class="head no_bottom_margin" id="sec1title">IntroductionThe cancer stem cell theory holds that there is a small sub-population of cells within a tumor that drives tumorigenesis and is capable of repopulating the tumor bulk from one cell (, , , ). Although this theory remains controversial (, ), it has become widely accepted and there are numerous assays for elucidating the stem-like character of cancer cells. One property of breast cancer stem cells is that they can survive detachment from the extracellular matrix (, ). Detached stem cells also retain the capability of proliferating (, ). As such, the sphere formation of suspended cells is hypothesized to be a method to assay for the self-renewal potential in vitro (). This property, first investigated in neural cells, was further adapted for mammary epithelial cells and termed the mammosphere assay (). In brief, suspended cells are cultured in serum-free media containing growth factors. The fraction that survives to form spheroid colonies (mammospheres) is deemed more stem-like. This is often followed by monitoring the alterations in sphere formation following treatment (, , , href="#bib35" rid="bib35" class=" bibr popnode">Reynolds et al., 2017). Treatments that lower the sphere-forming efficiency (SFE, [spheres/cells seeded]*100) of a population are hypothesized to have reduced the stem-like sub-population of the cells.MCF-7 human breast carcinoma cells are widely used in the mammosphere assay (href="#bib1" rid="bib1" class=" bibr popnode">Akrap et al., 2016, href="#bib10" rid="bib10" class=" bibr popnode">Fu et al., 2016, href="#bib11" rid="bib11" class=" bibr popnode">Grimshaw et al., 2008, href="#bib15" rid="bib15" class=" bibr popnode">Guttilla et al., 2012, href="#bib16" rid="bib16" class=" bibr popnode">Hinohara et al., 2012, href="#bib25" rid="bib25" class=" bibr popnode">Manuel Iglesias et al., 2013, href="#bib47" rid="bib47" class=" bibr popnode">Zhang et al., 2011). These luminal-type cells have been observed to make very cohesive, easily defined spheres. However, the problem is SFEs are quite disparate between reports and have ranged from 1% to 20% depending on the conditions (href="#bib6" rid="bib6" class=" bibr popnode">de la Mare et al., 2013, href="#bib27" rid="bib27" class=" bibr popnode">Montales et al., 2012, href="#bib29" rid="bib29" class=" bibr popnode">Morrison et al., 2012). Many factors could be contributing to these discrepancies, including growth media composition, counting procedures, and variability between different human operators performing the same assay. Of utmost importance, however, is the seeding density (href="#bib37" rid="bib37" class=" bibr popnode">Shaw et al., 2012). Due to the mobile nature of cells in suspension, cells drift and collide, leading to an aggregation tendency that is proportional to the cell density (href="#bib41" rid="bib41" class=" bibr popnode">Tolbert et al., 1980). This is problematic because clonality is an integral concept to the mammosphere assay (href="#bib37" rid="bib37" class=" bibr popnode">Shaw et al., 2012). Mammospheres should arise from a single cell to effectively measure stem-like propagation.Attempts to address aggregation have been reported (href="#bib25" rid="bib25" class=" bibr popnode">Manuel Iglesias et al., 2013, href="#bib33" rid="bib33" class=" bibr popnode">Patel and Rameshwar, 2013, href="#bib36" rid="bib36" class=" bibr popnode">Rota et al., 2012, href="#bib37" rid="bib37" class=" bibr popnode">Shaw et al., 2012). There is no common protocol, however, and seeding densities up to 100,000 cells/mL have been reported. Varying densities can lead to large differences in SFE (href="#bib37" rid="bib37" class=" bibr popnode">Shaw et al., 2012) and beg the question of how to interpret results. If a drug treatment lowers the sphere count in an experiment, can that result be interpreted as an effect on SFE or aggregation? To completely remove results confounded by aggregates we visually tracked 1,823 verified single cells over the course of 14 days, monitoring the cell count, sphere size, and morphology.
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