Effect of 2-methoxyestradiol on mammary tumor initiation and progression

Abstract

Background: The anti-cancer agent 2-methoxyestradiol (2-ME) has been shown to have anti-proliferative and anti-angiogenic properties. Previously, the effect of 2-ME on early- and late-stage breast cancer (BC) was investigated in vivo using a transgenic mouse model (FVB/N-Tg(MMTV-PyVT)) of spontaneous mammary carcinoma. Anti-tumor effects were observed in late-stage BC with no effect on early-stage BC. Given the contrasting results obtained from the different BC stages, we have now investigated the effect of 2-ME when administered before the appearance of palpable tumors.

Methods: Each mouse received 100 mg/kg 2-ME on day 30 after birth, twice per week for 28 days, while control mice received vehicle only. Animals were terminated on day 59. Lung and mammary tissue were obtained for immunohistochemical analysis of CD163 and CD3 expression, and histological examination was performed to analyze tumor necrosis. Additionally, blood samples were collected to measure plasma cytokine levels.

Results: 2-ME increased tumor mass when compared to the untreated animals (p = .0139). The pro-tumorigenic activity of 2-ME was accompanied by lower CD3+ T-cell numbers in the tumor microenvironment (TME) and high levels of the pro-inflammatory cytokine interleukin (IL)-1β. Conversely, 2-ME-treatment resulted in fewer CD163+ cells detectable in the TME, increased levels of tumor necrosis, increased IL-10 plasma levels, and low IL-6 and IL-27 plasma levels.

Conclusion: Taken together, these findings suggest that 2-ME promotes early-stage BC development.

Keywords: 2‐methoxyestradiol; breast cancer; in vivo; metastasis; tumor growth.

FIGURE 1

The time taken for mice to develop palpable mammary tumors. In the 2‐methoxyestradiol (2‐ME) and control groups, seven mice as opposed to three mice in the control group developed tumors earlier, respectively.

FIGURE 2

(A) No difference was observed in tumor volumes in the 2‐methoxyestradiol (2‐ME) treated and control groups (p = .3319). (B) A significantly greater tumor mass was observed in 2‐ME treated mice (p = .0139; p < .05) (N = 9 in each group).

FIGURE 3

(A) Blue boundaries surround the tumor and necrotic regions are surrounded by red boundaries and arrows. Histology images showcasing various sizes (1, 2 mm and 800 μm) along with a scale bar. (B) More extensive (p = .1031) necrotic regions were observed in mice that were treated with 2‐methoxyestradiol (2‐ME) (N = 4).

FIGURE 4

(A) CD163+ cells stained dark brown with red boundaries. IHC images accompanied by a 100 μm scale bar for reference. (B) A lower number of CD163+ cells (p = .1965) was detected in the 2‐ME group while (C) no change was observed in the lungs (p = .3450). N = 5 in each group.

FIGURE 5

(A) CD3 immunohistochemistry in mammary and lung tissue. Immunohistochemistry (IHC) images with a scale bar representing 100 μm. (B) Significantly lower number of CD3+ T cells were observed in mammary tissue in the 2‐methoxyestradiol (2‐ME) group (p = .0217) (C) and lower number of CD3+ T cells were detected in the lung tissues of the same group. n = 5 in each group.

FIGURE 6

Cytokine profiles of 2‐methoxyestradiol (2‐ME) treated and control group (n = 5 in each group). IL‐6 was significantly higher (p = .0057; p = .05), and IL‐27 (p = .3317) were notably higher in the control group.