Na Hee Lee; Byungchan Kim; Keon Hee Yoo

doi:10.4143/crt.2025.583

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Original Article Cytokine-Primed Mesenchymal Stromal Cells Enhance Antitumor Immunity–Associated Gene Expression without Promoting Acute Myeloid Leukemia Cell Growth: Na Hee Lee¹, Byungchan Kim², Keon Hee Yoo³; DOI: https://doi.org/10.4143/crt.2025.583
Published online: September 11, 2025

¹Department of Pediatrics, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam, Korea

²Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, Korea

³Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Correspondence: Keon Hee Yoo, Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea
Tel: 82-2-3410-3532 E-mail: hema2170@skku.edu

• Received: June 2, 2025 • Accepted: September 10, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract

Purpose
Mesenchymal stromal cells (MSCs), while regarded as promising immunomodulatory tools, have raised concerns in recent studies for their potential to promote tumor growth or facilitate immune evasion. These risks are particularly relevant in hematologic malignancies, where MSCs have been used to mitigate graft-versus-host disease or support hematopoietic engraftment. This study aimed to evaluate the effects of cytokine-primed MSCs on acute myeloid leukemia (AML) cells, with a focus on both safety and immunomodulatory efficacy.
Materials and Methods
Wharton’s jelly–derived MSCs were primed with interferon-γ (IFN-γ), interleukin-6, lipopolysaccharide, and tumor necrosis factor-α, followed by co-culture with AML cell lines. AML cell viability was measured by Cell Counting Kit-8 assay. RNA sequencing and quantitative real-time polymerase chain reaction were performed to assess gene expression profiles under each priming condition.
Results
Cytokine-primed MSCs did not result in significant changes in AML cell viability (p > 0.05), supporting the safety of this approach. Meanwhile, IFN-γ priming significantly upregulated genes involved in immune modulation and apoptosis, including IDO1, TNFSF10, ICAM1, and chemokines CXCL9, CXCL10, and CXCL11.
Conclusion
Cytokine priming, particularly with IFN-γ, enhanced the immunomodulatory gene expression of MSCs without promoting AML cell proliferation. Although direct cytotoxic effects were not observed in co-culture experiments, the absence of increased leukemic cell growth under any priming condition supports the biological safety of this approach. These findings provide a strong foundation for further in vivo studies to assess the therapeutic applicability of primed MSCs in hematologic malignancies while ensuring oncologic safety.
Key words: Acute myeloid leukemia, Mesenchymal stem cells, IFN-γ priming, IDO1, TNFSF10

Introduction

Mesenchymal stromal cells (MSCs) have emerged as promising candidates in cellular therapy due to their regenerative capacity and potent immunomodulatory functions. Clinically, MSCs have been employed to mitigate graft-versus-host disease (GVHD), support hematopoietic engraftment, and manage post-transplant complications in hematologic malignancies. Their immune-regulatory effects, including T-cell suppression, cytokine modulation and extracellular vesicles as additional mediators of these functions, have established MSCs as valuable tools in the supportive care of hematologic malignancies [1-5].

However, the clinical utility of MSCs is complicated by their functional heterogeneity. Studies have reported that MSCs may promote tumor progression by modulating the tumor microenvironment, suppressing anti-tumor immunity, and facilitating immune evasion [6-8]. These risks necessitate refinement of MSC-based therapies to preserve their therapeutic benefits while minimizing oncogenic potential.

To address these concerns, various preconditioning or “priming” strategies have been explored to enhance the immunomodulatory and anti-tumor properties of MSCs. Notably, interferon-γ (IFN-γ) or tumor necrosis factor-α (TNF-α) priming have been shown to enhance MSC immunomodulatory and anti-tumor functions. IFN-γ priming increases indoleamine 2,3-dioxygenase 1 (IDO1) expression, promoting T-cell suppression, whereas TNF-α priming upregulates TNF-related apoptosis-inducing ligand (TRAIL) and adhesion molecules, contributing to tumor cell apoptosis and immune cell recruitment [9-11].

Despite these advances, the impact of cytokine-primed MSCs on hematologic malignancies such as acute myeloid leukemia (AML) remains poorly defined. AML is an aggressive disease with high relapse rates and limited therapeutic options, necessitating the development of novel supportive strategies that do not compromise anti-leukemic immunity [12]. In this context, elucidating how primed MSCs interact with leukemic cells at both functional and molecular levels is critical.

This study aims to investigate the impact of cytokine priming MSCs in enhancing anti-tumor immunity-associated gene expression while maintaining tumor safety in AML. By analyzing gene expression profiles and AML cell viability in co-culture, we sought to determine whether specific priming conditions can improve the therapeutic applicability of MSCs without promoting tumor growth. These findings are intended to inform the development of MSC-based strategies optimized for use in hematologic malignancies.

Materials and Methods

1.Culture of Wharton’s jelly–derived MSCs

Human Wharton’s jelly (WJ)–derived MSCs were provided by the Samsung Stem Cells and Regenerative Medicine Institute (Seoul, Korea). Cells were cultured in α-minimum essential medium (α-MEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution. Cells were maintained until 70% confluency, passaged with 0.05% trypsin-EDTA, and replated at a density of 3,000-5,000 cells/cm².

2. Flow cytometric immunophenotyping of MSC surface markers

Antibodies against CD14, CD19, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR (BD Biosciences) were used. For immunophenotyping, cells were resuspended in 0.2 mL of 0.2% bovine serum albumin in Hank’s balanced salt solution and incubated with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies at 4°C for 30 minutes in the dark. Flow cytometry was conducted using FACSverse (BD Biosciences), and data were analyzed with BD suite software.

