Bacteria-mediated cancer imaging and immunotherapy is emerging fields. However, most of the study related to targeting and monitoring of therapeutic response after bacterial therapy was done with tissue analysis after sacrificed the animal. To evaluate the theranostic efficacy of fluorescence encoded bacteria in vivo, the tumor should be made on the subcutaneous area due to the limitation of light penetration of the tissue material. However, the subcutaneous xenograft model doesn’t reflect the actual tumor microenvironment. In this study, we monitored theranostic bacteria in orthotopic tumor model with lab-built wide-field fluorescence endoscopy (WFE) and commercialized confocal endomicroscopy (CFEM) in vivo. First, orthotopic mouse colon cancer models were made by using endoscopic cancer cell implantation methods. Seconds, fluorescence emitting theranostic bacteria was constructed by using Salmonella strain. The bacteria were injected into the orthotopic colon tumor-bearing mice via tail vein injection. Finally, we serially monitored fluorescence signal emitted from theranostic bacteria with WFE and CFEM. Multiscale fluorescence image showed the accumulation of bacteria within the tumor area compared to normal tissue contrast. We expected that this endoscopic fluorescence imaging approaches can be used in the direct monitoring of bacterial immunotherapy in vivo.
Cerenkov luminescence (CL) is generated when a charged particle moves faster than the speed of light in dielectric media. Recently CL imaging becomes an emerging technique with the use of radioisotopes. However, due to relatively weak blue light production and massive tissue attenuation, CL has not been applied widely. Therefore, we attempted to shift the CL emission to more near infrared (NIR) spectrum for better tissue penetration by using Cerenkov Radiation Energy Transfer (CRET). Gold nanoclusters were conjugated with NIR dye molecules (AuNc-IR820 and AuNc-ICG) to be activated with ultraviolet light. We found optimal conjugate concentrations of AuNc-NIR conjugates by spectroscopy system to generate maximal photon emission. When exposed by ultraviolet light, the emission of NIR light from the conjugates were verified. In quantitative analysis, AuNc-NIR conjugates emit brighter light signal than pure AuNc. This result implies that NIR fluorescent dyes (both IR820 and ICG) can be excited by the emission from AuNc. Following the above baseline experiment, we mixed F-18 fluorodeoxyglucose (F-18 FDG) radioisotope to the AuNc- NIR conjugates, to confirm NIR emission induced from Cerenkov radiation. Long pass filter was used to block Cerenkov luminescence and to collect the emission from AuNc-NIR conjugates. Instead of one long exposure imaging with CCD, we used multiple frame scheme to eliminate gamma radiation strike in each frame prior to combination. In summary, we obtained NIR emission light from AuNc-NIR conjugated dyes that is induced from CL. We plan to perform in vivo small animal imaging with these conjugates to assess better tissue penetration.
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