The Soft Agar Colony Formation Assay

Summary

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Abstract

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

Introduction

The soft agar colony formation assay is a technique widely used to evaluate cellular transformation in vitro. Historically, another assay, the clonogenic assay, described by Puck et al. in 1956 was used to evaluate the ability of cells to form colonies¹. In this technique, cells were dispersed onto a culture plate and grown in the presence of 'feeder' cells or conditioned medium to provide necessary growth factors. The limitation of this technique was that it only provided information regarding colony formation. Normal cells are prevented from anchorage-independent growth, due to a particular type of apoptotic death, called anoikis....

Protocol

1. Preparation of Materials and Reagents

1. Label each well of a tissue culture treated 6-well plate appropriately for each cell line or condition being investigated.
2. Prepare 2x cell culture medium by dissolving 1 g of powder medium and 0.2 g of sodium bicarbonate in de-ionized water to a final volume of 50 ml.
3. Pass this medium through a 0.2 μm filter to sterilize.
4. Add additional components needed for normal culture of the cell line of interest. For example, grow CMT 167 cell line in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin solution. Warm medium to 37 °C in hot water bath prior to use.

Results

The expression of Wnt7A and Fzd9 in CMT167 cells is effective in tumor suppression as illustrated by our soft agar colony formation assay. Preliminarily, we used Q-PCR to show that Wnt7A and Fzd9 mRNA are expressed in low levels in CMT167 cells. CMT167 cells showed low levels of endogenous Wnt7A and Fzd9 when compared to MLE-12 cells, an SV40-transformed murine lung epithelial cell line (Figure 1). We then transfected CMT167 cells with two retroviral overexpression vectors expressing human constructs of .......

Discussion

In vitro confirmation of tumor suppressive function of signaling proteins is difficult. One of the most rigorous assays available to investigate this property is the soft agar colony formation assay. Here, we have illustrated the soft agar colony formation assay using a murine lung carcinoma cell line stably overexpressing Wnt7a and Fzd9 compared to its parental CMT167 cell line.

There are several important points to consider regarding the soft agar assay. The most critical step in th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cancer Cell Line of Interest	Sigma-Aldrich	10032302	CMT-167 Cells
Powdered RPMI 1640 Medium	Gibco	31800-089	Used to prepare 2x cell culture medium.
Liquid RPMI 1650 Medium	Cellgro	10-040-CV	Referred to as 1x cell culture medium.
Fetal Bovine Serum	HyClone	SH30910.03	Used to supplement cell culture medium.
Penicillin/Streptomycin	CellGro	30-002-Cl	Used in cell culture medium.
Difco Noble Agar	BD Biosciences	214230	Used to prepare 1.0% and 0.6% agar.
Sodium Bicarbonate	Fisher	BP-328-1	Used in 2x cell culture medium.
Trypsin	Cellgro	25-050-Cl
Sterile Bottle-Top Filters	Fisher	09-761-126	Used to sterile filter 2x medium.
Lipofectamine Reagent	Invitrogen	18324-020	Used in PPAR-RE luciferase assay.
6-well Plates Tissue-culture Treated
37 °C/5% CO2 Incubator
Chemi-Doc Imager	Bio-Rad		Used to take pictures of colonies.
Quantity One Software	Bio-Rad		Used to count cell colonies.
15 ml Conical Tubes
50 ml Conical Tubes
250 ml Erlenmeyer Flasks
Microwave
5 ml Serological Pipettes
Pipette Aid
Micropipette
Hemacytometer w/ cover slip
Pipette Tips
Inverted Light Microscope
Centrifuge
Heat-Resistant Gloves
Saran Wrap
Ice Bucket