Name: fibroblast-derived 3d matrix system applicable to endothelial tube formation assay
Uploaded: 2019-12-26T12:00:00
Description: Watch this Scientific Journal Video about Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay at JoVE.com

The development of this protocol was supported by PI12/02037, PI15/02101, PI17/01847, PI18/01034 and RD12/0036/0041 from the Instituto de Salud Carlos III; by the Fondo Europeo de Desarrollo Regional (FEDER); by &#34;CIBER de C&#225;ncer&#34;, CB16/12/00273 and CB16/12/00446, from the Instituto de Salud Carlos III-FEDER; and by the Fundaci&#243;n Cient&#237;fica AECC (a multifaceted approach to target pancreatic cancer). Cristina Pe&#241;a is a recipient of a Miguel Servet Contract from the Instituto de Salud Carlos III. M. Eaude helped with the English text. We thank lab members for help and advice throughout this research.

Human tissue samples were obtained with the approval of the Research Ethics Board of the Hospital Ram&#243;n y Cajal, Madrid.
1. Preparation of Solutions
<ol>
	<li>Prepare 0.2% gelatin solution: add 1 g of gelatin to 500 mL of PBS. Autoclave the solution and keep at 4 &#176;C. Filter with a 0.22 &#181;m filter before use.</li>
	<li>Prepare 1% glutaraldehyde: Add 1 mL of 25% glutaraldehyde stock solution to 24 mL of PBS. Filter with a 0.22 &#181;m filter before use.</li>
	<li>Prepare 1 M etha...

<ol>
	<li>Frantz, C., Stewart, K. M., Weaver, 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=The+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> Journal of Cell Science. 123 (24), 4195-4200 (2010).</li><li>Kular, J. K., Basu, S., Sharma, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+Structure,+composition,+age-related+differences,+tools+for+analysis+and+applications+for+tissue+engineering.">The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering.</a> Journal of Tissue Engineering. 5, (2014).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+function+of+fibroblasts+in+cancer.">The biology and function of fibroblasts in cancer.</a> Nature Reviews Cancer. 16 (9), 582-598 (2016).</li><li>Erdogan, B., Webb, D. J. <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+modulate+growth+factor+signaling+and+extracellular+matrix+remodeling+to+regulate+tumor+metastasis.">Cancer-associated fibroblasts modulate growth factor signaling and extracellular matrix remodeling to regulate tumor metastasis.</a> Biochemical Society Transactions. 45 (1), 229-236 (2017).</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-kB-Dependent+Manner.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kB-Dependent Manner.</a> Cancer Cell. 17 (2), 135-147 (2010).</li><li>Herrera, 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=Colon+Cancer-associated+Fibroblast+Establishment+and+Culture+Growth.">Colon Cancer-associated Fibroblast Establishment and Culture Growth.</a> Bio-protocol. 6 (7), e1773 (2016).</li><li>Herrera, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+activation+on+3D-matrices+derived+from+PDGF-BB-stimulated+fibroblasts+is+mediated+by+Snail1.">Endothelial cell activation on 3D-matrices derived from PDGF-BB-stimulated fibroblasts is mediated by Snail1.</a> Oncogenesis. 7 (9), 76 (2018).</li><li>MacColl, E., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Metalloproteinases+as+Regulators+of+Vein+Structure+and+Function:+Implications+in+Chronic+Venous+Disease.">Matrix Metalloproteinases as Regulators of Vein Structure and Function: Implications in Chronic Venous Disease.</a> The Journal of Pharmacology and Experimental Therapeutics. 355 (3), 410-428 (2015).</li><li>Shian, S. G., Kao, Y. R., Wu, F. Y. H., Wu, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Invasion+and+Angiogenesis+by+Zinc-Chelating+Agent+Disulfiram.">Inhibition of Invasion and Angiogenesis by Zinc-Chelating Agent Disulfiram.</a> Molecular Pharmacology. 64 (5), 1076-1084 (2003).</li><li>Neri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+invasion+driven+by+extracellular+matrix+remodeling+is+dependent+on+the+properties+of+cancer-associated+fibroblasts.">Cancer cell invasion driven by extracellular matrix remodeling is dependent on the properties of cancer-associated fibroblasts.</a> Journal of Cancer Research and Clinical Oncology. 142 (2), 437-446 (2016).</li><li>Du, P., Subbiah, R., Park, J. H., Park, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+morphogenesis+of+human+umbilical+vein+endothelial+cells+on+cell-derived+macromolecular+matrix+microenvironment.">Vascular morphogenesis of human umbilical vein endothelial cells on cell-derived macromolecular matrix microenvironment.</a> Tissue Engineering. (Part A), (2014).</li><li>Bignon, 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=Lysyl+oxidase-like+protein+2+regulates+sprouting+angiogenesis+and+type+IV+collagen+assembly+in+the+endothelial+basement+membrane.">Lysyl oxidase-like protein 2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane.</a> Blood. 118 (14), 3979-3989 (2011).</li><li>Huang, L., Xu, A. M., Liu, S., Liu, W., Li, T. J. <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+in+digestive+tumors.">Cancer-associated fibroblasts in digestive tumors.</a> World Journal of Gastroenterology. 20 (47), 17804-17818 (2014).</li><li>Herrera, A., Herrera, M., Pe&#241;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=The+emerging+role+of+Snail1+in+the+tumor+stroma.">The emerging role of Snail1 in the tumor stroma.</a> Clinical and Translational Oncology. 18 (9), 872-877 (2016).</li><li>Sheng, Y., Fei, D., Leiiei, G., Xiaosong, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+Scaffolds+for+Tissue+Engineering+and+Regenerative+Medicine.">Extracellular Matrix Scaffolds for Tissue Engineering and Regenerative Medicine.</a> Current Stem Cell Research &amp; Therapy. 12 (3), 233-246 (2017).</li><li>Castell&#243;-Cros, R., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromagenesis+during+tumorigenesis:+characterization+of+tumor-associated+fibroblasts+and+stroma-derived+3D+matrices.">Stromagenesis during tumorigenesis: characterization of tumor-associated fibroblasts and stroma-derived 3D matrices.</a> Methods in Molecular Biology. 522, 275-305 (2009).</li></ol>

