Preclinical Models to Study Obesity and Breast Cancer in Females: Considerations, Caveats, and Tools

Abstract

Obesity increases the risk for breast cancer and is associated with poor outcomes for cancer patients. A variety of rodent models have been used to investigate these relationships; however, key differences in experimental approaches, as well as unique aspects of rodent physiology lead to variability in how these valuable models are implemented. We combine expertise in the development and implementation of preclinical models of obesity and breast cancer to disseminate effective practices for studies that integrate these fields. In this review, we share, based on our experience, key considerations for model selection, highlighting important technical nuances and tips for use of preclinical models in studies that integrate obesity with breast cancer risk and progression. We describe relevant mouse and rat paradigms, specifically highlighting differences in breast tumor subtypes, estrogen production, and strategies to manipulate hormone levels. We also outline options for diet composition and housing environments to promote obesity in female rodents. While we have applied our experience to understanding obesity-associated breast cancer, the experimental variables we incorporate have relevance to multiple fields that investigate women's health.

Keywords: Adipose tissue; Breast cancer; Carcinogen; Diet; Menopause; Obesity; Ovariectomy; Preclinical model; Thermoneutrality; Tumor subtype.

Figure 1.. Considerations for preclinical modeling of obesity and breast cancer.

Each factor, including the presence of estrogen, tumor subtype, rodent strain, immune function, housing temperature, and diet influences and/or is influenced by obesity. Representative images of Lean and Obese female rats (top; white) and mice (bottom; black) derived from these models are shown.

Figure 2.. Diet-induced separation of obesity and metabolic dysfunction in mice.

(A) Body fat percentage measured by qMR in Rag1-null C57Bl/6 mice fed low-fat/low-sucrose (LFLS), low-fat/high-sucrose (LFHS), or high-fat/high-sucrose (HFHS) diets. Note the variability in body fat across the HFHS group. (B) Body fat percentage in LFLS, LFHS, and HFHS fed mice, showing the obesity resistant (OR) and obesity prone (OP) groups separately. (C) Fasting insulin and (D) glucose measured in mice on LFLS, LFHS, or HFHS diets. (E) HOMA-IR calculated from fasting insulin and glucose measures in mice on LFLS, LFHS, or HFHS diets. Bars represent mean and SEM. Data were analyzed by unpaired t-test.

Figure 3.. Diet-induced separation of obesity prone and resistant rats.

(A) Body weight for lean and obese rats all on the same HF diet (46% kcal fat) demonstrates the differential predisposition to obesity in the outbred Wistar strain. Graph represents weekly body weights from 288 rats (mean +/− SEM) based on classification as obesity resistant (OR) or obesity-prone (OP) using % body fat at 18 weeks of age. (B) Frequency distribution of % Body Fat at 18 weeks of age (N=288 rats). Color coding shows selection of OR (n=105 rats; 36%) and OP (n=91 rats; 32%) from the entire cohort; Middle Tertile animals (n=92 rats; 32%) were moved to other studies. (C) Body Composition in mature Lean and Obese Rats (mean = 22 weeks of age). (D) Loss of circulating hormones following OVX induces rapid weight gain over approximately 3–4 weeks in both lean and obese rats, with no difference in the cumulative weight gained between lean and obese groups. All data is presented as mean and SEM.

Figure 4.. Effect of thermoneutral housing on body composition in Rag1-null C57Bl/6 female mice.

(A) diagram of cage placement on warm water blankets. Each cage is housed half on, and half off, the blanket. (B) Temperatures of the blanket surface, the bottom of the warmed cage, and the bottom of cages housed at ambient vivarium temperature, measured with an infrared thermometer. The red box indicates the mouse thermoneutral zone. (C) Body mass of LFLS and HFHS Rag1-null females. The vertical line indicates the initiation of thermoneutral housing; N=16 per group. (D) Body fat percentage, (E) Fat mass, and (F) lean mass measured before (pre) and after (post) warming with qMR in mice fed LFLS or HFHS diets; N=16 per group. Data were analyzed by 2-way ANOVA testing for main effects of diet or time (panel c) or diet and warming (panels d-f) and for an interaction.

Figure 5.. Effect of thermoneutral housing on body composition in wild type C57Bl/6 females.

(A) Body mass of LFLS and HFHS wild type C57Bl/6 females. The vertical line indicates the initiation of thermoneutral housing. (B) Body fat percentage in mice fed LFLS or HFHS diets measured after 9 weeks on study via qMR. Fat mass (C) and lean mass (D) in g are shown. N=19 LFLS, N=18 HFHS. Data were analyzed by 2-way ANOVA (panel a) testing for main effects of diet and time, or by unpaired t-tests (panels b-d).

Figure 6.. Effect of oral E2 administration in mice.

(A) Gonadal fat pad mass and (B) uterine mass expressed as mg per g body mass, and (C) percentage of mammary epithelial cells positive for the progesterone receptor (PR) by IHC in OVX mice or OVX mice supplemented with 0.5 μM E2 in drinking water. For a and b, N=10 OVX and N=5 OVX+E2. For c N=6 OVX and N=6 OVX+E2. Data were analyzed by unpaired t-test.

Figure 7.. General Overview of Models.

Key features of studies done in (A) mice and (B) rats are shown. The grey box in each diagram represents the period following estrogen loss when weight gain and insulin resistance increase. OVX-ovariectomy; EWD-estrogen withdrawal; LFLS-low fat/low sucrose; LFHS-low fat/high sucrose; HFHS-high fat/high sucrose.