The Impact of M2-Polarized Macrophages in Hedgehog Signaling Activity and Proliferation of Triple Negative Breast Cancer Cells

Hedgehog (Hh) signaling activity is highly upregulated in about 30-40% of tumor specimens and it has been associated with poor prognosis in breast tumors of the triple-negative sub-type. Hh signaling activity has been linked with the phenotype of tumor-associated macrophages (TAMs) in tumors, yet its oncogenic role within the context of triple negative breast cancer (TNBC) is not well-understood. We hypothesize that TAMs support the oncogenic activity of Hh signaling in TNBC in a paracrine manner. Hh signaling activity was examined in monocytes, TAMs, and MDA-MB-231 (TNBC cell line). Analysis of gene expression levels showed no significant difference in the levels of GLI1and SMOgenes in TAM monocultures +/- SHH ligand, indicating absence of canonical Hh signaling activity. Protein levels of the SHH ligand were only found expressed in TNBC cells. Macrophage cell retention and tumor cell growth was examined in monoculture and a co-culture system at increasing ratios, 2:1 and 1:1 TAM to TNBC ratios. NVP-LDE225 (NVP) was used as a pharmacological suppressor of the Hh pathway. Macrophage retention in the co-cultures were analyzed in comparison to the TAM monocultures, these demonstrated no statistical difference across all conditions. Initial examination of growth rates indicates that NVP does not affect the TAM and TNBC monocultures; having a non-significant difference when analyzed through a One-Way ANOVA. For both co-culture ratios, Tukey’s multiple comparisons test indicated that the TAM promotes tumor cell proliferation, and this effect was independent of NVP treatment indicating that the Hh pathway is not involved in this paracrine interaction. Future work will seek to examine this paracrine interaction in the presence of fibroblasts which are a well-known cell target of Hh inhibitors. This will allow to determine the impact of TAM in the oncogenic activity of the Hh pathway within the context of the tumor-stroma interaction.