Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor diffuse large B-cell lymphoma

doi:10.21203/rs.3.rs-4220323/v1

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Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor diffuse large B-cell lymphoma

https://doi.org/10.21203/rs.3.rs-4220323/v1

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The pathogenesis of MYC and BCL2 double expressor diffuse large B-cell lymphoma (DE-DLBCL) remains unclear. To investigate how MYC and BCL2 contribute to tumor aggressiveness, we analyzed tumors from 14 patients each with DE- and non-DE-DLBCL patients by whole transcriptome sequencing. Validation was performed using publicly available datasets, tumor tissues from 126 patients, DLBCL cell lines, and a syngeneic mouse lymphoma model. Our transcriptome analysis revealed significantly elevated mRNA levels of C-C motif chemokine ligand 2 (CCL2) and C-C chemokine receptor type 2 (CCR2) in DE-DLBCLs compared to non-DE-DLBCLs (Padj < 0.05). Transcriptomic analysis with public datasets and immunohistochemistry corroborated these findings, indicating heightened M2 macrophage presence but diminished T-cell infiltration in DE-DLBCLs compared to non-DE-DLBCLs (all, P < 0.05). MYC^high/BCL2^high DLBCL cells showed higher CCL2 secretion than MYC^low/BCL2^low cells. MYC and BCL2 increased CCL2 secretion by upregulation of nuclear factor-κB p65 in DLBCL cells, and the CCL2 promoted M2 polarization of macrophages. In a mouse lymphoma model, CCL2 contributed to the immunosuppressive microenvironment and tumor growth. We demonstrated that the increased CCL2/CCR2 axis confers aggressiveness to DE-DLBCL by increasing M2 polarization and can be a potential therapeutic target.

Biological sciences/Cancer/Tumour immunology

Biological sciences/Cancer/Oncogenes

Biological sciences/Immunology/Chemokines

MYC

BCL2

Diffuse large B-cell lymphoma

CCR2

CCL2

M2 macrophages

Diffuse large B-cell lymphoma (DLBCL) exhibits significant heterogeneity in terms of pathological features, biology, and clinical behavior [1]. Despite this diversity, conventional rituximab-cyclophosphamide, hydroxydaunoirubicin, oncovin, and prednisone (R-CHOP) chemoimmunotherapy, comprising rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, has consistently been applied, leading to variable responses among patients. Many efforts have been made to predict the prognosis of patients with DLBCL, based on clinical, histopathological, and molecular/cytogenetic characteristics.

MYC proto-oncogene encodes a transcription factor that plays various roles in cell proliferation and growth, DNA replication, protein biosynthesis, and regulation of metabolism [2]. MYC alterations and overexpression have been identified in many tumors and are associated with aggressive behaviors [3, 4]. BCL2 is an anti-apoptotic molecule that is frequently overexpressed and genetically altered in various tumors [5]. In malignant lymphoma, BCL2 overexpression acts synergistically with MYC and other oncogenes, promoting lymphoma progression and resistance to chemotherapy [6]. Double-expressor lymphoma refers to a subset of DLBCLs that show the overexpression of both MYC and BCL2 using immunohistochemistry. Double-expressor lymphoma accounts for 25–30% of DLBCL cases and is associated with the non-GCB subtype, older age, and a higher Ki-67 index. Although they do not form a distinct clinicopathological entity, MYC/BCL2 double expression (DE) serves as an independent poor prognostic marker in patients with DLBCL treated with R-CHOP [7–9]. However, the mechanisms underlying the aggressiveness of DE-DLBCL remain unclear, and there are no differences in the management of patients with and without DE-DLBCL [10]. Several clinical trials are ongoing for DE-DLBCL (NCT03036904 and NCT02213913) [11]; however, targeted therapeutic strategies for this aggressive DLBCL subset remain elusive. Understanding the pathobiology of DE-DLBCL is crucial for developing novel therapeutic strategies.

