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.
Highlights
The soft agar colony formation assay is a technique widely used to evaluate cellular transformation in vitro
We used Q-PCR to show that Wnt7A and Fzd[9] mRNA are expressed in low levels in CMT167 cells
As our previous work has shown, peroxisome proliferatoractivated receptor γ (PPARγ) is a downstream effector of Wnt7A/Fzd[9] signaling
Summary
The soft agar colony formation assay is a technique widely used to evaluate cellular transformation in vitro. Transformed cells have the capability to grow and divide without binding to a substrate To capitalize on this concept, researchers developed the soft agar colony formation assay. In the traditional soft agar colony formation assay, cells are grown in a layer of soft agar mixed with cell culture medium that rests on another layer of soft agar, mixed with cell culture medium, but containing a higher concentration of agar This prevents cells from adhering to the culture plate, yet allows transformed cells to form visible colonies. Wnts signal through several non-canonical pathways, for example, the planar cell polarity pathway, which regulates elements involved in cytoskeletal structure[7], and the Wnt-calcium pathway, which regulates release of calcium from the endoplasmic reticulum[8] Wnt ligands exert their activity through binding Frizzled receptors. Overexpression of Wnt7A and Fzd[9] were confirmed by quantitative-PCR (Q-PCR) and the functionality of Wnt7A and Fzd[9] overexpression was confirmed through downstream activation of PPARγ
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