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 (passage up to 25) in the spheroid formation solution. If preparing one 96 well plate, prepare an excess of spheroid formation solution, 10 mL and add 75,000 cells, keeping in mind that each spheroid is composed by 750 cells, and that 100 &#181;L are distributed per each well of the plate.</li>
	<li>If cytokines or integrin inhibitors are included in the study, add these compounds to the cell suspension, in order to embed them into the spheroids during their formation. Note that iNFs are stimulated with 10 ng/mL of TGF-&#946;1 in order to trigger differentiation. Where appropriate, add inhibitor compounds together with TGF-&#946;1, such as 20 &#181;g/mL BM2 or 10 &#181;g/mL of lebein 1.</li>
	<li>Prepare CAF homospheroids in the same way but without the TGF-&#946;1 stimulus, since they are already differentiated. Prepare the integrin &#945;3 KO CAF homospheroids without the addition of any compound.</li>
	<li>Distribute the spheroid formation solution onto a round bottom 96 multi-well plate by adding 100 &#181;L of the solution in each well. Every time the spheroid solution is distributed in the wells, mix well by pipetting up and down, several times.</li>
	<li>Place the plate in the cell culture incubator for 24 h to allow one spheroid to be formed in each well.</li>
	<li>Collect the spheroids after about 24 h and use them for different downstream purposes: immunofluorescent staining, real time (RT) q-PCR and flow cytometry. To detect expression of intra- and extracellular antigens, including deposition of ECM proteins within the spheroid, use immunofluorescent staining. After disintegration of spheroids into single cells, quantify membrane-anchored receptor by flow cytometry. To detect gene activation and transcriptional changes, use RT q-PCR of mRNA isolated from the spheroid cells.</li>
</ol>
2. Immunofluorescent staining of spheroids
<ol>
	<li>Collect the spheroids after about 24 h into one 1.5 mL reaction tube per experimental condition or per protein to be analyzed. For example, place the spheroids of iNFs stimulated with TGF-&#946;1 in a tube different from the control iNFs, which were not treated. To avoid rupture of spheroids due to too strong shear forces, cut the end of the pipette tip to enlarge the orifice. Include at least 5-10 spheroids in one reaction tube, to allow replicates and take into account the possible losses during the washing steps.</li>
	<li>Centrifuge the spheroids with a bench top centrifuge for about 30-60 s at 1,000 x g. Carefully remove the methylcellulose-containing supernatant by pipetting, without disturbing the pelleted spheroids.</li>
	<li>Wash with 50 &#181;L of 1x PBS. Repeat the centrifugation step and carefully remove the PBS by pipetting, as described in 2.2.</li>
	<li>Depending on the protein to be stained, fix with 50 &#181;L of 4% paraformaldehyde in PBS for 20 minutes at room temperature (for &#945;SMA, NG2 and integrin &#945;3&#946;1) or with 50 &#181;L of methanol for 10 min at -20 &#176;C (for laminin-332 subunits).</li>
	<li>Repeat the washing step as explained in steps 2.2-2.3.</li>
	<li>Permeabilize the cells with 0.1% Triton-X in PBS for 4 min at room temperature.</li>
	<li>Repeat the washing step as explained in steps 2.2-2.3 three times.</li>
	<li>Incubate the spheroids in PBS containing 5% FBS and 2% BSA, for 1 h at RT to block non-specific protein interaction sites.</li>
	<li>Incubate the spheroids with 30 &#181;L of primary antibodies in PBS, 2.5% FBS and 1% BSA at 4 &#176;C overnight. The following primary antibodies were used: anti-&#945;SMA Cy-3 conjugated and anti-NG2 at 5 &#181;g/mL; BM2 anti-laminin &#945;3, 6F12 anti-laminin &#946;3 and anti-laminin &#947;2 20 &#181;g/mL.</li>
	<li>Repeat the washing step as explained in steps 2.2-2.3 three times.</li>
	<li>Incubate the spheroids with 30 &#181;L of secondary antibodies in PBS, 2.5% FBS and 1% BSA at RT for 90 min. The following secondary antibodies used were: Alexa 488 anti-rabbit and anti-mouse (5 &#181;g/mL).</li>
	<li>Repeat the washing step as explained in steps 2.2-2.3 three times.</li>
