Fig 1: VSV-M51R-hCXCL9 Expresses hCXCL9 Chemokine In Vitro and In Vivo(A) Human CXCL9 expression from VSV-M51R-hCXCL9 was tested in vitro in FaDu-Luc cells. Supernatants of FaDu-Luc cells were collected 48 h postinfection with VSV-M51R-hCXCL9, VSV-M51R-hCXCLi, (MOI 0.01) or after mock infection, and were assayed by ELISA (first panel). Data are presented as average concentration + standard deviation. Intratumoral and plasma hCXCL9 concentrations at various time points following virus injection were determined in FaDu-Luc tumor-bearing mice by ELISA (n = 3 mice/group for each time point). Values are presented as average chemokine concentration in pg/mL + standard deviation (second and third panels). Significance was determined by t test (*p < 0.05, **p < 0.01). (B) Human T cells were activated, and CXCR3 expression was tested by flow cytometry. Fluorescence minus one-stained cells are shown in red, and CXCR3-stained cells are shown in blue. FaDu-Luc tumors were harvested and processed 2 and 4 days after intratumoral injection of VSV-M51R-hCXCL9, VSV-M51R-hCXCLi, or PBS treatment (corresponding, respectively, to 1 and 3 days post adoptive T cell transfer). Flow data were gated on live hCD45+ or hCD3+ cells. T cell numbers are presented as average number of immune cells ± standard deviation (n = 3 mice/group for 48 h; n = 4 for PBS treated, n = 3 for VSV-M51R-hCXCL9 treated, and n = 2 for VSV-M51R-hCXCLi at 96 h).
Fig 2: CXCL9 Expressed from VSV-mCXCL9 Is Biologically Active(A) Murine CXCL9 secretion was evaluated in vitro in the LM2 non-small cell lung cancer cell line. Supernatants of VSV-infected LM2 cells (MOI 0.1) were collected 24 h postinfection, and chemokine concentration was determined by ELISA. Concentrations are presented as average concentration + standard deviation. (B) Chemotactic activity of virally encoded mCXCL9 was assessed in an in vitro migration assay adapted from Campanella et al.29 Numbers of migrated cells are presented as average percent increase in migration compared with mock treated + standard deviation. Significance was determined by paired two-tailed t test (**p < 0.01).
Fig 3: Generation of a Tumor to Blood CXCL9 Chemokine Gradient after Intratumoral VSV-mCXCL9 AdministrationIntratumoral and serum mCXCL9 protein concentrations were determined after intratumoral injection of VSV-mCXCL9 or VSV-GFP in LM2 tumor-bearing mice by ELISA (n = 3 mice/group for each time point). Values are presented as average chemokine concentration in pg/mL + standard deviation. Significance was determined by paired two-tailed t test (**p < 0.01).
Fig 4: Murine and Human CXCL9 Transgenes Engineered in Recombinant VSVs Do Not Alter the Growth Kinetics or Viral Killing Ability In VitroSchematic depiction of the genomes of recombinant VSVs encoding murine (A) or human (B) CXCL9, CXCLi, and GFP. (C) Replication kinetics of VSVs encoding murine or human CXCL9 were performed in a multistep viral growth curve in Vero cells. Data are shown from duplicate experiments as average titer + standard deviation. (D) Viability of VSV-mCXCL9-, VSV-mCXCLi-, and VSV-GFP-infected Vero and murine LM2 tumor cells was assessed at 24 and 48 h postinfection at an MOI of 10. (E) Viability of VSV-M51R-hCXCL9- and VSV-M51R-hCXCLi-infected Vero and human FaDu-Luc tumor cells was measured at 24 and 72 h postinfection at an MOI of 10. Data are presented as duplicate experiments as average percent viability compared with mock-infected cells + standard deviation. Significance was determined by paired t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Supplier Page from Sino Biological, Inc. for Mouse CXCL9 / MIG Gene ORF cDNA clone in cloning vector