The tumor microenvironment (TME) plays an important role in tumor progression and affects the effectiveness of cancer therapies. Understanding the tumor immune microenvironment is essential in immunotherapy. T-cell-inflamed tumors exhibit a favorable prognosis, while T-cell-non-inflamed and/or immunosuppressive cell-rich tumors have a worse prognosis and show resistance to immunotherapies in lymphomas [12–14]. The immune landscape of tumors is primarily influenced by the local chemokine milieu [15]. Additionally, oncogenic alterations contribute to shaping the tumor immune microenvironment [16]. In lymphomas, alterations in PTEN, EZH2, TP53, and MYC are associated with poor immune cell infiltration and activation [12]. In contrast, mutations leading to constitutive activation of the nuclear factor-kappa B (NF-κB) pathway, microsatellite instability, and/or the presence of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) mutational signatures are associated with an inflamed TME of lymphoma [12]. However, the impact of the DE of MYC and BCL2 on the immune environment of lymphomas remains unclear.

This study aimed to evaluate the underlying pathophysiology of DE-DLBCL, focusing on the immune landscape, and to propose potential treatment targets.

Patients

The patients for whole transcriptomic analysis (RNA sequencing) included 14 patients with nodal DE-DLBCL and 14 patients with nodal non-DE-DLBCL, who were newly diagnosed with DLBCL and treated with R-CHOP therapy between 2014 and 2017 at Seoul National University Hospital (SNUH). For immunohistochemistry validation, a separate cohort of 126 patients with DLBCL, NOS treated with R-CHOP therapy from 2013 to 2020 at SNUH was employed. The clinicopathological features of these patients are shown in Supplementary Table S1 and S2. Patients with specific DLBCL types, including primary mediastinal large B-cell lymphoma, primary central nervous system lymphoma, Epstein-Barr virus-positive DLBCL, T-cell/histiocyte-rich large B-cell lymphoma, and immunodeficiency-associated DLBCL, were excluded. This study adhered to the ethical guidelines outlined in the Declaration of Helsinki of the World Medical Association. This study was approved by the Institutional Review Board of SNUH (H-1609-129-795).

Immunohistochemistry

Tumor microarrays were constructed using representative formalin-fixed paraffin-embedded tissues. Immunohistochemistry (IHC) was performed using a panel of antibodies, listed in Supplementary method Table S1.

The cell of origin was determined using the IHC-based Han algorithm. The MYC/BCL2 DE status was defined as the expression of both MYC (in ≥ 40% of tumor cells) and BCL2 (in ≥ 50% of tumor cells). Major histocompatibility complex (MHC) class molecule expression was assessed on a scale of 0 to 3, and a score of 0 and 1 was designated as "low expression.” Aperio ImageScope software (Aperio Technologies, Vista, CA, USA) was used to automatically count the number of CD3⁺, CD4⁺, CD8⁺, and FOXP3⁺ tumor-infiltrating lymphocytes per mm², as well as the positive pixel count of CD68 and CD163 per mm².

RNA sequencing (RNA-seq) and bioinformatics analyses

Total RNA was extracted from formalin-fixed, paraffin-embedded tissues, and 1 µg of total RNA was used for cDNA library construction. The library was sequenced using an Illumina HiSeq2500 sequencer (Illumina Inc., San Diego, CA, USA). Gene expression levels were quantified using Cufflinks v2.1.1, using the Ensembl release 77 gene annotation database. Gene set enrichment analysis, differentially expressed gene (DEG) analysis, and immune cell deconvolution analysis were performed. The detailed methods are described in Supplementary methods.

Validation using public databases

Three publicly available DLBCL datasets were used for external validation. Clinical and mRNA expression data from Schmitz et al. [17] were obtained from the National Cancer Institute Genomic Data Commons (https://gdc.cancer.gov/about-data/publications/DLBCL-2018; acquired on January 10, 2020). Data from studies by Sha et al. [18] (GSE117556) and Painter et al. [19] (GSE181063) were downloaded from the Gene Expression Omnibus database.

In the datasets from Schmitz et al. and GSE181063, cases with mRNA values above the median for MYC and BCL2 were categorized as "DE" (double-expressor) and other cases as "non-DE.” In the case of GSE117556, the data provided on DE status were directly used for analysis.

Cell lines and reagents

The following cell lines were used in this study: HBL-1, OCI-LY10, OCI-LY3, HT, DOHH2, SUDHL-10, SUDHL-8, SUDHL-5, SUDHL-4, OCI-LY8, OCI-LY1, U-2932, TMD8 (human B-cell lymphoma), THP-1 (human monocyte), and A20 (mouse B-cell lymphoma). OCI-LY3 and OCI-LY10 cells were cultured in IMDM (Biowest, Nuaillé, France) supplemented with 20% fetal bovine serum and 100 U/mL penicillin/streptomycin. The other cell lines were cultured in RPMI 1640 (Biowest) supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin. Cell lines were procured from the American Type Culture Collection (Manassas, VA, USA) or the Korean Cell Line Bank (Seoul, Republic of Korea).

