Fig 1: HVEM is highly expressed on MM cell lines and primary myeloma cells(A and C) Indicated cancer cell lines were stained with antibodies for (A) NECTIN-1 (CD111) and (C) HVEM (CD270). Overlay images show relative surface NECTIN-1 levels and HVEM levels on different cell lines with respect to their individual isotype control. (B, D, and E) Distribution of (B) NECTIN-1, (D) HVEM, and (E) BCMA expression in 768 newly diagnosed patients with MM from the CoMMpass trial. Density plots of expression are expressed in log2(FPKM+1). The first quartile (blue), median (gray), and third quartile (red) of expression are denoted by dashed lines. (F) Representative flow cytometry dot plot demonstrating gating strategy for plasma cells (CD138+, CD38+) and HVEM staining in plasma cells versus non-plasma cells versus unstained cells. (G) Mean florescence intensity of HVEM in plasma cells versus non-plasma cells from 10 myeloma BM aspirates. (H) Cancer types are sorted by median expression for HVEM and include data from the Genomic Data Commons. Boxplots show the median and first and third quartiles of expression, with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. LAML, acute myeloid leukemia; DLBC, lymphoid neoplasm diffuse large B cell lymphoma; ALL, acute lymphoblastic leukemia; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; COAD, colon adenocarcinoma; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; LGG, brain lower grade glioma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma. See also Figures S2 and S3 and Table S1. Data are reported as mean ± SD; n=10, p=0.0002.
Fig 2: Oncolytic herpes simplex virus 1 (oHSV-1) infects MM cell lines and primary myeloma cells(A) MM cell line MM.1S was infected with GFP-expressing oHSV-1 at a multiplicity of infection (MOI) of 0.1. Twenty-four hours and 48 h post-infection (p.i.), cells were observed under a fluorescence microscope, which showed greater infection and hence GFP expression over time. (B) MM.1S and U266 cell lines were infected with differently modified oHSV-1 (HSVQ referred to here as oHSV-1, RAMBO, and rQnestin34.5) at an MOI of 0.1. Twenty-four hours after treatment, GFP+ cells were analyzed by flow cytometry, and high infection efficiency was observed independently of the genetic modifications of the backbone oHSV-1 vector for both cell lines. (C) Several MM cell lines (U266, RPMI 8226, NCI-H929, and LP1) were infected with oHSV-1 at an MOI of 0.1 for 24 h. Cells observed under a fluorescence microscope showed significant GFP+ signals in all MM cell lines tested even at low an MOI of 0.1. (D) Primary CD138+ MM cells and CD138- fractions isolated from bone marrow (BM) aspirates of MM patients were infected with oHSV-1 at an MOI of 10. Forty-eight hours after infection, cells were observed under a fluorescence microscope for the presence of GFP. (E) Flow cytometry analysis showing reduction in surface NECTIN-1 expression in MM cell lines (MM.1S, RPMI 8226, KMS11, and L363) upon oHSV-1 treatment (MOI of 0.1) for 24 h. See also Figure S1. Data are reported as mean ± SD of three to four experiments. *p < 0.05, **p < 0.001.
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