---
title: "Macrophage phenotypic shifts in 3D co-culture alter TNBC immune evasion and chemotherapy response"
id: "plos-one-13-bidirectional-phenotypic-transition-in-macrophages-influences-triple-negative"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-13-bidirectional-phenotypic-transition-in-macrophages-influences-triple-negative"
content_type: "clinical_feed_article"
specialty: "Oncology"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0356198"
published_at: "2026-08-14T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Macrophage phenotypic shifts in 3D co-culture alter TNBC immune evasion and chemotherapy response
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-13-bidirectional-phenotypic-transition-in-macrophages-influences-triple-negative
- **Specialty:** [Oncology](https://medichelpline.com/clinical-feed/oncology.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0356198)
- **Published At:** 2026-08-14T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and influence triple-negative breast cancer (**TNBC**) progression, immune evasion, and therapeutic resistance. This study models TAM–TNBC interactions using 3D co-culture spheroids of MDA-MB-231 cancer cells and THP-1-derived polarized macrophages. - The authors generated M1-like and M2-like macrophages from THP-1 monocytes and combined them with MDA-MB-231 cells to form 3D spheroids intended to better mimic the tumor microenvironment than 2D monolayers. - Evaluations included cell viability, proliferation, epithelial–mesenchymal transition (**EMT**) markers, cancer stem cell (CSC) dynamics, drug efficacy assays with **doxorubicin** and **paclitaxel**, flow cytometry phenotyping, RNA sequencing (RNA-seq), pathway enrichment, and CIBERSORTx deconvolution to estimate cell-type fractions. - Key findings: M2-like macrophages increased MDA-MB-231 viability in 3D spheroids, whereas both M1- and M2-like macrophages increased chemosensitivity of co-cultured spheroids to doxorubicin and paclitaxel. - Macrophage phenotypes were not stable in co-culture: both M1 and M2 macrophages lost polarization and developed a mixed M1–M2 phenotype when cultured with cancer cells in 3D. RNA-seq deconvolution supported bidirectional phenotype transitions. - The extent of phenotype switching was asymmetric: a smaller fraction of M1-like macrophages shifted toward M2-like phenotype compared with a larger fraction of M2-like macrophages shifting toward M1-like in the 3D co-cultures. - Compared with 2D cultures, MDA-MB-231 cells in 3D spheroids showed expected mesenchymal features, many differentially expressed genes (DEGs), and enrichment of pathways including both tumor-promoting and tumor-suppressing signals. - Co-culture with either M1- or M2-like macrophages induced only partial EMT changes in cancer cells; co-cultured cancer spheroids displayed a coexistence of pro-inflammatory and anti-inflammatory DEGs. - The study establishes an effective 3D co-culture platform for probing dynamic, reciprocal interactions between TNBC cells and macrophages and for improving preclinical in vitro assessment of treatment responses. Data and RNA-seq code are publicly available (GEO GSE304137 and GitHub repository reported).
## Clinical Analysis & Structured Key Points
Bidirectional phenotypic transition in macrophages influences triple-negative breast cancer cell immune evasive patterns and response to chemotherapies in a co-culture spheroid model | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract 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. Citation: Cheng C, McGregor BA, Hur J, Combs CK (2026) Bidirectional phenotypic transition in macrophages influences triple-negative breast cancer cell immune evasive patterns and response to chemotherapies in a co-culture spheroid model. PLoS One 21(8): e0356198. https://doi.org/10.1371/journal.pone.0356198 Editor: Xiaozhe Han, Nova Southeastern University, UNITED STATES OF AMERICA Received: March 31, 2026; Accepted: July 28, 2026; Published: August 14, 2026 Copyright: © 2026 Cheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The raw and processed RNA-seq data supporting the conclusions of this article are available in the NCBI Gene Expression Omnibus database under GEO Accession ID GSE304137. The code used for the RNA-seq analysis presented in this manuscript is available at https://github.com/UND-CDAC/tnbc-3d-coculture-rnaseq . All other data generated or analyzed during this study are included in this article and its supplementary materials. Funding: Financial support was provided by the National Science Foundation Experimental Program to Stimulate Competitive Research Grant 1946202 to CKC. The North Dakota Flow Cytometry and Cell Sorting Core (NDFCCS) at UND was supported by NIH/NIGMS grant P20GM113123, DaCCoTA CTR NIH grant U54GM128729, and UND SMHS funds. The Computational Data Analysis Core at UND was supported by NIH/NIGMS grant P20GM113123. The INBRE Microscopy Core at UND was supported by NIH/NIGMS grant P20GM103442. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer malignancy among females worldwide [ 1 ]. The World Health Organization (WHO) reported that 2.3 million women were diagnosed with breast cancer, with 670,000 global deaths in 2022 [ 2 ]. Triple-negative breast cancer (TNBC) is a highly aggressive subtype of BC characterized by the absence of receptors for estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER-2), which limits the efficacy and application of hormone therapies and immunotherapies, leaving conventional therapies as the essential treatment option [ 3 – 5 ]. In addition, the unique tumor microenvironment (TME) of TNBC, including a high level of tumor-infiltrating immune cells, especially tumor-associated macrophages (TAMs), amplifies the complexity of this subtype of breast cancer and contributes to poor prognosis [ 6 ]. As the most abundant immune population in the TME, macrophages comprise over 50% of hematopoietic cells and play a pivotal role in cancer cell survival and progression [ 7 ]. These macrophages play a dual role in cancer development, with an M1-like pro-inflammatory and immunostimulatory phenotype exhibiting anti-tumor properties, while an M2-like anti-inflammatory, angiogenic, and immunosuppressive phenotype promotes tumor growth and immune evasion [ 8 , 9 ]. In TNBC, M2-like TAMs play predominant roles from tumor initial onset to metastasis, including angiogenesis, invasion, and metastasis promotion, as well as suppression of anti-tumor immune responses and reduction of chemo- and radiotherapy efficacy [ 10 , 11 ]. In addition, studies have demonstrated that TNBC cells possess a strong ability to evade immune clearance via different patterns, such as regulating immune checkpoints, altering epigenetic and metabolic profiles, changing genomic architecture, and recruiting suppressive immune cells, of which the most important ability is to induce macrophages to polarize into an M2-like phenotype, further facilitating tumor cells escape from the immune response [ 12 , 13 ]. Increasing evidence indicates that TAM-targeting therapies, including inhibiting macrophage recruitment, eliminating TAMs, and repolarizing TAMs, have improved therapeutic effects in cancer treatment. Furthermore, a combination of chemotherapies and TAM-targeting therapies has shown promising outcomes in both preclinical and clinical cancer research with a goal of overcoming TAM-mediated resistance [ 11 – 14 ]. Therefore, due to their high plasticity and complex transition abilities, it is essential to precisely understand how tumor cells interact with these macrophages to determine the mechanisms underlying cancer cell growth, invasion, and metastasis, and to identify candidates for targeted therapies [ 13 ]. In vitro 2D cell culture models have been used for decades in cancer research. However, the complex interactions between various cell types within the TME make it challenging to mimic the in vivo tumor environment in 2D cell culture monolayers [ 14 ]. Thus, many studies have emphasized the use of 3D cell culture models, which offer unique opportunities to study cell-cell, cell-matrix, and spatial-organizational interactions that influence cellular behavior and responses to therapeutics [ 15 ]. Due to these characteristics, 3D tumor cell spheroids are now widely used as an in vitro model for investigating various aspects of cancer biology and testing anti-tumor drugs [ 16 ]. Given the significant importance of TAMs in tumor development and treatment resistance, it is crucial to include them in the in vitro models. Despite attempts to co-culture macrophages and tumor cells in 3D models, challenges still exist in the current co-culture systems, including a lack of standardization, cell population bias, analytical hurdles, and difficulties in multi-cell maintenance. A detailed characterization of the structure and biological characteristics of co-culture spheroids requires further analysis [ 17 , 18 ]. This study aimed to gain better insight into the interaction between polarized macrophages and triple-negative cancer cells in our newly established 3D co-culture spheroids, which mimic aspects of the TME. To achieve this goal, we investigated changes in drug response, cell viability, cell proliferation, epithelial-mesenchymal transition (EMT), and cancer stem cell (CSC) population dynamics in cancer cells when co-cultured with polarized macrophages. We also assessed the phenotype changes of macrophages when they were co-cultured with the cancer cells. In addition, we performed RNA sequencing (RNA-seq) analysis paired with CIBERSORTx deconvolution to identify differentially expressed genes, enriched pathways, and estimate cell type changes in the co-cultured spheroids. To establish a relatively standardized and stable co-culture system and better understand the roles of macrophages in chemoresistance, THP-1-derived macrophages and a more aggressive TNBC cell line, MDA-MB-231, were