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2929 Hodgkin Lymphoma Reed-Sternberg Cells Induce Immunosuppressive and Pro-Angiogenic Phenotype of Tumor-Associated Macrophages in a Paracrine Manner

Program: Oral and Poster Abstracts
Session: 622. Lymphoma Biology—Non-Genetic Studies: Poster III
Hematology Disease Topics & Pathways:
Diseases, Hodgkin Lymphoma, Biological Processes, Lymphoid Malignancies, immune mechanism, microenvironment, pathogenesis
Monday, December 7, 2020, 7:00 AM-3:30 PM

Maciej Szydlowski, PhD1*, Michał Pawlak, PhD1*, Filip Garbicz, MD1,2*, Patryk Górniak, PhD1*, Justyna Żurańska, PhD3*, Magdalena Król, PhD4*, Bartłomiej Taciak4*, Maciej Białasek4*, Anna Szumera-Ciećkiewicz, MD, PhD5*, Kamil Sokół5*, Monika Stańczak, Msc5*, Daria Owczarek5*, Monika Prochorec-Sobieszek, MD, PhD5*, Anna Polak, PhD1*, Emilia Bialopiotrowicz, PhD1*, Michał Kurlapski6*, Jan Zaucha, MD PhD6*, Paweł Wołkow, PhD3* and Przemyslaw Juszczynski, MD, PhD1*

1Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland
2Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland
3Center for Medical Genomics OMICRON, Jagiellonian University Medical College, Cracow, Poland
4Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland
5Department of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland
6Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland

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

Disclosures: Zaucha: Cellgene: Other: travel, accomodations, expenses; Abbvie: Honoraria; Sandoz: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, accomodations, expenses; Takeda: Consultancy, Honoraria, Other: travel, accomodations, expenses; BMS: Consultancy; Novartis: Consultancy. Juszczynski: Ryvu Therapeutics: Other: member of advisory board.

*signifies non-member of ASH