Information on the reagents used is listed in Supplementary method Table S2

MYC/BCL2 overexpression and knockdown

Cells were transfected with MYC-expressing plasmid vector, BCL2-expressing plasmid vector, MYC siRNAs, or BCL2 siRNAs using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Information on the plasmids and siRNAs used is provided in Supplementary method Table S3.

To generate stable mouse cell lines, pLVX-mCherry-mMyc and pLVX-EGFP-mBcl2 were packaged into lentiviral particles using Lenti-X-293T packaging cells co-transfected with the viral packaging plasmids pVSV-G, BH10, and pcREV, and viral supernatants were harvested 32–40 h after transfection. A20 cells were infected with polybrene-supplemented lentiviral supernatants and sorted using a FACSAria Ⅲ cell sorter (BD Biosciences, Franklin Lakes, NJ, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total mRNA was extracted from cells and subjected to qRT-PCR analysis, as detailed in the Supplementary methods using primers with the sequences listed in Supplementary method Table S4.

Western blotting

Western blotting was performed as detailed in the Supplementary methods section using the antibodies listed in Supplementary method Table S5.

Immunofluorescence staining

Immunofluorescence staining was performed as described in Supplementary methods.

Flow cytometry

Flow cytometric analysis was performed as detailed in the Supplementary methods section using the antibodies listed in Supplementary method Table S6.

Enzyme-linked immunosorbent assay

CCL2 in culture supernatant was measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Migration assay

Cell migration was detected using 8-µm pore Transwells (SPL Life Sciences, Gyeonggi-do, Republic of Korea). THP-1 cells were stimulated with 200 µg/mL phorbol myristate acetate for 24 h to induce into macrophages. These cells were then seeded in the upper well of the Transwell chamber, while the lower compartment was filled with DLBCL cells, either in the presence or absence of an anti-CCL2 neutralizing Ab (nAb). After 24 h of incubation, the cells in the upper well were wiped with a wet cotton swab, and the cells on the lower side of the filter were fixed and stained with crystal violet. The cells were counted microscopically and quantified using ImageJ software.

Mice and in vivo experiments

Six- to seven-week-old specific-pathogen-free female BALB/c mice were purchased from Orient Bio (Gyeonggi-do, Republic of Korea) and maintained under specific pathogen-free conditions in accordance with the animal care guidelines approved by the Institutional Animal Care and Use Committee of Seoul National University (Approval No. SNU-220303−5) and SNUH (Approval No. 22−0082-S1A0(1)).

Stable MYC/BCL2-overexpressing-A20 cells and their respective control cells were subcutaneously implanted (5 × 10⁵ cells/mouse) into the flanks of female BALB/c mice. After cell implantation, the primary tumor volume was measured once every two to three days using calipers and calculated using the following the formula: tumor volume (mm³) = (longest diameter × shortest diameter²)/2. For clodronate liposomes treatment, clodronate or control liposomes (6.5 uL/g body weight) were administered intraperitoneally (i.p) three times per week to mice. For the anti-CCL2 nAb treatment, anti-CCL2 or control IgG antibodies (10 mg/kg body weight) were administered i.p three times per week.

Statistical analysis

Continuous values, including immune cell infiltration and Ki-67 index, according to various clinical parameters, including DE status, were compared using the Mann-Whitney U and Kruskal–Wallis tests. Continuous values from the in vitro study were analyzed using unpaired two-tailed Student’s t test and one-way analysis of variance, followed by Dunnett’s multiple comparison test. Categorical values, including MHC molecule expression and other clinicopathological parameters according to DE status, were compared using Pearson’s chi-squared test, Fisher’s exact test, and linear-by-linear association. Survival analysis was conducted using the Kaplan-Meier method and log-rank test. Univariate and multivariate survival analyses were performed using Cox proportional hazards models. All P values were obtained from two-sided tests, and P value of < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 21 (IBM Corp) and GraphPad Prism 9 statistical software (GraphPad Software, San Diego, CA, USA).

Data sharing statement

RNA-seq datasets are available under the accession number GSE252690.