used in this study. By understanding the macrophage-TNBC axis, combined standard chemotherapies, such as doxorubicin and paclitaxel, with TAM-targeting agents could possibly reprogram the tumor microenvironment and re-sensitize the TNBC cells to the drugs. With these insights, our study may identify new targets for TNBC treatment by considering the complexity of its microenvironment. Materials and methods Cell culture The triple-negative breast cancer cell line MDA-MB-231 and the human THP-1 cell line were obtained from ATCC (Manassas, VA, USA). Cells were maintained in D-MEM/F-12 medium (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc., MA, USA). The cells were incubated at 37 °C in a humidified incubator with 5% CO 2 . Macrophage differentiation and polarization THP-1 cells (passages 5–10) were stimulated with 100 nM phorbol-12-myristate 13-acetate (PMA) (Sigma-Aldrich, Saint Louis, MO, USA) in D-MEM/F-12 medium for 24 hours. Afterward, cells were activated for 24 hours into M1-like macrophages by treatment with 50ng/mL IFN-γ (R&D Systems Inc., Minneapolis, MN, USA) or into M2-like macrophages by treatment with 50ng/mL IL-4 (R&D Systems Inc., Minneapolis, MN, USA). 3D Co-cultured spheroids 10,000 MDA-MB-231 cells (passages 9–15) were seeded into each well of a Nunclon Sphera-treated U-shaped-bottom microplate, which is coated with a hydrophilic polymer. (Thermo Fisher Scientific Inc., MA, USA). After 24 h, polarized M1-like or M2-like macrophages were washed three times with sterile PBS to remove polarizing cytokines, and then 5,000 cells were collected and seeded into each well of the microplate containing cancer cells for co-cultures. The co-culture seeding ratio of macrophages to cancer cells (1:2) was determined to better mimic the in vivo TME, based on the observation that TAMs typically comprise 10–50% of the TNBC tumor mass [ 13 ]. Mono- and co-cultures were incubated for an additional 4 days at 37 °C in a humidified incubator with 5% CO 2 before analysis or drug treatment ( S1 Fig ). A 4-day co-culture was selected based on an optimization assay to allow the macrophage infiltration into the spheroids (preliminary data not shown). Spheroid morphology and structure were monitored under an Olympus FV3000 Laser Scanning Confocal Microscope. Spheroids were validated for uniformity based on the relatively well-defined spherical shape and size variation <10% across wells before further experiments were performed. Drug treatment and cell viability assay Doxorubicin (DOX) (CAS# 25316409, Sigma-Aldrich, Saint Louis, MO, USA) and paclitaxel (PTX) (CAS# 33069624, Selleck Chemicals, Houston, TX, USA) were dissolved in DMSO to make stock solutions and diluted with culture media to the desired concentrations for treatment. The final DMSO concentration including the vehicle control was < 0.1% (v/v). Based on our preliminary dose-dependent treatment data in MDA-MB-231 2D and 3D cultures, 50 μM was selected for further analysis to maximize treatment effects ( S2 Fig ). Mono-culture 3D spheroids of cancer cells and co-culture 3D spheroids of cancer cells and macrophages were treated with 50 μM doxorubicin or paclitaxel for 48 hours. For comparison, MDA-MB-231 cells were seeded into standard 96-well plates and incubated for 5 days to form 2D monolayer cultures. Afterward, 2D cultured cells were treated with 50 μM doxorubicin or paclitaxel for 48 hours. Additional comparisons in MDA-MB-231 and M1/M2 macrophages 2D co-cultures were also performed for reference ( S2 Fig ). The viability of 2D- and 3D-cultured cells was measured using WST-1 (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s instructions. Cells without treatment were used as media controls. Briefly, 20 μL of WST-1 reagent was added to each well. The plates were incubated at 37°C for 4 hours, and the absorbance was measured at 450 nm using the ELx800 plate reader (Bio-Tek Instruments, Winooski, VT, USA). To prevent chemical intrinsic absorbance from interfering with the assay results, background absorbance was corrected by including cell-free blanks (wells containing corresponding chemicals and WST reagent without cells). Experiments were performed in seven biological replicates. Fluorescence imaging Fluorescent images of cells were recorded by confocal laser scanning microscopy (CLSM). 