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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 alterations2,3, including the activation of oncogenes and inactivation of tumor suppressor genes3,4. 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 composed of different cell populations, including undifferentiated CSCs and bulk tumor cells displaying some differentiation traits, which constitute a hierarchical structure reminiscent of the organization of the epithelium in a normal colon crypt6,7.

CSCs are considered to be responsible for tumor appearance8, its maintenance and growth, metastatic capacity, and resistance to conventional therapies6,7. Within tumors, cancer cells, including CSCs, display a high level of heterogeneity and complexity in terms of their distinct mutational and epigenetic profiles, morphological and phenotypic differences, gene expression, metabolism, proliferation rates, and metastatic potential9. Therefore, to better understand cancer biology, tumor progression, and acquisition of resistance to therapy and its translation into effective treatments, human preclinical models capturing this cancer heterogeneity and hierarchy are important10,11.

In vitro 2D cancer cell lines have been used for a long time and provide valuable insights into tumor development and the mechanisms underlying the efficacy of therapeutic molecules. However, their limitation with respect to the lack of the phenotypic and genetic heterogeneity found in the original tumors is now widely recognized12. Moreover, nutrients, oxygen, pH gradients, and the tumor microenvironment are not reproduced, the microenvironment being especially important for the maintenance of different cell types including CSCs11,12. To overcome these main drawbacks, several 3D models have been developed to experimentally address and reproduce the complexity and heterogeneity of cancers. In effect, these models recapitulate tumor cellular heterogeneity, cell-cell interactions, and spatial architecture, similar to those observed in vivo12,13,14. Primary tumor organoids established from fresh tumors, as well as cell line-derived spheroids, are largely employed15,16.

Spheroids can be cultured in a scaffold-free or scaffold-based manner to force the cells to form and grow in cell aggregates. Scaffold-free methods are based on the culture of cells under non-adherent conditions (e.g., the hanging-drop method or ultra-low attachment plates), whereas scaffold-based models rely on natural, synthetic, or hybrid biomaterials to culture cells12,13,14. Scaffold-based spheroids present different disadvantages as the final spheroid formation will depend on the nature and composition of the (bio)material used. Although the scaffold-free spheroid methods available so far do not rely on the nature of the substrate, they generate spheroids that vary in structure and size17,18.

This work was aimed at designing a robust and reproducible 3D culture system of spheroids, which are homogenous in size, composed of Caco2 colon adenocarcinoma cells to study CSC biology. Caco2 cells are of particular interest owing to their capacity to differentiate over time19,20, strongly suggesting a stem-like potential. Accordingly, long-term culture of the spheroids revealed the presence of different CSC populations with different responses to chemotherapy.

