(F-225) HER2+ breast cancer drives the in-vitro expression of Tumor-Associated Macrophage Markers in the Tumor Microenvironment

HER2+ breast cancer accounts for 15-20% of all breast cancer cases1,2. Monocytes present in the tumor microenvironment (TME) can differentiate into a spectrum of tumor-suppressive macrophages and tumor-promoting macrophages (Tumor-Associated Macrophages: TAMs)3, which promote tumor growth, angiogenesis, metastasis, and resistance to HER2-targeted therapies4. TAMs express CD206, CD163, and CD2045, however the mechanisms and subtypes of HER2+ breast cancer cells that reprogram macrophages into TAMs are still unclear. In this study, we present a systematic experimental protocol and analysis of TAM marker expression in HER2+ breast cancer-monocyte cocultures with a focus on breast cancer heterogeneity, macrophage survival factors, coculture ratios and duration .

Human pan-monocytes were isolated from healthy donor PBMCs. After differentiation using Macrophage Colony Stimulating Factor (M-CSF), the monocyte-derived macrophages were exposed to either HER2+ breast cancer cells in coculture, or HER2+ tumor conditioned medium (TCM) to generate TAMs. Six different HER2+ cell lines were tested, including HCC1954, BT474, SKBR3, HCC1569, HCC1419, and EFM192. M-CSF dosages were tested between 5 ng/mL-50 ng/mL. Macrophage differentiation time periods varied between 2 and 5 days, while polarization time periods varied between 3 days and 5 days. Cells were permeabilized and stained for CD206, CD163 and CD204 antibody markers utilizing immunofluorescent staining and flow cytometry. TAM marker intensity was quantified utilizing CellProfiler and R, graphs were created with GraphPad Prism 9, and data was statistically analyzed using two-sided Student’s T-Tests and One-Way ANOVA statistical tests.

Results: Conditioned media harvested from cell lines HCC1954 and BT474 yielded promising levels of the CD206 TAM marker in macrophages throughout many experimental conditions. Three experiments of note include 5ng/mL M-CSF for 2 days + 3 day exposure to CM (Figure 1A), 25ng/mL M-CSF for 2 days + 5 day exposure to CM (Figure 1B), and 50 ng/mL M-CSF for 5 days + 3 day exposure to CM (Figure 1C). In each figure, CD206 intensity increased in the wells exposed to HCC1954 72hr CM (red plots) compared to the control well (blue plots), suggesting increased levels of TAMs. In addition, previous studies have shown that BT474 do not effectively reprogram TAMs in macrophages; this is shown with nonsignificant differences in Figures 1A and 1B (green plots), however synergistic reprogramming was observed at a higher M-CSF dose (Figure 1C). Furthermore, increases in other TAM markers were detected in similar conditions, including CD204 and CD163 (data not shown). 

Discussion/Conclusion: Our results have shown that HCC1954 CM elicits a significant increase in TAM macrophage markers on monocyte-derived macrophages in vitro. This significant increase despite experimental conditions varying widely in time and M-CSF concentration is promising for future analysis on the functional effects of macrophages on cancer cell growth and drug response. Planned future experiments include measuring the infiltrative ability of macrophages in a simulated TME when exposed to HCC1954 and BT474, and identifying signaling mechanisms that play a role in tumor-macrophage crosstalk.

1      Ban, M., Petric Mise, B. & Vrdoljak, E. Early HER2-Positive Breast Cancer: Current Treatment and Novel Approaches. Breast Care (Basel) 15, 560-569, doi:10.1159/000511883 (2020).

2      Harbeck, N. et al. Breast cancer. Nat Rev Dis Primers 5, 66, doi:10.1038/s41572-019-0111-2 (2019).

3      Noy, R. & Pollard, J. W. Tumor-associated macrophages: from mechanisms to therapy. Immunity 41, 49-61, doi:10.1016/j.immuni.2014.06.010 (2014).

4      Huang, X., Cao, J. & Zu, X. Tumor-associated macrophages: An important player in breast cancer progression. Thorac Cancer 13, 269-276, doi:10.1111/1759-7714.14268 (2022).