Name: three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation
Uploaded: 2016-08-18T15:00:00
Description: Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

NOTE: No ethics consideration since animal and human cancer cells were purchased or kindly provided to us.
1. Making Collagen Plugs (Pseudo-primary Tumor)
<ol>
	<li>Prepare a collagen dispersion. Type I collagen from rat tail tendons (RTT) can be either extracted and sterilized as previously reported17, or purchased. Disperse freeze-dried RTT collagen (3.25-3.50 mg/ml in 0.02 N acetic acid) using a blender (high-speed setting; five 2 min runs) for a uniform mixing.</li>
	<li>Harvest (trypsin-EDTA, usu...

<ol>
	<li>Alizadeh, A. M., Shiri, S., Farsinejad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+review:+from+bench+to+bedside.">Metastasis review: from bench to bedside.</a> Tumour Biol. 35 (9), 8483-8523 (2014).</li><li>Roudsari, L. C., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+influence+of+angiogenesis+in+in+vitro+cancer+model+systems.">Studying the influence of angiogenesis in in vitro cancer model systems.</a> Adv Drug Deliv Rev. , (2016).</li><li>Bill, R., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+EMT+in+breast+cancer+metastasis:+Correlation+or+causality.">The relevance of EMT in breast cancer metastasis: Correlation or causality.</a> FEBS Lett. 589 (14), 1577-1587 (2015).</li><li>Kimlin, L. C., Casagrande, G., Virador, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Three-Dimensional+(3D)+Models+in+Cancer+Research:+an+Update.">In Vitro Three-Dimensional (3D) Models in Cancer Research: an Update.</a> Mol Carcinog. 52 (3), 167-182 (2013).</li><li>Obenauf, A. C., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+at+a+Distance+Organ-Specific+Metastasis.">Surviving at a Distance Organ-Specific Metastasis.</a> Trends Cancer. 1 (1), 76-91 (2015).</li><li>Sykes, J. A., Fogh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+Tumor+Cells+from+Fibroblasts.">Separation of Tumor Cells from Fibroblasts.</a> Human Tumor Cells In Vitro. 1, 1-22 (1975).</li><li>Lang, S. H., Stark, M., Collins, A., Paul, A. B., Stower, M. J., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Prostate+Epithelial+Morphogenesis+in+Response+to+Stroma+and+Three-Dimensional+Matrigel+Culture.">Experimental Prostate Epithelial Morphogenesis in Response to Stroma and Three-Dimensional Matrigel Culture.</a> Cell Growth Differ. 12 (12), 631-640 (2001).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+Oncogenesis+of+MCF-10A+Mammary+Epithelial+Acini+Grown+In+Three-Dimensional+Basement+Membrane+Cultures.">Morphogenesis and Oncogenesis of MCF-10A Mammary Epithelial Acini Grown In Three-Dimensional Basement Membrane Cultures.</a> Methods. 30 (3), 256-268 (2003).</li><li>Shaw, L. M. <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+assays.">Tumor cell invasion assays.</a> Methods Mol. Biol. 294, 97-105 (2005).