3D mono-cultured and co-cultured cell spheroids were formed and treated as described above. MDA-MB-231 cells were seeded in a 4-chamber glass-bottom cell culture dish (Cellvis, Mountain View, CA, USA) at a density of 2.5 × 10 5 cells/well and treated with 50 μM doxorubicin or paclitaxel for 48 hours for comparison. After treatment, the viability and morphology of the cells were assessed using the Cyto3D™ Live-Dead Assay Kit (TheWell Bioscience, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions. 2 µL of Cyto3D reagent was added to every 100 µL of media in each well. Cells were cultured at 37°C for 10 minutes and then imaged under a confocal microscope (Olympus FV3000 Laser Scanning Confocal Microscope). The live cells were excited at (Ex/Em) 494/517 nm, while the dead cells were excited at (Ex/Em) 535/617 nm. Doxorubicin was not included due to its intrinsic fluorescence at (Ex/Em) 480/595 nm, which can overlap with the dead-cell marker. To confirm the distribution of different types of cells in the 3D spheroids, MDA-MB-231 cells (CellTracker Green CMFDA, Thermo Fisher Scientific), M1 macrophages (CellTracker Red CMTPX, Thermo Fisher Scientific), and M2 macrophages (CellTracker Deep Red, Thermo Fisher Scientific) were stained according to the manufacturer's instructions before seeding to form 3D spheroids. Cells were observed under the confocal microscope after co-culturing. To evaluate drug penetration into the 3D spheroids, 50 μM doxorubicin and Oregon Green 488-conjugated paclitaxel were used to treat mono-cultured and co-cultured cells for 48 hours. After treatment, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 10 minutes. Nuclear DNA was stained with Hoechst 33342 (Thermo Fisher Scientific Inc., MA, USA). After three additional washes with PBS, the cells were imaged using an Olympus confocal microscope. All spheroids were transferred to glass-bottom cell culture dishes using cut-edge pipette tips before imaging under a confocal microscope. Experiments were performed in three biological replicates. Flow cytometry Phenotypic changes in MDA-MB-231 cells and macrophages in mono-cultured and co-cultured spheroids were assessed by multicolor flow cytometry (BD FACS Symphony A3). 2D and 3D cell samples were dissociated using TrypLE Express Enzyme (1x) (Thermo Fisher Scientific Inc., MA, USA), neutralized with serum-supplemented media, and centrifuged for 5 min at 1500 rpm. After washing with PBS (Ca2 + -/ Mg2 + - free), cells were harvested and resuspended in viability dye (Ghost dye 510, 1:1000; Tonbo Biosciences, CA, USA) prepared in PBS (Ca2 + -/ Mg2 + - free). After incubation for 30 minutes at 4°C, the cells were centrifuged and washed with FACS buffer (Ca2 + - and Mg2 + -free PBS supplemented with 2% fetal bovine serum). Then, the cells were incubated with Human TruStain FcX-Fc Receptor Blocking Solution (BioLegend, CA, USA) at a 1:50 dilution for 10 minutes on ice. Cells were stained with anti-human antibodies against CD45 (HI30), CD24 (IT2.2), CD44 (BJ18), CD324 (67A4), laminin (Polyclonal), fibronectin (1G10F9), vimentin (O91D3), CD325 (8C11), α-SMA (1A4/asm-1), β Catenin 1(15B8), FSP1 (NJ-4F3-D1), CD120b (3G7A02), CD284 (HTA125), CD16 (3G8), CD209 (9E9A8), CD206 (MMR 15−2), CD38 (HIT2), CD86 (ML5), CD14 (63D3), CD319 (162.1), CD80 (2D10), CD163 (GHI/61), CD68 (Y1/82A), CD32 (FUN-2), CD93 (VIMD2). Laminin and α-SMA antibodies were purchased from Bio-Techne, USA. Fibronectin antibody was purchased from Proteintech, USA. All other antibodies used in this study were purchased from BioLegend, USA. Detailed information on antibodies is summarized in S1 Table . For proliferation analysis, Tag-it Violet™ Proliferation and Cell Tracking Dye (BioLegend, CA, USA) was used according to the manufacturer’s instructions. Briefly, cells were incubated with dye for 20 minutes at 37°C and kept in the dark. After washing with pre-warmed media, cells were ready for downstream applications or analysis. Doublet exclusion was performed using FSC-A versus FSC-H to isolate single cells, and a viability dye was utilized to exclude dead cells as described above. Viable cells were identified as Ghost Dye negative, and dead cells were identified as Ghost Dye positive. Single-stained, unstained, and fluorescence one minus (FMO) controls were used to set compensation and manual gating, ensuring that shifts in autofluorescence or cell granularity did not affect the specificity of the gating. To account for potential variations in macrophage activation, the gating was anchored on specific phenotypic marker CD45 rather than scatter properties alone. Macrophages were identified as the CD45 + leukocyte population, whereas cancer cells were gated as the CD45- fraction. The percentage of viable cells within the target population was calculated using the following formula: Cell Viability (%) = (Ghost Dye negative cells/ Total number of cells) × 100. Data were collected on a BD FACS Symphony A3 and analyzed using FlowJo software (BD Life Sciences, Ashland, OR) ( S3 Fig ). Experiments were performed in three biological replicates. RNA extraction and sequencing Cells were washed in PBS and lysed using RNAzol RT according to the RNAzol® RT Column Kit (Molecular Research Center, Cincinnati, OH) protocol. Briefly, the lysate was mixed with water and centrifuged at 12,000 × g to remove DNA and proteins. The lysate was then diluted at a 1:1 ratio with isopropanol and added to a column, followed by another 12,000 × g centrifugation. RNA was then washed twice with 100% ethanol to remove impurities. RNA was then eluted using R
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