Matrices can be generated with immortalized fibroblasts or primary fibroblast cultures. Fibroblasts are easy to maintain in in vitro culture with a high growth rate and stress resistance. They can even be isolated from post-mortem tissue3, although contamination could be a restriction, depending on the tissue origin.
The 3D-matrix model here offers a novel and effective system to successfully study ECM composition and fiber orientation. It is also suitable for the analy...

The extracellular matrix (ECM) is a dynamic structure present in all types of tissue. It consists of proteins and polysaccharides that create a net of fibers crucial for cell adhesion, migration, and communication1. ECM composition varies depending on the tissue. While type-I collagen is the most prevalent structural protein, collagen types II, III, V and XI can also be found in various tissues2. Fibronectin generated by fibroblasts is needed for cell adhesion2. Moreover, there are other structural molecules like elastin, laminin and surface receptors called integrins that mediate fiber assembly and are specific to the different ECM tissues2. The ECM plays an important role as a cell scaffold and can also be involved in both physiological and pathological processes1. Abnormalities in the ECM are observed in pathologies such as cancer, which alters ECM composition and/or its organization. In tumors, the ECM represents the non-cellular component of the tumor microenvironment (TME), a complex milieu of cell components such as fibroblasts, immune cells, endothelial cells, pericytes and a variety of soluble factors. It is known that the TME promotes cancer progression and metastasis; cancer-associated fibroblasts, as a predominant cell type in the tumor stroma, take part in this process3. Unlike normal fibroblasts, CAFs are in permanent activation, showing increased secretion of ECM proteins and growth factors (e.g., Transforming Growth Factor-&#946;, TGF-&#946;), as well as a higher expression of some markers, such as &#945;-smooth muscle actin (&#945;-SMA) and fibroblast activation protein (FAP)4. However, CAFs are a heterogeneous cell population, showing different levels of activation or marker expression5. It can be assumed then that the composition and structure of fibroblast-derived matrices will depend on fibroblast status and characteristics.
In this context, the goal of this methodology is to establish an appropriate in vitro model for ECM generation by fibroblasts equivalent to the in vivo ECM setting. We propose this approach as an in vitro translational methodology for further studies of tumor cell functions, like chemoresistance or migration, mediated by ECM. As our group has published elsewhere, CAFs can be obtained from fresh tissue samples, but it has to be noted that the CAFs&#39; survival in culture is limited and their cell passage number is reduced6. In addition, CAF primary cultures established from patients&#39; samples can be used for matrix generation. Manipulation of gene expression in fibroblasts is also an interesting way to produce varied in vitro matrices to assess the possible effects on matrix composition, fiber orientation, etc. Along these lines, our group has recently reported the role of Snail-expressing fibroblasts in the composition and fiber orientation of various derived matrices7.
Furthermore, CAFs and ECM are involved in the vascular system, both in vessel generation and as part of the vase outer layer8. ECM remodeling induces angiogenesis; matrix metalloproteinases (MMPs) seem to be the most important enzyme type contributing to this process9,10. The tissue vascularization of primary cells that generate the ECMs, ECM macromolecules, residual growth factors included in the ECM, matrix elasticity, and matrix thickness are described as factors involved in endothelial cell activation11. In tumors, hypoxia increases ECM stiffness and endothelial sprout generation12. Moreover, CAFs secrete vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) that stimulate angiogenesis in tumor stroma13. In this field, in vitro matrix generation could be used to study angiogenesis processes or MMP action under different experimental conditions. Thus, the in vitro reproduction of the most analogous in vivo matrix could be a valuable tool to investigate ECM&#39;s role in angiogenesis or micro-environmental cell interactions.
