-air+interface.">Epidermal morphogenesis and induction of the 67 kD keratin polypeptide by culture of human keratinocytes at the liquid-air interface.</a> Exp. Cell Res. 159 (2), 536-539 (1985).</li><li>Pruni&#233;ras, M. M., R&#233;gnier, M. M., Woodley, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Cultivation+of+Keratinocytes+with+an+Air-Liquid+Interface.">Methods for Cultivation of Keratinocytes with an Air-Liquid Interface.</a> J. Invest. Dermatol. 81 (1 Suppl), 28-33 (1983).</li><li>Prunieras, M., R&#233;gnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+procedure+for+culturing+human+epidermal+cells+on+allogenic+or+xenogenic+skin:+preparation+of+recombined+grafts.">New procedure for culturing human epidermal cells on allogenic or xenogenic skin: preparation of recombined grafts.</a> Ann. Chir. Plast. 24 (4), 357-362 (1979).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nature. 9 (12), 1392-1400 (2007).</li><li>Bhowmick, N. A., Neilson, E. G., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer+initiation+and+progression.">Stromal fibroblasts in cancer initiation and progression.</a> Nature. 432 (7015), 332-337 (2004).</li><li>Erez, N., Truitt, M., Olson, P., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-&#954;B-Dependent.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-&#954;B-Dependent.</a> Manner. Cancer Cell. 17 (2), 135-147 (2010).</li><li>Harkness, R. D., Marko, A. M., Muir, H. 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Cell Res. 202 (1), 1-8 (1992).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BD+Biosciences+-+Discover+Labware.">BD Biosciences - Discover Labware.</a> SPC-356230 Rev 5.0 at Rev 5.0. , .</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Ng, Y. Z., Dayal, J. H., South, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Predisposition+to+Cutaneous+Squamous+Cell+Carcinoma.">Genetic Predisposition to Cutaneous Squamous Cell Carcinoma.</a> , .</li><li>Christiano, A. M. A., Greenspan, D. S. D., Lee, S. S., Uitto, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+human+type+VII+collagen.+Complete+primary+sequence+of+the+alpha+1(VII)+chain+and+identification+of+intragenic+polymorphisms.">Cloning of human type VII collagen. Complete primary sequence of the alpha 1(VII) chain and identification of intragenic polymorphisms.</a> J. Biol. Chem. 269 (32), 20256-20262 (1994).</li><li>Hovnanian, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+linkage+of+recessive+dystrophic+epidermolysis+bullosa+to+the+type+VII+collagen+gene.">Genetic linkage of recessive dystrophic epidermolysis bullosa to the type VII collagen gene.</a> J. Clin. Invest. 90 (3), 1032-1036 (1992).</li><li>Ryyn&#228;nen, M. M., Ryyn&#228;nen, J. J., Sollberg, S. S., Iozzo, R. V. R., Knowlton, R. G. R., Uitto, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+linkage+of+type+VII+collagen+(COL7A1)+to+dominant+dystrophic+epidermolysis+bullosa+in+families+with+abnormal+anchoring+fibrils.">Genetic linkage of type VII collagen (COL7A1) to dominant dystrophic epidermolysis bullosa in families with abnormal anchoring fibrils.</a> J. Clin. Invest. 89 (3), 974-980 (1992).</li><li>Ng, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-derived+dermal+matrix+drives+development+of+aggressive+cutaneous+squamous+cell+carcinoma+in+patients+with+recessive+dystrophic+epidermolysis+bullosa.">Fibroblast-derived dermal matrix drives development of aggressive cutaneous squamous cell carcinoma in patients with recessive dystrophic epidermolysis bullosa.</a> Cancer Res. 72 (14), 3522-3534 (2012).</li><li>Larouche, D., Paquet, C., Fradette, J., Carrier, P., Auger, F. A., Germain, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.">Chapter 15.</a> Methods Mol. Biol. 482, 233-256 (2009).</li><li>Pouliot, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstructed+human+skin+produced+in+vitro+and+grafted+on+athymic+mice).">Reconstructed human skin produced in vitro and grafted on athymic mice).</a> Transplantation. 73 (11), 1751-1757 (2002).</li><li>Purdie, K. J., Pourreyron, C., South, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+squamous+cell+carcinoma+lines.">Isolation and culture of squamous cell carcinoma lines.</a> Methods Biol. 731, 151-159 (2011).</li><li>Pinnell, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+collagen+biosynthesis+by+ascorbic+acid:+a+review.">Regulation of collagen biosynthesis by ascorbic acid: a review.</a> Yale J. Biol. Med. 58 (6), 553-559 (1985).</li><li>Hata, R., Senoo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-ascorbic+acid+2-phosphate+stimulates+collagen+accumulation,+cell+proliferation,+and+formation+of+a+three-dimensional+tissuelike+substance+by+skin+fibroblasts.">L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts.</a> J. Cell. Physiol. 138 (1), 8-16 (1988).</li></ol>

The authors have nothing to disclose.

