Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Summary

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Abstract

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Introduction

Both non-transformed (normal) and transformed cells can readily proliferate in a 2D monolayer culture. This form of adherent cell growth is quite dissimilar from that which occurs in vivo where, in the absence of mitogenic stimulation, cells do not often rapidly divide within their microenvironment. The soft agar assay on the other hand is distinct from 2D culture systems because it quantifies tumorigenicity by measuring a cell’s ability to proliferate and form colonies in suspension within a semi-solid agarose gel¹. In this setting, non-transformed cells are unable to rapidly propagate in the absence of anchorage to the extracellular matrix ....

Protocol

1. Preparation of 3% 2-Hydroxyethyl Agarose

1. Into a clean, dry 100 ml glass bottle, add 0.9 g of 2-hydroxyethyl agarose (Agarose VII) followed by 30 ml of distilled water.
2. Microwave the mixture for 15 sec and gently swirl. Repeat this step at least three more times until the agarose powder fully dissolves.
3. Autoclave the solution-containing bottle for 15 min.
4. Allow the agarose solution to cool down to RT before further use. Store the solution at RT.

2. Preparation of the Bottom Layer: 0.6% Agarose Gel

1. Pre-warm several 5 ml and 10 ml pipettes in a 37 °C incub....

Results

The soft agar colony formation assay can be used for a broad range of applications documenting the tumorigenicity of cancer cells. A major advantage of this technique is that the semi-solid matrix selectively favors the growth of cells that can proliferate in an anchorage-independent manner. This trait is mainly exhibited by cancer cells but not by normal cells. We primarily use this technique to test the efficacy of tumor growth inhibition by drugs and to test for the effect of overexpression or depletion of our genes o.......

Discussion

The rate of colony formation in soft agar varies depending on the cell type⁹. Therefore, the number of cells to start with should be optimized and adjusted accordingly. A suggested starting range is between 5 x 10² to 1 x 10⁴ cells per well using a 6-well plate. In addition, colony size varies depending on the growth rate of each cell. Therefore, a predefined a cut-off for colony size is needed to annotate individual colonies for downstream quantitative analyses. Here, colonies larger tha.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Zeiss Axiopot	Carl Zeiss Microscopy	1021859251
Inverted Microscope	Olympus	CKX41
DMEM/F-12	Lonza BioWhittaker	12-719F
HyClone Donor Equine Serum	Fisher Scientific	SH30074.03
Penicillin Streptomycin	Life Technologies	15140-122
2-Hydroxyethylagarose: Type VII, low gelling temperature	Sigma-Aldrich	39346-81-1