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

Summary

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.

Abstract

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 α3β1 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' integrin α3β1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

Introduction

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 "reactive stroma", 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 α-smooth muscle actin (αSMA)⁵ or neural/glial antigen 2 (NG2)⁶.

The most suitable 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 tension^5,7. 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 αSMA-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 α3 subunit to examine the differentiation process and heterospheroids of CAFs or integrin α3 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 α3 chain¹⁰, or lebein 1, a snake venom derived compound that blocks the laminin-binding integrins α3β1, α6β1 and α7β1^11,12.

For the invasion assay, cells had been transduced with lentivirus containing cDNA encoding either mCherry (iCAFs and integrin α3 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.

Protocol

1. 3D spheroids as an in vitro model for fibroblast/CAF differentiation using different TGF-β1 inhibiting compounds of cell-matrix interaction

1. 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.
2. Resuspend freshly thawed immortalized normal fibroblasts (iNFs), immortalized CAF (iCAFs) or integrin α3β1 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 µL are distributed per each well of the plate.
3. 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-β1 in order to trigger differentiation. Where appropriate, add inhibitor compounds together with TGF-β1, such as 20 µg/mL BM2 or 10 µg/mL of lebein 1.
4. Prepare CAF homospheroids in the same way but without the TGF-β1 stimulus, since they are already differentiated. Prepare the integrin α3 KO CAF homospheroids without the addition of any compound.
5. Distribute the spheroid formation solution onto a round bottom 96 multi-well plate by adding 100 µL 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.
6. Place the plate in the cell culture incubator for 24 h to allow one spheroid to be formed in each well.
7. 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.

2. Immunofluorescent staining of spheroids

1. 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-β1 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.
2. 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.
3. Wash with 50 µL of 1x PBS. Repeat the centrifugation step and carefully remove the PBS by pipetting, as described in 2.2.
4. Depending on the protein to be stained, fix with 50 µL of 4% paraformaldehyde in PBS for 20 minutes at room temperature (for αSMA, NG2 and integrin α3β1) or with 50 µL of methanol for 10 min at -20 °C (for laminin-332 subunits).
5. Repeat the washing step as explained in steps 2.2-2.3.
6. Permeabilize the cells with 0.1% Triton-X in PBS for 4 min at room temperature.
7. Repeat the washing step as explained in steps 2.2-2.3 three times.
8. Incubate the spheroids in PBS containing 5% FBS and 2% BSA, for 1 h at RT to block non-specific protein interaction sites.
9. Incubate the spheroids with 30 µL of primary antibodies in PBS, 2.5% FBS and 1% BSA at 4 °C overnight. The following primary antibodies were used: anti-αSMA Cy-3 conjugated and anti-NG2 at 5 µg/mL; BM2 anti-laminin α3, 6F12 anti-laminin β3 and anti-laminin γ2 20 µg/mL.
10. Repeat the washing step as explained in steps 2.2-2.3 three times.
11. Incubate the spheroids with 30 µL 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 µg/mL).
12. Repeat the washing step as explained in steps 2.2-2.3 three times.
13. Incubate the cells with 30 µL of DAPI staining solution for 4 min at room temperature.
14. Repeat the washing step as explained in steps 2.2-2.3.
15. Place the spheroids on a glass slide, in a drop of PBS, using a glass Pasteur pipet.
16. 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.
17. 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:
    
18. Determine the total corrected fluorescence (TCF) of the spheroids as the TCCF normalized to the area of spheroid and the number of stacks.
    

3. RT q-PCR of homospheroids

1. 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.
2. 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.
3. Dissociate the spheroids with 250 µL of 4 mg/mL collagenase B, 50 µg/mL DNase I, 2% BSA, 1 mM CaCl₂ in PBS at 37 °C for about 30-45 min by gently vortexing and pulsing intermittently for a few seconds every 5 min.
4. Repeat the washing step as explained in step 3.2.
5. Isolate the total RNA from the cells of disintegrated spheroids using a commercial RNA isolation kit, according to the manufacturers’ instructions.
6. Reverse transcribe the isolated mRNA into cDNA with a commercial kit, according to manufacturers’ instructions.
7. Quantify the transcription levels in replicates by real-time PCR. The primers used were: α-SMA: Fw 5′-CCGACCGAATGCAGAAG GA-3′, Rev 5′ ACAGAGTATTTGCGCTCCGAA-3′¹⁵; Laminin α3 chain: Fw 5′-GCTCAGCTGTTTGTGGTTGA-3′, Rev 5′-TGTCTGCATCTGCCAATAGC-3′¹⁶; Laminin β3 chain: Fw 5′-GGCAGATGATTAGGGCAGCCGAGGAA-3′ Rev 5′-CGGACCTGCTGGATTAGGAGCCGTGT-3′¹⁷; Laminin γ2 chain: Fw 5′-GATGGCATTCACTGCGAGAAG-3′ Rev 5′-TCGAGCACTAAGAGAACCTTTGG-3′ ¹⁶; NG2: Fw 5′-CTGCAGGTCTATGTCGGTCA-3′ Rev 5′-TTGGCTTTGACCCTGACTATG-3′¹⁸; integrin α3 subunit: Fw 5’-AGGGGACCTTCAGGTGCA-3’ Rev 5’-TGTAGCCGGTGATTTACCAT-3’¹⁹; TOP-1: Fw 5′-CCAGACGGAAGCTCGGAAAC-3′ Rev 5′-GTCCAGGAGGCTCTATCTTGAA-3′²⁰.
8. Correct the Ct values for the Ct-value of an endogenous reference gene such as TOP-1 using the equation: 2^{- ΔΔCt}. ΔΔCt = ΔCt (target sample) – ΔCt (reference sample) and ΔCt= Ct of target gene or reference gene – TOP-I.