Due to the nature of this experiment, the total time required for completion can be up to two months. Throughout this time, utmost care and sterile tissue culture practices must be employed to prevent microbial contamination.

Aside from its role as a cofactor in the synthesis of hydroxyproline and hydroxylysine of collagens, ascorbic acid stimulates collagen specific mRNA expression in fibroblasts22. The ascorbic acid of choice here is the more stable L-ascorbic acid 2-phosphate23. Refeeding is advised three times a week but this will invariably be dependent on the cells being studied. The initial fibroblast cultures will consume more nutrients as the weeks pass, and more frequent refeeding with larger media volumes may be advisable. It is important to be gentle when changing media and care should be taken to minimize disturbances to the cell layer during native matrix production.

The matrix should become visible usually after 2 weeks, especially around the circumference of the well. Infrequently, the matrix will free itself without any mechanical disruption. This is a reflection of the intrinsic remodeling of the matrix and tensile strength, which pulls the matrix inwards, but may also be a sign that the circumference of the native matrix was disturbed during media changes.

When releasing and freeing the matrix cell layer from the plate, one must be careful not to poke holes through the matrix. Small blunt cell scrapers may also be used in lieu of 1 ml micropipette tips. Placement of the released native matrix onto a Nylon mesh first (being sure to lay the native matrix flat) makes for much easier handling and subsequent lifting to the air-liquid interface.

This protocol is the first to describe the use of native fibroblast matrix as a dermal substrate to model the microenvironment in tumor cell assays in order to obtain more accurate and physiologically relevant data.

The use of biomaterial in 3D tissue culture has enabled researchers to study cell behavior in the laboratory under physiological conditions more akin to an in vivo environment than that of one recapitulated with 2D adhesion and a plastic substrate. In particular, great strides forward have been made in modeling stratified epithelia with the adoption of 3D culture methods at the air-liquid interface1-4. Such techniques faithfully mimic keratinocyte differentiation and tumor cell invasion enabling greater flexibility and fidelity for researchers studying these processes. The choice of biomaterial substrate to mimic the stromal environment has primarily involved the use of type I collagen, Engelbreth-Holm-Swarm mouse sarcoma matrix and de-epidermized dermis. For example, cancer-associated fibroblasts have been shown to contribute toward cancer invasion5, initiation, and progression via stromal-epithelial interactions6,7 when grown in such substrates.

The gold standard for mimicking the stromal environment in skin, the largest and most widely studied stratified epithelia using such techniques, is regarded to be de-epidermized human dermis (DED). Preparation of DED involves the removal of the epidermis via trypsinization or physical disassociation from human cadaver skin3,4. However, access to such skin can be very difficult for laboratories not associated with clinical institutions, and diseased dermis is near impossible to obtain. As an alternative, laboratories frequently use a combination of type I collagen (isolated from rat tails) and/or Engelbreth-Holm-Swarm mouse sarcoma matrix.

After the discovery in 1927 by Nageotte8 that collagen can easily be isolated using acetic acid and salt precipitation, its application to tissue culture was subsequently pioneered by Huzella and colleagues9. Collagen coating proved to be superior to glass for cell culture of 29 strains and tissue explants as interrogated by Ehrmann and Gey9. Currently, the major type of collagen used in tissue culture is isolated from rat-tail tendons, and is usually bought from commercial sources. However, the drawback for faithful substrate recapitulation is that the rat tail collagen is not identical to human collagen, or the human dermis, where type I and III collagens are present as major constituents, and isolated rat tail collagen is invariably fragmented.

Engelbreth-Holm-Swarm mouse sarcoma matrix is a gelatinous protein mixture secreted by cultured Engelbreth-Holm-Swarm mouse sarcoma cells10. The major constituents are laminin, type IV collagen, heparin sulfate proteoglycan, entactin and nidogen and the exact ratios of these proteins will vary from batch to batch. Aside from structural proteins, this matrix also contains significant levels of growth factors such as transforming growth factor &#946;, epidermal growth factor, insulin-like growth factor 1, bovine fibroblast growth factor, and platelet-derived growth factor which would alter cellular behavior11,12. Pointing towards the sheer complexity of Engelbreth-Holm-Swarm mouse sarcoma matrix, a total of 1,851 proteins were identified in a recent proteomic study13. In light of the rich and complex nature of this matrix, caution has been advised when interpreting and comparing different experiments with the use of it11.

