In vivo imaging of orthotopic prostate cancer with far-red gene reporter fluorescence tomography and in vivo and ex vivo validation

Abstract

Fluorescence gene reporters have recently become available for excitation at far-red wavelengths, enabling opportunities for small animal in vivo gene reporter fluorescence tomography (GRFT). We employed multiple projections of the far-red fluorescence gene reporters IFP1.4 and iRFP, excited by a point source in transillumination geometry in order to reconstruct the location of orthotopically implanted human prostate cancer (PC3), which stably expresses the reporter. Reconstruction was performed using a linear radiative-transfer-based regularization-free tomographic method. Positron emission tomography (PET) imaging of a radiolabeled antibody-based agent that targeted epithelial cell adhesion molecule overexpressed on PC3 cells was used to confirm in vivo GRFT results. Validation of GRFT results was also conducted from ex vivo fluorescence imaging of resected prostate tumor. In addition, in mice with large primary prostate tumors, a combination of GRFT and PET showed that the radiolabeled antibody did not penetrate the tumor, consistent with known tumor transport limitations of large (∼150 kDa) molecules. These results represent the first tomography of a living animal using far-red gene reporters.

Fig. 1

IFP1.4 gene reporter fluorescence tomography six weeks after orthotopic cell injection with in vivo and ex vivo validation. (a) to (d) show the mapped fluorescent photon distribution on the mouse surface at four different views. (e) shows the trimodal ($\text{fluorescence}/\mu \mathrm{PET}/\mu \mathrm{CT}$) tomographic image results. Blue represents skeletal information from CT images; yellow represents PET imaging information; and red represents the reconstructed results of fluorescence tomography. The artifacts on the mouse surface are removed for better demonstration. (f) and (g) show the ex vivo white and fluorescent images of the prostate with tumor in ex vivo experiments.

Fig. 2

PC3-iRFP cell imaging and stability analysis. (a) and (b) are the white and fluorescent images of the transfected PC3 cells. (c) is the stability analysis of the transfected PC3 cells by using flow cytometry. “Alexa Fluor 700-A” means that the fluorescent signals are collected by using Alexa Fluor 700-A filters. The table shows the mean channel intensity of each sample.

Fig. 3

iRFP gene reporter fluorescence tomography five weeks after orthotopic cell injection with in vivo and ex vivo validation. (a) shows the trimodal ($\text{fluorescence}/\mu \mathrm{PET}/\mu \mathrm{CT}$) tomographical results. Blue represents skeletal information from CT images; yellow represents the PET imaging information; and red represents the reconstructed results of fluorescence tomography. The artifacts on the mouse surface are removed for better demonstration. (b) and (c) show the white and fluorescent images of the prostate with tumor in ex vivo experiments.

Fig. 4

iRFP gene reporter fluorescence tomography overlaid on CT and PET at different tumor stages. (a) and (b) are reconstructed results 4 and 10 weeks after cell implantation, respectively. Blue represents the skeletal information from CT images; yellow represents PET imaging information; and red represents the reconstructed results of fluorescence tomography. The artifacts on the mouse surface are removed for better demonstration. (c) In situ white light image for euthanized mouse depicted in (b) (the liver and intestine were removed).

Fig. 5

Fluorescence imaging comparison between tumor- [(a) and (b)] and nontumor- (c) bearing mice and fluorescence tomography with nontumor-bearing mouse (d). The images were acquired from the ventral side of the mice. Blue represents skeletal information from CT images; yellow represents PET imaging information; and red regions are the reconstructed results of fluorescence tomography.