(629a) Radiation Damaged Adipose Tissue Cells Metabolically Influence the Breast Cancer Tumor Microenvironment

Background and Motivation: Triple negative breast cancer (TNBC) patients have poor prognoses, high locoregional recurrence rates, and low post-recurrence survival rates. Due to the receptor status of TNBC, treatment for this subtype lacks targeted therapies 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 primary tumor site following radiation therapy due to chemotactic cues from the damaged normal tissue. Regardless of the mechanism of recurrence, the interactions of tumor cells and radiation-damaged normal tissue cells may help elucidate mechanisms that can be targeted to prevent recurrence in TNBC patients.

Altered metabolic profiles of cancer cells have been attributed to radioresistance and the formation of circulating tumor cells. Both radioresistant cancer cells and circulating tumor cells have been characterized by increased reliance on lipid metabolism; however, the metabolic alterations of normal tissue cells following radiation therapy have been significantly less studied. Since it has been identified that radiation damaged cells can impact non-irradiated cells through radiation-induced bystander effects, we hypothesized that metabolic responses to radiation damage by cells of the normal tissue could impact recruited or residual cancer cells as well as the post-treatment wound healing response, which may impact TNBC recurrence.

Our current work explores how specific cells within mammary adipose tissue, such as fibroblasts and adipocytes, metabolically respond to radiation damage, how this response impacts the local microenvironment and the wound healing process, and how these mechanisms impact TNBC recurrence.

Methods: For fibroblast experiments, murine NIH 3T3 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 Nile Red and Perilipin-2 immunofluorescence staining. Fatty acid uptake experiments were performed using BODIPY-labeled palmitate. 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 Bafilomycin A1. Conditioned media (CM) from fibroblasts was collected at 2- and 7-days post-IR and utilized in invasion and radiation-induced bystander effect assays. Luciferase-labeled 4T1 TNBC cells were used in a transwell migration and invasion assay with CM from irradiated fibroblasts as the chemoattractant. Additionally, TNBC cells were exposed to fibroblast CM for 24 hours, at which point the effect of secreted factors from irradiated fibroblasts on the induction of autophagy in TNBC cells was evaluated.

For adipocyte experiments, murine 3T3-L1 pre-adipocytes were differentiated into adipocytes, exposed to 10 Gy of IR, and evaluated against control cells receiving no radiation. Glucose and fatty acid transporter expression was evaluated through immunofluorescence staining. Conditioned media (CM) from adipocytes was collected at 2- and 7-days post-IR and utilized in proliferation assays. Luciferase-labeled 4T1 cells were exposed to adipocyte CM for 48 hours and proliferation was determined from bioluminescence measurements. At 5-days post-IR, RAW 264.7 macrophages were co-cultured in a transwell system with irradiated and control adipocytes for 48 hours, and macrophage metabolic activation was evaluated through immunofluorescence staining of perilipin-2.

Results: To begin exploring how radiation therapy impacts the lipid metabolism of fibroblasts, we visualized lipid droplets within the cells up to 7-days post-IR. Both Nile Red and Perilipin-2 immunofluorescence staining indicated that irradiated fibroblasts demonstrate increased lipid accumulation up to 7-days post-IR (p<0.05). Fatty acid uptake experiments utilizing BODIPY-labeled palmitate demonstrated that irradiated fibroblasts did not have increased fatty acid uptake from the extracellular environment, indicating that lipid accumulation was due to fatty acids being generated and transformed into triglycerides within the cell. LysoTracker Red DND-99 live cell imaging indicated increased lysosomal staining through both increased intensity and area, which we hypothesized was due to increased autophagic flux. Increased autophagy was confirmed via increased LC3B puncta analysis and inhibition of autophagic flux through Bafilomycin A1 incubation suggested lipid droplet accumulation in fibroblasts was due to autophagy and the recycling of organelles damaged by IR. To evaluate if irradiated fibroblasts could influence autophagy in 4T1 TNBC cells, CM from irradiated fibroblasts was collected at 2- and 7-days post-IR and exposed to 4T1 cells for 24 hours. LysoTracker Red DND-99 staining and LC3B puncta analysis indicated that fibroblast secreted factors increased autophagic flux in 4T1 cells. Interestingly, irradiated fibroblast secreted factors also increased the invasive index of 4T1 cells when fibroblast CM was utilized in a transwell invasion assay (p<0.05). Whether or not the increased autophagic flux in TNBC cells due to irradiated fibroblast CM has a direct influence on the invasive capacity of TNBC cells is currently being evaluated.

Irradiated adipocytes evaluated at 7-days post-IR demonstrated increased membrane expression of glucose transporters I and IV (p<0.05) and increased expression of fatty acid translocase (CD36) compared to unirradiated controls. 4T1 cells exposed to CM collected from irradiated adipocytes at this same 7-day timepoint showed a significant increase in proliferation when compared to that of control CM (p < 0.05). Additionally, RAW 264.7 macrophages co-cultured with irradiated adipocytes exhibited over a 2-fold increase in expression of perilipin-2 compared to those co-cultured with control adipocytes, indicating increased metabolic activation. Metabolically activated macrophages have been demonstrated to increase the stemness of breast cancer cells. These results suggest adipocytes respond metabolically to radiation damage and may promote the proliferation of TNBC cells directly or through the metabolic activation of macrophages, supporting a pro-tumor niche. This hypothesis is currently being evaluated using murine bone-marrow derived macrophages, primary adipocytes, and 4T1 TNBC cells in tri-culture experiments.

Conclusions: Our study demonstrates a burgeoning link between the metabolic activation of irradiated adipose tissue cells and TNBC recurrence. This work underscores the importance of evaluating individual cellular responses to radiation damage as a consequence of primary tumor treatment to better understand how wound healing impacts recurrence. Ultimately, our work will lead to specific mechanisms of metabolic crosstalk between radiation damaged cells and TNBC cells that can be targeted to improve radiation therapy and ultimately help decrease the likelihood of recurrence in TNBC patients.