3. Differentiation assays

Osteogenic, adipogenic, and chondrogenic differentiation of MSCs was induced using StemPro kits. Osteogenic differentiation was confirmed by alkaline phosphatase expression after 14-21 days of culture. The medium was replaced every 3 days. Adipogenic differentiation was assessed by staining neutral lipid vacuoles with Oil Red O (Sigma-Aldrich) after 14-21 days of culture at 100% confluency. Chondrogenic differentiation was verified by staining the extracellular matrix with alcian blue (Sigma-Aldrich) after 14-21 days of culture. Cells were seeded at 2×10⁵ per tube and centrifuged before staining.

4. Establishment of primed MSCs

MSCs were cultured in T75 flasks with or without 200 IU/mL IFN-γ (Immukin, Boehringer Ingelheim), 100 ng/mL interleukin 6 (IL-6; Peprotech), 100 ng/mL lipopolysaccharide (LPS; eBioscience), or 100 ng/mL TNF-α (Peprotech) for 24 hours. MSCs were seeded at 1×10³ cells/well in 96-well plates in α-MEM (Biowest) supplemented with 10% FBS (Biowest).

5. RNA sequencing

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen). Following phase separation with chloroform, RNA was purified using RNeasy spin columns (QIAGEN) and quantified. The RNA library was constructed using the Illumina TruSeq Stranded mRNA Sample Prep Kit. Sequencing was conducted on an Illumina NovaSeq platform with paired-end 2×100 bp reads. Data preprocessing included adapter trimming with Trimmomatic, alignment to the GRCh37 reference genome using HISAT, and analysis using the SAMtools, StringTie, edgeR, or DESeq2. Significant genes were filtered by |fold change| ≥ 2 and p-value < 0.05, using the Benjamini-Hochberg method to control the false discovery rate. Hierarchical clustering was performed on the significant genes, and heat maps were generated to visualize the gene expression patterns. Gene enrichment analysis was performed using gProfiler and R 3.6.0 (R Foundation for Statistical Computing).

6. Quantitative real-time polymerase chain reaction analysis of immune-related genes

Total RNA was extracted from primed MSCs using TRIzol and the RNeasy Mini Kit. The priming conditions included IL-6, LPS, and TNF-α at concentrations of 50, 100, and 200 ng/mL, and IFN-γ at 200, 400, and 800 IU/mL. Quantitative real-time polymerase chain reaction assays for IDO1, C-X-C motif chemokine ligand 10 (CXCL10), intercellular adhesion molecule 1 (ICAM1), transforming growth factor alpha (TGFA), transforming growth factor beta 1 (TGFB1), IL-6, C-X-C motif chemokine ligand 6 (CXCL6), and C-X-C motif chemokine ligand 9 (CXCL9) were performed using the PrimeScript First Strand cDNA Synthesis Kit. Gene expression levels were measured using Gel Doc (Bio-Rad Laboratories) and calculated using delta-delta Ct method. Each condition was analyzed in technical triplicates and independently repeated three times.

7. Co-culture of leukemic cells with MSCs

AML cell lines HL-60 (ATCC CCL240), MOLM-14 (ATCC), MV-4-11 (ATCC), and THP-1 (ATCC) (1,000 cells/well) were seeded into 96-well plates. Immediately after seeding, MSCs were plated at 1,000 cells/well and co-cultured with leukemic cells at the same density (1,000 cells/well) for 48 hours using MSC^Naive or each pretreated MSC (MSC^IFN-γ, MSC^IL-6, MSC^LPS, and MSC^TNF-α). The co-culture was conducted directly, without Transwell inserts to facilitate cell-to-cell interactions.

8. Cell viability assay using absorptiometric analysis

After 48 hours of co-culture, AML cells, excluding MSCs, were transferred to new 96-well plates. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay; the cells were treated with 10 μL CCK-8 reagent (Dojindo Laboratories), incubated for 1 hour, and absorbance was measured at 450 nm. This experiment was conducted in triplicates and repeated three times. The absorbance values for each co-culture condition were normalized relative to the AML cells cultured alone, which were set at 100% for comparative analysis.

9. Statistical analysis

Statistical analysis was performed using one-way ANOVA or the Kruskal-Wallis tests, depending on data distribution. Normalized ratios and p-values were analyzed, with p < 0.05 considered statistically significant. The magnitude of changes in cell viability was assessed by calculating Cohen’s d, based on pooled standard deviation. All analyses were conducted using SPSS ver. 18 (SPSS Inc.) and GraphPad Prism (GraphPad Software Inc.).

Results

1. Characteristics of WJ-derived MSC

The human WJ-derived MSCs exhibited a fibroblast-like, spindle-shaped morphology (Fig. 1). Flow cytometry analysis of the expressed surface antigens confirmed that the cells were uniformly positive for typical MSC antigens (CD44, CD73, CD90, and CD105) (Fig. 2A) and negative for hematopoietic lineage markers (CD14, CD19, CD34, CD45, and HLA-DR) (Fig. 2B). MSCs showed osteogenic, adipogenic, and cartilage-like phenotypes after inducing differentiation with the appropriate media for 14-21 days (Fig. 3).

2. Effect of IFN-γ on MSC gene expression

RNA sequencing analysis was conducted to investigate molecular changes in MSCs following IFN-γ priming. Hierarchical clustering revealed 1,493 genes with significant differential expression (fold change ≥ 2, p < 0.05) between MSC^Naive and MSC^IFN-γ, indicating marked transcriptional reprogramming (Fig. 4).

Gene Ontology (GO) analysis (Table 1) revealed significant involvement of immune-related pathways. Notably, “defense response” (GO:0006952; 267/1,956 genes, p < 0.001), “immune response” (GO:0006955; 298/2,544 genes, p < 0.001), and “regulation of cytokine production” (GO:0001817; 104/123 genes, p < 0.001) were prominently enriched, reflecting the enhanced immunomodulatory profile of IFN-γ primed MSCs. In addition, GO analysis revealed enrichment in pathways associated with “cell death” (GO:0008219; 134/202 genes, p < 0.001) and “regulation of apoptotic process” (GO:0042981; 92/137 genes, p < 0.001), suggesting increased expression of genes involved in apoptotic signaling.

