Presentation: 2025 ND EPSCoR Annual conference 

October 21, 2025, NDSU Memorial Union, Fargo, North Dakota

Identifying Macrophage and Triple Negative Breast Cancer (TNBC) Phenotype Changes in a 3D Co-culture Model

A limitation of in vitro 3D cell culture models for TNBC research is often the lack of immune cells, which play important roles in regulating pro- and anti-tumorigenic responses during cancer development. We optimized a co-culture 3D spheroid model of differentiated M1- or M2-like macrophages from THP-1 cells and triple-negative MDA-MB-231 cells in our study. Gene expression profiles, cell fractions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway enrichment of cancer cells and macrophages were analyzed using RNA sequencing. We found that MDA-MB-231 cells in 3D spheroids showed numerous differentially expressed genes (DEGs) and various involved pathways compared to 2D monolayer cultures. Deconvolution analysis of the RNA-seq results showed a dramatic change in gene expression of both types of macrophages in the 3D co-cultured spheroids. For instance, a reduced number of DEGs was observed between M1- and M2-like macrophages when co-cultured with the cancer cells, and both pro- and anti-inflammatory mRNA changes were observed. A small fraction of M1-like macrophages switched to an M2-like phenotype, while a much larger fraction of M2-like macrophages switched to an M1-like phenotype in the 3D co-cultures. In conclusion, our 3D co-culture model recapitulated aspects of the complex multi-cellular environment of tumor development. The instability of macrophage gene expression when co-cultured with MDA-MB-231 cells suggests a malleable immune phenotype influenced by cancer cell interaction. Developing immunotherapies targeting macrophage phenotype transitions could be a potential adjunct strategy to improve the outcome of TNBC.