P831: novel immunosuppressive myeloid populations in the bone marrow (bm) and osteolytic lesions (ol) of multiple myeloma (mm) patients

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Multiple Myeloma (MM) patients experience an elevated risk of infection and disease progression due to immunosuppression. However, the mechanistic drivers of immunosuppression in the MM tumor microenvironment (TME), osteolytic lesions (OL), and bone marrow (BM) of these patients are not fully understood. Aims: In this prospective diagnostic study, we aimed to investigate the differences in myeloid cell populations from biopsy samples collected from different locations from MM patients (OL and BM) compared to the BM of healthy controls (HC). Methods: We obtained paired BM/TME and OL samples via imaging-guided biopsy from 12 MM patients in addition to BM aspirates from 3 HCs. Samples were never frozen, and were immediately processed and subjected to Ficoll-Paque gradients for isolation. CD11b+CD3-CD56-CD138- myeloid cells were isolated by fluorescence-activated cell sorting (SONY sorter), and single cell RNA sequencing was immediately carried out. Results: After QC and normalization, the following approximate number of myeloid cells were analyzed from: 1) MM BM: 60,000, 2) MM OL: 60,000, and 3) HC BM: 20,000 respectively. Using known marker gene expression and principle-component analysis, cell groups were identified (Table 1). We found 2 major myeloid cell categories of macrophages and neutrophils, defined by their gene expression. Distinct neutrophil development clusters were identified and confirmed by pseudo-time trajectory analysis. In the HC BM, major populations were neutrophil precursors and immature neutrophils. Importantly, 3 new neutrophil types comprise a major percentage of cells from BM and OL biopsies from MM patients. These neutrophil types were early, immunosuppressive, and inflammatory neutrophils. The immunosuppressive neutrophil expansion in OLs was significantly increased compared to MM BM and HC BM. We also found 5 macrophage subtypes: M1, inflammatory, monocytic, M2, and tumor associated. There were significant decreases in inflammatory and M1 macrophages in OL compared to HC BM. Additionally, results from in vitro T cell suppression assays found that both immature and mature neutrophil populations isolated from MM patient samples were shown to be more immunosuppressive than those from HC BM. Summary/Conclusion: We identified an increase in novel subpopulations of immunosuppressive and inflammatory neutrophils and found a significant reduction of M1 and inflammatory macrophages in the BM, and especially in OL, of MM patients. These populations may therefore represent a target for novel therapeutic strategies designed to overcome immunosuppression and improve patient outcomes. Immature neutrophil MMP9, S100A12, MME, PADI4, HMGB2, PGLYRP1, MMP8 Early neutrophil FCGR3B, NAMPT, CMTM2, MME, RGS2, Camp, FAM129A, CSF3R Immunosuppressive Neutrophil FCGR3B, NAMPT, CMTM2, MME, RGS2, Camp, CXCR2, NEAT1, FAM129A, TLR2, CSF3R, Fut4 (CD15), Sell, FCGR3A Classical M2 macrophages CD14, CD74, VCAN, HLA-DRB1, S100A10, CD36, CYP1B1, LGAS1, HLA-DPA1, NAP1L1, HLA-DPB1, CSF1R, CD33 Monocytic macrophages TLR2, CD33, HLA-DRB1, S100A10, CD36, CYP1B1, LGAS1, CSF1R, CSF3R, FCGR3A, CD14 M1 macrophages LCN2, CEACAM8, RETN, LTF, OLMF4, DEFA3, DEFA4, AZU1, MS4A3, STMN1, CSF1R, Adgre1, Mrc1, CD86 Inflammatory neutrophil FCGR3B, NAMPT, CMTM2, MME, RGS2, Camp, TLR2, IFIT1, IFIT2, IFIT3, RSAD2, ISG15, Fut4, Sell, FCGR3a Inflammatory Macrophages HLA-DRB1, S100A10, CD36, LGAS1, CD33, CD74, VCAN, HLA-DRB1, Azu1, HIST1H4C,MPO, RNSE2, STMN1, CSF3R, Sell CD16+ HLA-DR+ Tumor associated macrophages CD33, CD74, VCAN, HLA-DRB1, CDKN1C, FCGR3A, HLA-DPA1, NAP1L1, HLA-DPB1 CD16- HLA-DR+ Tumor associated macrophages CD74, VCAN, HLA-DRB1, HLA-DPA1, NAP1L1, HLA-DPB1, CSF3R, Sell, CD14 Keywords: Multiple myeloma, Macrophage, Neutrophil