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  • br Figure CF hNIS Kills Colon Cancer in


    Figure 2. CF33-hNIS Kills Colon Cancer in a Dose-Dependent Manner with Viral Efficacy Unaltered by tk Deletion
    (A) Cytotoxicity of CF33-hNIS against HCT116 as compared with CF33 backbone virus (#33), CF33-GFP, which shares the same backbone of CF33-hNIS virus but has GFP inserted at tk locus (#33-GFP). (B) Cytotoxicity against HT29. For cytotoxicity experiments, cells Methylpiperidino pyrazole were infected at MOI 1 and MOI 0.01. Bars indicate SD. NIS, sodium iodide symporter.
    with established HT29 and HCT116 flank xeno-grafts that had been treated with either intratu-moral PBS, CF33-luciferase (another described CF33 derivative with deleted tk locus),14 or CF33-hNIS. A representative set of images in mice bearing HCT116 xenografts is shown in Figure 4A. In Figure 4B, we show serial images taken over several days after the initial viral in-jection and demonstrating an increase in signal on day 15 (Figure 4B, middle panel) compared with day 7 (Figure 4B, left panel). This suggests an increase in viral replication and accumula-tion in the tumor, which has been confirmed in models correlative with viral titer using similar CF33-luciferase.15 Non-injected contra-
    lateral tumors also demonstrated hNIS expression and I-124 uptake commensurate with viral infection (Figure 4B, right panel, day 22). Expected basal hNIS expression also resulted in I-124 uptake in thy-roid, stomach, and isotope excretion in the Methylpiperidino pyrazole in addition to tumor tissues. No I-124 tumor uptake was noted in PBS or CF33-luciferase control mice.
    CF33-hNIS Induces Tumor Regression in HCT116 Human Colon Cancer Xenografts
    We next examined the effect of CF33-hNIS on tumor growth. HT29-and HCT116-induced flank xenografts were implanted into athymic nude female mice. When tumors reached an average volume of 150 mm3, they received intratumoral PBS or CF33-hNIS injections at 1 105 PFU. CF33-hNIS caused tumor growth abrogation and later regression in animals with HCT116-derived xenografts that was noticeable around days 10–15 and that was sustained from then up to the end of the study period (day 55). CF33-hNIS also promoted tumor regression and growth abrogation of HT29-derived tumors in some mice, although the observed aggregate effect was not statistically significant (Figure 5B). More specifically, heterogeneity of HT29-based xenograft formation does adversely affect our ability to draw conclusions regarding viral efficacy. For example, in the treat-ment group, one outlier that did not appear to experience uniform tumor infection grew quite large. In the same HT29 group, there was a PBS control mouse that had very little tumor growth, thus bringing these curves closer together (Figure 5). In repeat HT29 ex-periments, heterogeneity of xenograft formation resulted in similar
    Figure 3. CF33-hNIS Induces Caspase-Independent Immunogenic Cell Death
    (A) Cells were infected at MOI 5. At 18 and 48 h post-infection, cells were stained for Annexin V, caspase-3, and propidium iodide, fixed, and analyzed by flow cytometry. (B)
    For calreticulin detection, cells were infected with CF33 or CF33-hNIS (MOI 5) for 16 h. 1 106 cells were resuspended in PBS containing 2% FBS and stained with antibody against calreticulin or an isotype control antibody (EPR3924; Abcam). After staining, cells were analyzed by flow cytometry. (C) Cells were left uninfected or received CF33-hNIS (MOI 5). After 16 h of incubation, supernatants were collected and ATP was measured using ATP determination kit. Bars indicate SD. Statistical significance was determined by Student’s t test; **p < 0.01, ****p < 0.0001. (D) Cells were infected with CF33-NIS (MOI 5), and supernatants were collected at 24, 48, and 72 h post-infection. Supernatants were concentrated using centrifugal filters. Proteins in the supernatants were resolved by SDS-PAGE, and HMGB1 was detected by western blot analysis. Representative western blots are shown. CRT, calreticulin; HMGB1, high-mobility group box 1 protein; NIS, sodium iodide symporter.