<i>In vitro</i> and <i>in vivo</i> peptide sequence validation.

Abstract

<p>(A) An ELISA compares the binding of phage displaying the peptides to CAFs versus normal fibroblasts (MRC5). The first three sequences were selected using our selectivity analysis; whereas, the next six sequences were found using a traditional phage display approach. The dashed line indicates a fold change of 1.2. (B) Flow cytometry was performed by binding fluorescently-labeled phage to cells with a live-dead violet stain. Data was gated on cell population, live cells, and phage positive cells. (C) An ELISA compares binding of phage to HPSC and MRC5. Statistical significance was measured with a student t-test between HPSC and MRC5 where ^#p<0.01 and *p<0.02. (D) An ELISA compares binding of phage to HPSC and BXPC3. Statistical significance was measured with a student t-test between HPSC and BXPC3 where *p<0.02 and ^Φp<0.06. (E) Fluorescently-labeled phage were injected into mice bearing subcutaneous admix CAF/BXPC3 tumors or BXPC3-only tumors (n = 6 tumors per group) and tumor accumulation was measured on an FMT using a region-of-interest around the tumor area. Statistical significance was determined using student’s t-test of each type of displayed peptide versus KE with ^#p<0.01 and *p<0.02. (F) FMT images of mice with admix CAF/BXPC3 tumors scanned 4 h post-injection. Tumor regions have been circled with dashed lines. (G) Tumor sections of admix tumors were fixed, sectioned, and stained with anti-αSMA (green), then mounted with prolong gold anti-fade with DAPI (blue). The fluorescent labeling of the phage is colored red. Mander’s correlation coefficients (M) are indicated at the bottom of each image. For each phage type, images are representative of two tumors, three tumor sections each. Scale bars, 10 um.</p