Consistent with these findings, RNA-seq identified the upregulation of key genes with functional relevance to tumor–immune interactions (Table 2). Tumor necrosis factor ligand superfamily member 10 (TNFSF10), which encodes TRAIL, a key mediator of tumor cell apoptosis via death receptor activation, was significantly increased, providing direct evidence of IFN-γ–induced apoptotic gene activation. In addition, chemokines including CXCL9, CXCL10, and CXCL11, together with ICAM1, were strongly upregulated and enriched in immune-related pathways (e.g., GO:0006952, GO:0006955; p < 0.001), implying enhanced recruitment and engagement of cytotoxic T-cells at tumor sites. Toll-like receptor 3 (TLR3) was also upregulated, indicating increased innate immune sensing capacity. Moreover, IFN-γ priming upregulated genes associated with immunosuppressive functions, including IDO1, tumor necrosis factor receptor superfamily member 14 (TNFRSF14), tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), and programmed death-ligand 1 (PDL1). Specifically, IDO1 was enriched in the “immune system process” (GO:0002376; p < 0.001) and “positive regulation of the biological process” (GO:0048518; p < 0.001), consistent with enhanced immunosuppressive functions. Similarly, TNFRSF14, TNFRSF1B, and PDL1 have been linked to pathways that “modulate immune responses” (GO:0006955; p < 0.001), underscoring their potential roles in shaping the tumor microenvironment.

3. Changes in immune-modulating cytokine expressions

The quantitative real-time polymerase chain reaction (qPCR) analysis of immune-related cytokines in MSCs under various priming conditions revealed significant changes in gene expression levels (Table 3, S1 Fig.). IDO1 expression was notably increased by IFN-γ priming in a concentration-dependent manner (p < 0.05), and was also elevated by TNF-α and IL-6 priming (p < 0.05), while LPS induced the strongest response overall (p < 0.05). These results suggest that MSCs modulate immune responses through IDO1 expression in response to various inflammatory stimuli.

TGFA expression was significantly increased by IL-6 and TNF-α priming at all concentrations tested (p < 0.05), with particularly marked induction at higher doses. LPS and IFN-γ priming also robustly upregulated TGFA expression (p < 0.05). In contrast, TGFB1 expression was modestly but significantly increased by IL-6 priming at all concentrations tested (p < 0.05), whereas TNF-α at 200 ng/mL induced numerically higher but statistically non-significant changes (p=0.918).

IL-6 expression was significantly increased by LPS priming at all tested concentrations (p < 0.001), whereas IL-6 or TNF-α priming did not induce significant changes. In contrast, IFN-γ priming led to a consistent reduction in IL-6 expression, with statistical significance at higher doses (p < 0.05). CXCL10 expression was significantly upregulated by LPS priming at all tested concentrations (p < 0.001), as well as by TNF-α and IFN-γ priming, particularly at higher concentrations (p < 0.001). IL-6 priming induced a modest increase in CXCL10 expression, but this was not statistically significant (p=0.191). This finding suggests that MSCs enhance immune cell recruitment via CXCL10 secretion in response to inflammatory signals.

CXCL9 expression was significantly upregulated by LPS, TNF-α, and IFN-γ priming, with LPS producing the highest induction (p < 0.001). CXCL6 expression increased with LPS priming at 50 and 100 ng/mL (p < 0.05) but decreased in response to IL-6, TNF-α, and IFN-γ priming (p < 0.05). ICAM1 expression was strongly upregulated by LPS priming at all concentrations (p < 0.001), while TNF-α and IL-6 induced only modest increases at 200 ng/mL (p=0.049 and p=0.002, respectively). In contrast, IFN-γ priming reduced ICAM1 expression, with significant decreases at 200 and 400 IU/mL (p=0.010 and p=0.036), differing from RNA sequencing findings.

These results revealed that IFN-γ, TNF-α, and LPS priming induced significant changes in the expression of immunomodulatory genes in MSCs, with responses varying depending on the priming type and concentration. Notably, IFN-γ priming upregulated immune-suppressive genes such as IDO1 while also enhancing the expression of chemotactic cytokines, including CXCL10 and CXCL9.

4. Co-culture and absorptiometric analysis

Four AML cell lines (HL60, MOLM-14, MV-4-11, and THP-1) were cultured alone and co-culture with MSC^Naive, MSC^IFN-γ, MSC^IL-6, MSC^LPS, and MSC^TNF-α. The relative viability of each AML cell line, normalized to monocultured AML cells (set at 100%), is presented in Table 4 and Fig. 5.

In HL60 cell line, all co-culture conditions resulted in a slight reduction in viability compared to the control, with MSC^IFN-γ showing the lowest value (94.91%). However, the differences were not statistically significant (p=0.794).

MOLM-14 and MV-4-11 cells exhibited modest increases in viability, particularly when co-cultured with MSC^IL-6 and MSC^TNF-α (up to 106.28% and 109.44%, respectively), yet no condition yielded a significant change (p=0.697 and p=0.744, respectively).

In THP-1 cell line, co-culture with all MSC groups slightly increased viability (101.17%-104.12%), with no significant changes observed across conditions (p=0.765).

Although some priming conditions slightly increased or decreased the cell viability, the overall effect was relatively slight and not statistically significant across all tested conditions.

Cohen’s d values were calculated to assess the magnitude of viability changes across priming conditions. Most comparisons showed small or negligible effect sizes (|d| < 0.3), indicating minimal biological impact. A moderate effect was observed only in HL60 cells co-cultured with IFN-γ–primed MSCs (d=–1.21), although this did not reach statistical significance. These findings further support the safety of cytokine priming in terms of AML cell growth.

