1. Assay Set-up
<ol>
	<li>Thaw a small aliquot (&lt;500 &mu;l) of Matrigel on ice at 4 &deg;C (approximately 2 hr).</li>
	<li>Prepare gel recipe (see example volumes in table below): 10x PBS (1x in total volume), 1N sodium hydroxide (equivalent to 0.023 volumes of added collagen, or per the collagen manufacturer's recommendations, as appropriate), Matrigel and collagen type I to final concentrations of 1 mg/ml and 1.3 mg/ml respectively (other matrix formulations may be used depending on cell type and experiment).</li>...

<ol>
	<li>Cichon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+influences+that+drive+progression+from+benign+breast+disease+to+invasive+breast+cancer.">Microenvironmental influences that drive progression from benign breast disease to invasive breast cancer.</a> J. Mammary Gland. Biol. Neoplasia. 15, 389-3897 (2010).</li><li>Proia, D. A., Kuperwasser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroma:+tumor+agonist+or+antagonist.">Stroma: tumor agonist or antagonist.</a> Cell Cycle. 4, 1022-1025 (2005).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment+and+progression.">Tumor microenvironment and progression.</a> J .Surg. Oncol. 103, 468-474 (2011).</li><li>Provenzano, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC Med. 6, 11 (2008).</li><li>Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Butler, T. P., Grantham, F. H., Gullino, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+transfer+of+fluid+in+the+interstitial+compartment+of+mammary+tumors.">Bulk transfer of fluid in the interstitial compartment of mammary tumors.</a> Cancer Res. 35, 3084-3088 (1975).</li><li>Dafni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+vascular+endothelial+growth+factor+165+drives+peritumor+interstitial+convection+and+induces+lymphatic+drain:+magnetic+resonance+imaging,+confocal+microscopy,+and+histological+tracking+of+triple-labeled+albumin.">Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin.</a> Cancer Res. 62, 6731-6739 (2002).</li><li>Chary, S. R., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+interstitial+convection+and+diffusion+of+albumin+in+normal+and+neoplastic+tissues+by+fluorescence+photobleaching.">Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 5385-5389 (1989).</li><li>Heldin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+interstitial+fluid+pressure+-+an+obstacle+in+cancer+therapy.">High interstitial fluid pressure - an obstacle in cancer therapy.</a> Nat. Rev. Cancer. 4, 806-813 (2004).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li><li>Shields, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chemotaxis+as+a+mechanism+of+tumor+cell+homing+to+lymphatics+via+interstitial+flow+and+autocrine+CCR7+signaling.">Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling.</a> Cancer Cell. 11, 526-538 (2007).</li><li>Shieh, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+invasion+is+promoted+by+interstitial+flow-induced+matrix+priming+by+stromal+fibroblasts.">Tumor cell invasion is promoted by interstitial flow-induced matrix priming by stromal fibroblasts.</a> Cancer Res. 71, 790-800 (2011).</li><li>Shi, Z. D., Wang, H., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycans+mediate+interstitial+flow+mechanotransduction+regulating+MMP-13+expression+and+cell+motility+via+FAK-ERK+in+3D+collagen.">Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.</a> PLoS One. 6, e15956 (2011).</li><li>Fleury, M. E., Boardman, K. C., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+morphogen+gradients+by+subtle+interstitial+flow+and+matrix+interactions.">Autologous morphogen gradients by subtle interstitial flow and matrix interactions.</a> Biophys J. 91, 113-121 (2006).</li><li>Miteva, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmural+flow+modulates+cell+and+fluid+transport+functions+of+lymphatic+endothelium.">Transmural flow modulates cell and fluid transport functions of lymphatic endothelium.</a> Circ. Res. 106, 920-931 (2010).</li><li>Ng, C. P., Hinz, B., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+fluid+flow+induces+myofibroblast+differentiation+and+collagen+alignment+in+vitro.">Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro.</a> J. Cell. Sci. 118, 4731-4739 (2005).</li><li>Kunder, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+particles+deliver+peripheral+signals+to+remote+lymph+nodes.">Mast cell-derived particles deliver peripheral signals to remote lymph nodes.</a> J. Exp. Med. 206, 2455-2467 (2009).</li><li>Helm, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+interstitial+flow+and+VEGF+directs+capillary+morphogenesis+in+vitro+through+a+gradient+amplification+mechanism.">Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 15779-15784 (2005).</li><li>McGuire, P. G., Seeds, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+plasminogen+activator+with+a+reconstituted+basement+membrane+matrix+and+extracellular+macromolecules+produced+by+cultured+epithelial+cells.">The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells.</a> J Cell Biochem. 40, 215-227 (1989).</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=Isolation+and+characterization+of+type+IV+procollagen,+laminin,+and+heparan+sulfate+proteoglycan+from+the+EHS+sarcoma.">Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.</a> Biochemistry. 21, 6188-6193 (1982).</li><li>Haessler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+dynamics+of+breast+cancer+cells+in+a+tunable+3D+interstitial+flow+chamber.">Migration dynamics of breast cancer cells in a tunable 3D interstitial flow chamber.</a> Integr. Biol. (Camb). , (2011).</li><li>Polacheck, W. J., Charest, J. L., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+flow+influences+direction+of+tumor+cell+migration+through+competing+mechanisms.">Interstitial flow influences direction of tumor cell migration through competing mechanisms.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 11115-11120 (2011).</li></ol>

No conflicts of interest declared.

Here we have described a methodology for quantifying the effect of interstitial flow on tumor cell invasion, using cells embedded in a 3-D matrix within a cell culture insert. This and similar methods have been used to study the effect of interstitial flow on a variety of cell types 13-15, 17. Our approach partially mimics the matrix microenvironment of the tumor by using type I collagen and Matrigel which contain proteins found in the basement membrane of epithelial tissue and the surrounding stroma 21-2...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Collagen (Rat Tail)</td>
			<td>BD</td>
			<td>354236</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Millicell cell culture insert</td>
			<td>Millipore</td>
			<td>PI8P01250</td>
			<td>8 &mu;m pore diameter, polycarbonate membrane</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD</td>
			<td>354234</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>100M-8202</td>
			<td>10x for preparing gel solution, 1x for washing steps</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, 1.0N Solution</td>
			<td>Sigma Aldrich</td>
			<td>S2770</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>DMEM 1X</td>
			<td>CellGro</td>
			<td>10-013-CV</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>511150</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>CellGro</td>
			<td>30002CI</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-500 ml</td>
			<td>0.5% in PBS</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>04042-500</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Deionized Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>4',6-diaminido-2-phenylindole (DAPI)</td>
			<td>MP Biomedicals</td>
			<td>0215757401</td>
			<td>1 mg/ml stock solution</td>
		</tr>
		<tr>
			<td>Mounting Solution</td>
			<td>Thermo Scientific</td>
			<td>TA-030-FM</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CellGro</td>
			<td>25-052-CI</td>
			<td>Keep sterile</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

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

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 ...

three-dimensional-cell-culture-model-for-measuring-effects

Research

JoVE Journal

Bioengineering

16.4K 조회수.  Drexel University. 침입형 유체의 흐름은 고체 종양에서 상승되며 종양 세포 침공을 조절하실 수 있습니다. 여기에서 우리는 매트릭스에 포함된 세포에 침입형 유체 흐름을 적용한 다음 세포 침입에 미치는 영향을 측정할 수있는 기법을 설명합니다. 이 기술은 쉽게 다른 시스템을 연구하는 데 적용할 수 있습니다.