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In This Article

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

Summary

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Abstract

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Introduction

The use of multicellular spheroid models in cancer biology is several decades old1,2, but has gained substantial momentum in recent years. In large part, this reflects increased awareness of how strongly the phenotype of cancer cells is dependent on their microenvironment and specific growth conditions. The microenvironment in solid tumors is fundamentally different from that in corresponding normal tissues. This includes physico-chemical conditions such as pH, oxygen tension, as well as interstitial pressure, concentration gradients of soluble factors such as nutrients, waste products, and secreted signaling ....

Protocol

1. Generation of Spheroids

  1. Preparing cell suspensions for spheroid formation
    NOTE:
    Different cell lines have very different adhesion properties and the most suitable spheroid formation protocol must be established in each case. We have found that MCF-7 and BxPC-3 cells are suitable for spontaneous spheroid formation, while MDA-MB-231, SKBr-3, Panc-1 and MiaPaCa require the addition of reconstituted basement membrane to successfully form spheroids. Only MDA-MB-231 and BxPC-3 cells .......

Representative Results

Spheroid growth assays based on the spheroid formation protocol schematically illustrated in Figure 1A and Figure 1B, were used as a starting point for analysis of the effects of anti-cancer drug treatments in a 3D tumor mimicking setting. The ease with which spheroids are formed is cell line specific, and some cell lines require supplementation with rBM in order to form coherent spheroids22. The concentr.......

Discussion

The use of 3D cancer cell spheroids has proven a valuable and versatile tool not only for anticancer drug screening, but also for gaining mechanistic insight into the regulation of cancer cell death and viability under conditions mimicking those in the tumor microenvironment. This is particularly crucial as the accessibility, cellular uptake, and intracellular effects of chemotherapeutic drugs are profoundly impacted by the physico-chemical conditions in the tumor, including pH, oxygen tension, tortuosity, and physical a.......

Acknowledgements

We are grateful to Katrine Franklin Mark and Annette Bartels for excellent technical assistance and to Asbjørn Nøhr-Nielsen for performing the experiments in Figure 1D. This work was funded by the Einar Willumsen Foundation, the Novo Nordisk Foundation, and Fondation Juchum (all to SFP).

....

Materials

NameCompanyCatalog NumberComments
2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI)Invitrogen# C10595 For staining nuclei
5-Fluorouracil (5-FU)Sigma-Aldrich#F6627Component in chemotherapeutic treatment
5-(N-ethyl-isopropyl) amiloride (EIPA)Life Technologies#E3111Inhibitor of NHE1
Antibody against PARP and cPARPCell signaling#9542Used in western blotting
Antibody against Ki-67Cell signaling#9449Used for IHC
Antibody against p53Cell Signaling #2524 Used for IHC
Antibody against β-actinSigma A5441Used in western blotting
BactoagarBD Bioscience#214010Used for agarose gel preparation
Benchmark protein ladderInvitrogen#10747-012Used for SDS-PAGE
Bio-Rad DC Protein Assay kitBio-Rad Laboratories#500-0113, #500-0114, #500-0115  Used for protein determination from lysates
Bürker chamberMarienfeld610311For cell counting 
BX63 epifluoresence microscopeOlympusUsed for fluorescent imaging
CellTiter-Glo 3D Cell Viability AssayPromega#G9681Used for the cell viability assay
CisplatinSigma-Aldrich#P4394 Component in chemotherapeutic treatment
Corning Spheroid Microplate, 96 well, Black with clear round bottom,  Ultra-low attachment, With lid, SterileCorning#4520Used for growing spheroids with luminescence measurements as end point
Corning 96 well, clear round bottom,  Ultra-low attachment microplate, With lid, SterileCorning#7007Sufficient for spheroid growth without luminescence measurements as end point
Criterion TGX Precast GelsBio-Rad5671025Used for SDS-PAGE
DoxorubicinAbcam#120629Component in chemotherapeutic treatment
FLUOStar Optima Microplate readerBMG LabtechUsed for recording luminescence 
Formaldehyde VWR Chemicals #9713.1000 Used for cell fixation
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane MatrixGibco#A1413202Keep at 4 °C to prevent solidification. Referred to as rBM in the protocol.
Heat-inactivated FBSSigma#F9665Serum for growth media
ImageJNIHScientific Image analysis
Medim Uni-safe casetteMedim Histotechnologie10-0114Used for storage of embedded spheroids
Mini protease inhibitor cocktail tabletsRoche Diagnostics GmBH # 11836153001Used for lysis buffer preparation
MZ16 microscopeLeicaUsed for light microscopic images
NuPAGE LDS 4x Sample Buffer Invitrogen#NP0007Used for western blotting
Pierce ECL Western blotting substrateThermo scientific#32106Used for western blotting
Ponceau SSigma-Aldrich#P7170-1LUsed for protein band staining
Prism 6.0GraphpadScientific graphing and statistical software
Propidium iodide (1mg/ml solution in water)Invitrogen P3566Light sensitive 
Sterile reservoirs, multichannelSPL lifesciences21002Used for seeding cells for spheroid formation
Superfrost Ultra-Plus Adhesion slide Menzel-Gläser#J3800AMNZMicroscope glass slide used for embedding
TamoxifenSigma-Aldrich#T5648Used as chemotherapeutic treatment
Trans-blot Turbo 0.2 µm nitrocellulose membranesBio-Rad#170-4159Used for western blotting
Tris/Glycine/SDS running buffer Bio-Rad #161 0732Used for SDS-PAGE
Trypsin-EDTA solutionSigma#T4174 Cell dissociation enzyme

References

  1. Sutherland, R. M. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science. 240 (4849), 177-184 (1988).
  2. Mueller-Klieser, W., Freyer, J. P., Sutherland, R. M.

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