br Fig Xenograft tumor models
Fig. 5. Xenograft tumor models were gen-erated by subcutaneous injection of Mia PaCa2 or patient-derived PDAC cell line (PDCL-15), both stably expressing GLuc-2A-sr39TK. 50ul of blood were collected on day 2 and 7 after inoculation of PDAC cells. Serum GLuc levels increased with tumor growth (A) and correlation between serum GLuc levels and minute tumor volume was highly significant (B; R = 0.93, p = 0.001). Optical imaging of PDAC tumors demon-strated strong signals in PDCL-15 and Mia PaCa2 tumors expressing GLuc-2A-sr39TK and no signal in control tumors (C) with a highly significant correlation of tumor vo-lume and bioluminescence emitted photons (D; R = 0.92; p = 0.0005). In a second study, PDCL-15 Echinomycin stably expressing GLuc-2A-sr39TK were implanted in the right flank and control patient derived PDAC cells (PDCL-15) were implanted in the left flank. In two weeks, [18F]FHBG microPET/CT was performed and demon-strated highly specific imaging of the minute PDCL-15 tumor with no imaging of the control cells and no background noise (E; orange circles), which correlated to bioluminescence imaging with coelenter-azine, (F; orange circles). However, stan-dard [18F]FDG microPET/CT imaging re-vealed non-specific imaging of the both PDCL-15 tumors expressing GLuc-2A-sr39TK and control PDCL-15 tumors with extensive non-specific background noise (G; orange circles). These data demonstrate that GLuc-2A-sr39TK is highly sensitive and specific to localize minute PDAC tumors using microPET/CT imaging, and support
the hypothesis that GLuc-2A-sr39TK can be used as a precision diagnostic platform for minute human PDAC. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
localization of PDAC, we performed transient transfection of PDAC cells with a CMV-driven GLuc-2A-sr39TK vector, which resulted in similar levels of secreted GLuc to that of CMV-GLuc (Fig. 4B) and equivalent expression of sr39TK to that of CMV-sr39TK (Fig. 4C). sr39TK activity was not aﬀected by the expression of GLuc-2A-sr39T (Fig. 4D). These data demonstrate eﬀective cleavage and separate expression of GLuc and sr39TK in vitro.
Human PDAC xenograft mouse models were generated using en-gineered patient-derived PDAC cell line-15, (PDCL-15CMV−GLuc−2A-sr39TK), Mia PaCa2CMV−GLuc−2A-sr39TK, or non-transfected Mia PaCa2
cells (6 mice/group: 3 males/3 females). Serologic GLuc levels were detected at 10 mm3 (1 mm diameter) PDAC tumor volume, whereas GLuc levels were undetectable in control mice (Fig. 5A). Rising ser-ologic GLuc levels highly correlated with increasing PDAC tumor vo-lume (Fig. 5B). GLuc bioluminescent imaging signals were highly pro-portional to tumor volume (R = 0.92; p = 0.005) (Fig. 5C and D). These data demonstrated remarkable sensitivity of serologic and optical imaging GLuc for detection and localization of minute human PDAC in vivo.
To evaluate microPET/CT imaging of human PDAC tumors, 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. When tumors reached 10 mm diameter, defined as a minute human PDAC tumor, mice underwent [18F]FHBG microPET/CT (PDAC-specific imaging) and [18F]FDG microPET/CT imaging (standard, non-specific imaging), as well as bioluminescence imaging. Three hours after in-jection of the specific TK probe, [18F]FHBG, microPET/CT revealed highly specific imaging of minute PDAC tumors only in the right flank
(Fig. 5E), which was consistent with results seen with bioluminescence imaging following injection of coelenterazine (Fig. 5F). Conversely, injection of [18F]FDG microPET/CT, resulted in non-specific signals seen in both tumors and normal tissues (Fig. 5G). Therefore, expression of GLuc-2A-sr39TK in human PDAC tumors was sensitively and speci-fically detected by microPET/CT imaging, which correlated with op-tical imaging. These data support the hypothesis that PDAC expressing dual reporter genes could be used to detect and localize minute human PDAC tumors in mice using serologic and optical imaging GLuc and sr39TK/microPET/CT imaging, respectively.