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  • br Optical imaging and serum GLuc measurement br

    2021-03-03


    2.6. Optical imaging and serum GLuc measurement
    Animal experiments were performed in accordance with IACUC guidelines. Ten to 8-week-old male Nude/Nude mice (Charles River Laboratories, Inc. Wilmington, MA) were subcutaneously implanted with 1 × 106 Mia PaCa2 or Capan2 SPDP stably expressing GLuc-2A-sr39TK with Matrigel. Optical imaging was performed with an IVIS CCD camera (IVIS Lumina II, Perkin Elmer, Waltham, MA) on day 8, 14, 21, and 30. Mice were anesthetized with isoflurane, and then given 100 μg/ 50 μl of water-soluble coelenterazine per mouse (Nanolight Technology, Pinetop, AZ) via tail vein and immediately imaged and analyzed using the Living Image Software (Caliper Life Sciences). For measurement of serum GLuc levels, 50 μl blood per mouse were col-lected from xenograft tumor mice via tail vein on days 2, 4, 7, 14, 21, and 30 after cancer cell implantation and serum was extracted; 50 μl serum was used for evaluation of GLuc levels.
    2.7. MicroPET/CT imaging
    Engineered patient-derived PDAC cell line-15 (PDCL-15CMV−GLuc−2A-sr39TK) cells versus control PDCL-15WT cells were im- planted in the right and left flanks, respectively, of the same mouse. On day 8, tumors reached 10 mm, defined as a minute human PDAC tumor, 
    animals were optically imaged followed by injection with 200 μCi [18F] FHBG via the tail vein. Following an hour of substrate uptake, the entire animal was scanned for 20 min in a micro-PET/CT scanner (Genisys, Sofie Biosciences, CA), and imaged for 15 min at 50 keV, 325 μA, and 196 total angles of rotation in two bed positions. After 24 h, mice were injected with 200 μCi [18F]FDG via the tail vein, and micro-PET/CT scanning was performed. All microPET/CT data analyses and 3-D images were compiled using AMIDE.
    2.8. Targeting AAV2 detection virus delivery and tumor imaging
    Ten 6 to 8-week-old nude mice were engrafted subcutaneously with 1 × 106 PANC-1 tumor cells. When tumors reached 4.5 mm, defined as
    a minute human PDAC tumor, 8 × 1011 genome copies (gc) of AAV2WT-CMV-GLuc-2A-sr39TK, AAV2RGD-CMV-GLuc-2A-sr39TK or AAV2RGD-BIRC5-SPTSTA-GLuc-2A-sr39TK were injected via tail vein and luciferase signals were detected by optical imaging 1-week post-injection.
    2.9. Statistical analysis
    Data from western blot, reporter assays, and immunohistochemistry experiments were analyzed by two-tailed t-test in Excel. All error bars indicate s.d. unless stated otherwise. Organoid size and shape mea-surements were analyzed by t-test using GraphPad Prism. One-way ANOVA was used to compare and correlate multiple groups using GraphPad Prism 6.
    3. Results
    3.1. AAV2RGD-BIRC5-SPTSTA-GLuc-2A-sr39TK is highly efficient and specific for detection and localization of minute human PDAC tumors in mice
    AAV2RGD-BIRC5-SPTSTA-GLuc-2A-sr39TK vector is a viral vector that is highly specific for PDAC, as the engineered viral surface RGD spe-cifically binds to integrin on PDAC cell surface and the TSTA enhanced BIRC5 super promoter is only activated in the PDAC cells. In addition, enhanced super-promoter contributes to highly sensitive detection of
    BIRC5 expression. To evaluate the sensitivity and specificity of AAV2RGD-BIRC5-SPTSTA-GLuc-2A-sr39TK to detect minute PDAC tumors
    in xenograft mice, 1 × 106 PANC1 cells were implanted into the flank of nude mice and allowed to grow for two weeks to a size of 4.5 mm, defined as a minute human PDAC tumor. Control mice were non-tumor
    bearing. PANC-1 cells were chosen, as they express BIRC5 and integrin and harbor KrasG12D and p53R273H mutations. AAV2RGD-BIRC5-SPTSTA- GLuc-2A-sr39TK (versus control vectors AAV2RGD-CMV-GLuc-2A-sr39TK or AAV2WT-CMV-GLuc-2A-sr39TK) was systemically admini-strated via tail vein injection (2 × 1011 vp; n = 10/group; 5 males; 5 females). Imaging studies and serologic GLuc levels were performed at
    1 week; no toxicity was observed in any group. AAV2RGD-BIRC5-SPTSTA-GLuc-2A-sr39TK resulted in a strong signal from a 4.5 mm PANC-1 tumor with complete absence of non-specific signals (Fig. 1 A1; red circle). Control AAV2RGD-CMV-GLuc-2A-sr39TK resulted in significantly less tumor signaling with extensive non-specific signaling (Fig. 1 A2; red circle). Control vector AAV2WT-CMV-GLuc-2A-sr39TK resulted in negligible tumor signaling with extensive, diffuse and non-specific signals (Fig. 1 A3; yellow circle). Correspondingly, quantification of bioluminescence images revealed high, low, and minimal photon levels for tumor imaging, respectively (Fig. 1 B1, B2 and B3), as well as ab-
    sent, moderate and high levels of photons for background signals in mice given AAV2RGD-BIRC5-SPTSTA-GLuc-2A-sr39TK, AAV2RGD-CMV-