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 h). Although the exact stiffness of the spheroid used in these studies was not measured, Ito et al. evaluated the stiffness of human umbilical vein endothelial cells and mesenchymal stem cell heterospheroids using a robot integrated microfluidic chip22. The average stiffness-index of the heterospheroids was measured 1 and 3 days after assembly. The size of the spheroids was different, the MSC seemed to increase their aggregation capacity, which resulted in a decrease in size over time (from 135.5 &#181;m to 99.8 &#181;m)21,22. Consequently, the stiffness of the spheroid also increased from 5.0 x 10-3 to 7.5 x 10-3 Pa22. However, the stiffness of the spheroids composed of a heterotypic population of CAFs and AsPC-I might possess mechanical properties and stiffness values different from the ones of homospheroids made of a single population of fibroblasts/CAFs. This is still unknown and might be different from what was described by Ito et al22. The stiffness also depends on the amount and cross-linkage of cell-produced and deposited ECM, on the different cell-cell interactions within the spheroid, on the number of cells within the spheroid or on the duration of culture.
Like any other model, the experimental design involves some variables that need optimization such as the number of cells necessary to form the spheroids, the time points of treatment and microscopic imaging. The described experimental design was optimized with the aim of having a spheroid size, which would have minimal influence in the diffusion of nutrients and oxygen, as there is a diffusion limit due to mass transport limitations9. For example, the transport of oxygen in spheroids with a size greater than 150-200 &#181;m is significantly affected, as well as the diffusion of glucose and lactate, in aggregates bigger than 300 &#181;m9,18. Spheroids with diameters larger than 400-500 &#181;m usually present a concentrically layered structure consisting of a necrotic core surrounded by a viable layer of quiescent cells and an outer rim of proliferating cells9,24. To avoid the limited diffusion of compounds, and to eliminate the problem of the inaccessible spheroid core, cells were treated with TGF-&#946;1, BM2 and lebein-1 prior to spheroid formation, when the cells are individually suspended in the spheroid formation solution. Moreover, to allow the availability of oxygen and nutrients to most of the cells in the spheroid and to prevent the formation of a necrotic core, the number of cells per spheroid was reduced to 750-1000 cells, which form spheroids with a diameter of 140-200 &#181;m.
It is important to thoroughly mix the different cell types in the spheroid formation solution, so the spheroids are composed by approximately the same number of cells, avoiding significant size differences between the spheroids. Nevertheless, sometimes some spheroids can be slightly bigger than the other and that will result in more Z-stacks acquired in the microscope. Some spheroids might also deviate from the perfectly spherical form, but this is also taken in account when the ROI of the spheroid is delineated. These differences of size and shape are accounted in the normalized quantification values as explained in the next paragraph. Another important factor regarding the shape of the spheroid is the cell type. For example, fibroblasts and CAFs form spheroids with a nearly spherical form, while AsPC-I and PANC-I cells form a more disperse aggregate of cells.
For imaging the spheroids, cryosections of fixed spheroids can also be used, however the integrity of the spheroid may be compromised upon sectioning, depending on the cell type and the immunostaining protocol. Alternatively, the staining of the whole spheroid in its 3D structure, proved to be a simpler option. Acquiring microscopic Z-stacked pictures of the spheroid as a 3D structure takes on average 5-15 min per spheroid.
The immunofluorescent signals in images of spheroids are difficult to quantify as they represent planes of 3D structures. The method used was based on a previously described one, where the authors extrapolated and considered the spheroid to be a cell14. In this way, the fluorescence signal is corrected using Equation 1.
As background, the integrated signal density of a cell-free region of interest (ROI) was determined. The measured integrated signal density is the sum of the intensity values of the pixels within the selected ROI. Since spheroids are 3D structures, confocal images from different stacks are acquired. To process such images one can measure the fluorescent signal in each stack individually or use the sum of all stacks into a Z-projection. All the quantifications can be performed using ImageJ. The total corrected fluorescence (TCF) of the spheroids is determined as the normalized TCCF, considering the area of spheroid and the number of stacks, using Equation 2. This accounts for the varying sizes of spheroids and the discrepancy in the spherical shape. Analyzing the immunofluorescent staining of the spheroids using this method can take up to 5 min per spheroid.
Similarly, invasion of cells from spheroids into the surrounding stromal matrix can be analyzed by different algorithms. A straightforward method was previously described by Nowicki et al, in which the invasive cancer cells were counted23. In order to discriminate the invasive cells from the ones that do not invade, it is important to establish a spatial limit beyond which the cells are considered to have left the spheroid structure. Therefore, the starting point is the limit that corresponds to the perimeter of the spheroid images acquired at the initial time point (time 0), when the spheroids were embedded into the gel. The cell that invades the gel the furthest is considered the maximal distance an invasive cell can reach, and thus, it defines the outer rim of the invasion region. This method counts the invasive cancer cells that are present in the invading area, using the counting tool from ImageJ, and the ROI corresponding to the spheroid at time 0 h is added to the ROI manager and then transposed to the image of the same exact spheroid acquired 48 h or 72 h later. For this reason, it is important that the spheroid remains as close to the center of the planes as possible, during the different times of acquisition. Counting the number of cells can take up to 5 min per spheroid.
Another important consideration is to distinguish the different cell types in the heterospheroid. This can be performed using live cell fluorescent dyes, which can be problematic when the cell type has a high cell division rate or if the experiment is to be performed for long periods of time. The more robust and reliable way to distinguish the two different populations is to develop cell lines expressing fluorescent proteins such as mCherry and GFP. The delivery of the expression vectors can be performed using lentivirus, which results in stable expression of these proteins.

