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The Impact of M2-polarized macrophages on the oncogenic activity of hedgehog signaling in triple negative breast cancer cells

Undergraduate #2
Discipline: Biological Sciences
Subcategory: Cancer Research
Session: 1
Room: Council

Alexxa C. Cruz-Bonilla - University of Puerto Rico, Mayaguez


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) through qRT-PCR and Western Blot. The qRT-PCR results showed no significant difference in the levels of GLI1 and SMO genes in TAM monocultures +/- SHH ligand, indicating the absence of canonical Hh signaling activity. The SHH ligand was only expressed in tumor cells but not in TAMs. Tumor cell growth was examined in a co-culture system when increasing TNBC to a set TAM quantity, with a 2:1 and 1:1 ratio. These ratios were also observed under -/+ NVP-LDE225 (NVP) treatment; a Hh pathway inhibitor. Macrophage retention in the co-cultures was analyzed in comparison to the TAM monocultures, these demonstrated no statistical difference across all conditions. All experiment replicates of n=3 that were performed demonstrated replicability amongst all samples. 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. Utilizing Dunnet’s multiple comparison test, the results showed a decreasing tendency in the tumor proliferation rates between the co-cultures treated -/+ NVP and when compared to the TNBC monocultures. In the absence of NVP, both 2:1 and 1:1 co-culture, the difference in proliferation rate was significantly higher (p<0.0001) than in the TNBC monocultures. Where the 1:1 co-culture -NVP conditions had a p<0.0001 and +NVP a p=0.0020, decreasing the proliferation rate when in treatment. Overall, results pointed to an increase in TNBC growth when in direct contact with TAMs and lower proliferation rates in co-cultures under NVP treatment. Utilizing the same parameters established for the co-culture system, a tri-culture model with a fibroblast cell line is currently being tested. Future work also includes replicating into the three-dimensional platform to better understand the role of cell-to-cell contact and the metabolic processes that occur in vivo.

Funder Acknowledgement(s): National Institutes of Health

Faculty Advisor: Prof. Maribella Domenech, maribella.domenech@upr.edu

Role: This research has been conducted solely by myself over a four-year span in unison with the Principle Investigator, Prof. Maribella Domenech.

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This material is based upon work supported by the National Science Foundation (NSF) under Grant No. DUE-1930047. Any opinions, findings, interpretations, conclusions or recommendations expressed in this material are those of its authors and do not represent the views of the AAAS Board of Directors, the Council of AAAS, AAAS’ membership or the National Science Foundation.

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