Discussion

This study evaluated whether cytokine priming could enhance the therapeutic profile of MSCs while maintaining tumor safety in AML. Although co-culture experiments did not reveal measurable reductions in AML cell viability, IFN-γ priming elicited significant transcriptional activation of immune- and apoptosis-related genes, suggesting enhanced immunomodulatory potential without evidence of promoting leukemic proliferation.

The dual role of MSCs in cancer has been a subject of ongoing debate. Clinically, MSCs have been used as important supportive therapies for hematologic malignancies. They play a crucial role in mitigating GVHD owing to their immunosuppressive properties [1,13]. Additionally, co-transplantation with MSCs promotes hematopoietic engraftment in clinical settings. Lee et al. [3] reported that patients receiving cord blood transplantation with third-party cord blood MSCs had faster neutrophil and platelet engraftment (19 and 47 days, respectively) compared to historical controls, with similar GVHD incidences [2]. The immunosuppressive properties of MSCs, while beneficial for GVHD mitigation, may inadvertently impair graft-versus-leukemia activity and raise relapse risk [6,14]. Consequently, although MSCs are recognized for their role in managing cancer-related complications, concerns remain regarding their potential to impair anti-leukemic immune surveillance and increase relapse risk in hematologic malignancies. This clinical paradox is paralleled by mechanistic evidence from experimental studies, showing that MSCs can either support leukemic cell survival or exert tumor-suppressive effects depending on the context [8,15].

In addition, MSCs have also been reported to contribute to tumor progression by fostering an immune-suppressive microenvironment, in part through Notch signaling, adhesion molecules, and cytokine/chemokine networks [6-8,15]. These pathways can be further reprogrammed by AML blasts, thereby promoting leukemic persistence [15]. Conversely, MSCs also exhibit anti-tumor properties by enhancing immune surveillance through the secretion of cytokines such as IFN-γ, TNF-α, and IL-6, which activate immune cells and promote an anti-tumor immune microenvironment. In addition, MSC-derived factors like Dickkopf-1, a soluble inhibitor of the Wnt/β-catenin pathway, have been implicated in suppressing leukemic proliferation [7,8,15-18]. This dual role underscores the complexity of MSC-based therapies influenced by both the tumor microenvironment and external modulation such as cytokine priming.

To address this variability, various priming strategies have been employed to modulate MSC behavior and reduce heterogeneity. Several studies have demonstrated that cytokine priming enhances MSC immunomodulatory and anti-tumor functions by activating specific pathways. For instance, IFN-γ enhances MSC antigen-presenting capabilities and induces IDO synthesis, while promoting TRAIL expression to induce tumor cell apoptosis [11,19]. TNF-α upregulates IDO, prostaglandin E2 (PGE2), and hepatocyte growth factor (HGF) to modulate T-cell suppression and macrophage polarization [10,11]. LPS priming has been associated with increased secretion of IL-6, TNF-α, and vascular endothelial growth factor, contributing to immune activation and modulation of the tumor niche [11].

This study investigated MSC priming with IFN-γ, IL-6, LPS, and TNF-α to address MSC heterogeneity and enhance their therapeutic efficacy against hematologic malignancies. Although measurable reductions in AML cell viability were not observed in co-culture experiments, none of the priming conditions promoted leukemic cell growth, supporting the safety of this approach. Importantly, molecular analyses using RNA sequencing and qPCR offered valuable insights into the potential mechanisms underlying the influence of primed MSCs on the tumor microenvironment.

Notably, RNA sequencing of IFN-γ primed MSCs in our study revealed marked upregulation of immune- and apoptosis-related genes, including TNFSF10, CXCL9, CXCL10, CXCL11, ICAM1, IDO1, and TLR3. These changes align with pathways identified in our GO analysis (Table 1) and can be integrated into the model illustrated in Fig. 6: (1) TRAIL-mediated extrinsic apoptosis in AML cells via death receptor–caspase signaling [18-20], (2) chemokine (CXCL9-11) driven recruitment of cytotoxic T-cells and NK cells, supported by ICAM1-facilitated adhesion [21,22], and (3) IDO1-dependent immunometabolic regulation via tryptophan catabolism and kynurenine pathway activation, which may exert immunoregulatory effects by depleting tryptophan, potentially restraining leukemic growth while also modulating T-cell responses in a context-dependent manner [23]. In addition, TLR3 upregulation may enhance innate immune signaling within primed MSCs [24]. Together, these transcriptional changes highlight potential mechanisms by which primed MSCs could suppress AML, either directly through apoptosis induction or contributing to immune control, as illustrated in Fig. 6. While these findings suggest a shift toward an anti-tumor phenotype accompanied by immunoregulatory programs, confirmation at the protein level and functional levels will be critical to determine whether these transcriptional programs translate into functional effects without promoting AML proliferation, and further studies will be required to address this gap.

These transcriptomic findings were supported by qPCR, which confirmed significant upregulation of IDO1 and CXCL9 following IFN-γ priming. ICAM1 expression, however, showed inconsistent patterns between the two platforms, possibly reflecting post-transcriptional regulation or platform-specific sensitivity. To clarify these discrepancies and establish the functional relevance, future studies should include protein-level validation using methods such as Western blotting or enzyme-linked immunosorbent assay.

