Bidirectional phenotypic transition in macrophages influences triple-negative breast cancer cell immune evasive patterns and response to chemotherapies in a co-culture spheroid model

Tumor-associated macrophage (TAM) infiltration is a critical characteristic of triple-negative breast cancer (TNBC) related to drug resistance and poor prognosis. Integrating macrophages into TNBC spheroids is crucial to improve the accuracy of 3D in vitro models that mimic the complexity of the tumor microenvironment (TME) and assess treatment response. However, this remains challenging since the reciprocal effects of these two cell types on each other are not fully understood. In this study, we used the TNBC cell line, MDA-MB-231, and polarized M1-like or M2-like macrophages derived from THP-1 monocytes to establish 3D co-culture spheroids to examine bidirectional interactions between these cells and responses to chemotherapy. Drug efficacy, epithelial-mesenchymal transition (EMT) in cancer cells, macrophage phenotypes, and RNA sequencing, including pathway enrichment analysis, were performed in 3D spheroids. CIBERSORTx deconvolution of RNA sequencing results facilitated the separation of cell types within mixtures to estimate their corresponding cell fractions. We observed that M2 macrophages increased the viability of MDA-MB-231 cells in 3D spheroids, while both M1 and M2 macrophages increased the chemosensitivity of 3D spheroids to doxorubicin and paclitaxel. Interestingly, instead of maintaining their phenotypes, both M1 and M2 macrophages lost some polarization and formed a mixed M1-M2 phenotype when co-cultured with MDA-MB-231 cells in 3D spheroids, a phenomenon further supported by RNA-seq deconvolution analysis. However, the fraction of M1-like macrophages shifting to M2-like was much lower than the fraction of M2-like macrophages shifting to M1-like in the 3D co-cultures. Compared with 2D cultures, an expected mesenchymal transition, numerous differentially expressed genes (DEGs) and various pathways, including both tumor-promoting and tumor-suppressing genes, were observed in 3D spheroid MDA-MB-231 cells. However, both M1- and M2-like macrophages induced only partial EMT phenotype changes of cancer cells in co-cultures. Furthermore, a coexistence of pro-inflammatory and anti-inflammatory DEGs was observed in both M1 and M2-like co-cultured cancer spheroids. In conclusion, our findings present an effective 3D co-culture system of breast cancer cells and integrated macrophages for studying dynamic cellular phenotype changes and reciprocal interactions in a heterogeneous environment to mimic aspects of the TME and enhance the accuracy of preclinical in vitro treatment response studies.