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. M., Neuberger, 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+metabolism+of+collagen+and+other+proteins+of+the+skin+of+rabbits.">The metabolism of collagen and other proteins of the skin of rabbits.</a> Biochem. J. 56 (4), 558-569 (1954).</li><li>Ehrmann, R. L., Gey, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growth+of+cells+on+a+transparent+gel+of+reconstituted+rat-tail+collagen.">The growth of cells on a transparent gel of reconstituted rat-tail collagen.</a> J. Natl. Cancer Inst. 16 (6), 1375-1403 (1956).</li><li>Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane+complexes+with+biological+activity.">Basement membrane complexes with biological activity.</a> Biochemistry. 25 (2), 312-318 (1986).</li><li>Vukicevic, S., Kleinman, H. K., Luyten, F. P., Roberts, A. B., Roche, N. S., Reddi, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+multiple+active+growth+factors+in+basement+membrane+Matrigel+suggests+caution+in+interpretation+of+cellular+activity+related+to+extracellular+matrix+components.">Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.</a> Exp. 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-...

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 border="0" cellpadding="0" cellspacing="0" width="352">
	<colgroup>
		<col span="4" style="width:66pt" width="88" />
	</colgroup>
	<tbody>
		<tr height="64" style="height:48.0pt">
			<td class="xl63" height="64" style="height:48.0pt;width:66pt" width="88">L-Ascorbic acid 2-phosphate</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">A8960</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl65" height="42" style="height:31.5pt;width:66pt" width="88">DMEM with l-glutamine,</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">Life Technologies</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">11995-073</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl63" height="106" style="height:79.5pt;width:66pt" width="88">4,500 mg/L d-glucose, 110 mg/L sodium pyruvate</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl63" height="85" style="height:63.75pt;width:66pt" width="88">100x Penicillin-Streptomycin</td>
			<td class="xl63" style="width:66pt" width="88">Life Technologies</td>
			<td class="xl63" style="width:66pt" width="88">15070</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Vaseline</td>
			<td class="xl63" style="width:66pt" width="88">VWR</td>
			<td class="xl63" style="width:66pt" width="88">PROL28908.290</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Clonal cylinders</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">Z370789</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="148" style="height:111.0pt">
			<td class="xl63" height="148" style="height:111.0pt;width:66pt" width="88">Nylon Net Filter Disc Hydrophilic 100um 25um diameter 100/pk</td>
			<td class="xl63" style="width:66pt" width="88">Millipore</td>
			<td class="xl63" style="width:66pt" width="88">NY1H02500</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="127" style="height:95.25pt">
			<td class="xl63" height="127" style="height:95.25pt;width:66pt" width="88">Bent stainless steel wire mesh support</td>
			<td class="xl63" style="width:66pt" width="88">Made in house</td>
			<td class="xl63" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88">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 m...

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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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

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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 ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

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 ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...