Whole transcriptome analysis reveals differentially activated biological pathways and gene expression in DE-DLBCL compared with non-DE-DLBCL

RNA-seq analysis of 14 nodal DE-DLBCL and 14 nodal non-DE-DLBCL tissues revealed that DE-DLBCL upregulated the pathways associated with MYC target responses, unfolded protein responses, various mitochondria-related biological processes, and the cell cycle/apoptosis pathway (Fig. 1A; Supplementary Data 1). DEG analysis revealed 128 genes that met the stringent criteria (Padj, FDR < 0.05, fold change > 2), of which 18 were associated with immune responses (Fig. 1B; Supplementary Data 2). DE-DLBCL showed higher expression of anti-inflammatory and immunoglobulin genes than non-DE-DLBCL. In contrast, the expression levels of certain genes, such as ITGAM (CD11b) (primarily expressed in monocytes, macrophages, and granulocytes), ITGAX (CD11c) (predominantly expressed in dendritic cells, monocytes, macrophages, and granulocytes), and MHC II molecules (HLA-DRB5 and HLA-DRB1) (which are primarily found in antigen-presenting cells), were high in non-DE-DLBCL. DE-DLBCL cells displayed significantly higher expression of CCL2 and its receptor CCR2, which are key molecules involved in monocyte recruitment and M2 macrophage polarization. These observations suggest a distinct immune profile in DE-DLBCL.

The immune profile is distinctive between DE-DLBCL and non-DE-DLBCL, and M2 macrophages are elevated in DE-DLBCL

To investigate the immune cell composition, we conducted an immune cell deconvolution analysis of the whole transcriptome data using CIBERSORTx. There was a tendency toward a higher ratio of M2 macrophages/macrophages (M0 + M1 + M2 or M1 + M2) in DE-DLBCL than in non-DE-DLBCL (Supplementary Figure S1A). We extended the analysis to other large publicly available DLBCL datasets for validation (Schmitz et al. 2018, GSE181063, and GSE117556). Across all datasets, B cell populations were increased, whereas CD8⁺ T cells and various CD4⁺ T-cell subsets tended to be decreased in DE-DLBCL compared with non-DE-DLBCLs (Fig. 1C; Supplementary Figure S1B and C). Among the myeloid populations, the ratio of M2 macrophages/macrophages was consistently elevated in DE-DLBCL relative to non-DE-DLBCL (Fig. 1C; Supplementary Figure S1B and C).

The immune cell infiltration was validated in an extended IHC cohort. Given the variations in immune cell infiltration according to the tumor site (Supplementary Figure S2) in the IHC cohort and the tumor site (nodal) in our RNA-seq cohort, we restricted our comparison of immune cell infiltration to nodal cases. In nodal DLBCLs, a consistent immune cell infiltration pattern was observed between the transcriptome and IHC cohorts. Specifically, DE-DLBCL exhibited an increase in CD163⁺ M2 macrophages and a decrease in T-cell infiltration compared with non-DE-DLBCL (Fig. 1D). CD4⁺ T cells were significantly decreased, and CD8⁺ T cells and regulatory T cells (FOXP3⁺ cells, Tregs) tended to decrease in DE-DLBCL. The ratio of CD8⁺ T cells to Tregs was not significantly different between the DE-DLBCL and non-DE-DLBCL (Fig. 1D). MHC expression was significantly lower in extranodal tumors than in nodal tumors (Supplementary Figure S2S-U). In nodal cases, the expression of MHC class I molecules tended to be lower in DE-DLBCLs than in non-DE-DLBCLs, although the difference was not statistically significant (Supplementary Figure S3A-C). The Ki-67 index was higher in DE-DLBCLs (Supplementary Figure S3D and E), which was consistent with the increased cell cycle signature and elevated B cell population in DE-DLBCLs from our RNA-seq cohort and public datasets.

The comparison between non-GCB and GCB DLBCLs revealed decreased infiltration of CD8⁺ T cells and lower expression of MHC II molecules in non-GCB DLBCLs than in GCB DLBCLs (Supplementary Figure S4A–J). In nodal DLBCL cases, a significant reduction in the number of CD4⁺ T cells was observed in non-GCB DLBCL compared with GCB DLBCL (Supplementary Figure S4J–T).

