A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Summary

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Abstract

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

Introduction

Colorectal cancer (CRC) remains the second leading cause of cancer-associated deaths in the world¹. The development of CRC is the result of a progressive acquisition and accumulation of genetic mutations and/or epigenetic alterations^2,3, including the activation of oncogenes and inactivation of tumor suppressor genes^3,4. Moreover, non-genetic factors (e.g., the microenvironment) can contribute to and promote oncogenic transformation and thus participate in the evolution of CRCs⁵. Importantly, CRCs are com....

Protocol

NOTE: The details of all reagents and materials are listed in the Table of Materials.

1. Spheroid formation

1. Spheroid culture media
   1. Prepare basal medium consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 4 mM L-alanyl-L-glutamine dipeptide.
   2. Prepare DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen/Strep) in basal medium from step 1.1.1.
   3. Prepare DMEM/basement membrane matrix medium containing 2.5% basement membrane matrix, 10% FBS, and 1% Pen/Strep in basal medium from step 1.1.1.
2. Preparati....

Results

As the lack of homogeneity in the size of spheroids is one of the main drawbacks of currently available 3D spheroid culture systems¹³, the aim of this work was to set up a reliable and reproducible protocol to obtain homogenous spheroids. First, to establish ideal working conditions, different numbers of Caco2 cells were tested, ranging from 50 to 2,000 cells per microwell/spheroid using dedicated plates (Table 1). In effect, each well in these pla.......

Discussion

In vitro 3D models overcome the main experimental drawbacks of 2D cancer cell cultures, as they appear to be more reliable in recapitulating typical tumoral features including microenvironment and cell heterogeneity. Commonly used 3D models of spheroids are scaffold-free (cultured in low-attachment conditions) or scaffold-based (using biomaterials to culture cells). These methods present different disadvantages as they depend on the nature of the scaffold used or give rise to spheroids that are variable in structure and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
37 µm Reversible Strainer, Large	STEMCELL Technologies	27250	To be used with 50 mL conical tubes
5-Fluorouracil	Gift from Pharmacy of the Centre Leon Berard (CLB)	-	stock solution, 5 mg/100 mL; final concentration, 50 µg/mL
Agarose	Sigma	A9539
Aggrewell 400 24-well plates	STEMCELL Technologies	34411	1,200 microwells per well for spheroid formation and growth
Anti Caspase3 - Rabbit	Cell Signaling	9661	dilution 1:200
Anti Musashi-1 (14H1) - Rat	eBioscience/Thermo Fisher	14-9896-82	dilution 1:500
Anti-Adherence Rinsing Solution x 100 mL	STEMCELL Technologies	07010
Anti-CD133 (13A4) - Rat	Invitrogen	14-133-82	dilution 1:100
Anti-CD44 -Rabbit	Abcam	ab157107	dilution 1:2000
Anti-PCNA - Mouse	Dako	M0879	dilution 1:1000
Anti-β-catenin - Mouse	Santa Cruz Biotechnology	sc-7963	dilution 1:50
Black multiwell plates	Thermo Fisher Scientific	237108
Citric Acid Monohydrate	Sigma	C1909
CLARIOstar apparatus	BMG Labtech		microplate reader
Dako pen			marker pen to mark circles on slides for creating barriers for liquids
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488	Thermo Fisher Scientific	A21202	dilution 1:1000
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 568	Thermo Fisher Scientific	A10037	dilution 1:1000
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Thermo Fisher Scientific	A21206	dilution 1:1000
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568	Thermo Fisher Scientific	A10042	dilution 1:1000
Dulbecco's Modified Eagle Medium (DMEM) Glutamax (L-alanyl-L-glutamine dipeptide)	Gibco	10569010
Fetal Bovine Serum (FBS)	Gibco	16000044
Fluorogel mounting medium with DAPI	Interchim	FP-DT094B
Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568	Thermo Fisher Scientific	A11077	dilution 1:1000
ImageJ software			Spheroid image analysis
Irinotecan	Gift from Pharmacy CLB	-	stock solution, 20 mg/mL; final concentration, 100 µg/mL
iScript reverse transcriptase	Bio-Rad	1708891
Leucovorin	Gift from Pharmacy CLB	-	stock solution, 50 mg/mL; final concentration, 25 µg/mL
Matrigel Basement Membrane Matrix	Corning	354234	Basement membrane matrix
Nucleospin RNA XS Kit	Macherey-Nagel	740902 .250
Oxaliplatin	Gift from Pharmacy CLB	-	stock solution, 100 mg/20 mL;final concentration, 10 µg/mL
Penicillin-streptomycin	Gibco	15140130
Phosphate Buffer Saline (PBS)	Gibco	14190250
SYBR qPCR Premix Ex Taq II (Tli RNaseH Plus)	Takara	RR420B
SYTOX- Green	Thermo Fisher Scientific	S7020	nucleic acid stain; dilution 1:5000
Trypsin-EDTA (0.05 %)	Gibco	25300062
Zeiss-Axiovert microscope			inverted microscope for acquiring images of spheroids