While these gene-level findings suggest a shift toward an anti-tumor phenotype, our co-culture experiments did not show a measurable reduction in AML cell viability unlike previous studies that reported tumor suppression following cytokine priming [19,25]. This discrepancy may reflect several limitations of our study. First, our in vitro co-culture system lacks the complexity of the tumor microenvironment, including interactions with angiogenesis, stromal elements, and immune cell components that are essential to fully recapitulate in vivo responses. Although priming-induced gene expression endows MSCs with the capacity to modulate immune responses and enhance cell-to-cell interactions in specific immune environments, these effects may be constrained in co-culture settings due to the characteristics of AML cells or inhibitory signals within the tumor microenvironment [26,27]. Second, the 48-hour co-culture duration, though commonly used in similar experimental settings, may have been insufficient for the full manifestation of MSC-secreted immunoregulatory factors such as IL-10 and PGE2 [28,29]. Third, priming with individual cytokines, while effective in driving transcriptional changes, may not be sufficient to fully optimize the anti-tumor functionality of MSCs. Indeed, TNF-α acts synergistically with IFN-γ by amplifying IDO1 activity and TRAIL-mediated caspase signaling, leading to leukemic growth inhibition [25,30]. In parallel, co-stimulation co-activates STAT1 and nuclear factor κB, markedly increasing CXCL9/10/11 and ICAM1 expression to enhance immune cell recruitment and adhesion [30]. Thus, our Fig. 6 model may represent a core framework of IFN-γ–induced reprogramming that is further strengthened by TNF-α, reinforcing the anti-tumor and immunomodulatory functions of MSCs.

Taken together, our findings highlight the importance of exploring more physiologically relevant models to further elucidate the dual immunomodulatory and cytotoxic roles of primed MSCs in the leukemic microenvironment. Future studies should incorporate complex in vivo systems and extended co-culture durations that better reflect niche dynamics, including angiogenesis, stromal interactions, and immune cell infiltration. In particular, transwell-based co-culture systems, 3D spheroid models, or organotypic cultures may help recapitulate spatial organization, paracrine signaling, and localized cellular crosstalk among MSCs, AML cells, and immune subsets such as T-cells and NK cells. At this stage, one key limitation is that our study addressed transcriptional and qPCR changes without confirming protein expression, and future research must integrate protein-level validation to ensure these molecular signatures translate into functional anti-leukemic activity.

Despite these limitations, our findings revealed that none of the priming conditions, including IFN-γ, TNF-α, IL-6, or LPS, resulted in increased viability of AML cells across multiple cell lines. This contrasts with earlier reports suggesting tumor-promoting effects of unprimed MSCs in various malignancies, and provides supportive evidence for the safety of cytokine-primed MSCs in translational applications [6-8,15].

In conclusion, this study demonstrated that cytokine priming induced gene expression changes in MSCs that could enhance their immune regulatory functions and cell-to-cell interactions, both of which are essential for anti-tumor activity. Notably, these effects were observed without evidence of promoting tumor growth, addressing a key concern in the therapeutic application of MSCs. These findings provide foundational insights into the efficacy and safety of MSC-based therapies for hematologic malignancies. Future studies should build on these results by exploring tailored priming strategies and leveraging more complex in vivo models and incorporating protein-level confirmation to fully realize the therapeutic potential of MSCs in cancer treatment.

Electronic Supplementary Material

Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).

crt-2025-583_S1_Fig.pdf

NOTES

Ethical Statement

This study was approved by the Institutional Review Board of Samsung Medical Center (IRB No. 2015-10-025), and informed consent was obtained for sample collection.

Author Contributions

Conceived and designed the analysis: Lee NH, Yoo KH.

Collected the data: Lee NH, Kim B.

Contributed data or analysis tools: Lee NH, Kim B, Yoo KH.

Performed the analysis: Lee NH, Kim B.

Wrote the paper: Lee NH, Yoo KH.

Conflicts of Interest

Conflict of interest relevant to this article was not reported.

Funding

This study was supported by Samsung Medical Center Grant #SMO 125037.

Fig. 1.

Morphological appearance of mesenchymal stromal cells (MSCs) without (A) or with interferon-γ (IFN-γ) priming (B) (×100). (A) Naive MSCs display a typical spindle-shaped, fibroblast-like appearance with elongated cell bodies and bipolar projections, arranged in parallel or swirling patterns upon adherence to plastic surfaces. (B) MSCs primed with IFN-γ (200 IU/mL for 24 hours) retain this typical morphology, indicating that cytokine stimulation does not alter the defining structural features of MSCs.

Fig. 2.

Flow cytometric analysis of positive surface markers in mesenchymal stromal cells (MSCs). (A) Flow cytometry analysis demonstrating high expression of canonical MSC markers CD44, CD73, CD90, and CD105, confirming the mesenchymal phenotype of the cultured cells. (B) Flow cytometry confirming lack of expression of hematopoietic (CD14, CD34, CD45), B-cell (CD19), and MHC class II (HLA-DR) markers in Wharton’s jelly–derived MSCs, consistent with International Society for Cell & Gene Therapy–defined criteria for MSC identification. Blue dashed line indicates x-axis zero reference.

Fig. 3.

Differentiation of naive mesenchymal stromal cells (MSCs) into adipocytes (A), chondrocytes (B), and osteocytes (C). MSCs were successfully induced to differentiate into: adipocytes (A), visualized by Oil Red O staining of lipid vacuoles, chondrocytes (B), confirmed by Alcian blue staining of glycosaminoglycans, osteoblasts (C), demonstrated by Alizarin red staining of calcium deposits.

Fig. 4.

Hierarchical clustering heat map illustrating differential gene expression in interferon-γ (IFN-γ) primed mesenchymal stromal cells (MSCs) (IC). Unsupervised hierarchical clustering of genes significantly modulated by IFN-γ priming in MSCs (fold change ≥ 2, p < 0.05), based on RNA sequencing data. The heat map illustrates a clear separation between naive and IC, indicating distinct transcriptional prof iles. Color scale represents normalized expression values (yellow: high expression; blue: low expression).

Fig. 5.

