Journal: Vaccines
Article Title: Modified Hematopoietic Stem Cell-Derived Dendritic Cell Therapy Retained Tumor-Inhibitory Function and Led to Regression of Primary and Metastatic Pancreatic Tumors in Humanized Mouse Models
doi: 10.3390/vaccines13111131
Figure Lengend Snippet: LVV Optimization. ( A ) Flow cytometry results of feasibility study testing the effect of varying MOIs (5, 10, and 20) and promoter strength (EFS vs. EF1α) on MFI of CD93 and CD40L expression illustrated in raw MFI, difference in MFI between the EFS and EF1α viruses at indicated MOI, and fold change, n = 4. EFS virus results are identified as blue points whereas EF1α virus results are identified as red points. ( B ) MFI results of CD93 and CD40L with EFS (EFS_CD93_CD40L_CXCL13) and six EF1α viruses (all possible combinations of transgene orientations) illustrated as MFI mean ± SD on the left and MFI fold changes relative to the EFS control virus on the right graph, n = 4, MOI = 5. EFS virus is identified by the red dashed box on left graph and results are normalized to said group on the right graph by a red dashed line when compared by fold change. ( C ) Percentage of 7AAD-positive cells corresponding to each experimental condition on left graph, n = 4. Lentivirus yield from two rounds of large-scale packaging on right graph, n = 4. ( D ) CXCL13 and CD40L protein concentration in cell supernatant determined by ELISA, n = 2. Protein concentration fold changes relative to the virus with EFS promoter. ( E ) Vector maps illustrating 1st Gen EFS control and 2nd Gen LVV that were selected for use in future in vitro and in vivo experiments. Star annotations highlight modifications to vector construct, such as EF1α promoter from EFS, new transgene orientation with CD40L in 1st ORF, CD93 in 2nd ORF, and CXCL13 in 3rd ORF, oPRE operator from WPRE, and kanamycin antibiotic resistance from ampicillin. “(ns)” stands for nonsynonymous describing the type of gene variant.
Article Snippet: Propidium iodide or 7AAD (Miltenyi Biotec) was used to evaluate cell viability.
Techniques: Flow Cytometry, Expressing, Virus, Control, Protein Concentration, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, In Vitro, In Vivo, Construct, Variant Assay
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