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 (ECM) and undergo apoptosis, a process known as anoikis. In contrast, cells that have undergone malignant transformation lose their anchorage dependence due to activation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and Rac/Cdc42/PAK. Therefore, these cells are able to grow and form colonies within the semi-solid soft agar matrix².

A common use of the soft agar assay is to test whether specific compounds, such as PADI inhibitors, are able to suppress tumor growth in vitro. In general, colony count or colony sizes are quantitative read-outs from the assay that can be compared between control and treatment groups to assess differences in cellular tumorigenicity. Therefore, if one finds that colony formation is inversely correlated with increasing drug concentration, then a conclusion could be drawn that the drug is an effective inhibitor of tumorigenicity in vitro. On the other hand, if the drug does not affect colony formation, the drug is either not at the appropriate dosage or it is not an effective tumorigenic inhibitor. Aside from using a soft agar assay to test the anti-tumor effect of a drug, this assay can also be used to probe the relationship between a specific gene and tumorigenesis. For example, the effect of suppressing PADI2 expression on tumorigenicity can be addressed by PADI2-specific siRNA treatment.

PADIs are calcium-dependent enzymes that post-translationally modify proteins by converting positively charged arginine residues into neutrally charged citrulline in a process known as citrullination or deimination^3-5. We have recently found that peptidylarginine deiminase 2 (PADI2) may function as a novel breast cancer biomarker and that PADI inhibitors represent candidate therapies for early stage breast cancers⁶. For example, we have previously demonstrated that a “pan-PADI” inhibitor, Cl-amidine, suppresses the proliferation of breast cancer cells using 2D monolayers and that the inhibitor suppressed the growth of 3D tumor spheroids⁶. In this report, we extend these studies, and highlight the utility of the soft agar assay, by testing the efficacy of a new PADI inhibitor, BB-CLA, in suppressing the growth of MCF10DCIS breast cancer colonies⁷. We note that we used MCF10DCIS cells for this experiment because they are oncogenic derivatives of non-transformed human MCF10A cells and because they contain high steady state levels of PADI2 protein⁸. We hypothesize that PADI2 enzymatic activity plays a key role in the tumorigenicity of this cell line and that BB-CLA-mediated inhibition of PADI2 activity will suppress cancer progression.

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 incubator to prevent the agarose from solidifying in the pipette when handling.
2. Partially loosen the bottle lid and microwave the pre-made 3% 2-hydroxyethyl agarose solution for 15 sec. Then, gently swirl the solution and microwave for another 15 sec. CAUTION: Be careful when swirling the agarose solution because the solution rises up when exposed to air and can spill over.
3. If there is residual solid gel in the bottle, microwave for a few more seconds.
4. Keep the bottle containing the agarose solution in a 45 °C water bath during the next steps to prevent the agarose solution from solidifying prematurely.
5. Warm MCF10DCIS media in a 37 °C water bath. Note: MCF10DCIS media consists of DMEM/F12, 5% horse serum, 5% penicillin streptomycin.
6. Transfer 3 ml of the 3% agarose solution using the pre-warmed pipettes into a sterile 50 ml conical tube.
7. Immediately add 12 ml of warm MCF10DCIS media and gently invert the conical tube to mix the agarose with the media. Try not to form any bubbles as it will interfere with the colony counting later.
8. Gently add 2 ml of this mixture into each well of a 6-well culture plate without forming any air bubbles.
9. Incubate the 6-well culture plate horizontally on a flat surface at 4 °C for 1 hr to allow the mixture to solidify.
10. After the mixture solidifies, place the plate into a 37 °C incubator for 30 min. The bottom layer is now ready for use.

3. Preparation of the Cell-containing Layer: 0.3% Agarose Gel

1. Trypsinize MCF10DCIS cells and dilute them to a cell concentration of 4 x 10⁴ /ml.
2. Take 2 ml of the 3% agarose using pre-warmed pipettes and transfer into a sterile 50 ml conical tube.
3. Immediately add 8 ml of MCF10DCIS media to the conical tube and gently invert to mix the agarose with the media. Avoid forming any bubbles.
4. Take 2 ml of the MCF10DCIS cells (4 x 10⁴ /ml) and treat with BB-CLA (0 µM (DMSO) or 1 µM).
5. In a 1:1 dilution, mix the cells with the 0.6% agarose.
6. Take 1 ml of the cell-agarose mixture and gently add onto the bottom layer of the 6-well culture plate (2 x 10⁴ cells/ml).
7. Place the 6-well culture plate horizontally on a flat surface at 4 °C for at least 15 min to allow the top layer to solidify.
8. After the mixture solidifies, place the plate into a 37 °C incubator for a week before adding the feeding layer.

4. Preparation of the Feeder Layer: 0.3% Agarose Gel

1. Microwave the pre-made 3% 2-Hydroxyethyl agarose solution for 15 sec. gently swirl the solution and microwave for another 15 sec.
2. Equilibrate the agarose solution bottle in a 45 °C water bath.
3. Warm the MCF10DCIS media in a 37 °C water bath.
4. Mix 1 ml of 3% agarose solution with 9 ml of warm MCF10DCIS media into a 50 ml conical tube and gently invert to mix the agarose with the media. Avoid forming air bubbles.
5. Treat the mixture with BB-CLA (0 µM (DMSO) or 1 µM).
6. Gently add 1 ml of this mixture (without forming bubbles) into each well of the 6-well culture plate containing the bottom and soft layers.
7. Place the 6-well culture plate horizontally on a flat surface at 4 °C for at least 15 min to allow the mixture to solidify.
8. After the feeder layer solidifies, place the plate into a 37 °C incubator.
9. Repeat this feeding procedure weekly by overlaying 1 ml of 0.3% agarose/medium/treatment solution onto the existing feeder layer to replenish the cells with new media until colony formation is observed. Note: Agar in the soft and feeder layers is very soft and, therefore, the added nutrients from the feeder layer will readily diffuse into the cell-containing layer to reach the cells.

5. Data Collection

1. After 2.5 weeks of cell growth in the soft agar, count the number of colonies in each well using a light microscope. To facilitate quantification, print a grid onto a transparency and attach the grid to the 6-well plate to help locate where the cells are during counting. Since colony size (as quantified by the diameter of each colony) will vary, predefine a reference colony size to determine which colonies will be scored. For example, include colony sizes of 70 µm or larger in the data analysis.
2. Store the samples at 4 °C to prevent further colony formation and for future counting. Seal the 6-well culture plate with Parafilm to prevent the gels from drying out.

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

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