(533c) Understanding the Roles of Lean and Obese Adipose Tissue on Triple Negative Breast Cancer Recurrence

Background: Triple negative breast cancer (TNBC) patients have poor prognoses, high locoregional recurrence rates, and low post-recurrence survival rates, and these statistics are exacerbated for patients with obesity. Our group’s previous work in a pre-clinical model has shown that recurrence in TNBC may be facilitated by circulating tumor cells (CTCs) recruited to the primary tumor site following radiation therapy due to chemotactic cues from the damaged normal tissue. Our current work explores how specific cells within adipose tissue, such as adipocytes, respond to radiation damage, how these cells impact immune cell recruitment and the wound healing process, and how these processes change in the context of obesity and impact TNBC recurrence.

Methods: For in vitro experiments, murine 3T3-L1 pre-adipocytes were differentiated into adipocytes, which were then exposed to 10 Gy of ionizing radiation (IR) and evaluated against control cells receiving no radiation. Conditioned media (CM) from adipocytes was collected at 2 and 7 days post-IR and utilized in proliferation and invasion assays. Luciferase-labeled 4T1 cells were exposed to adipocyte CM for 48 hours and proliferation was determined from bioluminescence measurements. For co-culture proliferation studies, luciferase-labeled 4T1 cells were co-cultured with irradiated adipocytes for 48 hours. Additionally, 4T1 cells were used in a transwell invasion assay with CM from irradiated adipocytes as the chemoattractant.

For in vivo experiments, 6-week-old female BALB/c mice were fed a high-fat diet (60% kcal from fat) to induce obesity and were compared to control groups fed a sucrose-matched low-fat diet (10% kcal from fat) for 15 weeks. The inguinal mammary fat pads (MFPs) were resected for further analysis. MFPs from both diet-induced obesity (DIO) and lean groups were subjected to either 0 or 20 Gy of ex vivo IR and incubated for 2 days in serum-free media for CM collection. MFPs were then fixed, processed, and stained for hematoxylin and eosin (H&E) analysis. Cytokine expression in CM between the groups was determined via a murine 31-plex Luminex assay.

Results: 4T1 cells exposed to CM collected from adipocytes 7 days post-IR showed a significant increase in proliferation when compared to that of control CM (p < 0.05), which was confirmed in co-culture studies. This result was interesting especially since there was no difference in proliferation between TNBC cells exposed to CM from either irradiated or control adipocytes at the 2-day time point. Additionally, an increase in migration, but not invasion, was observed when TNBC cells were exposed to CM from radiation-damaged adipocytes at 2 days post-IR.

Obese mice showed an over 2-fold increase in MFP weight (p < 0.001) and microvesicular steatosis compared to lean mice. After ex vivo radiation, MFPs from DIO mice exhibited differences in adipocyte morphology, including decreased density and variable size, and decreased ECM deposition compared to lean MFPs, suggesting that IR promotes distinct microenvironmental changes in obese tissues. Cytokine analysis of CM collected from irradiated DIO compared to lean MFPs showed a greater than 2-fold upregulation in post-IR secretion of CXCL-2, CXCL-10, and IL-6 and an almost 2-fold downregulation of IL-5 and IL-7 secretion. This altered secretion profile highlights the unique radiation damage response in obese MFPs and suggests that irradiated obese MFPs may influence immune cell recruitment dynamics, which will be studied in future experiments.

Conclusions: Our study demonstrates the potential role of radiation-damaged adipocytes and obese mammary adipose tissue in the recruitment and proliferation of CTCs following radiotherapy. This work underscores the importance of evaluating individual cell and whole tissue responses to radiation damage in the microenvironment as a consequence of primary tumor treatment to better understand how wound healing impacts recurrence. These results suggest that the damaged stroma may facilitate tumor regrowth, which merits further exploration and could lead to new insights into TNBC recurrence mechanisms.