Occupational Exposure to Pesticides Affects Pivotal Immunologic Anti-Tumor Responses in Breast Cancer Women from the Intermediate Risk of Recurrence and Death

Abstract

Breast cancer risk stratification is a strategy based using on clinical parameters to predict patients' risk of recurrence or death, categorized as low, intermediate, or high risk. Both low and high risk are based on well-defined clinical parameters. However, the intermediate risk depends on more malleable parameters. It means an increased possibility for either suboptimal treatment, leading to disease recurrence, or systemic damage due to drug overload toxicity. Therefore, identifying new factors that help to characterize better the intermediate-risk stratification, such as environmental exposures, is necessary. For this purpose, we evaluated the impact of occupational exposure to pesticides on the systemic profile of cytokines (IL-12, IL-4, IL-17A, and TNF-α) and oxidative stress (hydroperoxides, total antioxidants, and nitric oxide metabolites), as well as TGF-β1, CTLA-4, CD8, and CD4 expression, investigated in tumor cells. Occupational exposure to pesticides decreased the levels of IL-12 and significantly increased the expression of TGF-β1 and CTLA-4 in the immune infiltrate. Nevertheless, we observed a decrease in CTLA-4 in tumor samples and CD8 in infiltrating cells of intermediate overweight or obese patients with at least one metastatic lymph node at the diagnosis. These findings indicate that occupational exposure to pesticides changes the molecular behavior of disease and should be considered for intermediate-risk stratification assessment in breast cancer patients.

Keywords: breast cancer; cytokine; inflammation; oxidative stress; pesticide exposure; risk stratification.

Figure 1

Study design. Six hundred sixty-four patients were screened. Three hundred forty-two were diagnosed with breast cancer. Based on risk stratification and pesticide exposure data, 130 patients were included and categorized into intermediate-risk exposed to pesticides (n = 77) and intermediate-risk unexposed to pesticides (n = 53). The in situ analysis was randomly selected by lot (n = 80).

Figure 2

Oxidative stress analysis of plasma samples of exposed and unexposed breast cancer patients from intermediate-risk stratification. (A) Plasma hydroperoxide levels, measured in RLU = relative light units. (B) Total plasma antioxidant capacity (TRAP), measured in nM of Trolox. (C) Oxidation index in arbitrary units. (D) Levels of nitric oxide metabolites, measured in μM. Results are represented in box plots (min–max). Analyses were performed with GraphPad Prism 7.0 (GraphPad Software. San Diego, CA, USA).

Figure 3

Cytokine profile of exposed and unexposed breast cancer patients from intermediate-risk stratification. (A) Interleukin 4. (B) Interleukin 17-A. (C) TNF-α. (D) Interleukin 12. All measured in pg/mL. Results are represented in box plots (min–max). * Indicates statistically significant difference (p < 0.05). Analyses were performed with GraphPad Prism 7.0 Software (San Diego, CA, USA).

Figure 4

Heatmap: correlation analysis of clinicopathological data according to systemic levels of IL-12 of exposed and unexposed breast cancer patients from intermediate-risk stratification. (A) Exposed group. (B) Unexposed group. The analysis was performed with R programming language (R Development Core Team. 2021). Red squares indicate positive correlations. Blue squares indicate the negative ones. A more intense color indicates a stronger correlation.

Figure 5

Immunostaining of TGF-β1 (A,B), CTLA-4 (C,D), CD8+ lymphocytes (E,F), and CD4+ lymphocytes (G,H) in breast tumor biopsies from intermediate-risk patients exposed (IE) or unexposed (INE) to pesticides. Labeling was evaluated in breast tumors and infiltrating leukocytes (400×). The images sequentially represent (horizontal view) DAPI labeling, marker labeling, and the merge of DAPI + marker. The resulting images were merged in ImageJ to generate the final images. For all images: immunostaining in green for Alexa Fluor (positive staining), red for Texas Red (positive staining), and blue for DAPI (negative counterstaining). The yellow arrows indicate the labeled areas.

Figure 6

Pesticide-induced immune deregulation may worsen the prognosis in women with breast cancer from the intermediate risk of death and recurrence. Women diagnosed with breast cancer occupationally exposed to pesticides exhibit several significant alterations in the tumor microenvironment, affecting its immunosurveillance. Decreased TCD8+ lymphocyte infiltrate and increased expression of negative regulators of tumor-driven immune responses such as TGF-β and CTLA-4 are found in tumors from pesticide-exposed women compared to the unexposed ones. In addition, reduced levels of circulating interleukin 12 are reported, reducing the anti-tumor arsenal of pesticide-exposed women. This immunocompromised scenario may result in a worse clinical prognosis since women from the exposed group have more metastasis than the unexposed ones.