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&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td><td>SIGMA-ALDRICH</td><td>D9542-10</td><td></td></tr><tr><td>6F12 anti-Laminin &amp;beta;3 subunit Mouse (monoclonal)</td><td></td><td></td><td>Homemade</td></tr><tr><td>A3IIF5 Anti-&amp;alpha;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 &amp;gamma;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-&amp;alpha;-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&#039;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 &amp;alpha;3 subunit Mouse (monoclonal)</td><td></td><td></td><td>Kindly provided by Prof. Patricia Rousselle, CNRS, Lyon</td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</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&#039;s Lab</td></tr><tr><td>Paraformaldehyde</td><td>Riedel-de Ha&amp;euml;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&amp;oacute;rtex</td><td>Scientific Industries</td><td></td><td>Vortex-Genie 2</td></tr><tr><td>&amp;mu;-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 (Figure 1A), as well as the immunofluorescence quantification (Figure 1B) and the transcriptional levels of the integrin &#945;3 subunit gene by qPCR (Figure 1C). This panel of results demonstrates that integrin &#945;3 is up-regulated in iCAFs, as compared to the normal counterpart. This proved that integrin &#945;3&#946;1 can be considered a marker for pancreatic fibroblasts differentiation. This was also demonstrated by flow cytometry studies13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60122/60122fig1.jpg" /> 
Figure 1. Expression of integrin &#945;3 subunit by iNFs and iCAFs. The integrin &#945;3 subunit is upregulated by iCAFs, reflecting its potential as a differentiation marker for pancreatic CAFs. (A) Immunofluorescent staining of homospheroids of normal pancreatic fibroblasts (iNFS) compared to the homospheroids of iCAF shows an increased signal of the integrin &#945;3 subunit in the differentiated cells. (B) The fluorescence signal of the cells in the spheroid was quantified with the Z-stack images of the 3D spheroid, using the ImageJ software. Means &#177; SEM of three independent experiments are shown and compared by t-test (*, p &#60; 0.1). (C) The cells obtained from the dissociated spheroids were analyzed for their transcriptional levels of integrin &#945;3 subunit. Means &#177; SEM of &#916;&#916;Ct-values as fold changes from two independent experiments are shown and compared by t-test. Although not reaching significance level of 0.1, the transcriptional levels of integrin &#945;3-encoding mRNA are higher in iCAFs than in iNFs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60122/60122fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Heterospheroids</td>
		</tr>
		<tr>
			<td>Cells type (passage up to 25)</td>
			<td>Number of cells</td>
			<td>Medium (1 part methylcellulose +&#8230;)</td>
		</tr>
		<tr>
			<td>mCherry-iCAFs + GFP-AsPC-I</td>
			<td rowspan="4">400 CAF + 400 pancreatic cancer cells</td>
			<td rowspan="2">1,5 parts cell MEM with 10% of FBS and 1% pen/strep + 1,5 parts of RPMI with 10% of FBS and 1% pen/strep</td>
		</tr>
		<tr>
			<td>mCherry-&#945;3KO-iCAFs + GFP-AsPC-I</td>
		</tr>
		<tr>
			<td>mCherry-iCAFs + GFP-PANC-I</td>
			<td rowspan="2">1,5 parts cell MEM with 10% of FBS and 1% pen/strep + 1,5 parts of DMEM with 10% of FBS and 1% pen/strep</td>
		</tr>
		<tr>
			<td>mCherry-&#945;3KO-iCAFs + GFP-PANC-I</td>
		</tr>
		<tr>
			<td colspan="3">Homospheroids</td>
		</tr>
		<tr>
			<td>Cell type (passage up to 25)</td>
			<td>Number of cells</td>
			<td>Medium (1 part methylcellulose +&#8230;)</td>
		</tr>
		<tr>
			<td>mCherry-iCAFs</td>
			<td rowspan="4">400 cells</td>
			<td rowspan="2">3 parts MEM supplemented with 10% of FBS and 1% pen/strep</td>
		</tr>
		<tr>
			<td>mCherry-&#945;3KO-iCAFs</td>
		</tr>
		<tr>
			<td>GFP-AsPC-I</td>
			<td>3 parts RPMI with 10% of FBS and 1% pen/strep</td>
		</tr>
		<tr>
			<td>GFP-PANC-I</td>
			<td>3 parts DMEM with 10% of FBS and 1% pen/strep</td>
		</tr>
	</tbody>
</table>
Table 1. Cell composition of hetero- and homospheroids. The number of cells necessary to assemble the hetero- and homospheroids as well as the corresponding medium which should be used for the spheroid formation solution are summarized in the following table.
Supplemental Figure 1. Sequential steps for quantification of the immunofluorescent staining of a protein of interest in spheroids, using ImageJ. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60122/Supplemental_Figure_1.zip">Please click here to download this file.</a>
Supplemental Figure 2. Sequential steps for quantification of the number of invading cancer cells in the invasion assay, using ImageJ. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60122/Supplemental_Figure_2.eps">Please click here to download this file.</a>

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 ...

a-3d-spheroid-model-as-more-physiological-system-for-cancer

Nanyang Technological University

Research

JoVE Journal

Cancer Research

8.8K Views.  University of Münster. Het kweken van fibroblasten in sferoïde recapituleren tumorweefsel veel beter dan het veelgebruikte 2D-systeem op stijf plastic. Het maakt het mogelijk om fibroblasten differentiatie in een tumor-achtige omgeving te bestuderen. In deze methode heeft de stijfheid van het plastic werk van de celcultuur geen artefactuele invloed op fibroblastdifferentiatie.In plaats daarvan kan de invloed van celmatrix interactie op tumor geïnduceerde differentiatie van fibroblast in vitro worden geanal...

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

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medicine

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 Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

3D Models in Cancer Biology: Invasion Dynamics in Heterospheroids

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

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

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

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

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