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. Preparation of plates for spheroid formation
    1. Warm basal and DMEM/basement membrane matrix medium at room temperature (RT) for approximately 20 min.
    2. Pretreat the wells of a 24-well plate dedicated to spheroid formation by adding 500µL of anti-adherence rinsing solution to each well.
      NOTE: In these plates, each well consists of 1,200 microwells.
    3. Centrifuge the plate at 1,200 × g for 5 min in a swinging bucket rotor with adaptors for plates.
      NOTE: If only one plate is used, prepare an additional standard plate filled with water to counterbalance the weight.
    4. Rinse each well with 2 mL of warm basal medium, and aspirate the medium from the wells.
    5. Observe the plate under the microscope to ensure that bubbles have been completely removed from the microwells. If bubbles remain trapped, centrifuge again at 1,200 × g for 5 min to eliminate the bubbles.
    6. Repeat the rinsing steps 1.2.4-1.2.5.
    7. Add 1 mL of warm DMEM/basement membrane matrix medium to each well.
  3. Generation of spheroids
    1. Grow the Caco2 cells in a 2D monolayer in DMEM medium supplemented with 10% FBS and 1% Pen/Strep at 37 °C in a humidified atmosphere containing 5% CO2 (hereafter referred to as 37 °C/5% CO2).
      NOTE: The maximum number of cell passages to be used is 80.
    2. When 80% of confluency is reached, wash the cells with phosphate-buffered saline (PBS) 1x (5 mL for a 10 cm dish), add trypsin-ethylenediamine tetraacetic acid (EDTA) (2 mL for a 10 cm dish), and incubate for 2-5 min at 37 °C/5% CO2.
    3. Check cell detachment under the microscope, and neutralize the trypsin by adding 4 mL of DMEM complete medium per 10 cm dish.
    4. Count cells using a hemocytometer to determine the total number of cells.
    5. Centrifuge the cell suspension at 1,200 × g for 5 min. Discard the supernatant, and resuspend the pellet in an appropriate volume of DMEM/basement membrane matrix medium.
    6. Refer to Table 1 to determine the number of cells required per well to achieve the desired number of cells per microwell. Alternatively, calculate the number of cells using the following formula for a 24-well plate, considering each well contains 1,200 microwells
      Required number of cells per well = Desired number of cells per microwell × 1,200
    7. Add the required volume of the cell suspension to each well to achieve the desired cell number in a final volume of 1 mL.
    8. Add 1 mL of DMEM/basement membrane matrix medium to each well to reach the final volume of 2 mL per well (see also step 1.2.7).
      NOTE: Be careful not to introduce bubbles into the microwells.
    9. Centrifuge the plate immediately at 1,200 × g for 5 min to capture the cells in the microwells. If necessary, counterbalance the centrifuge with a standard plate filled with water.
    10. Observe the plate under the microscope to verify that the cells are evenly distributed among the microwells.
    11. Incubate the plate at 37 °C/5% CO2 for 48 h without disturbing the plate.
      NOTE: According to the original protocol21, although many cell lines can form spheroids within 24 h, some require a longer incubation time. In this protocol, 48 h are sufficient for spheroid formation.
  4. Harvesting the spheroids from the microwells
    1. Warm the basal and DMEM/basement membrane matrix medium at RT for approximately 20 min.
    2. Using a serological pipette, remove half of the culture medium (1 mL) from each well.
    3. Add the medium back onto the surface of the well to dislodge the spheroids from the microwells.
      NOTE: Do not touch or triturate the spheroids.
    4. Place a 37 µm reversible strainer (or a 40 µm standard strainer) on the top of a 15 mL conical tube to collect the spheroids.
      NOTE: If using a 40 µm standard strainer, place it upside down.
    5. Gently aspirate the dislodged spheroids (from step 1.4.3), and pass the spheroid suspension through the strainer.
      NOTE: The spheroids will remain on the filter; single cells will flow through with the medium.
    6. Using a serological pipette, dispense 1 mL of the warm basal medium across the entire surface of the well to dislodge any remaining spheroids and recover them on the strainer.
    7. Repeat this washing step 1.4.6 twice.
    8. Observe the plate under the microscope to ensure that all spheroids have been removed from the microwells. Repeat the wash if necessary (steps 1.4.6-1.4.7).
    9. Invert the strainer, and place it on a new 15 mL conical tube. Collect the spheroids by washing the strainer with DMEM/basement membrane matrix medium.
      NOTE: The collected spheroids are ready for downstream applications and analyses.
  5. Long-term culture of spheroids
    1. Prepare 1.5% agarose solution in basal medium, and sterilize it by autoclaving (standard cycle).
    2. While the agarose solution is warm and still liquid, coat the wells of standard culture plates or dishes, as described in Table 2.
      NOTE: Warming the dish/plate in the oven will facilitate the coating step. Coated dishes/plates can be left at RT for up to 10 days in a sterile environment and protected from the light.
    3. Seed the harvested spheroids (from step 1.4.9) in the agarose-coated plates, and add DMEM/basement membrane matrix medium to achieve the final volume depending on the size of the plate.
      NOTE: To avoid spheroid aggregates, seed them at the optimal density of 22 spheroids/cm2. Observe that the coating is not perfectly flat, and it rises towards the edge, creating a light concavity at the center of the plate. If the number of spheroids is too high, they are more likely to adhere to each other.
    4. Incubate the plate at 37 °C/5% CO2 until recovery of the spheroids for specific analyses.
  6. Treatment of spheroids with chemotherapeutic drugs
    1. Plate spheroids from step 1.5.4, and grow them for 2 days. Starting from day 3 (D3), treat them with FOLFIRI (5-Fluorouracil, 50 µg/mL; Irinotecan, 100 µg/mL; Leucovorin, 25 µg/mL) or with FOLFOX (5-Fluorouracil, 50 µg/mL; Oxaliplatin, 10 µg/mL; Leucovorin, 25 µg/mL) chemotherapeutic regimen combinations routinely used to treat CRC patients22,23,24,25, or maintain them in (control) not-treated (NT) condition.
    2. Collect the spheroids after 3 days of treatment by using a pipette with the tip cut off (1,000 µL tip), ensuring that each condition is represented by at least three replicates. Centrifuge them at 1,000 × g for 3 min, and then remove the supernatant.
    3. Fix the pellets in 2% paraformaldehyde (PFA) for histological analysis (see section 3), or use the pellets for RNA extraction (see section 4).
    4. To analyze cell death, incubate the spheroids from step 1.6.1 in black culture well plates in DMEM/basement membrane matrix medium for 30 min with a nucleic acid stain (1:5000 dilution) that does not permeate live cells, but penetrates the compromised membranes of dead cells26. Measure the accumulation of fluorescence with a microplate reader.

