Name: a 3d spheroid model as a more physiological system for cancer-associated fibroblasts differentiation and invasion in vitro studies
Uploaded: 2019-08-08T15:00:00
Description: Watch this Scientific Journal Video about A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies at JoVE.com

We acknowledge Barbara Schedding&#39;s help in preparing the BM2 and lebein-1. We acknowledge &#192;gnes Noel for sharing her expertise in spheroid assays. We thank Sonja Schelhaas and Michael Sch&#228;fers for their help in handling lentiviral transfection under S2 conditions. We acknowledge Sabine von R&#252;den&#39;s assistence in preparing CAFs from pancreatic cancer tissue.
The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union&#39;s Seventh Framework Programme FP7/2007-2013/ under the REA grant agreement n&#9702; (316610) to J.A.E. Moreover, J.A.E. and A.C.M.C. was financially supported by the Deutsche Forschungsgemeinschaft (DFG) within the Cells-in-Motion Cluster of Excellence (EXC 1003-CiM). This project was also supported by Wilhelm Sander Stiftung (grant: 2016.113.1 to J.A.E.).

1. 3D spheroids as an in vitro model for fibroblast/CAF differentiation using different TGF-&#946;1 inhibiting compounds of cell-matrix interaction
<ol>
	<li>Mix 1 part of 6 mg/mL methylcellulose solution and 3 parts of cell culture media, MEM supplemented with 1% of heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin to obtain the spheroid formation solution.</li>
	<li>Resuspend freshly thawed immortalized normal fibroblasts (iNFs), immortalized CAF (iCAFs) or integrin &#945;3&#946;1 KO iCAF (pass...

<ol>
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To develop an appropriate in vitro model to study CAF differentiation is a challenging task. After employing different approaches, we concluded that a 3D spheroid model is the more practical, physiological and clinically relevant model, in which the interplay between pancreatic carcinoma cells with immortalized CAFs can be studied. This model prevented spontaneous differentiation of fibroblasts, due to artefactual stressors such as stiffness of the cell culture plastic, at least in short-term culture conditions (up to 48...

