Soft Agar Colony Formation Assay as a Hallmark of Carcinogenesis

Soft agar colony formation assay is established to estimate the anchorage-independent growth ability of cells. In this assay, a bottom layer of agar with complete media is poured and solidified first, followed by an upper layer containing a specified number of cells suspended in medium-agar mixture. After two weeks of incubation, the number of colonies will be counted, serving as an indicator of malignancy of tumor cells.

Background

Anchorage-independent growth is an ability of cells to grow independently on a solid surface, and is considered as a hallmark of carcinogenesis (de Larco and Todaro, 1978). Soft agar colony formation assay is a well-established method to evaluate cellular anchorage-independent growth for the detection of the tumorigenic potential of malignant cells ( Roberts et al., 1985 ), which is developed from plate colony formation assay described by Puck et al. in 1956 where cells were seeded on to a culture plate to assay the ability of cells to form colonies ( Puck et al., 1956 ). The limitation of plate colony formation assay is that it only displays cellular abilities for anchorage-dependent growth, by which normal cells can escape from anoikis (a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix) and survive ( Taddei et al., 2012 ). In contrast, malignant cells are capable of proliferating and growing without attachment to a substrate. Therefore, soft agar colony formation assay is developed to characterize this ability in vitro (Hamburger and Salmon, 1977; Yuan et al., 2017 ). The soft agar colony formation assay has been widely adapted for researches on cell differentiation, transformation and tumorigenesis as well as the efficacy evaluation of anti-tumor treatment.