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 ethanolamine: prepare ethanolamine solution with sterile H2O. Filter with a 0.22 &#181;m filter before use. 
	NOTE: As ethanolamine is provided with a security cap, a needle and syringe will be needed.</li>
	<li>Prepare ascorbic acid: add 0.1 mL of 50 mg/mL stock solution ascorbic acid (light-sensitive) to 100 mL of medium.</li>
	<li>Prepare lysis buffer: prepare PBS 0.5% Triton 100X with 20 nM of NH4OH. Add NH4OH right before use.</li>
	<li>Prepare PBS Pen/Strep: dilute Pen/Strep stock solution to 100 U/mL Pen and 100 &#181;g/mL Strep.</li>
	<li>Prepare DMEM with 10% FBS: Add 10% FBS to 500 mL of DMEM. Supplement with 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 0.1 mg/mL Normocin and 0.25 &#181;g/mL amphotericin B.</li>
	<li>Prepare 2% BSA heat denatured: add 2 g of BSA to 100 mL of sterile water. Warm in boiling water for 7 min.</li>
	<li>Prepare FBS with antibiotics: supplement FBS with 200 U/mL penicillin, 200 &#181;g/mL streptomycin, 100 &#181;g/mL gentamicin and 2.5 g/mL amphotericin B.</li>
</ol>
2. Cell Culture Preparation
<ol>
	<li>Immortalized fibroblasts cell line
	<ol>
		<li>Culture recombinant telomerase transfected immortalized human foreskin fibroblasts (BJ-hTERT, ATCC CRL-4001) in DMEM with 10% FBS and maintain at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Endothelial cells
	<ol>
		<li>Culture human umbilical vein endothelial cells (HUVECs, ATCC PCS-100-013) in EBM-2 medium containing 2% FBS and maintain at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Fibroblast primary culture 
	NOTE: For CAF establishment and culture, the protocol previously published by our group was followed6.
	<ol>
		<li>Briefly, cut tissue samples into small pieces of approximately 2-3 mm3 and seed in FBS with high concentration of antibiotics.</li>
		<li>When the first fibroblasts appeared, replace medium with FBM medium for cell maintenance. 
		NOTE: Depending on tissue origin, cell cultures can be contaminated easily. Wash samples in PBS supplemented with antibiotics, with highly contaminated tissue (e.g., colon6) and shake for 30-45 min.</li>
	</ol>
	</li>
</ol>
3. Fibroblast-derived 3D Matrices (Adapted from Castell&#243;-Cros and Cukierman16)
<ol>
	<li>Add 2 mL of 0.2% gelatin solution to each well of a 6-well plate and incubate for 1 h at 37 &#176;C or overnight at 4 &#176;C.</li>
	<li>Aspirate gelatin and wash it in 2 mL of PBS.</li>
	<li>Add 2 mL of 1% glutaraldehyde and incubate for 30 min at RT. Glutaraldehyde will cross-link gelatin.</li>
	<li>Aspirate glutaraldehyde and wash wells in 2 mL of PBS for 5 min. Repeat 3 times.</li>
	<li>Add 2 mL of 1 M ethanolamine and incubate for 30 min at RT. Ethanolamine will act to block the remaining glutaraldehyde.</li>
	<li>Aspirate ethanolamine and wash wells in 2 mL of PBS for 5 min. Repeat 3 times.</li>
	<li>Add 1 mL of DMEM with 10% FBS. If the medium immediately turns pink, remove the medium, wash in 2 mL of PBS and add again 1 mL of DMEM with 10% FBS.</li>
	<li>Seed 1 mL of fibroblast suspension, with 5 x 105 cells in each well. Total volume in each well will be 2 mL.</li>
	<li>Culture cells until 100% confluence is reached. Then remove the medium and replace with DMEM with 10% FBS with 50 &#181;g/mL ascorbic acid and additional treatment if used.</li>
	<li>Replace medium with fresh DMEM with 10% FBS and 50 &#181;g/mL ascorbic acid every 2 days for 6 days. 
	NOTE: If additional treatment is used (e.g., PDGF-BB, TGF-&#946; and/or other grow factors), add them the same days as ascorbic acid treatment is added.</li>
	<li>Two days after the last ascorbic acid treatment, remove the medium and wash in 2 mL of PBS.</li>
	<li>Slowly add 1 mL of lysis buffer, pre-heated at 37 &#176;C, to each well. Incubate for 5-10 min at RT until fibroblasts are lysed (observable under the microscope).</li>
	<li>Carefully and without removing lysis buffer, add 2 mL of PBS. Then aspirate approximately 2.5 mL of PBS. Repeat twice for a total of three washes.</li>
	<li>Eventually, remove 2.5 mL of PBS and add 2 mL of PBS with Pen/Strep (100 U/mL and 100 &#181;g/mL, respectively). Seal with film and keep at 4 &#176;C for up to 3 months. 
	NOTE: Depending on the experiment, long-term storage of the generated matrices may affect results. We recommend using matrices as soon as possible, and even more so if the experiment involves cell seeding, due to possible protein degradation. If matrices are used for structural assays, such as collagen observation, matrices can be fixed and stored for longer periods. This protocol is indicated for a 6-well culture plate. Other plates can be used, but reactive volumes and cell suspension concentration need to be recalculated according to well area.</li>
</ol>
4. Tube Formation Assay
<ol>
	<li>Grow HUVEC cells in EBM-2 2% FBS until maximum confluence.</li>
	<li>Replace medium with EBM-2 without FBS for 8 h.</li>
	<li>Prepare matrices before seeding cells.
	<ol>
		<li>Remove matrices from the refrigerator and place for 1 h at RT.</li>
		<li>Block matrices by adding 2 mL of heat-denatured 2% BSA. Incubate 1 h at 37 &#176;C.</li>
		<li>Aspirate BSA and wash in 2 mL of PBS.</li>
	</ol>
	</li>
	<li>Seed 2 x 105 HUVEC cells in FBS-depleted medium on each well of a 6-well plate previously coated with 3D fibroblast-derived matrices.</li>
	<li>Incubate endothelial cells at 37 &#176;C for 16 h.</li>
	<li>Examine tube-like structure formation under a standard bright field microscope at 20-40x magnification.</li>
</ol>

<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>

The authors declare no conflict of interest.

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...

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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 ...

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

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Cancer Research

7.3K Views.  Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), CIBERONC. 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).

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

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 ...

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 ...

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

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

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

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

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...