The tumor microenvironment is a very complex niche and extremely important for the maintenance and progression of the tumor cells1. It is formed not only by the cancer cells but also by stromal fibroblasts. The tumor cells are surrounded by a stroma that is specific and different from the stroma of normal tissues2. Laminin-332 is an extracellular matrix protein ectopically expressed in the stroma of different tumors, such as of pancreatic adenocarcinoma3. Moreover, the biochemical composition of the ECM and also its biophysical properties, such as rigidity and tension, change within the tumor bulk4. This tumor stroma, or &#34;reactive stroma&#34;, is caused by an adaptation of fibroblasts to the neighboring cancer cells and by the recruitment of other very important players that develop a favorable and supportive environment for tumor progression. The differentiation of stromal fibroblasts results in cancer-associated fibroblasts (CAF). These cells can be identified using different markers such as &#945;-smooth muscle actin (&#945;SMA)5 or neural/glial antigen 2 (NG2)6.
The most suitable&#160;in vitro model to recapitulate the tumor microenvironment (TME) with CAFs is difficult to select. The method to mimic physiological parameters of the TME in a cost-efficient and reproducible way must be considered for such a model system. Within the TME, different processes, such as proliferation, differentiation, migration and invasion of the different cell types occur. These cellular processes can be performed individually with different methods. However, the experimental conditions must consider the cellular interactions with the tumor stroma ECM, since the stiffness of the substratum influences the CAF differentiation process. R.G. Wells commented on the impact of matrix stiffness on cell behavior and highlighted that cytoskeletal organization and differentiation status observed in in vitro cultured cells might be artefactual7. Different stimuli seem to be involved in CAF differentiation, including mechanical tension5,7. To avoid this, 2D soft substrates could be possible approaches for differentiation studies, as they circumvent the problem of the stiff culture dish plastic. A soft 2D surface, on which fibroblasts can be grown, can be collagen-I coated polyacrylamide gels, whereby the gel stiffness can be manipulated by the concentration of polyacrylamide and the gel cross-linker. The adhesion and formation of &#945;SMA-rich stress fibers are enhanced in fibroblasts along with the gel stiffness8. These results stress the importance of soft substrate scaffolds for more physiological in vitro differentiation models. However, in our hands the experimental reproducibility and imaging of these gels were challenging. To overcome these shortcomings, we changed the 2D soft substrate system for a 3D spheroid model for differentiation and invasion studies. This model is more clinically relevant and, similar to an in vitro organoid, recapitulates in vivo cell-cell interactions, ECM production and deposition, as well as cell behavior9.
Spheroids are formed when cells lack a substrate to adhere to. When the cells are left without an adhesive surface, they aggregate to form a more or less spherical structure. If the spheroids are composed of one type of cell, they are called homospheroids; if composed of two or more different cell types they form heterospheroids.
Among the different methods for spheroid preparation, we perform the protocol using non-adherent round bottom 96-well plates. It is very effective with respect to the costs. Here, we produce both homospheroids of fibroblasts, CAF or CAFs lacking the integrin &#945;3 subunit to examine the differentiation process and heterospheroids of CAFs or integrin &#945;3 KO CAFs and pancreatic duct carcinoma cells (AsPC-I and PANC-I) to study the invasion into the surrounding matrix.
The aim for these studies was to use primary CAFs isolated from human pancreatic carcinoma biopsies. However, the biopsies to obtain the cells are scarce and for this reason, the CAFs used in these studies have been immortalized using lentivirus containing HTERT. They are called iCAFs, and their normal counterparts, primary human pancreatic fibroblasts, are termed iNFs. The human pancreatic fibroblasts and the pancreatic duct carcinoma cells, AsPC-I and PANC-I, are commercially available.
This protocol was used to study the effect of the laminin-332-integrin interaction in the CAF differentiation process. To prove specificity of this interaction and its function, inhibitor compounds were used: BM2, a monoclonal antibody that blocks the integrin binding site the laminin-332 &#945;3 chain10, or lebein 1, a snake venom derived compound that blocks the laminin-binding integrins &#945;3&#946;1, &#945;6&#946;1 and &#945;7&#946;111,12.
For the invasion assay, cells had been transduced with lentivirus containing cDNA encoding either mCherry (iCAFs and integrin &#945;3 KO iCAFs) or GFP (AsPC-I and PANC-I) to distinguish the different cell types in the heterospheroids. The transduction of the cells to immortalize them and/or to label them with fluorescent protein (mCherry and GFP) expression is described in a previous study13, that should be consulted for further information.

