(176t) The Metabolic Impact of Radiation Damage within the Tumor Microenvironment

Authors: 
Corn, K., Vanderbilt University
Rafat, M., Vanderbilt University
Background

Triple negative breast cancer (TNBC) is an invasive and highly metastatic subtype of breast cancer that lacks expression of the estrogen and progesterone hormone receptors and the human epidermal growth factor 2 receptor. Without these receptors, designing targeted therapeutics is difficult, and treatment instead relies on conventional cytotoxic chemotherapy followed by adjuvant radiation therapy after a mastectomy or lumpectomy. TNBC generally has a poor prognosis and high locoregional recurrence rate. Patients with TNBC have the highest rate of recurrence within the first 5 years after diagnosis, and post-recurrence survival is decreased in TNBC compared to other types of breast cancers. Our group’s previous work has shown that recurrence in TNBC is facilitated by circulating tumor cells (CTCs) recruited to the primary tumor site following radiation therapy due to chemotactic cues from the damaged tissue. These CTCs are preceded by a large influx of macrophages, which is abrogated by the presence of CD8+ T cells. Our current work explores the cellular mechanisms of irradiated adipose tissue that may facilitate the recruitment of CTCs and the proliferation of re-seeded cancer cells.

Methods

Adipose tissue is comprised of numerous cell types, among them adipocytes, resident macrophages, pre-adipocytes, and fibroblasts. We explored the impact of radiation damage on stromal cells through evaluating lipid droplet formation in murine NIH 3T3 fibroblasts, 3T3-L1 pre-adipocytes, and 3T3-L1 differentiated adipocytes. Cells were irradiated in vitro with 2, 6, or 10 Gy of ionizing radiation and evaluated against control cells receiving no radiation. Cells were stained with Oil Red O to identify storage of neutral lipids within cell bodies at 1, 2, and 3 days post-irradiation. Average lipid droplet sizes and total number of lipid droplets were normalized against cell count. Co-culture studies were performed to understand the impact of radiation-damaged stromal cells on cancer-cell proliferation in an in vitro model of CTC re-seeding and recurrence. Murine 4T1 TNBC cells were cultured with either irradiated or non-irradiated stromal cells in a transwell assay. 4T1 invasion and proliferation were analyzed.

Results

In evaluating the lipid droplet sizes of the stromal cells post-radiation, we observed an increase in the number and size of lipid droplets, which was dependent on both radiation dose and days following radiation damage. These results appeared to link radiation damage and metabolism through the increase in lipid content of the stromal cells. We hypothesized that these radiation-damaged stromal cells could provide a pro-tumorigenic environment following radiation therapy. If CTCs are recruited to the radiation-damaged tissue, as indicated by previous studies, this environment could allow for increased proliferation after CTC re-seeding. We tested this hypothesis by co-culturing murine 4T1 cells with either normal or radiation-damaged adipocytes and observed an increase in 4T1 invasion and proliferation following co-culture with radiation-damaged adipocytes.

Our study establishes an initial connection between radiation damage, stromal cell metabolism, and cancer recurrence and progression. This work underscores the importance of evaluating metabolic changes due to secondary damage to the microenvironment as a result of primary tumor treatment. These results suggest that damaged stroma may facilitate tumor regrowth, which merits further exploration and could lead to new insights into TNBC recurrence mechanisms.