Development and Characterization of a Preclinical Model of Breast Cancer Lung Micrometastatic to Macrometastatic Progression

Abstract

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500–1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30–60 micrometastases in the lung (with many animals also having 2–30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis.

A Small Molecule with Anticancer and Antimetastatic Activities Induces Rapid Mitochondrial-Associated Necrosis in Breast Cancer

“…To determine the effect of AG311 on necrosis in vivo, BALB/cJ mice were implanted with nonfluorescent 4T1 tumor cells. The experiment was performed as published (Bailey-Downs et al, 2014) with the following modifications: cell suspension (7500 cells/100 ml) in phosphate-buffered saline with 1 mM EDTA was implanted into the mammary fat pad #4 of 8-week-old female BALB/cJ mice. Three weeks after implantation when the average tumor size was ∼260 mm 3 , mice were separated based on matched tumor volumes (n 5 9-11).…”

“…Next, the efficacy of AG311 was examined in a TNBC allograft metastatic progression model in immune proficient mice previously developed in our laboratory (Bailey-Downs et al, 2014). Dual luciferase/GFP 4T1-tagged (4T1-luc2-GFP) cells were implanted in female BALB/cJ mice.…”

Inoculated Cell Density as a Determinant Factor of the Growth Dynamics and Metastatic Efficiency of a Breast Cancer Murine Model

“…Interestingly, Bailey-Down et al . [48] also showed that reducing the number of implanted 4T1 cells increased metastatic efficiency. Nevertheless, in animals inoculated with a cell density equal or lower than 1500, the tumor take rate was lower than 50% and macrometastases were barely observed at five weeks post-implantation [48], in contrast with results reported herein.…”

“…[48] also showed that reducing the number of implanted 4T1 cells increased metastatic efficiency. Nevertheless, in animals inoculated with a cell density equal or lower than 1500, the tumor take rate was lower than 50% and macrometastases were barely observed at five weeks post-implantation [48], in contrast with results reported herein. In the work of Bailey-Down et al ., the best compromise between tumor take rate (over 90%) and metastatic efficiency (67% in the lungs) was achieved upon inoculation of 7500 4T1 cells.…”

“…The absence of a vascular network or the existence of a poorly developed vasculature in lung metastases, as compared to the primary tumor, may justify the high metastatic burden despite treatment with Caelyx®. Nevertheless, and since colonization of the visceral organs by cancer cells in the 4T1 mammary carcinoma model was described to occur later than the lungs [19, 33, 48], it is possible that the liposomal chemotherapy tested herein, limited further dissemination/colonization of tissues of the peritoneal cavity by 4T1 cells, accounting for a lower incidence of metastasis in the mesentery. Extramedullary hematopoiesis has been shown to play a crucial role in the spontaneous metastization in the 4T1 mouse model [44, 56], by contributing to a receptive microenvironment for circulating tumor cells arrest, survival and proliferation in the lungs [44, 67, 68].…”