4. Flow cytometry analysis of integrin expression

1. 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.
2. 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.
3. Dissociate the spheroids as described in step 3.3.
4. Repeat the washing step as explained in step 4.2.
5. To block non-specific binding sites and to prevent binding of detection-relevant antibodies to Fcγ-receptors (CD16, CD32, and CD64), incubate the cells in 100 µL 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 °C with rotation.
6. In case of staining intracellular markers, fix/permeabilize the cells as described under step 2.2-2.5.
7. Incubate the cells with the primary antibodies in 100 µL of PBS containing 2% horse serum and 0.1% BSA for 30 min at 4 °C with rotation. Dilute the primary antibody to 10 µg/mL.
8. Wash with 100 µL of PBS + 0.1% BSA three times, with centrifugation steps in the top bench centrifuge at 1,000 x g in between.
9. Incubate the cells with the secondary antibodies in 100 µL of PBS containing 2% horse serum and 0.1% BSA for 30 min at 4 °C with rotation. Dilute secondary antibody to 10 µg/mL.
10. Repeat the washing step as explained in step 4.7.
11. 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.

5. Invasion assay using heterospheroids

1. 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.
2. Fill 100 µL 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.
3. Prepare 60 µL of gelatinous matrix mixture containing 2 mg/mL of rat tail collagen I, 30% of commercial gelifying matrix and 2.5 µg/mL of laminin-332 in a 1.5 mL reaction tube. Rapidly distribute 15 µL in 3 wells of the µ-slide angiogenesis chamber. Embed one spheroid in each well already containing the gel.
4. If necessary, rapidly adjust the location of the spheroid to the center of the well with a pipette under the microscope.
5. Repeat steps 5.2-5.3 for the next heterospheroids and repeat again for the homospheroids.
6. Incubate the gels in the incubator for 1 h to solidify and, then, gently overlay the gels with cell culture medium.
7. 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).
8. 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.

Results

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 α3 subunit of both immortalized normal fibroblasts and immortalized CAFs (

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)	SIGMA-ALDRICH	D9542-10
6F12 anti-Laminin β3 subunit Mouse (monoclonal)			Homemade
A3IIF5 Anti-α3 integrin subunit Mouse (monoclonal)			Kindly provided by Prof. M. Hemler, Dana Faber-Cancer Institute, Boston
Acetone	SIGMA-ALDRICH	32201
Albumin Fraction V - BSA	AppliChem	A1391
Alexa fluor 488 Goat (polyclonal) anti-Mouse	Invitrogen	A11029
Alexa fluor 488 Goat (polyclonal) Rabbit	Invitrogen	A11034
Anti-laminin γ2 subunit Mouse (monoclonal)	Santa Cruz	sc-28330
Anti-NG2 Rabbit (polyclonal) Millipore, AB5320	Millipore	AB5320
Anti-α-SMA-Cy3 Mouse (monoclonal)	SIGMA-ALDRICH	C6198
AsPC-1 cell line	ATCC		Kindly given by prof. Jorg Haier's Lab
Bench centrifuge	Fisher Scientific	50-589-620	Sprout
BM2 anti-laminin α3 subunit Mouse (monoclonal)			Kindly provided by Prof. Patricia Rousselle, CNRS, Lyon
Calcium Chloride (CaCl2)	Fluka	21074
Centrifuge	Thermo Scientific		Multifuge 1S-R
Centrifuge tubes 50 mL	Corning	430290
Collagenase B	Roche	11088831001
Collagen-I, rat tail	Gibco	A10483-01
Confocal microscope	Zeiss		LSM 700 and 800
DMEM (High glucose 4.5 g/L)	Lonza	BE12-604F
Dnase I	Roche	10104159001
Flow Cytometer	BD Biosciences		FACSCaliburTM
Gelifying matrix	ThermoFisher Scientific	A1413202	Matrigel, Geltrex
Goat IgG, isotype	DAKO	X 0907
Horse Serum	SIGMA-ALDRICH	12449-C
Human Primary Pancreatic Fibroblasts	PELOBiotech	PB-H-6201
Incubator	Heraeus	B6060
Laminin-332	Biolamina	LN332
MEM	SIGMA-ALDRICH	M4655
Microplate, 96 wells, U-bottom	Greiner Bio-One	650101
Microscope Slides	Thermo Scientific	J1800AMNZ
Mouse IgG, isotype	SIGMA-ALDRICH	I8765
Multi axle rotating mixer	CAT	RM5 80V
PANC-I	ATCC		Kindly given by Prof. Jorg Haier's Lab
Paraformaldehyde	Riedel-de Haën	16005
Penicillin/streptomycin	Gibco	15140-122
QuantiTect Reverse Transcription Kit	Qiagen	205310
Rat IgG, isotype	Invitrogen	10700
Reaction tubes, 1.5 mL	Greiner Bio-One	616201
Real-time PCR cycler	Qiagen		Rotor-Gene Q
RNeasy Mini Kit	Qiagen	74104
Rotor Gene SYBR Green PCR Kit	Qiagen	204074
RPMI	Lonza	BE12-702F	Add glucose to 4.5 g (0.2 um filter) and 1% sodium pyruvate
TritonX-100	SIGMA-ALDRICH	X100RS
Vórtex	Scientific Industries		Vortex-Genie 2
μ-Slide Angiogenesis, uncoated	Ibidi	81501