(2hb) Investigation of Breast Cancer Recurrence Mechanisms Following Radiotherapy of Mammary Gland Adipose Tissue: Evaluating Cellular Metabolism & 3D in Vitro Models

Background and Motivation: Triple negative breast cancer (TNBC) patients have high locoregional recurrence rates and low post-recurrence survival rates. Treatment for TNBC lacks targeted therapies due to its receptor status, and radiation therapy is routinely utilized in treatment plans. Locoregional recurrence may be facilitated by residual radioresistant cancer cells or the recruitment of circulating tumor cells to the radiation-damaged site following therapy. Regardless of the mechanism of recurrence, the interactions of TNBC cells and radiation-damaged normal tissue cells may help elucidate mechanisms that can be targeted to prevent recurrence.

Both radioresistant cancer cells and circulating tumor cells have been characterized by increased reliance on lipid metabolism; however, the metabolic alterations of the cells found in the mammary gland adipose tissue following radiation therapy have been significantly less studied. We hypothesized that metabolic responses to radiation damage by fibroblasts and adipocytes, two main cell types within mammary gland adipose tissue, may result in metabolic crosstalk with post-radiotherapy-recruited immune cells and TNBC cells, which could promote TNBC locoregional recurrence.

Methods: Murine NIH 3T3 fibroblasts and human reduction mammary fibroblasts were exposed to 10 Gy of ionizing radiation (IR) and evaluated against control cells receiving no radiation up to 7-days post-IR. Lipid accumulation was evaluated using Oil Red O and Perilipin-2 immunofluorescence staining. Post-IR fibroblast autophagy was evaluated utilizing a combination of LysoTracker Red DND-99 live cell staining and LC3B puncta analysis, and the impacts of autophagy inhibition were evaluated using the drug chloroquine. Mitochondrial analysis was performed through flow cytometry following MitoTracker Deep Red staining. Fibroblast metabolic rate assessment was conducted using modified glycolytic and mitochondrial stress tests on the Seahorse XFe96 analyzer in conjunction with chloroquine and etomoxir, the latter being an inhibitor of fatty acid oxidation. Conditioned media (CM) from fibroblasts was collected at 7-days post-IR and utilized in radiation-induced bystander effect assays. Luciferase-labeled 4T1 TNBC cells were exposed to fibroblast CM and evaluated in tumorsphere growth and scratch-wound assays. The impact of fibroblast-secreted lactate on TNBC cells in these assays was determined through blocking lactate uptake with the small molecule inhibitor AZD3965.

Radiation-damaged adipocytes were evaluated in 3-dimensional adipocyte spheroids generated from murine 3T3-L1 pre-adipocyte cells and primary murine mammary gland adipose tissue pre-adipocytes. Spheroids were formed by plating 20,000 cells in ultra-low attachment plates, and differentiating them in an adipogenic cocktail containing insulin, 3-isobutyl-1-methylxanthine, and dexamethasone for 30 days. Adipocyte spheroids were exposed to 20 Gy of IR and evaluated against spheroids receiving no radiation up to 14-days post-IR. Immunofluorescence staining and western blotting were used to evaluate expression of lipolytic markers. For co-culture studies, murine bone-marrow derived macrophages (BMDMs) and TNBC cells were utilized. BODIPY-C16, a fluorescent analog of palmitic acid, was utilized in lipolysis and fatty acid transfer studies.

Results: Irradiated fibroblasts displayed increased lipid accumulation through immunofluorescence staining of lipid droplets up to 7-days post-IR. Higher post-IR autophagic flux was responsible for this lipid accumulation. Interestingly, fibroblasts displayed higher ATP-linked respiration and aerobic glycolysis rates following radiation damage. Radiation-induced autophagy was necessary to maintain these higher metabolic rates, and irradiated fibroblasts also displayed increased levels of fatty acid oxidation. Increased aerobic glycolysis in irradiated fibroblasts led to a rise in lactate export. Culturing TNBC cells in irradiated fibroblast conditioned media led to decreased autophagy, increased migration, and increased tumorsphere outgrowth. These changes were determined to be caused by TNBC lactate uptake from irradiated fibroblast CM through lactate transporters MCT1 and MCT2, which was evaluated using the small molecule inhibitor AZD3965.