Relative cell viability of AML cell lines with different MSCs priming conditions. AML cell lines HL60 (A), MOLM-14 (B), MV-4-11 (C), and THP-1 (D) were co-cultured for 48 hours with naïve or cytokine-primed MSCs. Cell viability was measured using the Cell Counting Kit-8 assay and expressed relative to monoculture controls. No statistically significant differences were observed across conditions. AML, acute myeloid leukemia; IFN-γ, interferon-γ; IL-6, interleukin 6; LPS, lipopolysaccharide; MSC, mesenchymal stromal cell; TNF-α, tumor necrosis factor-α.

Fig. 6.

Proposed model of how IFN-γ priming reprograms MSCs to modulate the tumor microenvironment in AML. RNA sequencing revealed upregulation of TNFSF10, CXCL9, CXCL10, CXCL11, ICAM1, IDO1, and TLR3 following IFN-γ priming. These transcriptional changes suggest multiple mechanisms: TRAIL-mediated apoptosis via DR4/DR5 and caspase activation; CXCL9-11–driven recruitment of CXCR3⁺ T-cells and NK cells, enhancing chemotaxis and lysis; ICAM1-facilitated adhesion and cytotoxicity; IDO1-dependent tryptophan catabolism, depleting metabolites required for leukemic growth and influencing indirect NK cell activation and paradoxical T-cell traff icking, reflecting a dual immunoregulatory role; and TLR3-mediated innate immune activation. This model integrates transcriptomic changes observed in IFN-γ primed MSCs with their potential impact on immune modulation and leukemic cell death. AML, acute myeloid leukemia; CXCL10, C-X-C motif chemokine ligand 10; ICAM1, intercellular adhesion molecule 1; IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-γ; MSC, mesenchymal stromal cell; NK, natural killer; TLR3, Toll-like receptor 3; TNFSF10, tumor necrosis factor ligand superfamily member 10; TRAIL, TNF-related apoptosis-inducing ligand.

Table 1.

GO analysis based on cancer function-related genes upregulated in IFN-γ primed MSC

GO category	Biological process	p-value	Total No.	Intersection	Up regulation	Down regulation
GO:0006952	Defense response	1.31972E-34	1,956	267	233	34
GO:0006955	Immune response	1.34568E-27	2,544	298	266	32
GO:0001817	Regulation of cytokine production	6.48247E-17	847	123	104	19
GO:0042611	MHC protein complex	1.0948E-14	24	18	18	0
GO:0050776	Regulation of immune response	2.08585E-14	1,159	145	130	15
GO:0022610	Biological adhesion	6.42536E-08	1,509	152	90	62
GO:0008219	Cell death	5.22537E-06	2,306	202	134	68
GO:0042981	Regulation of apoptotic process	0.000380569	1,560	137	92	45
GO:0002253	Activation of immune response	0.02198115	566	53	50	3
GO:0016477	Cell migration	0.075179453	1,660	126	73	53
GO:0048771	Tissue remodeling	0.092424478	187	20	11	9

GO, gene ontology; IFN-γ, interferon-γ; MSC, mesenchymal stromal cells.

Table 2.

Comparison of quantitative expression of cancer function–related factors in naive MSC and IFN-γ priming MSC

Gene symbol	Fold change^a)	Log2 fold change	Basal (mean, TPM)	IFN-γ (mean, TPM)	BH-adjusted p-value^b)
IDO1	610,491.67	19.22	< 0.001	1,506.68	1.77497E-56
CXCL9	41,085.06	15.33	< 0.001	48.61	1.06737E-34
CXCL11	19,225.21	14.23	< 0.001	45.16	4.20726E-30
TNFSF10	8,492.13	13.05	< 0.001	30.20	4.03294E-25
CXCL10	1,865.88	10.87	< 0.001	62.98	2.99496E-13
CX3CL1	380.93	8.57	< 0.001	0.56	2.12163E-09
TNFSF13B	242.60	7.92	0.09	61.73	1.3394E-120
ICAM1	59.07	5.88	11.20	648.34	2.66471E-68
TLR3	49.10	5.62	0.41	15.97	5.1842E-110
TNFRSF14	31.42	4.97	1.07	17.15	3.70296E-10
TNFRSF1B	24.09	4.63	0.39	5.37	1.31785E-23
PD-L1	6.30	2.65	20.78	146.79	5.36233E-35

CX3CL1, C-X3-C motif chemokine ligand 1; CXCL9, C-X-C motif chemokine ligand 9; CXCL10, C-X-C motif chemokine ligand 10; CXCL11, C-X-C motif chemokine ligand 11; ICAM1, intercellular adhesion molecule 1; IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-γ; MSC, mesenchymal stromal cells; PD-L1, programmed death-ligand 1; TLR3, toll like receptor 3; TNFRSF14, TNF receptor superfamily member 14; TNFRSF1B, TNF receptor superfamily member 1B; TNFSF10, TNF superfamily member 10; TNFSF13B, TNF superfamily member 13b; TPM, transcripts per million.

^a) Fold change was derived from DESeq2 nbinom WaldTest results,

^b) p-value was calculated using the Benjamini–Hochberg (BH) method to control for errors from multiple comparisons.

Table 3.

Quantitative analysis of genes expression in MSCs under different cytokine priming conditions

Condition	IDO1	TGFA	TGFB1	IL-6	CXCL10	CXCL9	CXCL6	ICAM1
Control	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
IL-6 (ng/mL)
50	7.76	9.65	1.5	1.23	86.17	1.1	0.65	1.28
100	9.0	7.05	1.19	1.38	1.04	6.9	0.72	1.15
200	81.14	87.92	1.91	2.47	120.78	2.22	0.73	1.49
LPS (ng/mL)
50	397.94	16.52	0.75	20.74	36,912,582.06	19,709,200.6	2.44	92.08
100	648.33	19.21	0.83	27.28	75,935,372.63	35,173,011.01	2.26	139.8
200	834.7	69.73	0.74	30.64	83,178,563.64	60,433,193.8	2.14	151.7
TNF-α (ng/mL)
50	16.8	23.81	1.28	0.94	7,172.17	70,452.37	0.66	0.88
100	59.61	88.19	1.25	1.27	10,274.01	4,401.48	0.48	1.65
200	91.81	93.75	5.33	3.47	31,478.48	2,491.77	0.45	3.23
IFN-γ (IU/mL)
200	22.97	24.77	0.64	0.59	447.41	562.72	0.49	0.56
400	13.24	15.38	0.68	0.37	1,450.8	449.04	0.54	0.91
800	75.42	89.16	0.62	0.62	1,980.63	632.99	0.62	0.69