Taken together, transcriptome and IHC analyses showed a distinct immune cell composition associated with DE status, especially highlighting increased M2 macrophage infiltration, which is in line with the increased CCL2-CCR2 gene expression observed in our transcriptomic data

CCL2-CCR2 axis is related to the poor prognosis of DLBCLs

To assess the prognostic significance of the CCL2-CCR2 axis in DLBCLs, we conducted survival analyses based on CCL2 and CCR2 mRNA expression using publicly available datasets. In all datasets, DE-DLBCLs exhibited significantly worse progression-free survival (PFS) and overall survival (OS) than non-DE-DLBCLs (Schmitz dataset, PFS: P < 0.001, OS: P = 0.007; GSE117556, PFS: P < 0.001, OS: P < 0.001; GSE181063 OS: P = 0.008) (Supplementary Figure S5).

In the Schmitz cohort, increased CCL2 expression was significantly associated with poor PFS and OS (P = 0.014 and P = 0.009, respectively) (Fig. 2A and B). Increased CCR2 expression was also significantly associated with worse PFS and OS (P = 0.005 and P = 0.010, respectively) (Fig. 2C and D). The combined high expression of CCL2 and CCR2 showed a significant associated with decreased PFS and OS (P = 0.013 and P = 0.017, respectively) (Fig. 2E and F). In the GSE117556 and GSE181063 datasets, high expression of CCL2 and CCR2 was associated with poor prognosis (Supplementary Figure S6). The combined high expression of CCL2 and CCR2 was significantly associated with poor prognosis (Fig. 2G-I). These findings indicate that the CCL2/CCR2 axis may contribute to poor clinical outcomes in patients with DLBCL, particularly in those with DE-DLBCL.

CCL2 production is increased by MYC and BCL2 in DLBCL cells

In the IHC of DLBCL tissues, CCR2 expression was mainly observed in tumor-associated macrophages (TAMs) rather than in tumor cells. Only 2.4% (3 of 126) of cases exhibited high tumoral CCR2 expression (Supplementary Figure S7A and B). In flow cytometry, CCR2 expression was low, except in the case of OCI-LY3 cells (Supplementary Figure S7C). Given that tumor cells and TAMs are significant sources of CCL2 [20–22], we hypothesized that CCL2 is secreted from DE-DLBCL cells and affects CCR2-expressing macrophages. To verify this hypothesis, 11 DLBCL cell lines were screened for basal protein expression of MYC and BCL2. HT and SUDHL10 were selected as MYC^low/BCL2^low cell lines (non-DE-DLBCLs), and OCI-LY8 and DOHH2 were selected as MYC^high/BCL2^high cell lines (DE-DLBCL cells) (Supplementary Figure S8).

The mRNA expression and production of CCL2 was significantly higher in MYC^high/BCL2^high cells than in MYC^low/BCL2^low cells, both in the resting and interleukin (IL)-4/IL-13-activated states (Fig. 3A). At baseline, the CCL2 expression and its transcription factor, phosphorylated NF-κB p65, was also higher in MYC^high/BCL2^high cells than in MYC^low/BCL2^low cells (Fig. 3B). When either MYC- or BCL2-expression vectors were transfected into MYC^low/BCL2^low HT and SUDHL10 cells, CCL2 expression increased at the mRNA and protein levels. Moreover, when both the MYC- and BCL2-expression vectors were co-transfected, CCL2 expression increased additively (Fig. 3C-E). The levels of phosphorylated NF-κB p65 were also increased in MYC/BCL2-overexpressing cells compared with control (Fig. 3D). MYC/BCL2 overexpression-induced NF-κB p65 phosphorylation and CCL2 expression and secretion were restored by NF-κB inhibitor, JSH-23 treatment (Fig. 3F and G).

When MYC and BCL2 were inhibited in MYC^high/BCL2^high OCI-LY8 and DOHH2 cells using bioavailable inhibitors, that is JQ-1 and fimepinostat for MYC and venetoclax for BCL2, CCL2 expression decreased (Fig. 3H-K and Supplementary Figure S9). Consistently, transfection of siRNAs against MYC and BCL2 into MYC^high/BCL2^high cells decreased CCL2 expression and secretion (Supplementary Figure S10A-C). These findings indicate that MYC and BCL2 upregulate CCL2 expression and secretion through NF-κB activation in DLBCL cells.