</li><li>Nelson, C. M., Bissell, 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=Modeling+Dynamic+Reciprocity:+Engineering+Three-Dimensional+Culture+Models+of+Breast+Architecture,+Function,+and+Neoplastic+Transformation.">Modeling Dynamic Reciprocity: Engineering Three-Dimensional Culture Models of Breast Architecture, Function, and Neoplastic Transformation.</a> Semin Cancer Biol. 15 (5), 342-352 (2005).</li><li>Hedlund, T. E., Duke, R. C., Miller, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Spheroid+Cultures+of+Human+Prostate+Cancer+Cell+Lines.">Three-Dimensional Spheroid Cultures of Human Prostate Cancer Cell Lines.</a> Prostate. 41 (3), 154-165 (1999).</li><li>Le, V. M., Lang, M. D., Shi, W. B., Liu, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Collagen-Based+Multicellular+Tumor+Spheroid+Model+for+Evaluation+of+the+Efficiency+of+Nanoparticle+Drug+Delivery.">A Collagen-Based Multicellular Tumor Spheroid Model for Evaluation of the Efficiency of Nanoparticle Drug Delivery.</a> Artif. Cells Nanomed Biotechnol. 15, 1-5 (2014).</li><li>Neto, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Hanging+Spherical+Drop+System+for+the+Generation+of+Cellular+Spheroids+and+High+Throughput+Combinatorial+Drug+Screening.">A Novel Hanging Spherical Drop System for the Generation of Cellular Spheroids and High Throughput Combinatorial Drug Screening.</a> Biomater Sci. 3 (4), 581-585 (2015).</li><li>Janvier, R., Sourla, A., Koutsilieris, M., Doillon, C. J. <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+are+Required+for+PC-3+Human+Prostate+Cancer+Cells+to+Produce+Capillary-like+Formation+of+Endothelial+Cells+in+a+Three-dimensional+Co-culture+System.">Stromal Fibroblasts are Required for PC-3 Human Prostate Cancer Cells to Produce Capillary-like Formation of Endothelial Cells in a Three-dimensional Co-culture System.</a> Anticancer Res. 17 (3A), 1551-1557 (1997).</li><li>Doillon, C. J., Gagnon, E., Paradis, R., Koutsilieris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Culture+System+as+a+Model+for+Studying+Cancer+Cell+Invasion+Capacity+and+Anticancer+Drug+Sensitivity.">Three-dimensional Culture System as a Model for Studying Cancer Cell Invasion Capacity and Anticancer Drug Sensitivity.</a> Anticancer Res. 24 (4), 2169-2177 (2004).</li><li>Gobeil, S., Zhu, X., Doillon, C. J., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genome-Wide+shRNA+Screen+Identifies+GAS1+as+a+Novel+Melanoma+Metastasis+Suppressor+Gene.">A Genome-Wide shRNA Screen Identifies GAS1 as a Novel Melanoma Metastasis Suppressor Gene.</a> Genes Dev. 22 (21), 2932-2940 (2008).</li><li>Rajan, N., Habermehl, J., Cot&#233;, M. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+Of+Ready-To-Use,+Storable+And+Reconstituted+Type+I+Collagen+From+Rat+Tail+Tendon+For+Tissue+Engineering+Applications.">Preparation Of Ready-To-Use, Storable And Reconstituted Type I Collagen From Rat Tail Tendon For Tissue Engineering Applications.</a> Nat Protoc. 1 (6), 2753-2758 (2007).