	<li>Incubate the cells with 30 &#181;L of DAPI staining solution for 4 min at room temperature.</li>
	<li>Repeat the washing step as explained in steps 2.2-2.3.</li>
	<li>Place the spheroids on a glass slide, in a drop of PBS, using a glass Pasteur pipet.</li>
	<li>Image the spheroids by fluorescence microscopy in a laser scanning microscope using a magnification of 10x. Take different optical cross-section of the spheroid at different optical planes of a z-stack (15-20 stack planes). To establish the laser settings in the microscope, use a spheroid composed of cells negative for the antigen of interest or a spheroid stained only with the secondary antibody. Reuse the same settings for all the samples that will be compared.</li>
	<li>Analyze the microscopic images with ImageJ software as published previously14. The steps are summarized in Supplemental Figure 1. As background, determine the integrated signal density of a cell-free region of interest (ROI). Calculate the total corrected cell fluorescence (TCCF) as: 
	<img alt="Equation 1" src="/files/ftp_upload/60122/60122eq1.jpg" /></li>
	<li>Determine the total corrected fluorescence (TCF) of the spheroids as the TCCF normalized to the area of spheroid and the number of stacks. 
	<img alt="Equation 2" src="/files/ftp_upload/60122/60122eq2.jpg" /></li>
</ol>
3. RT q-PCR of homospheroids
<ol>
	<li>To perform RT-qPCR, collect at least 288 spheroids (corresponding to three 96-well plates) into a 50 mL reaction tube. Centrifuge for 5 min at 1,000 x g and carefully remove the methylcellulose-containing supernatant by pipetting without disturbing the pelleted spheroids.</li>
	<li>Wash with 1 mL of 1x PBS and transfer the spheroids to a 1.5 mL reaction tube. Centrifuge the spheroids with a bench top centrifuge for about 30-60 s at 1,000 x g. Carefully remove the PBS by pipetting, without disturbing the pelleted spheroids.</li>
	<li>Dissociate the spheroids with 250 &#181;L of 4 mg/mL collagenase B, 50 &#181;g/mL DNase I, 2% BSA, 1 mM CaCl2 in PBS at 37 &#176;C for about 30-45 min by gently vortexing and pulsing intermittently for a few seconds every 5 min.</li>
	<li>Repeat the washing step as explained in step 3.2.</li>
	<li>Isolate the total RNA from the cells of disintegrated spheroids using a commercial RNA isolation kit, according to the manufacturers&#8217; instructions.</li>
	<li>Reverse transcribe the isolated mRNA into cDNA with a commercial kit, according to manufacturers&#8217; instructions.</li>
	<li>Quantify the transcription levels in replicates by real-time PCR. The primers used were: &#945;-SMA: Fw 5&#8242;-CCGACCGAATGCAGAAG GA-3&#8242;, Rev 5&#8242; ACAGAGTATTTGCGCTCCGAA-3&#8242;15; Laminin &#945;3 chain: Fw 5&#8242;-GCTCAGCTGTTTGTGGTTGA-3&#8242;, Rev 5&#8242;-TGTCTGCATCTGCCAATAGC-3&#8242;16; Laminin &#946;3 chain: Fw 5&#8242;-GGCAGATGATTAGGGCAGCCGAGGAA-3&#8242; Rev 5&#8242;-CGGACCTGCTGGATTAGGAGCCGTGT-3&#8242;17; Laminin &#947;2 chain: Fw 5&#8242;-GATGGCATTCACTGCGAGAAG-3&#8242; Rev 5&#8242;-TCGAGCACTAAGAGAACCTTTGG-3&#8242; 16; NG2: Fw 5&#8242;-CTGCAGGTCTATGTCGGTCA-3&#8242; Rev 5&#8242;-TTGGCTTTGACCCTGACTATG-3&#8242;18; integrin &#945;3 subunit: Fw 5&#8217;-AGGGGACCTTCAGGTGCA-3&#8217; Rev 5&#8217;-TGTAGCCGGTGATTTACCAT-3&#8217;19; TOP-1: Fw 5&#8242;-CCAGACGGAAGCTCGGAAAC-3&#8242; Rev 5&#8242;-GTCCAGGAGGCTCTATCTTGAA-3&#8242;20.</li>
	<li>Correct the Ct values for the Ct-value of an endogenous reference gene such as TOP-1 using the equation: 2- &#916;&#916;Ct. &#916;&#916;Ct = &#916;Ct (target sample) &#8211; &#916;Ct (reference sample) and &#916;Ct= Ct of target gene or reference gene &#8211; TOP-I.</li>
</ol>
4. Flow cytometry analysis of integrin expression
<ol>
	<li>For flow cytometric analysis, collect at least 288 spheroids (corresponding to three 96-well plates) into a 50 mL reaction tube. Centrifuge for 5 min at 1,000 x g and carefully remove the methylcellulose-containing supernatant by pipetting.</li>
	<li>Wash with 1 mL of 1x PBS and transfer the spheroids to a 1.5 mL reaction tube. Repeat the centrifugation step and carefully remove the PBS by pipetting.</li>
