Estradiol increases ER-negative breast cancer metastasis in an experimental model

Abstract

Breast cancer (BC) is the most common cancer affecting women in the United States and metastatic breast cancer is the leading cause of death. The role estradiol plays in ER-positive BC is well-documented, but the way it contributes to ER-negative BC remains unclear. In the present study, we utilized an experimental model of BC metastasis into lung by injecting ER-negative murine 4T1 cells into mice via the lateral tail vein. A 56 % metastasis occurrence rate following the injection of 5 × 10(3) cells was observed, thus this cell number was selected to study the potential stimulatory effect of estradiol on ER-negative BC metastasis. Female ovariectomized mice were randomized into estradiol and control groups with 16 mice per group, and estradiol pellets were implanted subcutaneously in the estradiol group. Results demonstrated that estradiol accelerated BC metastasis as indicated by bioluminescent imaging. In addition, estradiol enhanced metastatic tumor colony formation and increased the size of tumor nodules in the lungs, which were due, in part, to the increase in proliferative cells in the metastatic tumors. In vitro, estradiol increased the motility and invasion of 4T1 cells, and the stimulatory effect on cell motility was not blocked by ICI 182, 780, confirming that ER was not involved in the process. Results from the present study suggest that estradiol plays a role in ER-negative BC metastasis in whole animals.

Fig. 1

Estradiol increased the motility of 4T1 cells in vitro via non-ER-mediated pathways. Wounded scratches were made on the confluent 4T1 cell monolayer and the migrated distance between the two edges of the scratch was calculated to indicate cell motility. a Estradiol (E2) at the indicated doses increased the motility of 4T1 cells, b The stimulatory effect of E2 (1 nM) on the motility of 4T1 cells was not blocked by the pure ER antagonist ICI 182,780 (ICI) at 1 µM. Data (mean ± SEM) are normalized to the untreated control cells (C) and are representative of 3 independent experiments.** Indicates significant difference at the level of 0.01 when compared to the untreated cells

Fig. 2

Estradiol (E2) increased the invasion of 4T1 cells. Murine 4T1 cells were incubated in biocoated invasion chambers for 24 h and invasiveness was evaluated by counting cells that travelled through the biocoated PET membrane per field. Data (mean ± SEM) are normalized to the untreated control cells (C) and are representative of two independent experiments. ** Indicates significant difference at the level of 0.01 when compared to the untreated cells

Fig. 3

Estradiol accelerated lung metastasis progression in mice receiving 5 × 10³ 4T1 cells via the lateral TV. a Estradiol decreased the half time for bioluminescent signals emitted from metastatic tumors on the lungs to reach saturation (P < 0.05). Half time for bioluminescent signals to reach saturation (ST_1/2) was recorded for each mouse. When bioluminescent signal from an individual mouse did not reach saturation by the end of the study, ST_1/2 for this mouse was set as 21 (the duration of the study). Mice without detected bioluminescent signals were excluded. Triangle: individual ST_1/2, dash median ST_1/2 for experimental groups, b Top panel a series of bioluminescent images of a mouse from the control group (CTL) recorded on D7, D11, D14, D18 and D21. Bottom panel a series of bioluminescent images of a mouse from the estradiol group (E2) recorded on D7, Dll, D14, D18 and D21. Bioluminescent signals from the estradiol mouse reached saturation faster than from the control mouse

Fig. 4

Estradiol (E2) increased metastatic tumor burden in the lungs in mice receiving 5 × 10³ 4T1 cells via the lateral TV. Lungs were embedded in paraffin, sectioned and stained with H&E. Metastatic tumor colonies were counted and tumor areas (percentage of the whole lung tissue) were measured in each lung section. Estradiol increased metastatic tumor colony formation (a) and metastatic tumor area (b) in the lung tissue, c Left panel a representative image of stained lung tissue in the control group with 1 tumor colony and smaller tumor area; Right panel a representative image of stained lung tissue in the estradiol group with two tumor colonies and larger tumor size. Data are presented as mean ± SEM. ** Indicates significant difference at the level of 0.01 when compared to the control group (CTL). T tumor colony; Scale bar 200 µm

Fig. 5

Estradiol (E2) increased the proliferation of the metastatic tumor cells in the lungs in mice receiving 5 × 10³ 4T1 cells via the lateral TV. a E2 increased Ki-67 expression in metastatic tumor cells in the lungs. Lungs were embedded in paraffin, sectioned and processed to examine Ki-67 expression. Positive (brown) and negative (blue) cells in the metastatic tumors were counted and the results are presented as the percentage of proliferating cells in a given area of metastatic tumors. Data are presented as mean ± SEM. ** Indicates significant difference at the level of 0.01 when compared to the control group (CTL). b Left panel, a representative image of stained metastatic tumors in the lungs from the control group (CTL) with less proliferative cells (brown); Right panel, a representative image of stained metastatic tumors in the lungs from the estradiol group (E2) with more proliferative cells. Magnification: 400×. (Color figure online)