</li><li>Horie, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Human+Lung+Cancer-associated+Fibroblasts+in+Three-dimensional+In+Vitro+Co-culture+Model.">Characterization of Human Lung Cancer-associated Fibroblasts in Three-dimensional In Vitro Co-culture Model.</a> Biochem Biophys Res Commun. 423 (1), 158-163 (2012).</li><li>Banyard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Genes+Regulating+Migration+and+Invasion+Using+a+New+Model+of+Metastatic+Prostate+Cancer.">Identification of Genes Regulating Migration and Invasion Using a New Model of Metastatic Prostate Cancer.</a> BMC Cancer. 30 (14), 387 (2014).</li><li>Palumbo, J. S., Degen, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+Tumor+Cell+Metastasis.">Fibrinogen and Tumor Cell Metastasis.</a> Haemostasis. 31, 11-15 (2001).</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+Stroma,+Tumor+Blood+Vessels,+and+Antiangiogenesis+Therapy.">Tumor Stroma, Tumor Blood Vessels, and Antiangiogenesis Therapy.</a> Cancer J. 21 (4), 237-243 (2015).</li><li>Luoto, K. R., Kumareswaran, R., Bristow, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Hypoxia+as+a+Driving+Force+in+Genetic+Instability.">Tumor Hypoxia as a Driving Force in Genetic Instability.</a> Genome Integr. 4 (1), 5 (2013).</li><li>Das, V., Bruzzese, F., Kone&#269;n&#253;, P., Iannelli, F., Budillon, A., Hajd&#250;ch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+In+Vitro+Tumor+Models+for+Drug+Screening.">Pathophysiologically Relevant In Vitro Tumor Models for Drug Screening.</a> Drug Discov Today. 20 (7), 848-855 (2015).</li><li>Longati, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Pancreatic+Carcinoma+Spheroids+Induce+a+Matrix-rich,+Chemoresistant+Phenotype+Offering+a+Better+Model+for+Drug+Testing.">3D Pancreatic Carcinoma Spheroids Induce a Matrix-rich, Chemoresistant Phenotype Offering a Better Model for Drug Testing.</a> BMC Cancer. 13 (95), (2013).</li><li>Tan, P. H., Chia, S. S., Toh, S. L., Goh, J. C., Nathanm, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Spatial+Configuration+of+Tumour+Cells+Confers+Resistance+to+Chemotherapy+Independent+of+Drug+Delivery.">Three-dimensional Spatial Configuration of Tumour Cells Confers Resistance to Chemotherapy Independent of Drug Delivery.</a> J Tissue Eng Regen Med. , (2013).</li><li>Koutsilieris, M., Reyes-Moreno, C., Choki, I., Sourla, A., Doillon, C., Pavlidis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+Cytotoxicity+of+Human+MCF-7+and+MDA-MB+231+Breast+Cancer+Cells+is+Altered+by+Osteoblast-Derived+Growth+Factors.">Chemotherapy Cytotoxicity of Human MCF-7 and MDA-MB 231 Breast Cancer Cells is Altered by Osteoblast-Derived Growth Factors.</a> Mol Med. 5 (2), 86-97 (1999).</li><li>Lang, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+Response+of+Cell+Invasion+to+Matrix+Stiffness+in+Three-Dimensional+Biopolymer+Networks.">Biphasic Response of Cell Invasion to Matrix Stiffness in Three-Dimensional Biopolymer Networks.</a> Acta Biomater. 13, 61-67 (2015).</li></ol>

