Triple-negative breast cancer (TNBC), accounting for ~15-20% of all breast cancer (BC) diagnoses worldwide, has the poorest prognosis, due to its great heterogeneity and limited treatment options, with a death rate in the metastatic setting disproportionately higher than any other BC subtypes. Analysis of the tumor microenvironment (TME) in TNBC have revealed a marked infiltration of tumor associated macrophages (TAMs), a heterogenous population with high plasticity. TAMs include both M1 (anti-tumor) and M2 (pro-tumor) phenotypes, with the latter predominating in TNBC. The M2-like TAMs contribute to the establishment of an immunosuppressive milieu, promoting tumor growth, metastasis, and resistance to therapy. During tumorigenesis, there is a complex and dynamic interplay between cancer cells and TAMs. Here, we aim to explore the crosstalk between chemoresistant cancer cells and TAMs within the TNBC microenvironment.

Cisplatin-resistant (Cis-Pt-R) and doxorubicin-resistant (Dox-R) cell lines were generated by chronic exposure of TNBC MDA-MB-231 cells to the chemotherapeutics. Human THP-1 monocytes were differentiated into M0 macrophages by incubation with PMA. Once adherent, cells were maintained in PMA-free medium to obtain resting macrophages. Conversely, murine RAW264.7 cells were directly used as M0 macrophages. For polarization experiments, human and murine M0 macrophages were treated with LPS and IFN-γ to induce the M1 phenotype, or IL-4/IL-10 to induce the M2 phenotype. To mimic the TNBC microenvironment in vitro, M0 macrophages were exposed to conditioned medium (CM) from TNBC 4T1 (murine setting) or Cis-Pt-R and Dox-R cells (human setting). In addition, to further investigate tumor-M2 like TAMs crosstalk within the TME, chemoresistant TNBC cells were co-cultured with TAMs. Macrophage phenotype and M1/M2 marker expression were validated by immunoblotting and confocal microscopy. Functional characterization was performed using ELISA assays.

TAMs generated using CM from chemoresistant TNBC cells showed a significant increase in the expression of M2-like TAMs (e.g. CD206, CD44) while M1-like TAMs marker levels (e.g. CD86) remained substantially unchanged. Quantification of inflammatory and anti-inflammatory cytokine production revealed M2 polarization. Furthermore, co-culture experiments confirmed that cultured with TAMs, chemoresistant TNBC cells promote the M2-polarization in TAMs. Studies are ongoing to assess the effects of polarized M2 macrophages in increasing EMT, stemness, and chemoresistance of TNBC cells.

Our findings show that chemoresistant TNBC cells actively modulate the TME by skewing macrophage differentiation toward the M2 phenotype, thus providing an opportune system to investigate therapeutic approaches targeting the crosstalk between tumor cells and TAMs.