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 bulk....

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, keepi....

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