As an important technical footnote, it is essential that no gap is present at the interface between the central and the peripheral gels. Otherwise, it might reduce the capacity of the cells to migrate/invade the fibrin gel. A space between the collagen and the fibrin gels may form during the first 24 hr of culture if thrombin has not been appropriately diluted. It is also possible that the cell line tested might lead the collagen gel to contract during culture, thereby causing a relatively large space to form between bot...

Investigating the fundamental and biomedical characteristics of cancer cell invasion/migration and subsequent metastasis establishment is the subject of an intense research1,2. Metastasis is the ultimate stage of cancer and its clinical management remains elusive. A better understanding of metastasis at the cellular and molecular levels will enable the development of more efficient therapies3.
Several properties of metastatic cells can be explored in vitro4 including their stemness and potential to acquire a transition state (e.g., epithelioid-mesenchymal transition) to migrate and invade within and from the primary tumor5. However, the in vitro assessment of invasion/metastasis processes has been a challenge since it virtually excludes the contribution of the blood/lymphatic circulation. Organotypic cultures that embed tumor fragments in collagen gels have previously been used to monitor cancer aggressiveness. Although the complexity of tumors is preserved (e.g., the presence of non-cancerous cells), tumor fragments are exposed to limited medium diffusion, to sampling variation, and to an overgrowth of stromal cells6. An alternative method consists in growing cancer cells within components of the extracellular matrix (ECM), which mimics the three-dimensional (3D) cell environment. The proliferation of breast cancer cell lines in a collagen gel and/or a basement membrane-derived matrix is amongst the best-characterized examples of 3D cell culture. By using specific 3D cell culture environments, the disorganized assembly observed for breast cancer cells grown under standard conditions can be reversed to the spontaneous formation of mammary acini and tubular structures7-10. Furthermore, the formation of multicellular tumor spheroids derived from adenocarcinoma cancer cells congregated using different techniques (e.g., hanging drops, floating spheroids, agar embedment) now constitutes the most commonly used 3D cell culture assay11-13. However, this assay is limited by the restricted set of cancer cell lines that can form spheroids and by the short period available to study cells in these conditions.
In this visualized technique, we herein introduce a sophisticated 3D cell culture assay where cancer cells of interest are embedded in a collagen gel to allow the in vitro formation of a pseudo-primary tumor that can be alternatively coated with a basement membrane-derived matrix. Once formed, the pseudo-primary tumor is then sandwiched in an acellular matrix (fibrin gel in the present case), which allows the cancer cells to cross the interface between the two matrix compartments (see Figure 1). Interestingly, secondary tumor-like structures originating from the pseudo-primary tumor along with aggressive cancer cells appear in the fibrin gel. Such a 3D culture system offers the flexibility required to investigate, for example, anticancer drugs, gene expression and cell-cell and/or cell-ECM interactions14-16.
<img alt="Figure 1" src="/files/ftp_upload/54322/54322fig1.jpg" /> 
Figure 1: Overview of the Method. Schematic summary of the method to generate the 3D cell culture system as a model for cancer studies. <a href="https://www.jove.com/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Freeze-dried collagen</td>
			<td>Sigma-Aldrich</td>
			<td>C7661</td>
			<td>from rat tail tendon (soluble dispersion) or home-made (see Rajan et al., ref.#14)</td>
		</tr>
		<tr>
			<td>Fibrinogen (freeze-dried)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F8630</td>
			<td>Type I-S, 65-85% protein with &#8805;75% of protein is clottable</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>EMD Chemicals Inc.</td>
			<td>605157</td>
			<td>&#160;Gibbstown, NJ; NIH units/mg dry weight&#160;</td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Previously from BD Biosciences</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma-Aldrich</td>
			<td>A6279&#160;</td>
			<td>&#160;solution at 5-10TIU/ml (Trypsin Inhibitor Unit)&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Micro-spoons</td>
			<td>Fisher Scientific</td>
			<td>2140115</td>
			<td>Fisherbrand Handi-Hold Microspatula</td>
		</tr>
		<tr>
			<td>96 well plate, round base</td>
			<td>Sarstedt</td>
			<td>3925500</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sarstedt</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s Medium</td>
			<td>Sigma Chemical, Co.</td>
			<td>D5546</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>CAA15-701</td>
			<td>FBS, Canadian origin.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Chemical, Co.</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution&#160;</td>
			<td>Sigma Chemical, Co.</td>
			<td>H8264</td>
			<td>HBSS</td>
		</tr>
	</tbody>
</table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

As previously mentioned, an interesting feature of this 3D cell culture assay is that cancer cells can not only migrate from the collagen plug to the adjacent fibrin gel, but also establish secondary tumors (e.g., satellite tumor-like structures). This can be directly observed with an inverted phase contrast microscope at low and high magnifications through the gel thickness, especially with a long working distance condenser (Figure 2). Using this 3D cell culture...

