Associate Professor University of Georgia, United States
Introduction:: Breast cancer is the second most lethal type of cancer in women between ages 40-50 or in their post-menopausal phase. It is characterized by three main hormones: estrogen (ER), progesterone (PR) and human epidermal growth factor receptor type 2 (HER2) categorizing breast cancer into different subtypes. Luminal A breast cancer is ER+, HER2- and/or PR+ and has the best prognosis of the various subtypes. Triple negative breast cancer (TNBC) is receptor negative, most aggressive and has the worst prognosis among cancer subtypes. The majority of breast cancer deaths in women are due to metastasis or the spread of the cancerous tumor cells to other organs in the body. Breast cancer metastasis is affected by numerous factors or conditions, including obesity. Adipocytes, or fat cells, have been reported to become dysfunctional in obese conditions, secreting several hormones and factors in the tumor microenvironment that promote cancer cell proliferation and tumor development. These factors are more likely to impact poor prognosis breast cancers, such as TNBC. Co-cultures can be used to identify the impact of individual adipocyte-secreted factors on breast cancer cell development in vitro. Our work focuses on assessing the effects of obese adipocytes on the behavior of metastatic breast cancer cells. An experiment was conducted with two TNBC (HCC-1806 and MDA-MB-231), a luminal A (MCF-7) and a benign (MCF-10A) breast cancer cell lines to observe the impact of different adipose conditions on the proliferation and migration rates of breast cancer cells.
Materials and Methods:: Mesenchymal stromal cells (D1 cells, ATCC) were cultured in either growth media (Low-glucose DMEM, FBS, P/S), lean adipogenic differentiation media (High-glucose DMEM, FBS, P/S, IBMX, Rosiglitazone, Indomethacin, Insulin, Dexamethasone), or obese adipogenic differentiation media (lean media + Bovine Serum Albumin + 700 mM stearic acid). The adipogenic media formulations were identified from preliminary studies where lean and obese adipocytes were observed and analyzed to identify concentrations of stearic acid to differentiate obese adipocytes. Cells were differentiated to an adipogenic state in HTC Transwell inserts (Corning) for 10 days. The four different breast cancer cell lines (HCC-1806, MDA-MB-231, MCF-7, MCF10A) were cultured in their appropriate medias in CytoViewZ plates (Axion Biosystems). The HTC inserts were added to the CytoViewZ plates 24 hours after seeding the cancer cells. After 48 hours of culturing, nutrient-reduced media was added to the cancer cultures. Then at 60 hours, a scratch was induced in the cancer cultures to observe migration rates via a wound healing assay as seen in Figure 1A. Data was collected using a non-destructive bioelectronic assay system (Axion Biosystems TrayZ), with impedance and barrier index data collected continuously in real-time throughout the culturing period.
Results, Conclusions, and Discussions:: Monitoring of the cell impedance data revealed a distinct impedance profile for each of the tested cell lines (Figure 1B). The benign tumor cell line (MCF-10A) had a different trajectory compared to the cancerous cell lines. Specifically, the impedance values were significantly lower than the MCF-7 cell impedance values, differentiating the behaviors of cancer cells vs. benign tumor cells. While the MDA-MB-231 cells had significantly lower impedance values compared to the HCC-1806 cells, both cancer cell lines followed the same trajectory, demonstrating similarities in the behaviors of different TNBC cell lines (Figure 1B). The presence of adipocytes in the cancer cell environment impacted the proliferation rates of the cancer cells, in different aspects. Both TNBC cell lines had similar proliferation rates in the presence of lean vs. obese adipocytes. Likewise, both the MCF-7 and MCF-10A cells exhibited significant impacts on their proliferation due to the different co-cultures prior to the scratch (Figure 1C). The MCF-10A cells had significantly higher impedance values compared to the other cell lines with co-cultures, supporting the impact of the adipocyte culture on tumor cells. Both the lean and obese cultures showed significant increases in impedance values compared to the control culture, confirming the impact of the presence of adipocytes on cancer cell cultures. Given the dysfunctional state of adipocytes observed under obese conditions, it was anticipated that alterations in cell behavior would result in different proliferation rates amongst different cancer subtypes. The findings from our analyses demonstrate visible differences between different subtypes and similar trajectories amongst different cell lines both characterized as TNBC. Preliminary studies showed slight differences in the response to lean and obese adipocytes, which translates to the slight differences in the impact observed on breast cancer cell proliferation. However, there are still limitations to be addressed to evaluate the cell-cell and cell-microenvironment interactions between cancer cells and adipocytes. Additionally, the differences in the responses between the cancer cell types can be better evaluated with other studies such as morphological studies to help predict the numerous factors that affect metastasis, which will be a focus of future work.
Acknowledgements (Optional): :
Many thanks to Axion Biosystems and Georgia Research Alliance for providing the Maestro Z to the Tissue RegenX Lab. Funding for this work provided by National Science Foundation CAREER Award #2145521.