The stimulation of cultured fibroblasts with ascorbic acid to enhance matrix deposition and generate an ECM is an accepted way of producing analogous in vivo matrices. Immortalized fibroblast cell lines are easily cultured and are activated by diverse growth factors, like PDGF-BB, Tumor Necrosis Factor-&#945; (TNF-&#945;) or TGF-&#946;14. Within the TME, CAFs synthesize type-I collagen and fibronectin as the main components of ECM4. Similarly, these components are found as major components of in vitro-generated fibroblast-derived matrices (Figure 1).
There are different in vitro methodologies to simulate in vivo ECM. The use of coated culture dishes with mixtures of ECM fibers was extended in past years, but this 2D approach needed improvement to 3D structures, such as cross-linked gels (e.g., Matrigel)1. The Matrigel-like setup has become the standard method for simulating a 3D matrix. Fibrin is also an alternative when generating matrices but fails in terms of strength and durability of the ECM1. Collagen used in combination with other ECM components amends some of the abovementioned issues. However, these collagen gels form a strong network with fibers that can be oriented, but are highly heterogeneous, which can be a problem in experiment repetitions1. Nevertheless, it must be assumed that, depending on the objective of the experiments, the use of Matrigel or other hydrogels is more appropriate (e.g., in matrix contraction studies in which gel contraction can be easily detected).
The potential immunogenicity of the generated matrices could be an issue in experiments with some cell types. Therefore, to reduce the possibility of immune responses due to ECM-generating cells when using our method, matrices are decellularized and washed, although cell fragment removal could not be total15. The ideal ECM needs to be compatible with cell culture and able to communicate and react to cell signals. Our procedure allows the introduction of changes without difficulty during ECM production (e.g., adding fibroblast-stimulating growth factors).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium hydroxide (NH4OH)</td>
			<td>Roth</td>
			<td>A990.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin-B</td>
			<td>Corning</td>
			<td>30-003-CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-014-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium (EBM)</td>
			<td>Lonza</td>
			<td>CC-3121</td>
			<td>add supplements before use</td>
		</tr>
		<tr>
			<td>Endothelial cell Basal Medium Supplements (EGM-2)</td>
			<td>Lonza</td>
			<td>CC-4176</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma</td>
			<td>411000-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biowest</td>
			<td>S181B-500</td>
			<td>heat-inactivated before use</td>
		</tr>
		<tr>
			<td>Fibroblast Growth Basal Medium (FBM)</td>
			<td>Lonza</td>
			<td>CC-3131</td>
			<td>add supplements before use</td>
		</tr>
		<tr>
			<td>Fibroblast Growth Medium Supplements and Growth Factors (FGM-2)</td>
			<td>Lonza</td>
			<td>CC-4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>Sigma</td>
			<td>G9391-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>g5882</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A92902-100G</td>
			<td>light sensitive</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>Invivogen</td>
			<td>3ANT-NR-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencillin/Streptomycin (Pen/Strep)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-040-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Roth</td>
			<td>3051</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant telomerase transfected immortalized human foreskin fibroblasts (BJ-hTERT)</td>
			<td>ATCC</td>
			<td>ATCC CRL-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Human umbilical vein endothelial cells (HUVECs)</td>
			<td>ATCC</td>
			<td>PCS-100-013</td>
			<td></td>
		</tr>
	</tbody>
</table>

fibroblast-derived 3d matrix system applicable to endothelial tube formation assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

PDGF-BB stimulated fibroblasts create a thicker ECM. Herrera et al. showed how PDGF-stimulated fibroblasts generated a thicker matrix as well as higher fiber orientation7. BJ-hTERT fibroblasts were incubated with or without PDGF and representative areas observed showed a more aligned cell distribution in matrices produced by PDGF-stimulated fibroblast (Figure 1a). Collagen I and fibronectin protein expression was incre...