MYC and BCL2 expression in DLBCL cells promotes M2 polarization of macrophages

We hypothesized that MYC and BCL2 overexpression in DLBCL cells promotes the differentiation of macrophages into immunosuppressive M2 macrophages via CCL2, thereby contributing to poor clinical outcomes. DLBCL cells were co-cultured with THP-1 cell-derived macrophages. For comparison, THP-1-derived macrophages were differentiated into M1 or M2 macrophages using IFN-γ + LPS or IL4 + IL13 treatments, respectively. The expression of M2 markers (CD206, CD163, ARG1, and TGFB1), but not M1 markers (CD11c, CD80, CD86, and NOS2), was increased in macrophages co-cultured with OCI-LY8 and DOHH2 (MYC^high/BCL2^high cells) compared with those co-cultured with HT and SU-DHL10 (MYC^low/BCL2^low cells) at the mRNA and surface protein expression levels (Fig. 4A and B).

MYC^low/BCL2^low cells were transfected with MYC- and/or BCL2-expression vectors and subsequently co-cultured with THP-1-derived macrophages, resulting in increased expression of M2 markers compared with those co-cultured with control cells (Fig. 4C and Supplementary Figure S11A-C).

Conversely, when MYC^high/BCL2^high cells were transfected with siRNAs targeting MYC and BCL2, the expression of M2 markers decreased in macrophages compared with those co-cultured with control cells. However, the expression of M1 markers was slightly increased in macrophages co-cultured with MYC/BCL2-knockdown cells (Fig. 4D and Supplementary Figure S11D-F). Collectively, these findings suggest that MYC and BCL2 expression in DLBCL cells induces macrophage polarization, particularly toward the M2 phenotype.

CCL2 secreted from DE-DLBCL cells promotes macrophage migration and M2 polarization

CCL2 is a chemoattractant for various immune cells, especially monocytes/macrophages [20]. To investigate the significance of CCL2 in macrophage recruitment and M2 polarization within the DE-DLBCL microenvironment, MYC^high/BCL2^high cells were co-cultured with THP-1-derived macrophages in the presence or absence of anti-CCL2 nAbs using a Transwell system. Macrophage migration toward MYC^high/BCL2^high cells was significantly inhibited by anti-CCL2 nAb (Fig. 5A).

Anti-CCL2 nAb treatment led to a significant decrease in the expression of M2 markers but an increase in M1 markers in macrophages co-cultured with MYC^high/BCL2^high cells (Fig. 5B–D). These findings indicate that CCL2 secreted from DE-DLBCL cells may play an important role in macrophage migration and M2 polarization.

CCL2 contributes to progression of DE-DLBCL and induces an immunosuppressive TME in vivo

To further determine whether CCL2 contributes to DE-DLBCL progression by increasing the number of M2 macrophages and inducing an immunosuppressive TME in vivo, MYC- and BCL2-overexpressing stable cell lines were established using A20 cells (Supplementary Figure S12A). These cells were subcutaneously injected into the flanks of mice. To evaluate whether macrophages are necessary for the aggressiveness of DE-DLBCLs, clodronate liposomes were injected intraperitoneally into mice (Fig. 6A). The growth of MYC/BCL2-overexpressing A20 cells was significantly higher than that of the control cells. However, macrophage depletion using clodronate liposomes significantly suppressed the growth of MYC/BCL2-overexpressing A20 cells (Fig. 6B), suggesting that macrophages play an important role in DE-DLBCL tumor progression.

To assess the role of CCL2 in vivo, an anti-CCL2 nAb was injected intraperitoneally into the mice (Fig. 6C). CCL2 depletion in tumor-bearing mice significantly suppressed the growth of MYC/BCL2-overexpressing A20 cells as well as control A20 cells (Fig. 6C). The TAM population (CD11b⁺F4/80⁺) was higher in MYC/BCL2-overexpressing tumors than in control tumors, which was restored by anti-CCL2 nAb. The surface expression of CD206 and CD163 on TAM from MYC/BCL2-overexpressing A20 tumors was significantly decreased, but the surface expression of CD11c and iNOS was slightly increased by anti-CCL2 nAbs (Fig. 6D and G). The population of monocytic myeloid-derived suppressor cells was also decreased by the anti-CCL2 nAb in MYC/BCL2-overexpressing A20 tumors (Supplementary Figure S12B and C). In contrast, tumor-infiltrating CD4⁺ and CD8⁺ T cells were increased, whereas FOXP3⁺ Tregs were decreased by anti-CCL2 nAbs (Fig. 6E and G). In particular, the population of IFN-γ-producing CD4⁺ and CD8⁺ T cells and cytotoxic CD8⁺ T cells (granzyme B⁺CD8⁺ T cells) increased after treatment with anti-CCL2 nAbs (Fig. 6F and G). These findings indicate that CCL2 secreted from DE-DLBCL cells promotes tumor progression by creating an immunosuppressive tumor immune microenvironment in vivo, and that CCL2 could be a therapeutic target in DE-DLBCL.