<table>
	<tbody>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>SIGMA-ALDRICH</td>
			<td>D9542-10</td>
			<td></td>
		</tr>
		<tr>
			<td>6F12 anti-Laminin &#946;3 subunit Mouse (monoclonal)</td>
			<td></td>
			<td></td>
			<td>Homemade</td>
		</tr>
		<tr>
			<td>A3IIF5 Anti-&#945;3 integrin subunit Mouse (monoclonal)</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. M. Hemler, Dana Faber-Cancer Institute, Boston</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>SIGMA-ALDRICH</td>
			<td>32201</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraction V - BSA</td>
			<td>AppliChem</td>
			<td>A1391</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488 Goat (polyclonal) anti-Mouse</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488 Goat (polyclonal) Rabbit</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-laminin &#947;2 subunit Mouse (monoclonal)</td>
			<td>Santa Cruz</td>
			<td>sc-28330</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NG2 Rabbit (polyclonal) Millipore, AB5320</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#945;-SMA-Cy3 Mouse (monoclonal)</td>
			<td>SIGMA-ALDRICH</td>
			<td>C6198</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1 cell line</td>
			<td>ATCC</td>
			<td></td>
			<td>Kindly given by prof. Jorg Haier&#39;s Lab</td>
		</tr>
		<tr>
			<td>Bench centrifuge</td>
			<td>Fisher Scientific</td>
			<td>50-589-620</td>
			<td>Sprout</td>
		</tr>
		<tr>
			<td>BM2 anti-laminin &#945;3 subunit Mouse (monoclonal)</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. Patricia Rousselle, CNRS, Lyon</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Fluka</td>
			<td>21074</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Multifuge 1S-R</td>
		</tr>
		<tr>
			<td>Centrifuge tubes 50 mL</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase B</td>
			<td>Roche</td>
			<td>11088831001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-I, rat tail</td>
			<td>Gibco</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 700 and 800</td>
		</tr>
		<tr>
			<td>DMEM (High glucose 4.5 g/L)</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>FACSCaliburTM</td>
		</tr>
		<tr>
			<td>Gelifying matrix</td>
			<td>ThermoFisher Scientific</td>
			<td>A1413202</td>
			<td>Matrigel, Geltrex</td>
		</tr>
		<tr>
			<td>Goat IgG, isotype</td>
			<td>DAKO</td>
			<td>X 0907</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>SIGMA-ALDRICH</td>
			<td>12449-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Primary Pancreatic Fibroblasts</td>
			<td>PELOBiotech</td>
			<td>PB-H-6201</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heraeus</td>
			<td>B6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin-332</td>
			<td>Biolamina</td>
			<td>LN332</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>SIGMA-ALDRICH</td>
			<td>M4655</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, 96 wells, U-bottom</td>
			<td>Greiner Bio-One</td>
			<td>650101</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG, isotype</td>
			<td>SIGMA-ALDRICH</td>
			<td>I8765</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi axle rotating mixer</td>
			<td>CAT</td>
			<td>RM5 80V</td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-I</td>
			<td>ATCC</td>
			<td></td>
			<td>Kindly given by Prof. Jorg Haier&#39;s Lab</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Riedel-de Ha&#235;n</td>
			<td>16005</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Reverse Transcription Kit</td>
			<td>Qiagen</td>
			<td>205310</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG, isotype</td>
			<td>Invitrogen</td>
			<td>10700</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes, 1.5 mL</td>
			<td>Greiner Bio-One</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR cycler</td>
			<td>Qiagen</td>
			<td></td>
			<td>Rotor-Gene Q</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor Gene SYBR Green PCR Kit</td>
			<td>Qiagen</td>
			<td>204074</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Lonza</td>
			<td>BE12-702F</td>
			<td>Add glucose to 4.5 g (0.2 um filter) and 1% sodium pyruvate</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>SIGMA-ALDRICH</td>
			<td>X100RS</td>
			<td></td>
		</tr>
		<tr>
			<td>V&#243;rtex</td>
			<td>Scientific Industries</td>
			<td></td>
			<td>Vortex-Genie 2</td>
		</tr>
		<tr>
			<td>&#956;-Slide Angiogenesis, uncoated</td>
			<td>Ibidi</td>
			<td>81501</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 results of this experimental design are published in Martins Cavaco et al.13, which is recommended for further reading on the conclusions that were drawn from these experiments.
Figure 1, a representative image of an immunofluorescent spheroid, shows the immunostaining of the integrin &#945;3 subunit of both immortalized normal fibroblasts and immortalized CAFs (<strong c...

Watch this Scientific Journal Video about A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies at JoVE.com

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

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 ...