Adipocyte spheroids demonstrated dynamic changes in total and phosphorylated adipose triglyceride lipase and perilipin-1 protein expression following radiation damage, suggesting increased post-IR lipolysis and release of free fatty acids. CM collected from irradiated adipocyte spheroids confirmed increased free fatty acid release, suggesting these fatty acids could influence immune cells and cancer cells that are recruited to the radiation damaged mammary gland tissue following therapy. To evaluate this hypothesis, we loaded adipocytes with BODIPY-C16, irradiated the spheroids, and tracked transfer of BODIPY-C16 to BMDMs and TNBC cells through fluorescence imaging and flow cytometry. Macrophages and breast cancer cells co-cultured with irradiated adipocytes demonstrated increased BODIPY-C16 uptake compared to those cells co-cultured with control adipocytes, suggesting macrophages and TNBC cells may become metabolically activated. Metabolically activated macrophages have been demonstrated to increase the stemness of breast cancer cells through enhanced cytokine secretion, and lipid uptake by cancer cells has been suggested to increase their proliferative capacity. These hypotheses are currently being evaluated in conjunction with the lipolysis inhibitor Atglistatin.

Conclusions: In these studies, we uncover how fibroblasts and adipocytes, two important cell types of mammary gland adipose tissue that are damaged during radiation therapy, respond metabolically following radiation damage. We demonstrate a burgeoning link between altered metabolic profiles of irradiated adipose tissue cells and TNBC recurrence. We highlight how evaluating metabolic crosstalk following cancer treatment between damaged adipose tissue cells, immune cells, and breast cancer cells can help to uncover targetable metabolic pathways that could improve outcomes of TNBC patients receiving radiation therapy. We are currently investigating the mechanisms evaluated in our in vitro models here through lean and obese in vivo models on TNBC recurrence to uncover how these mechanisms are influenced by diet-induced obesity.

Research Interests: After completing my training at Vanderbilt and obtaining my doctoral degree, I intend to enter a postdoctoral position in an established lab that investigates the role of metabolism and adipose tissue in cancer outcomes with translational relevance to prepare me for applying to tenure-track faculty positions and establishing my independent laboratory. My long-term research interests are to: (1) develop increasingly advanced models of adipose tissue to allow for the study of the tissue under various pathologies while minimizing the need for using mouse models; (2) utilize these models to understand how different adipose tissue depots and microenvironments influence communication between adipocyte, stromal, endothelial, immune, and cancer cells to promote the formation of a pre-metastatic niche; and (3) understand what metabolic and biological pathways are important in the contexts of different comorbidities such as diabetes, obesity, and chronic inflammation. Beyond my current area of research, I am also interested in studying other conditions such as aging and chronic stress/anxiety. Ultimately, through the development and study of these advanced adipose tissue models, I aim to discover druggable targets that can improve treatment outcomes for patients.

My current research has focused on understanding the impact of radiation therapy on mammary gland adipose tissue and how the metabolic response influences the generation of a pro-recurrent niche with a focus on both in vitro and in vivo models in the context of obesity. I am interested in expanding on this line of scientific questioning by exploring other adipose tissue depots or tissues that change in adiposity over time. For instance, bone marrow, a common site of cancer metastasis, increases in adiposity with age and osteoporosis. I am interested in how this change in adiposity influences tumor cell metastatic niche formation through both physical changes to the microenvironment as well as metabolic crosstalk between adipocytes, infiltrating tumor cells, and other cells of the bone marrow. Working to develop a 3D model of bone marrow that includes adipocyte spheroids and fluid flow would be an interesting engineering challenge. Utilizing this model to study metabolic crosstalk between cells and how cellular metabolism influences the establishment of a metastatic niche would be a unique research question. This thought process could easily be applied to other adipose tissue depots in the future.