ORIGINAL ABSTRACT

N-(phosphonomethyl)glycine, commonly known as glyphosate, is the most extensively used herbicide worldwide. Its widespread application in agriculture has led to continuous human exposure through food residues, contaminated water, and occupational contact. Although its carcinogenic risk remains inconclusive, mechanistic studies suggest endocrine-related signaling, oxidative stress, and epigenetic disruption may influence tumor biology. This study investigated whether prolonged exposure to glyphosate, at concentrations within the reported human exposure range, is associated with the acquisition of stem-like features and therapy tolerance in breast cancer cells. Estrogen receptor (ER)-positive MCF7 and triple-negative MDA-MB-231 cells were continuously exposed to 0.1 μM glyphosate for 8 weeks under non-cytotoxic conditions. Stemness and aggressiveness were assessed using mammosphere assays, soft-agar colony formation, migration assays, and doxorubicin dose-response testing, complemented by Western blot analysis of stemness-associated transcription factors. Chronic exposure increased mammosphere formation efficiency in both lines, accompanied by remodeling of stemness regulators, including increased Sox2 with reduced Nanog in MCF7 cells, alongside elevated β-catenin. Functionally, long-term exposure enhanced migratory behavior and anchorage-independent growth, and reduced doxorubicin sensitivity in MCF7 cells, with clonogenic persistence under doxorubicin challenge. These findings indicate that sustained exposure to glyphosate, at a concentration within the reported human exposure range, is associated with features consistent with a cancer stem-like and adaptive phenotype in breast cancer cells.