Investigating phenotypic plasticity due to toxicants with exposure disparities in primary human breast cells in vitro

Abstract

Introduction: Breast cancer is the second most diagnosed cancer, as well as the primary cause of cancer death in women worldwide. Of the different breast cancer subtypes, triple-negative breast cancer (TNBC) is particularly aggressive and is associated with poor prognosis. Black women are two to three times more likely to be diagnosed with TNBCs than white women. Recent experimental evidence suggests that basal-like TNBCs may derive from luminal cells which acquire basal characteristics through phenotypic plasticity, a newly recognized hallmark of cancer. Whether chemical exposures can promote phenotypic plasticity in breast cells is poorly understood.

Methods: To investigate further, we developed a high-content immunocytochemistry assay using normal human breast cells to test whether chemical exposures can impact luminal/basal plasticity by unbiased quantification of keratin 14 (KRT14), a basal-myoepithelial marker; keratin 8 (KRT8), a luminal-epithelial marker; and Hoechst 33342, a DNA marker. Six cell lines established from healthy tissue from donors to the Susan G. Komen Normal Tissue Bank were exposed for 48 hours to three different concentrations (0.1μM, 1μM, and 10μM) of eight ubiquitous chemicals (arsenic, BPA, BPS, cadmium, copper, DDE, lead, and PFNA), with documented exposure disparities in US Black women, in triplicate. Automated fluorescence image quantification was performed using Cell Profiler software, and a random-forest classifier was trained to classify individual cells as KRT8 positive, KRT14 positive, or hybrid (both KRT8 and KRT14 positive) using Cell Profiler Analyst.

Results and discussion: Results demonstrated significant concentration-dependent increases in hybrid populations in response to BPA, BPS, DDE, and PFNA. The increase in hybrid populations expressing both KRT14 and KRT8 is indicative of a phenotypically plastic progenitor-like population in line with known theories of carcinogenesis. Furthermore, BPA, BPS, DDE, and copper produced significant increases in cell proliferation, which could be indicative of a more malignant phenotype. These results further elucidate the relationship between chemical exposure and breast phenotypic plasticity and highlight potential environmental factors that may impact TNBC risk.

Keywords: breast cancer; disparities; environment; immunocytochemistry; phenotypic placticity; toxicology; triple negative breast cancer.

Figure 1

KCR 8195 classification intensity validation for (A) KRT8 intensity and (B) KRT14 intensity.

Figure 2

An example image series of KCR 8195 dosed with 1 μM of PFNA: (A) Nuclei. (B) KRT8. (C) KRT14 (D) Composite with KRT14 immunofluorescence shown in red, KRT8 immunofluorescence shown in green, with Hoechst shown in blue. Orange and yellow cells represent KRT8/KRT14 basoluminal hybrids. Scale bar represents 100 μm.

Figure 3

KCR 8195 combined populations (%) for each chemical compared to each associated control (0 μM concentration). Significance determined by Wilcoxon signed rank-sum tests and reported in Figures 4 , 5 and Supplementary Figures 12–15 .

Figure 4

Heatmap depicting the percent of the cells in each treatment condition, which are in a hybrid state, for organic chemical-treated cells only. Differences in hybrid percentages between a given treatment and the DMSO control were determined by Wilcoxon signed rank-sum tests and denoted by an * (p < 0.05). Increases in hybrid populations are represented by a black asterisk, while decreases in hybrid populations are represented by red asterisks.

Figure 5

Heatmap depicting the percent of the cells in each treatment condition, which are in a hybrid state, for metal-treated cells only. Differences in hybrid percentages between a given treatment and the water control were determined by Wilcoxon signed rank-sum tests and denoted by a * (p < 0.05). Increases in hybrid populations are represented by a black asterisk, while decreases in hybrid populations are represented by red asterisks.

Figure 6

Heatmaps depicting changes in cell counts (% relative to control) per sample for (A) DMSO solvent chemicals and (B) water solvent chemicals. Chemical concentrations are measured in μM—0.1 is equal to 100 nM. Differences in cell counts relative to control were determined by Wilcoxon signed rank-sum tests and denoted by a * (p < 0.05). Increases in cell counts are represented by a black asterisk, while decreases are represented by red asterisks.