The Soft Agar Colony Formation Assay

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.

H2O2 transit through the mitochondrial intermembrane space promotes tumor cell growth in vitro and in vivo

“…Colony formation on soft agar was carried out as described ( 51 ). Briefly a 1% base agar medium was prepared with 1% noble agar mixed with McCoy’s 5A medium containing 10% FBS and 1% penicillin/streptomycin plus other antibiotics used for selection.…”

Targeting epigenetic features in clear cell sarcomas based on patient-derived cell lines

“…This so-called anchorage-independent growth is characteristic for malignant transformation in cells and a major aspect for the tumor phenotype in general, especially with respect to metastatic potential [ 21 ]. Experiments were performed according to already published protocols [ 22 ]. In brief, in six-well plates MUG Lucifer prim and MUG Lucifer met were seeded in 0.3% agarose with medium at a density of 2000 cells per well on top of a bottom layer containing 0.6% agarose in medium.…”

The NRF2 antagonist ML385 inhibits PI3K‐mTOR signaling and growth of lung squamous cell carcinoma cells

“…A soft agar colony formation assay was used to assess anchorage independent growth of cancer cells 47 . Media were prepared as two separate 2× concentrated solutions with noble agar (Sigma‐Aldrich) (2% for the base layer and 0.8% for the cell growth layer), which were then diluted with 2× RPMI prior to being added to dishes.…”

“…A soft agar colony formation assay was used to assess anchorage independent growth of cancer cells. 47 Media were prepared as two separate 2× concentrated solutions with noble agar (Sigma‐Aldrich) (2% for the base layer and 0.8% for the cell growth layer), which were then diluted with 2× RPMI prior to being added to dishes. MGH7 cells (1 × 10 4 cells per 60 × 15 mm polystyren, petri dish, USA Scientific Inc) were seeded within 2 ml of 0.4% top agar medium on top of the solidified base layer in the presence of ML385 (5 μM) or DMSO.…”