This study demonstrated that the increased CCL2/CCR2 axis by MYC and BCL2 contributes to higher M2 polarization and lower T-cell infiltration in DE-DLBCL and is associated with poor clinical outcomes in patients using in vitro and in vivo experiments and patient tumor tissues. We propose that the MYC/BCL2-CCL2/CCR2-M2 polarization/immunosuppression axis is a novel mechanism underlying DE-DLBCL aggressiveness.

MYC and BCL2 are oncoproteins that contribute to tumorigenesis. They also have an impact on antitumor immunity. MYC in tumor cells is associated with immune suppression by affecting cytokine production [23, 24], inducing immune checkpoint molecules (e.g., CD47 and PD-L1) [23, 25], and modulating the TME into an immunosuppressive milieu by recruiting TAMs and excluding T cells and other lymphocytes [26–29]. In an MYC-induced pancreatic islet tumor model, MYC recruited mast cells by promoting cytokine production, including CCL2 and CCL5 [30]. BCL2 in tumor cells induced an immunosuppressive environment by upregulating M2 macrophages through the IL-1β axis in melanoma [31]. In this study, DE-DLBCLs showed unique transcriptomic profiles compared with non-DE-DLBCLs, including gene set enrichment of DE-DLBCL-involved pathways reported to be enriched in DHLs [29]. DEG analysis revealed that CCL2 and its receptor CCR2 were significantly upregulated in DE-DLBCLs. Unlike previous reports showing CCR2 expression in DLBCL cells [30, 32], CCR2 expression in lymphoma cells other than TAM was quite low in our DLBCL samples and undetectable using IHC. We also observed low surface expression of CCR2 in DLBCL cell lines, except in a few cell lines. In vitro and in vivo experiments showed that MYC and BCL2 in tumor cells led to the upregulation of CCL2 secretion through the NF-κB p65 activation, which might subsequently contribute to the immunosuppressive environment in DE-DLBCLs. While NF-κB is a well-known inducer of BCL2 expression, BCL2 can activate NF- κB [33–35]. This pathway, MYC/BCL2-NF- κB -CCL2 secretion-CCL2/CCR2 axis-M2 macrophages and the resulting immunosuppressive microenvironment, was first reported in DLBCLs in this study.

CCR2, the receptor for CCL2, is expressed by various cell types, including monocytes/macrophages, Tregs, T cells, mesenchymal cells, and tumor cells [22]. CCL2 is a prototype chemokine that binds to CCR2. In the TME, tumor cells secrete CCL2 and CCR2 + TAMs, and cancer-associated fibroblasts and adipocytes serve as additional sources of CCL2 [20]. The CCL2-CCR2 axis was primarily studied in solid tumors, where it recruits monocytes, promotes their survival and proliferation, induces their polarization into M2 macrophages, and is involved in monocytes/macrophages-mediated tumor metastasis and Tregs accumulation [36, 37]. CCL2-CCR2 signaling also has a direct impact on solid tumor cells, including tumor cell proliferation, invasion, and epithelial-mesenchymal transition, and predicts metastasis and poor survival [21, 38, 39]. Clinical trials targeting the CCR2-CCL2 axis have been conducted in solid tumors [40, 41]. However, the biological and functional roles of the CCL2-CCR2 axis in DLBCL remain unclear. The multifaceted impact of the CCL2-CCR2 axis on TME and its association with poor prognosis underscores its potential utility as a therapeutic target in DLBCL, particularly DE-DLBCL.

