Researchers Establish a New Standard for Studying Leukemia – Macrophage Interactions in vitro
A research team from the University Hospital of Cologne and the Center for Molecular Medicine Cologne (CMMC) has developed and systematically compared macrophage co-culture systems in chronic lymphocytic leukemia (CLL). The study “Comparative analysis of macrophage feeder systems reveals distinct behaviors and key transcriptional shifts in chronic lymphocytic leukemia cells via coculture” (https://doi.org/10.1002/hem3.70241) - published in HemaSphere - provides the first comprehensive analysis of these models and proposes a new experimental standard for reproducible investigations into leukemia-microenvironment interactions.
Background: Why the CLL Microenvironment Matters
The progression of chronic lymphocytic leukemia (CLL) depends not only on the malignant B cells themselves but also on their close interactions with the tumor microenvironment. Among the key players in this microenvironment are macrophages - immune cells that can either attack or support tumor cells depending on their polarization and local cues.
In CLL, blood monocytes can differentiate into so-called nurse-like cells (NLCs) that protect leukemic cells from apoptosis and contribute to drug resistance. However, researchers worldwide have used diverse macrophage co-culture systems to model these interactions, making it difficult to compare data or reproduce findings across laboratories.
To address this challenge, researchers from the Department I for Internal Medicine at the University Hospital of Cologne and the Center for Molecular Medicine Cologne (CMMC) undertook a systematic head-to-head comparison of macrophage-CLL co-culture systems. Their goal: to define robust experimental parameters and identify the biological pathways through which macrophages influence leukemia cells.
The Study: Comparing Human and Murine Macrophage Systems
The researchers evaluated several human and murine macrophage feeder systems, including:
- THP-1–derived macrophages, differentiated from a human monocytic leukemia cell line,
- Healthy donor–derived monocyte macrophages (HD-MDMs), generated from peripheral blood mononuclear cells,
- Nurse-like cells (NLCs), differentiated from patient-derived CLL peripheral blood mononuclear cells,
- Murine bone marrow–derived macrophages (BMDMs) and macrophage cell lines J774A.1 and MacCsf1r⁺/⁺.
Each system was tested for its ability to support CLL cell survival, perform spontaneous and antibody-dependent phagocytosis, and modulate leukemia cell responses to targeted therapies.
Key Experimental Findings
1. All macrophage feeders prolong CLL survival in vitro
Across all systems, macrophage feeders prevented apoptosis of primary CLL cells, maintaining cell viability for up to seven days. This effect was consistent across patient samples and independent of autologous versus allogeneic pairing.
2. Marked species-specific differences in phagocytosis
Murine macrophages exhibited strong spontaneous phagocytosis, with the J774A.1 line showing the highest activity, followed by MacCsf1r⁺/⁺ and BMDM.
In contrast, human macrophages - HD-MDMs, NLCs, and THP-1 - displayed lower spontaneous phagocytosis but were highly efficient in antibody-dependent phagocytosis (ADCP) when treated with the anti-CD20 antibody obinutuzumab.
This highlights that cross-species co-cultures must be interpreted with caution due to intrinsic immune function differences.
3. Distinct drug-response patterns
When treated with the targeted drugs ibrutinib (BTK inhibitor) and venetoclax (BCL2 inhibitor), CLL cells in co-culture displayed varying degrees of protection:
- THP-1 macrophages conferred partial resistance to both drugs, maintaining higher CLL viability.
- HD-MDMs and NLCs provided moderate protection but preserved drug sensitivity.
The team also tested the bispecific antibody epcoritamab, which activates T cells to eliminate CLL cells. Again, THP-1 feeders abrogated T-cell killing, whereas HD-MDMs and NLCs supported effective immune responses - further emphasizing model-specific effects.
Transcriptional and Signaling Insights
To uncover how macrophages influence CLL cells at the molecular level, the researchers performed bulk RNA sequencing of CLL cells after five days of co-culture.