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

The overall goal of this procedure is to sandwich collagen embedded cancer cells within a fibrin gel to generate a 3D culture system for the monitoring of satellite tumor invasiveness and formation. Our 3D picture system can offer new avenues for investigating various cell and many key variants during cancer cell apoptosis or migration or to evaluate the efficacy of anti-cancer cell loss. An attractive feature of this 3D cell culture model is the ability to change the concentration of the extra cellular matrix or use another extra cellular Matrix for the assembling of the 3D system.Demonstrating the procedure will be Marie-France Cote. Research assistant, my laboratory, and Audrey Turcotte, a grad student under my supervision. Begin by blending freeze dried rat tail tendon type one collagen for five two minute mixes at the high speed setting to uniformly disperse the collagen.While the collagen is being incorporated, harvest the tumor cells of interest with the appropriate enzyme detaching solution. Followed by Trypan blue exclusion in numeration. Dilute the dissociated cells to a 50, 000 cells per plug concentration in DMEM.When the collagen is ready, quickly add 1, 250, 000 cells to the collagen solution with pipetting to homogeneously disperse the cells. Immediately dispense 200 microliters of the single cell suspension into each well of a 96 well plate. Gently tapping the plate on the work area surface after each addition to remove any air bubbles and to distribute the solution evenly within the wells.When all of the cells have been added, place the plate in a 37 degree Celsius incubator for at least two hours, adding 100 microliters of culture medium to each well after the first two hours if the cells are to be cultured overnight. To prepare the first fibrin gel layer, bring the freeze dried fibrinogen to room temperature before opening the vial to avoid hydrate crystal formation. Then transfer the fibrinogen to a glass beaker and add 37 degrees Celsius HBSS drop wise to the powder to solubilize the fibrinogen fragments.Next, use a spatula to break down the larger protein pieces, agitating the beaker from time to time as appropriate to facilitate mixing. Pipette the suspension several times to dissolve the remaining protein, and filter sterilize the solution through a 22 micron filter. Then immediately overlay the surface of each well of a 24 well plate with 200 microliters of the fibrinogen, taking care to avoid air bubbles.Once all of the wells have been thoroughly coated, tilt the plate at a 45 angle, then drop 1.5 microliters of thrombin solution into the center of one well and gently swirl the plate horizontally for one to two seconds. After treating five more wells with thrombin in the same manner, place the plate in a stable horizontal position under a laminar flow hood for five to 10 minutes, until the solutions gel. For immediate placement of the collagen plug, carefully tilt the plate to confirm that the fibrin gels are completely polymerized.Then place the 96 well plate of collagen gel plugs next to the 24 well plate of fibrin gels and add one drop of HBSS into each collagen plug well. Next, use a micro spoon to transfer a single collagen plug into the center of each of the first fibrin gel layers in the 24 well plate. When all of the plugs have been transferred, add 300 microliters of fibrinogen solution to each well, followed by the addition of 2.25 microliters of thrombin as just demonstrated.To coat the collagen plugs with a thin layer of growth factor reduced basement membrane before their placement, transfer each plug into a 1.5 milliliter centrifuge tube on ice containing 100 microliters of pure growth factor reduced basement membrane. After two minutes of soaking, transfer a single coated collagen plug into the center of each fibrin layer and incubate the plugs at 37 degrees Celsius for five minutes to solidify the growth factor reduced basement membrane. Then add the second fibrin layer as just demonstrated.When the gels are fully polymerized, fill each well with 400 microliters of the appropriate culture medium and supplements. Next, add an Antifibrinolytic agent to the wells. And incubate the 3D cultures under the appropriate conditions for the tumor cell line being tested.On the appropriate cell feeding days, tilt the plate at a 30 to 35 degree angle and incline a pipette against the wall of each well to aspirate the medium without disturbing the gels. Then add 400 microliters of fresh cell culture medium and Antifibrinolytic agent to each gel culture. This 3D cell culture assay allows a direct visualization of cancer cell migration from the collagen plug into the adjacent fibrin gel for the establishment of secondary tumors, allowing a comparison of the behavior or known metastatic cells to that of non or weakly metastatic cells.For example, in this experiment, numerous satellite tumors dispersed randomly throughout the fibrin gel containing the metastatic cell line B16 F10 while no satellite tumors were seen for the weakly metastatic B16 F0 cell line. By contrast, these murine mammary gland carcinoma cells which are poorly metastatic but very invasive locally form a migration front that extends radially into the fibrin gel. After 14 days of culture, the mammary gland carcinoma cells pull away from the migratory front to form stellar shaped structures closely resembling mammary gland structures.Chemotherapeutic agents can be tested on the embedded cancer cells as in this representative experiment where an increasing amount of DNA fragmentation were observed concomitant with the increased incidents of chemotherapeutic agent induced cancer cell apoptosis. When mastered, this technique can be completed in approximately six hours or spread across two days. Depending on the initial collagen plug incubation period.While I'm attempting this procedure, it's important to prevent the formation of gaps at the interface between the central and the peripheral gels as they can reduce the ability of the cancer cells to invade the fibrin gel. Following this procedure, metallographic or Proteomic analysis can be performed in the satellite colonies, the collagen plugs, or the cancer cells to identify the differential expression of genes and proteins of interest. After its development and this technique paved the way to researchers in the field of cancer biology to explore the migration apoptosis metastatic suppressive gene expression can end angiogenesis to a variety of cancer cell lines.After watching this video, you should have a good understanding of how to embed cancer cells in a collagen gel and sandwich them within a fibrin gel to monitor the invasiveness and formation of satellite tumors.

Research

JoVE Journal

Medicine

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

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

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 ...

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 ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...