Impact of advanced age, diet-induced obesity, and intermittent calorie restriction on mammary tumor burden and tumor immune microenvironment
Creators: name: Craven, Dalton
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2021
Subjects: Breast--Cancer

description: Background: Breast cancer (BC) is the most commonly diagnosed and second leading cause of cancer-related death among women in the United States. In addition, advanced age and obesity are two major risk factors for BC mortality. This presents a significant public health concern as ~13% of adults worldwide are obese and ~60% of BC-related deaths occur in women aged 65 years or older. Obesity is characterized by a maladaptive metabolic state of chronic, low-grade inflammation. This inflammatory response is associated with an increase in circulating proinflammatory cytokines and a decline in immune function. Similar to obesity, advanced age is also associated with chronic, low-grade inflammation and reduced immune function. In our mouse models of triple negative breast cancer, we have demonstrated that, similar to obesity, advanced age accelerates mammary tumor growth. Given the increasing median age of our population and the increasing prevalence of obesity, interventions capable of reversing the protumor effects of advanced age and obesity are needed. Intermittent calorie restriction (ICR) may ameliorate the negative effects of obesity and advanced age on BC. Thus, we investigated whether advanced age- and/or obesity-related enhancement of mammary tumor growth was associated with reduced intratumoral T cells as well as changes in levels of circulating cytokines, and if these perturbations could be reverted with ICR. 
Methods: Tumors sections (3-5μm) from young control (5 months), young diet-induced obese (DIO; 5 months), and aged control (12 months) mice bearing metM-Wntlung tumors were stained for markers of proliferation (Ki67), apoptosis (cleaved caspase 3), and T cells (CD8 and CD3). In a subsequent study, young control (5 months), young DIO (5 months), aged control (15 months), and aged DIO (15 months) mice were fed 60% kcal fat (D12492) or 10% kcal fat (D12450J) diets for 15 weeks following which they were randomized to either remain on their baseline diet or switched to the ICR intervention (5 days 14% kcal reduction, 2 nonconsecutive days 70% kcal reduction per week). Following 9 weeks on ICR or baseline diet, serum samples were collected and E0771 cells were inoculated into the 4th mammary fat pad. After 4 weeks of tumor growth, mice were euthanized, and tumors were collected and weighed. Tumor sections (3-5μm) were stained for CD8. Serum cytokine concentrations were determined using a BIO-RAD Mouse Chemokine 31-Plex panel run on a BIO-RAD MAGPIX instrument. Histological staining was quantified using QuPath image analysis, and statistical analysis conducted by one-way or three-way ANOVA using GraphPad Prism (version 8/9). Results were considered significant where p<0.05. 
Results: In the initial study, intratumoral Ki67 was not altered between any groups while the number of cleaved caspase 3 positive cells increased and CD3 and CD8 positive cells decreased in both advanced age and DIO mice relative to control. Tumors from aged control and DIO mice were larger than control, and tumors from aged DIO mice were larger than all other tumors. In all groups ICR reduced tumor mass. Obesity and advanced age were associated with a reduction, but ICR with an increase, in intratumoral CD8+ cell count. Chemokine C-C motif ligand 24 (CCL24) was reduced across all ICR groups.
Conclusions: We conclude that accelerated tumor growth resulting from obesity and advanced age is associated with reduced intratumoral T cell numbers and promotion of systemic inflammation in a mouse model of breast cancer. Importantly, ICR limits these perturbations in both aged and obese animals. Further investigation into the impact of ICR on BC prevention and progression is warranted and may be a novel approach to improving BC outcomes.