Arsenic Exposure on Stem Cell Differentiation and Cellular Plasticity in Normal Mammary Cell Development

Abstract

Only an estimated 10-15% of breast cancers are linked to inherited genetic factors, leaving the remaining diagnoses potentially due to environmental exposures, such as toxicants. Inorganic arsenic is a known environmental toxicant linked to bladder, intestinal, and liver cancers. Recent studies estimate that 200 million people worldwide have been exposed to arsenic-contaminated drinking water alone, stressing its prevalence as a public health issue. Despite breast cancer accounting for nearly 30% of all cancer diagnoses, studies of mammary gland development and inorganic arsenic exposure are underdeveloped within the current literature. Our study aims to test the effect of arsenic exposure on the non-tumorigenic MCF10A breast cell line to study normal mammary stem cell proliferation, self-renewal capacity, and cellular plasticity. MCF10A cells were cultured for 3-5 days to achieve 90% confluency. Cells were plated in 10 µM, 1 µM, 100 nM, and 10 nM doses of inorganic arsenic, and were cultured for 7 days to form primary mammospheres. Primary mammospheres were collected and stained with Hoescht and fluorescently labeled keratin-8 and keratin-14 antibodies, or broken up and cultured into secondary mammospheres for 10 days. Both primary and secondary mammospheres greater than or equal to 40 µm were counted using the EVOS M7000 Imaging System; sphere-forming efficacy, size, and intensity were analyzed using the CellProfiler software. Compared to the control, the sphere-forming efficacy (SFE) of primary mammospheres in 10 µM arsenic concentrations demonstrated a median increase of 250% (p = 0.0046); SFE of secondary mammospheres for 1 µM, 0.1 µM, and 0.01µM arsenic concentrations demonstrated a median increase of 160% (p = 0.0048), 100% (p = 0.0046), and 200% (p = 0.0050), respectively. The intensity of the Hoescht DNA stain for a 10 µM arsenic concentration in primary and secondary mammospheres, compared to control, showed a median decrease of 29.41% (p = 0.015) and 9.59% (p = 0.041), respectively. Primary mammospheres in a 10 µM arsenic concentration demonstrated an average 15.79% (p = 0.0260) decrease in area, while those in 0.1 nM arsenic concentration showed a median 7.46% (p = 0.0022) increase in area compared to control. Secondary mammospheres in 1 µM, 0.1 µM, and 0.01 µM arsenic concentrations demonstrated an average 5.42% (p = 0.0022), 4.82% (p = 0.0260), 13.86% (p = 0.0022) increase in area, respectively, compared to control. Our preliminary data suggests that arsenic exposure affects normal breast stem cell proliferation and self-renewal capacity in vitro. Ongoing analyses of keratin-8 and keratin-14 immunofluorescence–which are epithelial cell markers–-will delineate the effect of arsenic concentration on cellular plasticity. Keratin-14 is an important marker of epithelial stem cell proliferation before differentiation and a marker of basal cells in the developed mammary gland. Keratin-8 maintains the structural stability of luminal epithelial cells and is also a marker of inflammatory stress response. Normal mammary stem cell populations that exhibit keratin-8 and keratin-14 hybrid morphology are indicative of an inability to properly differentiate, which often predicate aggressive breast cancer phenotypes.