	<li>Dissociate the spheroids as described in step 3.3.</li>
	<li>Repeat the washing step as explained in step 4.2.</li>
	<li>To block non-specific binding sites and to prevent binding of detection-relevant antibodies to Fc&#947;-receptors (CD16, CD32, and CD64), incubate the cells in 100 &#181;L of PBS containing 2% horse serum (or serum of another animal species, the antibodies of which will not be used in the experiment) and 0.1% BSA for 20 min at 4 &#176;C with rotation.</li>
	<li>In case of staining intracellular markers, fix/permeabilize the cells as described under step 2.2-2.5.</li>
	<li>Incubate the cells with the primary antibodies in 100 &#181;L of PBS containing 2% horse serum and 0.1% BSA for 30 min at 4 &#176;C with rotation. Dilute the primary antibody to 10 &#181;g/mL.</li>
	<li>Wash with 100 &#181;L of PBS + 0.1% BSA three times, with centrifugation steps in the top bench centrifuge at 1,000 x g in between.</li>
	<li>Incubate the cells with the secondary antibodies in 100 &#181;L of PBS containing 2% horse serum and 0.1% BSA for 30 min at 4 &#176;C with rotation. Dilute secondary antibody to 10 &#181;g/mL.</li>
	<li>Repeat the washing step as explained in step 4.7.</li>
	<li>Analyze 2.0 x 104 stained cells in a flow cytometer. Gate for single live cells via the FSC-SSC dot plot and analyze the fluorescence of the gated cells on the channel appropriate for the selected fluorophore.</li>
</ol>
5. Invasion assay using heterospheroids
<ol>
	<li>Prepare the spheroid formation solution for heterospheroids, containing the different cell types and corresponding mediums, as summarized in Table 1. The cells had previously been transduced with lentivirus containing vectors for either mCherry or GFP expression.</li>
	<li>Fill 100 &#181;L of the spheroid formation solution in each well of the plate and incubate the plate in the cell culture incubator for 24 h to allow one spheroid to be formed in each well.</li>
	<li>Prepare 60 &#181;L of gelatinous matrix mixture containing 2 mg/mL of rat tail collagen I, 30% of commercial gelifying matrix and 2.5 &#181;g/mL of laminin-332 in a 1.5 mL reaction tube. Rapidly distribute 15 &#181;L in 3 wells of the &#181;-slide angiogenesis chamber. Embed one spheroid in each well already containing the gel.</li>
	<li>If necessary, rapidly adjust the location of the spheroid to the center of the well with a pipette under the microscope.</li>
	<li>Repeat steps 5.2-5.3 for the next heterospheroids and repeat again for the homospheroids.</li>
	<li>Incubate the gels in the incubator for 1 h to solidify and, then, gently overlay the gels with cell culture medium.</li>
	<li>Image the spheroids by fluorescence microscopy in a laser scanning microscope. Take different optical cross-section of the spheroid at different optical planes of a z-stack and at different time points of invasion (e.g., 0 h, 24 h, 48 h and 72 h).</li>
	<li>Analyze the images by counting the number of invading cancer cells. Invading cells are those ones that have left the spheroid and entered the invasive area. The invasive area is defined as the ring area between the outer and inner perimeters given by the furthest invaded cell and by the spheroid border of the spheroid at the beginning of the invasion experiments, respectively, as outlined in Supplemental Figure 2.</li>
</ol>

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The authors declare no conflict of interest. This material reflects only the author&#39;s views and the European Union is not liable for any use that may be made of the information contained therein.

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 ...

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Cancer Research

8.7K Views.  University of Münster. 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.

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

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

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 ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

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 Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...