All values represent relative fold changes normalized to the control, calculated using the delta-delta Ct method. For genes with low baseline expression, fold changes may appear amplified and reflect relative induction. CXCL10, C-X-C motif chemokine ligand 10; ICAM1, intercellular adhesion molecule 1; IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-γ; IL-6, interleukin-6; LPS, lipopolysaccharide; MSC, mesenchymal stromal cells; TGFA, transforming growth factor alpha; TGFβ1, transforming growth factor beta 1; TNF-α, tumor necrosis factor-α.

Table 4.

Relative viability changes in AML cell lines co-cultured with cytokine-primed MSCs

	AML only	MSC^Naive	MSC^IFN-γ	MSC^IL-6	MSC^LPS	MSC^TNF-α	p-value
HL60	100	96.33	94.91	99.18	99.12	96.17	0.794
MOLM-14	100	100.16	101.32	106.28	101.63	105.39	0.697
MV-4-11	100	100.84	102.56	104.80	103.21	109.44	0.744
THP-1	100	102.07	101.17	103.98	103.19	104.12	0.765

Values are presented as percentage. AML, acute myeloid leukemia; HL-60, human promyelocytic leukemia; MOLM-14, monocytic leukemia-14; MSC^IFN-γ, mesenchymal stromal cells primed with interferon-γ; MSC^IL-6, mesenchymal stromal cells primed with interleukin-6; MSC^LPS, mesenchymal stromal cells primed with lipopolysaccharide; MSC^Naive, naive mesenchymal stem cells; MSC^TNF-α, mesenchymal stromal cells primed with tumor necrosis factor-α; MV-4-11, monocytic variant-4-11; THP-1, Tsuchiya human promyelocytic.

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Cytokine-Primed Mesenchymal Stromal Cells Enhance Antitumor Immunity–Associated Gene Expression without Promoting Acute Myeloid Leukemia Cell Growth

DOI: https://doi.org/10.4143/crt.2025.583

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Abstract
Introduction
Materials and Methods
Results
Discussion
Electronic Supplementary Material
NOTES
REFERENCES

Cytokine-Primed Mesenchymal Stromal Cells Enhance Antitumor Immunity–Associated Gene Expression without Promoting Acute Myeloid Leukemia Cell Growth

Fig. 1. Morphological appearance of mesenchymal stromal cells (MSCs) without (A) or with interferon-γ (IFN-γ) priming (B) (×100). (A) Naive MSCs display a typical spindle-shaped, fibroblast-like appearance with elongated cell bodies and bipolar projections, arranged in parallel or swirling patterns upon adherence to plastic surfaces. (B) MSCs primed with IFN-γ (200 IU/mL for 24 hours) retain this typical morphology, indicating that cytokine stimulation does not alter the defining structural features of MSCs.

Fig. 2. Flow cytometric analysis of positive surface markers in mesenchymal stromal cells (MSCs). (A) Flow cytometry analysis demonstrating high expression of canonical MSC markers CD44, CD73, CD90, and CD105, confirming the mesenchymal phenotype of the cultured cells. (B) Flow cytometry confirming lack of expression of hematopoietic (CD14, CD34, CD45), B-cell (CD19), and MHC class II (HLA-DR) markers in Wharton’s jelly–derived MSCs, consistent with International Society for Cell & Gene Therapy–defined criteria for MSC identification. Blue dashed line indicates x-axis zero reference.

Fig. 3. Differentiation of naive mesenchymal stromal cells (MSCs) into adipocytes (A), chondrocytes (B), and osteocytes (C). MSCs were successfully induced to differentiate into: adipocytes (A), visualized by Oil Red O staining of lipid vacuoles, chondrocytes (B), confirmed by Alcian blue staining of glycosaminoglycans, osteoblasts (C), demonstrated by Alizarin red staining of calcium deposits.

Fig. 4. Hierarchical clustering heat map illustrating differential gene expression in interferon-γ (IFN-γ) primed mesenchymal stromal cells (MSCs) (IC). Unsupervised hierarchical clustering of genes significantly modulated by IFN-γ priming in MSCs (fold change ≥ 2, p < 0.05), based on RNA sequencing data. The heat map illustrates a clear separation between naive and IC, indicating distinct transcriptional prof iles. Color scale represents normalized expression values (yellow: high expression; blue: low expression).

Fig. 5. Relative cell viability of AML cell lines with different MSCs priming conditions. AML cell lines HL60 (A), MOLM-14 (B), MV-4-11 (C), and THP-1 (D) were co-cultured for 48 hours with naïve or cytokine-primed MSCs. Cell viability was measured using the Cell Counting Kit-8 assay and expressed relative to monoculture controls. No statistically significant differences were observed across conditions. AML, acute myeloid leukemia; IFN-γ, interferon-γ; IL-6, interleukin 6; LPS, lipopolysaccharide; MSC, mesenchymal stromal cell; TNF-α, tumor necrosis factor-α.