Our laboratories have a keen interest in genetic skin diseases, particularly those with a predisposition to developing cutaneous squamous cell carcinoma (cSCC)14. In the case of recessive dystrophic epidermolysis bullosa (RDEB), a severe blistering disease with germline mutations in COL7A1 gene15-17, we have determined that the dermal microenvironment in these patients is tumor promoting18. During the course of this study we were unable to assess the tumor promoting properties of dermal fibroblasts embedded within collagen I/ Engelbreth-Holm-Swarm mouse sarcoma matrix and investigated ways of assessing cells&#8217; own, native matrix. To achieve this, we modified a previous technique from the laboratory of Lucie Germain working on human skin equivalents19,20. Germain&#8217;s technique was able to reconstruct human skin with well-organized basement membrane using primary human keratinocyte and fibroblast cultures in the absence of a synthetic or cadaveric scaffold.

In this paper the steps used to recapitulate the cutaneous tumor stromal microenvironment (native matrix) derived directly from primary stromal fibroblasts in vitro are described18. Native matrices produced by long-term culture of fibroblasts were used as a dermal equivalent to assay for cSCC cell invasion. We present data using native matrix derived from either the extracellular matrix secreted by RDEB fibroblasts (deficient in type VII collagen (C7)) or from RDEB fibroblasts retrovirally transduced with a type VII collagen expressing construct and demonstrate the profound effect of a single collagen on tumor cell invasion.

<table><tbody><tr><td>L-Ascorbic acid 2-phosphate</td><td>Sigma</td><td>A8960</td><td></td></tr><tr><td>DMEM with L-glutamine, 4,500 mg/L D-glucose, 110 mg/L sodium pyruvate</td><td>Life Technologies</td><td>11995-073</td><td></td></tr><tr><td>100x Penicillin-Streptomycin</td><td>Life Technologies</td><td>15070</td><td></td></tr><tr><td>Vaseline</td><td>VWR</td><td>PROL28908.290</td><td></td></tr><tr><td>Clonal cylinders</td><td>Sigma</td><td>Z370789</td><td></td></tr><tr><td>Nylon Net Filter Disc Hydrophilic 100 &amp;#956;m 25 &amp;#956;m diameter 100/pk</td><td>Millipore</td><td>NY1H02500</td><td></td></tr><tr><td>Bent stainless steel wire mesh support</td><td>Made in house</td><td></td><td>Dimensions were made so that the mesh would fit into 6-well plates</td></tr></tbody></table>

tissue engineering of tumor stromal microenvironment with application to cancer cell invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

This technique opens up the possibility of examining and comparing the invasive behavior of tumor cells (in this case cSCC) under different 3D stromal environments. &#160;Using this technique, not only C7-deficient native matrices that recapitulated the RDEB dermal environment can be generated, but also additional matrices that have been genetically engineered to over-express C718. As seen in Figure 2, invasion of RDEB cSCC keratinocytes was significantly retarded in C7-overexpressing native matrices compared to C7-deficient RDEB control. This invasion is visualized via standard histological methods, where the gels were fixed, embedded in paraffin blocks and then sectioned and immunostained. Suggested antibodies to use for staining are anti-keratin 14 (LL001) for cSCC cells and anti-vimentin (V9) for fibroblasts.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51321/51321fig1highres.jpg" width="500" /> 
Figure 1.&#160;Workflow of generation of fibroblast-derived native matrix and the tumor invasion assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51321/51321fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51321/51321fig2highres.jpg" width="500" /> 
Figure 2.&#160;Keratin 14 (LL001) staining of the cSCC invasion into the native matrix derived from control RDEB fibroblast deficient in C7 (ai &#38; bi), or in RDEB fibroblasts over-expressing full-length C7 expression (aii and bii), clearly demonstrates that the re-expression of C7 in extracellular matrix can retard cSCC keratinocyte invasion. Nuclei were stained with DAPI (in blue) and fibroblasts with vimentin (in green) in bi and bii.

Watch this Scientific Journal Video about Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion at JoVE.com

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.7K Views.  University of Dundee. Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Video: Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Cancer Research

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Medicine

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Tumor Cell Invasion Assay: A 3D In Vitro Native Matrix-based Method to Assess Epithelial-mesenchymal Cell Interactions

Source: Ng, Y. Z. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/51321"> Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion</a>. J. Vis. Exp. (2014)
This video describes the technique of utilizing a native matrix to study tumor cell invasion. The technique used in this assay provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix.

Source: Ng, Y. Z. et al.  Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion. J. Vis. Exp. (2014)
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Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

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Chapters in this video

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Tissue Engineering of a Human 3D in vitro Tumor Test System

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

Tumor Cell Invasion Assay: A 3D In Vitro Native Matrix-based Method to Assess Epithelial-mesenchymal Cell Interactions