Culturing fibroblasts in spheroid recapitulate tumor tissue much better than the commonly used 2D system on stiff plastic. It allows to study fibroblasts differentiation in a tumor-like environment. In this method, the rigidity of cell culture plastic work does not artifactually influence fibroblast differentiation.Instead, the influence of cell matrix interaction on tumor induced differentiation of fibroblast can be analyzed in vitro. To begin this procedure, obtain the cell culture media, which is MEM supplemented with 1%heat inactivated FBS and 1%penicillin streptomycin. Also, obtain a six milligram per milliliter methylcellulose solution.Mix one part of the methylcellulose with three parts of the cell culture media to prepare the spheroid formation solution. Resuspend freshly thawed immortalized normal fibroblasts or other cell types as listed in the text protocol in the spheroid formation solution. If cytokines or integrin inhibitors are included in the study, add these compounds to the cell suspension in order to imbed them into the spheroids during their formation.Where appropriate, add inhibitor compounds together with 10 nanograms per milliliter of TGF beta one to trigger differentiation in immortalized normal fibroblasts. Distribute 100 microliters of the spheroid formation solution into each well of a round bottom 96 multi-well plate. Pipette the solution in each well up and down several times to mix.Then, incubate the plate in the cell culture incubator at 37 degrees celsius with 5%carbon dioxide for 24 hours to allow one spheroid to be formed in each well. First, cut off the end of a pipette tip to enlarge the orifice. After the incubation is complete, use this cut pipette to collect the spheroids into one 1.5 milliliter reaction tube per experimental condition or per protein to be analyzed.Centrifuge the spheroids at 1000 times G for about 30 to 60 seconds. Carefully remove the methylcellulose containing supernatant by pipetting making sure to not disturb the pelleted spheroids. Special care is to be taken when transferring the spheroids to the reaction tubes.Make sure you don't have shears drift forces by cutting the tip. It's also important that you collect all the spheroids. During the washing step, make sure you don't lose the spheroids by aspiration.To wash the spheroids add 50 microliters of one X PBS. Centrifuge at 1000 times G for 30 to 60 seconds. Then carefully remove the PBS by pipetting.Depending on the protein to be stained, fix the spheroids either with 50 microliters of 4%paraformaldehyde and PBS for 20 minutes at room temperature. Wash the spheroids with PBS as previously described. Permeabilize the cells with 0.1%triton X and PBS for four minutes at room temperature.Then, wash the spheroids in PBS three times as previously described. Add PBS containing 5%FBS and 2%BSA to the spheroids and incubate at room temperature for one hour to block non-specific protein interaction sites. Now, incubate the spheroids with 30 microliters of primary antibodies and PBS with 2.5%FBS and 1%BSA at four degrees celsius overnight.The next day, wash the spheroids in PBS three times as described previously. After this, incubate the spheroids with 30 microliters of secondary antibodies and PBS with 2.5%FBS and 1%BSA at room temperature for 90 minutes. Wash the spheroids in PBS three times as previously described.Next, incubate the cells with 30 microliters of Dappy staining solution for four minutes at room temperature. Wash the spheroids in PBS one time as previously described. Using a glass Pasteur pipette, place the spheroids on a glass slide and a drop of PBS.Use a laser scanning microscope to image the spheroids by fluorescence microscopy at a magnification of 10 times. Take different optical cross section of the spheroid at different optical plains of the Z stack. A representative image of the immunofluorescent staining of the homo spheroids of normal pancreatic fibroblasts compared to the homo spheroids of immortalized cancer associated fibroblasts shows an increased signal for the alpha three subunit in the differentiated cells.The fluorescence signal of the cells in the spheroid are then quantified with the Z stack images of the 3D spheroid and the transcriptional levels of the integrin alpha three subunit gene are determined by qPCR. All of these results demonstrate that integrin alpha three is upregulated in immortalized cancer associated fibroblasts as compared to the normal counterpart. This proves that integrin alpha three beta one can be considered a marker for pancreatic fibroblasts differentiation.This method is ideally suited to capitulate self tissue generally found in organs and cancer masses where the extra cell matrix stiffness determines cell fate. Spheroids culture is a versatile model that can be combined with other analytical assays after spheroid dissociation such as quantity real time PCR and full cytometry. Paraformaldehyde used for fixation of the spheroids is a hazardous chemical agent.Gloves and eye wear should be worn for protection.

Research

JoVE Journal

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