Fig. 6. Proposed model of how IFN-γ priming reprograms MSCs to modulate the tumor microenvironment in AML. RNA sequencing revealed upregulation of TNFSF10, CXCL9, CXCL10, CXCL11, ICAM1, IDO1, and TLR3 following IFN-γ priming. These transcriptional changes suggest multiple mechanisms: TRAIL-mediated apoptosis via DR4/DR5 and caspase activation; CXCL9-11–driven recruitment of CXCR3+ T-cells and NK cells, enhancing chemotaxis and lysis; ICAM1-facilitated adhesion and cytotoxicity; IDO1-dependent tryptophan catabolism, depleting metabolites required for leukemic growth and influencing indirect NK cell activation and paradoxical T-cell traff icking, reflecting a dual immunoregulatory role; and TLR3-mediated innate immune activation. This model integrates transcriptomic changes observed in IFN-γ primed MSCs with their potential impact on immune modulation and leukemic cell death. AML, acute myeloid leukemia; CXCL10, C-X-C motif chemokine ligand 10; ICAM1, intercellular adhesion molecule 1; IDO1, indoleamine 2,3-dioxygenase 1; IFN-γ, interferon-γ; MSC, mesenchymal stromal cell; NK, natural killer; TLR3, Toll-like receptor 3; TNFSF10, tumor necrosis factor ligand superfamily member 10; TRAIL, TNF-related apoptosis-inducing ligand.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Cytokine-Primed Mesenchymal Stromal Cells Enhance Antitumor Immunity–Associated Gene Expression without Promoting Acute Myeloid Leukemia Cell Growth

GO category	Biological process	p-value	Total No.	Intersection	Up regulation	Down regulation
GO:0006952	Defense response	1.31972E-34	1,956	267	233	34
GO:0006955	Immune response	1.34568E-27	2,544	298	266	32
GO:0001817	Regulation of cytokine production	6.48247E-17	847	123	104	19
GO:0042611	MHC protein complex	1.0948E-14	24	18	18	0
GO:0050776	Regulation of immune response	2.08585E-14	1,159	145	130	15
GO:0022610	Biological adhesion	6.42536E-08	1,509	152	90	62
GO:0008219	Cell death	5.22537E-06	2,306	202	134	68
GO:0042981	Regulation of apoptotic process	0.000380569	1,560	137	92	45
GO:0002253	Activation of immune response	0.02198115	566	53	50	3
GO:0016477	Cell migration	0.075179453	1,660	126	73	53
GO:0048771	Tissue remodeling	0.092424478	187	20	11	9

Gene symbol	Fold change^a)	Log2 fold change	Basal (mean, TPM)	IFN-γ (mean, TPM)	BH-adjusted p-value^b)
IDO1	610,491.67	19.22	< 0.001	1,506.68	1.77497E-56
CXCL9	41,085.06	15.33	< 0.001	48.61	1.06737E-34
CXCL11	19,225.21	14.23	< 0.001	45.16	4.20726E-30
TNFSF10	8,492.13	13.05	< 0.001	30.20	4.03294E-25
CXCL10	1,865.88	10.87	< 0.001	62.98	2.99496E-13
CX3CL1	380.93	8.57	< 0.001	0.56	2.12163E-09
TNFSF13B	242.60	7.92	0.09	61.73	1.3394E-120
ICAM1	59.07	5.88	11.20	648.34	2.66471E-68
TLR3	49.10	5.62	0.41	15.97	5.1842E-110
TNFRSF14	31.42	4.97	1.07	17.15	3.70296E-10
TNFRSF1B	24.09	4.63	0.39	5.37	1.31785E-23
PD-L1	6.30	2.65	20.78	146.79	5.36233E-35

Condition	IDO1	TGFA	TGFB1	IL-6	CXCL10	CXCL9	CXCL6	ICAM1
Control	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
IL-6 (ng/mL)
50	7.76	9.65	1.5	1.23	86.17	1.1	0.65	1.28
100	9.0	7.05	1.19	1.38	1.04	6.9	0.72	1.15
200	81.14	87.92	1.91	2.47	120.78	2.22	0.73	1.49
LPS (ng/mL)
50	397.94	16.52	0.75	20.74	36,912,582.06	19,709,200.6	2.44	92.08
100	648.33	19.21	0.83	27.28	75,935,372.63	35,173,011.01	2.26	139.8
200	834.7	69.73	0.74	30.64	83,178,563.64	60,433,193.8	2.14	151.7
TNF-α (ng/mL)
50	16.8	23.81	1.28	0.94	7,172.17	70,452.37	0.66	0.88
100	59.61	88.19	1.25	1.27	10,274.01	4,401.48	0.48	1.65
200	91.81	93.75	5.33	3.47	31,478.48	2,491.77	0.45	3.23
IFN-γ (IU/mL)
200	22.97	24.77	0.64	0.59	447.41	562.72	0.49	0.56
400	13.24	15.38	0.68	0.37	1,450.8	449.04	0.54	0.91
800	75.42	89.16	0.62	0.62	1,980.63	632.99	0.62	0.69

	AML only	MSC^Naive	MSC^IFN-γ	MSC^IL-6	MSC^LPS	MSC^TNF-α	p-value
HL60	100	96.33	94.91	99.18	99.12	96.17	0.794
MOLM-14	100	100.16	101.32	106.28	101.63	105.39	0.697
MV-4-11	100	100.84	102.56	104.80	103.21	109.44	0.744
THP-1	100	102.07	101.17	103.98	103.19	104.12	0.765

Table 1. GO analysis based on cancer function-related genes upregulated in IFN-γ primed MSC

GO, gene ontology; IFN-γ, interferon-γ; MSC, mesenchymal stromal cells.

Table 2. Comparison of quantitative expression of cancer function–related factors in naive MSC and IFN-γ priming MSC

Fold change was derived from DESeq2 nbinom WaldTest results,

p-value was calculated using the Benjamini–Hochberg (BH) method to control for errors from multiple comparisons.

Table 3. Quantitative analysis of genes expression in MSCs under different cytokine priming conditions

Table 4. Relative viability changes in AML cell lines co-cultured with cytokine-primed MSCs