Tumor-associated macrophages (TAMs) are the most abundant immune population in many tumor microenvironments and substantially affect cancer progression, immune evasion, and treatment resistance. Triple-negative breast cancer (TNBC) lacks ER, PR, and HER2 receptors, limiting targeted therapy options and making conventional chemotherapy the mainstay of treatment. TNBC tumors often contain high levels of TAMs, and M2-like TAMs in particular have been implicated in angiogenesis, invasion, suppression of anti-tumor immunity, and reduced chemo- and radiotherapy efficacy. Given macrophage plasticity and the importance of cell–cell interactions in the tumor microenvironment, incorporating macrophages into 3D in vitro models may improve the physiological relevance of preclinical assays.
The study used the aggressive TNBC cell line MDA-MB-231 and THP-1 human monocytes to establish co-culture spheroids. Cells were maintained in D-MEM/F-12 medium with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO2. The 3D spheroid platform was chosen to capture cell–cell and cell–matrix interactions not represented in 2D monolayers and to allow examination of reciprocal phenotype changes between cancer cells and macrophages.
THP-1 cells were used as a source of macrophages and were differentiated and polarized to generate M1-like and M2-like phenotypes for co-culture with MDA-MB-231 spheroids. Specific differentiation and polarization reagents and concentrations were reported in the Methods section of the source article. The resulting M1- and M2-like macrophages were incorporated into 3D spheroids to investigate bidirectional interactions.
Co-culture spheroids were exposed to standard chemotherapies used in TNBC: doxorubicin and paclitaxel. Drug efficacy was assessed by measuring spheroid viability and chemosensitivity in the presence or absence of polarized macrophages. The study compared responses in 3D co-cultures to expectations from 2D culture systems.
The work evaluated cancer cell viability and proliferation within spheroids, changes in epithelial–mesenchymal transition (EMT) markers, and dynamics of cancer stem cell (CSC) populations when cancer cells were co-cultured with polarized macrophages. Flow cytometry and other phenotyping techniques were used to assess macrophage marker expression and cancer cell phenotypes within the spheroids.
Bulk RNA sequencing (RNA-seq) was performed on spheroid samples and analyzed for differentially expressed genes (DEGs) and enriched pathways. To estimate cell-type fractions within mixed samples and help resolve bidirectional phenotype transitions between macrophage subtypes, the authors applied CIBERSORTx deconvolution to the RNA-seq data. The raw and processed RNA-seq data are available at GEO under accession GSE304137, and code for the RNA-seq analysis is hosted at the reported GitHub repository.
In 3D spheroids, co-culture with M2-like macrophages increased viability of MDA-MB-231 cancer cells compared with cancer-only spheroids. Despite this pro-survival effect, both M1-like and M2-like macrophages increased the chemosensitivity of 3D spheroids to doxorubicin and paclitaxel. These results indicate that macrophage presence alters baseline viability and modulates treatment response in complex ways within the 3D tumor model.
When cultured with MDA-MB-231 cells in 3D spheroids, both M1- and M2-polarized macrophages lost some of their initial polarization and developed a mixed M1–M2 phenotype rather than maintaining distinct M1 or M2 states. RNA-seq deconvolution using CIBERSORTx supported these bidirectional phenotypic transitions. Importantly, the extent of switching was asymmetric: a smaller fraction of M1-like macrophages shifted toward an M2-like state than the fraction of M2-like macrophages that shifted toward M1-like characteristics in the co-cultures.
Compared with 2D cultures, MDA-MB-231 cells in 3D spheroids exhibited an expected mesenchymal transition and numerous differentially expressed genes, with enrichment of both tumor-promoting and tumor-suppressing pathways. However, when co-cultured with either M1- or M2-like macrophages, cancer cells showed only partial EMT phenotype changes. Co-cultured cancer spheroids contained a mixture of pro-inflammatory and anti-inflammatory DEGs, reflecting a complex and nonbinary impact of macrophages on cancer cell gene expression.
The findings demonstrate that integrating macrophages into 3D TNBC spheroids produces a dynamic environment where macrophage phenotypes shift and influence cancer cell survival, EMT-related programs, and response to chemotherapy. The asymmetric bidirectional transition of macrophage phenotypes and the coexistence of pro- and anti-inflammatory signals suggest that TAM-targeting strategies may need to account for macrophage plasticity and the heterogeneous nature of macrophage–cancer cell interactions. Combining standard chemotherapies such as doxorubicin and paclitaxel with TAM-targeting or repolarizing agents could be a rationale to reprogram the tumor microenvironment and potentially re-sensitize TNBC cells, although specific therapeutic strategies and efficacy were not directly tested beyond the chemotherapies reported.
The authors deposited raw and processed RNA-seq data in the NCBI Gene Expression Omnibus under accession GSE304137. The analysis code for RNA-seq is available at the study’s GitHub repository as reported in the source article. Other experimental data and supplementary materials were included in the publication.
Specific reagent concentrations and detailed stepwise protocols for macrophage polarization and spheroid formation are described in the Methods section of the source article; readers should consult the original publication for protocol replication. The study used THP-1-derived macrophages and a single TNBC cell line (MDA-MB-231), which may limit generalizability to primary macrophages or other TNBC models. The source article did not report in vivo validation within this manuscript.
This study establishes a reproducible 3D co-culture spheroid model combining MDA-MB-231 TNBC cells with THP-1-derived polarized macrophages. The model reveals bidirectional and asymmetric macrophage phenotype transitions, partial induction of EMT in cancer cells, coexistence of inflammatory and anti-inflammatory gene programs, and altered chemosensitivity to doxorubicin and paclitaxel. The platform can be used to study dynamic interactions within a heterogeneous tumor microenvironment and to improve preclinical evaluation of therapies that target both tumor cells and TAMs.