2. Monitoring spheroid growth

  1. Using an inverted microscope, acquire representative images of spheroids maintained under different conditions throughout the days in culture.
  2. Analyze the images by measuring three different representative diameters of each spheroid using appropriate software.
  3. Use the following formula to obtain the estimated sphere volume.
    figure-protocol-8848

    NOTE: The terms, d1, d2, and d3, are the three diameters of the spheroid.

3. Immunofluorescence (IF) and histological staining

  1. Fixation and paraffin embedding
    1. Collect the spheroids at selected time-points using a pipette with the tip cut off as described in step 1.6.2, and fix them for 30 min at RT in 2% PFA.
      NOTE: Alternatively, store the samples at this step at 4 °C until further use.
    2. For paraffin embedding, wash the spheroids 3x with PBS 1x, and resuspend them in 70% ethanol. After paraffin inclusion and sectioning, perform hematoxylin & eosin (H&E) staining for histological analysis.
  2. Immunolabeling of paraffin sections
    ​NOTE: Use 5-μm-thick sections for indirect immunostaining.
    1. Incubate slides at 60 °C for 2 h to melt the wax and improve deparaffinization.
    2. Wash the slides twice for 3 min in methylcyclohexane.
    3. Wash the slides for 3 min in 1:1 methylcyclohexane:100% ethanol.
    4. Wash the slides twice for 3 min in 100% ethanol.
      NOTE: Perform all manipulations for step 3.2.2 to step 3.2.4 in a chemical hood.
    5. Wash the slides for 3 min in 90% ethanol.
    6. Wash the slides for 3 min in 75% ethanol.
    7. Wash the slides for 3 min in 50% ethanol.
    8. Wash the slides under tap water.
    9. Rehydrate the slides in distilled water for 5 min.
    10. Prepare 700 mL of 0.01 M citrate buffer, pH 6.0, and add it to a suitable container (width: 11.5 cm, length: 17 cm, height: 7 cm); submerge the slides in it. Heat the container in the microwave for 9-10 min at 700 W, and when the boiling starts, decrease the power to 400-450 W. Incubate for an additional 10 min.
    11. Let the slides cool down in the buffer to RT for approximately 30-40 min.
    12. Wash the slides twice in PBS for 5 min.
    13. Draw a circle around the sections with a marker pen to create a barrier for liquids applied to the sections.
    14. Incubate each section with 50 µL of blocking buffer (10% normal goat serum, 1% bovine serum albumin (BSA), and 0.02% Triton X-100 in PBS) for at least 30 min at RT.
    15. Remove the blocking buffer, and add 50 µL of primary antibodies diluted in the incubation buffer (1% normal goat serum, 0.1% BSA, and 0.02% Triton X-100 in PBS). Incubate for 2 h at RT or overnight at 4 °C.
    16. Remove the primary antibodies, and wash the slides 3x in PBS for 5 min.
    17. Incubate the slides with 50 µL of fluorescent secondary antibodies diluted in the incubation buffer for 1 h at RT.
    18. Remove the secondary antibodies, and wash the slides 3x in PBS for 5 min.
    19. Add 50 µL of mounting medium with 4′,6-diamidino-2-phenylindole to each section, and place a glass coverslip over the section.

4. RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qRT-PCR)

  1. Collect spheroids at different time-points, each point represented by at least three replicates. Centrifuge them at 1,000 × g for 3 min, and then remove the supernatant.
    NOTE: Pellets can be directly used for RNA extraction or stored at -20 °C until further use.
  2. Isolate the total RNA using a commercial RNA isolation kit, according to the manufacturer's instructions.
  3. Reverse-transcribe 500 ng of each RNA sample into complementary DNA (cDNA) with a commercial kit according to the manufacturer's instructions.
  4. After reverse transcription, perform a PCR analysis on 1 µL of cDNA to amplify a housekeeping gene with primers located in different exons.
    NOTE: This step enables the verification of the absence of any genomic DNA contamination in the RNA preparations. For this protocol, peptidylprolyl isomerase B (PPIB) primers were used.
  5. Perform qPCR amplification on 4 µL of previously diluted cDNA (1:10 in RNAse-free double-distilled water) by using primers specific for the genes of interest. In each sample, quantify specific mRNA expression by using the ΔΔCt method and values normalized against a housekeeping gene levels.
    NOTE: For this protocol, β-actin (ACTB) was selected. Primers used in this protocol are listed in Table 3.

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 ...

Disclosures

The authors have nothing to disclose

Acknowledgements

We acknowledge the imaging and Anipath recherche histology platforms (CRCL, CLB). We are indebted to the pharmacy of the Centre Léon Bérard (CLB) Hospital for the kind gift of FOLFOX and FOLFIRI. We also thank Brigitte Manship for critical reading of the manuscript. The work was supported by the FRM (Equipes FRM 2018, DEQ20181039598) and by the Inca (PLBIO19-289). MVG and LC received support from the FRM and CF received support from ARC foundation and the Centre Léon Bérard.

Materials

NameCompanyCatalog NumberComments
37 µm Reversible Strainer, Large STEMCELL Technologies27250To be used with 50 mL conical tubes
5-FluorouracilGift from Pharmacy of the Centre Leon Berard (CLB)-stock solution, 5 mg/100 mL; final concentration, 50 µg/mL
Agarose SigmaA9539
Aggrewell 400 24-well platesSTEMCELL Technologies344111,200 microwells per well for spheroid formation and growth
Anti Caspase3 - RabbitCell Signaling9661dilution 1:200
Anti Musashi-1 (14H1) - RateBioscience/Thermo Fisher14-9896-82dilution 1:500
Anti-Adherence Rinsing Solution x 100 mLSTEMCELL Technologies07010
Anti-CD133 (13A4) - RatInvitrogen14-133-82dilution 1:100
Anti-CD44 -RabbitAbcamab157107dilution 1:2000
Anti-PCNA - MouseDakoM0879dilution 1:1000
Anti-β-catenin - MouseSanta Cruz Biotechnologysc-7963dilution 1:50
Black multiwell platesThermo Fisher Scientific237108
Citric Acid MonohydrateSigmaC1909
CLARIOstar apparatus BMG Labtechmicroplate reader
Dako penmarker pen to mark circles on slides for creating barriers for liquids
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA21202dilution 1:1000
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 568Thermo Fisher ScientificA10037dilution 1:1000
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA21206dilution 1:1000
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568Thermo Fisher ScientificA10042dilution 1:1000
Dulbecco's Modified Eagle Medium (DMEM) Glutamax (L-alanyl-L-glutamine dipeptide)Gibco10569010
Fetal Bovine Serum (FBS)Gibco16000044
Fluorogel mounting medium with DAPIInterchimFP-DT094B
Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568Thermo Fisher ScientificA11077dilution 1:1000
ImageJ softwareSpheroid image analysis
Irinotecan Gift from Pharmacy CLB-stock solution, 20 mg/mL; final concentration, 100 µg/mL
iScript reverse transcriptase Bio-Rad1708891
LeucovorinGift from Pharmacy CLB-stock solution, 50 mg/mL; final concentration, 25 µg/mL
Matrigel Basement Membrane MatrixCorning354234Basement membrane matrix
Nucleospin RNA XS KitMacherey-Nagel740902 .250
OxaliplatinGift from Pharmacy CLB-stock solution, 100 mg/20 mL;final concentration, 10 µg/mL
Penicillin-streptomycinGibco15140130
Phosphate Buffer Saline (PBS)Gibco14190250
SYBR qPCR Premix Ex Taq II (Tli RNaseH Plus)TakaraRR420B
SYTOX- GreenThermo Fisher ScientificS7020nucleic acid stain; dilution 1:5000
Trypsin-EDTA (0.05 %)Gibco25300062
Zeiss-Axiovert microscopeinverted microscope for acquiring images of spheroids

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