Watch this Scientific Journal Video about Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay at JoVE.com

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

This protocol make it possible to see different types of cultures and observe their interactions with extra-cellular matrix. The main advantage of this method is that the matrix you obtain is regenerated by fibroblast and it will be quite similar to the ones generated in vivo. We believe that this sickness come irrelevant when using fibroblast obtaining from baking dishes.Establishing a matrix from these cells could enlighten us how it can effect, for instance, persistent situation. These methods can also be applied to, for example, physical studies to survivors from matrix proprieties. Visualization of this protocol it's important to observe a pipetting technique of the steps that you have to regent.When trying these methods for the first time it will probably take more time than expected. Our advice is to prepare as much as you can in advance and using duration periods for getting ready for next steps. Begin by preparing 0.2%gelatin, 1%glutaraldehyde and one molar ethanolamine solutions according to the manuscript directions.Making sure to run each solution through a 0.22 micrometer filter prior to use. Next, prepare ascorbic acid and lysis buffer. Dilute the PBS pen-strep as described in the manuscript and prepare DMEM with 10%FBS.Prepare heat-denatured 2%BSA by adding 2 grams of BSA to 100 milliliters of sterile water and warming it in a boiling water bath for seven minutes. Then, supplement FBS with penicillin, streptomycin, gentamicin, and amphotericin B.Culture recombinant telomeres transfected immortalized human foreskin fibroblasts and DMEM with 10%FBS and maintained them at 37 degrees Celsius and 5%carbon dioxide. To establish the primary fibroblast culture, cut the tissue samples into small pieces of approximately two to three milliliters cubed and seed them in FPS with a high concentration of antibiotics.When the first fibroblasts appear, replace the medium with FBM medium for cell maintenance. Add two milliliters of 0.2%gelatin solution to each well of a six-well plate and incubate it for one hour for 37 degrees Celsius or overnight at four degrees Celsius. After the incubation, aspirate the gelatin and wash each well with two milliliters of PBS.Add two milliliters of 1%of glutaraldehyde to each well and incubate the plate at room temperature for thirty minutes which will cross-link the gelatin. Aspirate the glutaraldehyde and wash the wells in two milliliters of PBS for five minutes. To block the remaining glutaraldehyde, add two milliliters of one molar ethanolamine and incubate the plate at room temperature for thirty minutes.Aspirate the ethanolamine and repeat the washes with PBS. Add one milliliter of DMEM with 10%FPS. If the medium immediately turns pink, remove it, wash the wells once with two milliliters of PBS and add another milliliter of DMEM.Seed one milliliter of fibroblast suspension with 5 x 10 to the fifth cells in each well and culture the cells until 100%confluence is reached. Then remove the medium and replace it with DMEM with 10%FBS and 50 micrograms per milliliter ascorbic acid. Replace the medium every two days for six days.Two days after the last ascorbic acid treatment remove the medium and wash the wells with two milliliters of PBS. After the washes, slowly add one milliliter of preheated lysis buffer and incubate the plate at room temperature for five to ten minutes until the fibroblasts are lysed. Without removing the lysis buffer, carefully add two milliliters of PBS to each well and then aspirate approximately 2.5 milliliters.Repeat this process twice for a total of three washes. After the final wash, add two milliliters of PBS with pen-strep. Seal the plate with film and keep it at four degrees Celsius for up to three months.Grow HUVEC cells and EMB-2 with 2%FBS until maximum confluence. Then replace the medium with EBM-2 without FBS for eight hours. To prepare the matrices for cell seeding, remove them from the refrigerator and leave them at room temperature for one hour.Block the matrices by add two milliliters of heat-denatured 2%BSA and incubate them at 37 degrees Celsius for one hour. Then, aspirate the BSA and wash them with two milliliters of PBS. Seed 2 x 10 to the fifth HUVEC cells to each well of the plate coated with fibroblast-derived matrices.Incubate the cells at 37 degrees Celsius for 16 hours then examine the tube-like structures under a standard bright field microscope at 20-40x magnification. This protocol was used to examine the affects of PDGF treatment on fibroblast-derived matrix formation. The BGH third fibroblasts that were incubated with PDGF showed a more aligned cell distribution in matrices than those incubated without PDGF.Collagen 1 and fibronectin protein expression was increased in matrices derived from PDGF-stimulated fibroblasts and consequently matrix thickness was increased. Moreover, directionality histograms demonstrate that collagen 1 and fibronectin show parallel patterns. When HUVEC cells were seeded on to decellularized matrices derived from PDGF-stimulated fibroblasts they developed more capillary-like structures than under none stimulated conditions.Confirming that matrices derived from PDGF-BB stimulated fibroblasts promote endothelial cells activation. When performing this procedure, it's important to prepare solutions in advance and make sure the cells are ready before starting. Besides endothelial cells studies matrices can also be seeded with epithelial cells and the reaction to external factors like condition in media or cytotoxic drugs can be observed.Generation of extra-cellular matrix is crucial in the design of tumor macroenvironment experiments.

fibroblast-derived-3d-matrix-system-applicable-to-endothelial-tube

Research

JoVE Journal

Cancer Research

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living biobanks&#34; of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research.

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...