Hodgkin Lymphoma Reed-Sternberg Cells Induce Immunosuppressive and Pro-Angiogenic Phenotype of Tumor-Associated Macrophages in a Paracrine Manner

Increased frequency of tumor-associated macrophages (TAMs) predicts shortened survival of patients with classical Hodgkin lymphoma (cHL), suggesting their protumoral role in the disease. In vitro, malignant cells of cHL (Reed-Sternberg cells, RS) produce humoral factors that convert macrophages into TAMs capable of suppressing T-cell proliferation and exhibiting matrix remodeling activity that fosters lymphoma cell dissemination. Despite these features, phenotype and tumor-supportive activities of cHL-TAMs still remain to be elucidated. Herein, using an in vitro model of cHL-TAMs (RS-conditioned macrophages; RS-M), we provide more comprehensive insights into the biology of macrophages in cHL.

To confirm the ability of RS cells to polarize macrophages, THP1 cells and donor-derived CD14+ monocytes were first differentiated into M0 macrophages(Mϕ-0) using PMA and CSF-1, respectively. Obtained Mϕ-0 were nest cocultured for five days with L428 or L1236 RS cells under conditions prohibiting direct contacts (transwell system) and the expression of established M1/M2 polarization markers was assessed by flow cytometry. In comparison to control Mϕ-0, RS-M (L428- and L1236-conditioned: L428-M and L1236-M) exhibited increased surface levels of PD-L1 and M2-associated markers: CD163, CD206 and CD209, but failed to induce M1-specific expression of HLA-DR, indicating that RS cells alter macrophage phenotype in a paracrine manner. Next, using phospho-protein kinase arrays, we studied RS cell-triggered intracellular signaling in conditioned macrophages (L1236-M). In comparison to Mϕ-0, L1236-M exhibited increased phosphorylation levels of M2-specific transcription factors (TFs) and signaling intermediates (CREB1, AKT, STAT-3/6), M1/M2 TF cJun, but also exhibited elevated activity of the M1-specific STAT1 TF. To determine whether M1/M2 states exist within RS-M, we profiled transcriptomes of THP1 and CD14+-derived Mϕ-0 and RS-M by RNAseq, and performed Gene Set Enrichment Analysis (GSEA) using known M1/M2-macrophage signatures. Surprisingly, RS-M showed enrichment for both M1- and M2-signatures, indicating that RS-M phenotype is more complex and cannot be unequivocally classified as M1 or M2 state. Consistent with the transcriptional profiles of RS-M, THP1-derived L1236-M exhibited both enhanced glycolysis (typical for M1 macrophages) and oxidative phosphorylation (typical for M2). To look for genes and processes overrepresented in RS-M, we performed Gene Ontology Enrichment analysis, and found that THP1- and CD14+-derived RS-M similarly induced expression of genes involved in chemotaxis/immunomodulation (CCLs: 2, 5, 7, 8, 13, 17, 18 and 24), extracellular matrix organization (TGM2 and MMP-1, -7, -9 and -12), T cell repression (PD-L1) and angiogenesis (VEGFA, PDGFB, TIMP1, CHI3L1 and -2). Consistently, THP-1-derived L1236-M secreted higher levels of CCL-2, -5, -7 and -17, VEGF and CHI3L1 than Mϕ-0, as determined using cytokine arrays. Furthermore, L1236-M produced additional pro-angiogenic factors, Angiogenin and IL8, suggesting that cHL-TAMs support tumor growth by fostering angiogenesis. To verify this hypothesis, we incubated HUVEC endothelial cells in medium supplemented with VEGFA (10ng/ml), or THP1-derived Mϕ-0- or L1236-M-conditioned media and assessed blood vessel formation in a matrigel assay. In comparison to HUVEC cells grown in Mϕ-0-conditioned medium, addition of VEGFA (10ng/ml) increased number of master junctions by 169%, whereas L1236-M-conditioned medium by 187%, indicating that RS cells induce pro-angiogenic function in macrophages.

Together, our data indicate that RS cells determine TAM phenotype in a paracrine manner. Proteomic, transcriptional and metabolic profiles of the in vitro-generated cHL-conditioned TAMs indicate that these macrophages cannot be categorized into one of the two extreme M1/M2 polarization states. On the contrary, our data identify unique and disease-specific phenotype of cHL-TAMs, characterized by elevated expression of molecules involved in the recruitment and modulation of immune cell function, T-cell suppression, extracellular matrix remodeling and angiogenesis.

Study supported by National Centre of Science Poland grants: 2017/26/D/NZ5/00561, 2016/22/M/NZ5/00668 and 2018/31/N/NZ5/03214