MM tumor cells and MM-pDCs in the bone marrow microenvironment exhibit hypersialylation. (A) Hypersialylation in MM tumor cells results in strong interactions with Siglecs on immune cells in the microenvironment. (B) NESs that were significantly different between the MM-pDCs and normal pDCs based on GSEA MSigDB and WebGestalt analysis (false discovery rate <0.05; NES, ±1.5 as the cutoff). GSEA identified several downregulated (red) and upregulated (green) pathways. (C) Upregulation of SIGLEC6/CD327 in MM-pDCs (FC, 2.439; MM-pDC vs normal pDC; n = 3 each; adjusted P = .009283). (D) Total BM-MNCs were isolated from the BM of patients with relapsed/refractory MM and healthy donors (n = 3 each). pDCs were separated using CD304 magnetic beads on a Miltenyi autoMacs Neo machine and collected. MM.1S cells and BPDCN Cal-1 cell lines were used as controls. RNA was isolated using Qiagen RNeasy Mini kit. Poly RNA was subjected to Reverse Transcription Quantitative PCR (RT-qPCR) using Luna Universal 1-step RT-qPCR kit (New England Biolabs, Ipswich, MA) on an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific Inc). SIGLEC6/CD327 gene expression was quantified from raw data using ΔΔCT method, using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference. The bar graph denotes the relative expression of SIGLEC6 in different samples (normal, pDCs from normal healthy donor; MM-pDCs, pDCs from patients with relapsed/refractory with MM; Cal-1, BPDCN pDC cell line; MM.1S, dexamethasone-sensitive MM cell line; mean ± standard deviation [SD]; P < .05; all points done in triplicate). (E) CD303⁺/CD123⁺ MM-pDCs from MM BM-MNCs were examined by flow cytometry, and MFI of SIGLEC6/CD327 expression was determined using anti-Siglec-6 antibody (Ab) conjugated to Alexa Fluor 647. Panel shows representative histograms for SIGLEC6⁺ MM-pDCs (red), normal pDCs (black), and isotype control Ab conjugated to Alexa Fluor 647 (blue). (F-G) FC in MFI of biotinylated lectins from Sambucus nigra (SNA) and Maackia amurensis (MAL-II) bound to pDC (F) and MM.1R (G) cell surface. Fluorescence signal was detected by flow using secondary streptavidin conjugated to PE. The cells were first treated with biotinylated lectins (5 μg/mL; Vector Laboratories) for 45 to 60 minutes and washed, followed by staining with streptavidin-PE secondary (0.8-1.0 μg/mL) for 30 minutes. All samples were preblocked with Tru Fc blocker (BioLegend). 7-Aminoactinomycin D (7-AAD) dye was used to exclude dead cells, and signal was measured only on live cells. The data are presented as FC of biotinylated lectin–streptavidin-PE signal with respect to the fluorescence signal from cells treated with only secondary streptavidin-PE (mean ± SD; P < .05 for conjugated vs second signal). ER, Endoplasmic Reticulum; MFI, median fluorescence intensity; mRNA, messenger RNA; NES, normalized enrichment score; TLR10, Toll-like receptor 10.