In terms of T-cell infiltration, we observed a consistent tendency toward low T-cell infiltration in DE-DLBCL using both IHC and transcriptomic analyses. This phenomenon may be attributed to inhibition by increased M2 macrophages, immune exclusion, and a decrease in pro-inflammatory cytokines, as previously reported for MYC-related immune modulation [23, 24, 26–28]. DE-DLBCLs exhibit high proliferation, which was observed in this study by the enriched cell cycle pathway, increased B cell compartment in immune deconvolution, and elevated Ki-67 expression in IHC. High tumor cell proliferation may impede immune cell infiltration within the TME by immune exclusion and metabolic competition. While this study focused on two key immune cell subsets, T cells and macrophages, the rich and complex interactions among various cell types and cytokines/chemokines in the TME emphasize the need for further studies.

MYC and BCL2 targeting in DE-DLBCL may have a direct impact on immune cells, in addition to tumor cells. Overactivation of MYC in immune cell subsets has detrimental effects on pro-inflammatory mediator production and the tumor-killing activities of effector cells [42–44]. BCL2 is involved in the immunosuppressive phenotypes of T cells, dendritic cells, and macrophages [45–48]. Targeting MYC has been challenging and has shown limited efficacy and in vivo tolerability [49–51]. The BCL2 inhibitor venetoclax, despite its proven efficacy in leukemia, exhibits limited activity in patients with DLBCL with an ORR of only 18% [52] and is now being tested with several combination therapies [51–53]. Ongoing studies, such as the CAVALLI phase 2 trials investigating venetoclax plus R-CHOP as a first-line treatment for DLBCL, have shown promise, particularly in patients with BCL2 positive DLBCLs and double-expressor lymphomas [53]. It would be intriguing to evaluate the effects of MYC and BCL2 inhibitors in combination with chemoimmunotherapy on the immune microenvironment. Recently, immunotherapy using CAR T-cell therapy and a bispecific T-cell engager (bispecific antibody) has shown promising efficacy in patients with DLBCL and has been introduced into clinical practice [54, 55]. Thus, understanding the tumor immune microenvironment and its regulatory mechanisms is important in lymphoma. The findings of this study may provide valuable information for the management of patients with DE-DLBCL using chemoimmunotherapy and immunotherapy.

In summary, this study demonstrated that the increased CCL2/CCR2 axis contributes to the aggressiveness of DE-DLBCLs by promoting M2 polarization and an immunosuppressive microenvironment, and that targeting the CCL2/CCR2 axis could be a potential therapeutic strategy for DE-DLBCL.

Authorship Contributions

Y. K. J. and S. K. designed and supervised the project; S. K., H. J., and H. K.A. performed performed the experiments; S. K. and H. J. analyzed the results; S. K., B. H., K. C. L., Y. G. S., S. L., J. Y., J. K. and Y. K. J. contributed to the sample preparation and review of clinical data and pathology; S. K., H. J., and Y. K. J. wrote the manuscript; all authors read and approved the final manuscript.

Disclosure of Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgement

This work was supported by the Basic Science Research through the NRF funded by the Ministry of Education, Science and Technology Program (grant No.: NRF-2016R1D1A1B01015964), the Basic Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT (grant No.: RS-2023-00217571), Seoul National University Hospital Research Fund (grant No.: 03-2018-0260), Seoul National University Cancer Research Institute Research Program (grant No.: 0431-20230015), and the Development Fund from Seoul National University funded by HUNKIM FAMILY CHARITABLE FOUNDATION.

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There is NO conflict of interest to disclose.

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Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor diffuse large B-cell lymphoma

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Abstract

Figures

Introduction

Materials and Methods

Patients

Immunohistochemistry

RNA sequencing (RNA-seq) and bioinformatics analyses

Validation using public databases

Cell lines and reagents

MYC/BCL2 overexpression and knockdown

Quantitative real-time polymerase chain reaction (qRT-PCR)

Western blotting

Immunofluorescence staining

Flow cytometry

Enzyme-linked immunosorbent assay

Migration assay

Statistical analysis

Data sharing statement

Results

CCL2-CCR2 axis is related to the poor prognosis of DLBCLs

CCL2 production is increased by MYC and BCL2 in DLBCL cells

MYC and BCL2 expression in DLBCL cells promotes M2 polarization of macrophages

CCL2 secreted from DE-DLBCL cells promotes macrophage migration and M2 polarization

CCL2 contributes to progression of DE-DLBCL and induces an immunosuppressive TME in vivo

Discussion

Declarations

Authorship Contributions

Disclosure of Conflicts of Interest

Acknowledgement

References

Additional Declarations

Supplementary Files

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