Gene Set Enrichment Analysis (GSEA) identified shared upregulation of inflammatory response, IL-2/STAT5, IL-6/JAK/STAT3, and KRAS/MAPK signaling pathways across all human macrophage systems.
Importantly, these pathways are known drivers of leukemia cell survival, proliferation, and drug resistance.
Protein-level validation using immunoblotting and phospho-flow cytometry confirmed increased phosphorylation of STAT3, JAK1, MEK, ERK, and p38 MAPK, verifying activation of the JAK/STAT and MAPK cascades in cocultured CLL cells.
Interestingly, B-cell receptor (BCR) signaling was not activated - in some cases even downregulated - indicating that macrophage-mediated support occurs independently of direct BCR engagement.
Implications: A Framework for Reproducible Leukemia Research
“Our goal was to accelerate discovery through shared experimental standards,” said Dr. Phuong-Hien Nguyen from the Department I for Internal Medicine at the University Hospital Cologne, corresponding author of the study and principal investigator at the CMMC. “By systematically comparing co-culture systems, we identified key parameters that shape macrophage–CLL communication.”
“Who is Phuong-Hien Nguyen? - Learn more about her research activities
Dr. Viktoria Kohlhas (Department I for Internal Medicine at the University Hospital Cologne), co-first and co-corresponding author of the study, emphasized the collaborative nature of the project: “It’s exciting to see an idea I first developed, supported by funding from the CRC 1530, take shape through such a great team effort. I’m proud that our results will serve as a useful reference for others studying macrophage–leukemia interactions.”
Medical doctoral student and co-first author Hendrik Jestrabek (Department I for Internal Medicine at the University Hospital Cologne) added: “Being part of the research group and working under close supervision has been an inspiring and motivating experience. I’m thrilled that our findings are now published in HemaSphere and that I can continue developing this line of research.”
The study establishes a benchmark for future leukemia–microenvironment research. It demonstrates that HD-MDMs closely mirror the behavior of patient-derived NLCs and therefore represent a practical and accessible model for mechanistic studies, while THP-1 macrophages, though widely used, may overestimate drug resistance due to their tumor origin.
Cross-species (murine) models, while useful for mechanistic insight, should be applied with caution in translational research.
Outlook
By defining how different macrophage systems influence CLL cell behavior, this study lays the groundwork for harmonized experimental practices and improved reproducibility across laboratories.
The identification of JAK/STAT and MAPK signaling as central pathways in macrophage-mediated leukemia support also suggests potential targets for therapeutic intervention.
Future work will explore whether pharmacologic blockade of these pathways can disrupt the protective macrophage–leukemia dialogue and sensitize CLL cells to treatment.
The study was supported by funding from the Collaborative Research Center (CRC) 1530 and the CMMC.
Original Publication
https://onlinelibrary.wiley.com/doi/10.1002/hem3.70241
Viktoria Kohlhas*, Hendrik Jestrabek, Rocio Rebollido-Rios, Thanh Tung Truong, Anton von Lom, Rebekka Zölzer, Luca D. Schreurs, Duc Pham, Alexander F. vom Stein, Michael Hallek, and Phuong-Hien Nguyen*, Comparative analysis of macrophage feeder systems reveals distinct behaviors and key transcriptional shifts in chronic lymphocytic leukemia cells via coculture. HemaSphere. 2025 Oct 27;9(10):e70241. doi: 10.1002/hem3.70241. PMCID: PMC12558440. https://doi.org/10.1002/hem3.70241
Viktoria Kohlhas and Hendrik Jestrabek are joint first authors.
All authors do have a CMMC affiliation, corresponding authors are indicated with a star (*).
Scientific Contact
Dr. Phuong-Hien Nguyen
Hien.nguyen[at]uk-koeln.de
This text was jointly prepared by Dr. Phuong-Hien Nguyen, Dr. Viktoria Kohlhas, Hendrik Jestrabek (Department I for Internal Medicine at the University Hospital Cologne) and the press and communications team at the Center for Molecular Medicine Cologne (Dr. Debora Grosskopf-Kroiher and Kerstin Heber).