Near IR Heptamethine Cyanine Dye–Mediated Cancer Imaging

Kinetics and subcellular localization of the NIR dyes. A, confocal imaging shows significant uptake of IR-783 dye in ARCaP_M cells but not in normal human prostate epithelial P69 cells at 630× magnification. B, histogram shows differential and time-dependent uptake of IR-783 by human prostate cancer ARCaP_M cells and P69 cells. C, uptake of the IR-783 dye (20 μmol/L) by ARCaP_M cells can be abrogated by 250 μmol/L BSP. D, subcellular colocalization of the NIR hepatamethine cyanine dyes with lysosomes (Lyso) and mitochondrial (Mito) tracking dyes. ARCaP_M cells that were stained with IR-783 were stained with a lysosome-specific dye, Lyso Tracking Green DND-26, and a mitochondria-specific dye, Mito Tracker Orange CMTMROS (630×). Fluorescence imaging indicates that a large portion of the IR-783 was colocalized with these subcellular organelles.

Preferential uptake and retention of the hepatamethine cyanine dyes in human tumor xenografts. Mice bearing human prostate (ARCaP_M, orthotopic prostate tumor, p.o.), bladder (T24, s.c.), pancreatic (MIA PaCa-2, s.c.), and renal (SN12C, intraosseous to tibia, i.o) tumors were injected i.p. with IR-783 at a dose of 10 nmol/20 g. NIR imaging was done 24 hours later. Each mouse was subjected to fluorescence imaging (NIR) and X-ray imaging (X-ray) using the Kodak Imaging Station Imaging System, and the two images were superimposed (Merge) for tumor localization. After imaging, tissues with specific fluorescence signals were dissected, fixed in 10% formaldehyde, and subjected to histopathologic analysis by H&E staining (200×). In mice bearing s.c. tumors, we detected both tumors based on fluorescence imaging (see arrows).

Detection of tumor metastasis in mice and spontaneous tumors in transgenic animals. A, confirmation of the presence of bone metastatic prostate tumors in mice by NIR imaging after IR-783 i.p. injection at a dose of 10 nmol/20 g. a, the ARCaP_M human prostate cancer cell line was stably transfected with AsRed2 RFP. The clone being used in this study exhibited typical ARCaP_M cell morphology (bright field, 100×) and could emit intense red fluorescence. b, cells from this clone were inoculated orthotopically to athymic mice to produce both localized prostate tumor (thick arrow) and bone metastatic tumor (thin arrow), which were detected by IR-783 fluorescence imaging of the whole animal (left) and of the dissected skeletal bone (right). c, to confirm the detection of metastasis, marrow cells from the affected tibia/femur were cultured, and isolated cancer cells were found to express RFP. d, ARCaP_M cells in the metastatic tibial/femur tumor could also be seen in formaldehyde fixed sections, either by conventional H&E stain or directly by red florescence imaging. These analyses unanimously confirmed that the signals attained in IR-783 imaging reflect metastases of the orthotopic ARCaP_M tumor. B, detection of spontaneous prostate and intestine tumors in transgenic mouse models. a, whole body NIR fluorescent imaging of TRAMP mouse before dye injection, which revealed no background NIR fluorescence. b, whole body X-ray imaging of the animal. c, whole body NIR fluorescent imaging of TRAMP mouse revealed only tumor-positive signal after IR-783 i.p. injection at a dose of 10 nmol/20 g. d, fluorescence imaging picture of TRAMP mice merged with X-ray picture. e, the prostate tumor dissected from this TRAMP mouse showed a strong NIR signal even after fixation in 4% formalin solution for 3 weeks. f, the presence of tumor cells was confirmed by histopathology (H&E stain, 100×). C, detection of multiple intestinal neoplasia in Apc^Min/+ mice after the administration of IR-783 i.p. at a dose of 10 nmol/20 g with the Olympus OV110 imaging system. a, bright field photograph of a dissected intestine in the imaging chamber. b, NIR hepatamethine cyanine dye imaging of multiple tumors along the intestine, with two tumor nodules indicated with white arrows. c and d, these two nodules were excised, and adenoma was confirmed in these specimens by H&E staining (100×).

Distribution of heptamethine cyanine dye IR-783 and its metabolites in tissues; time course and concentration-dependent studies in normal and tumor-bearing mice. A, normal organs dissected at 0, 6, and 80 hours after IR-783 i.v. injection at a dose of 10 nmol/20 g were subjected to NIR dye imaging with a Kodak Imaging Station 4000 MM (see Materials and Methods). Note that at 80 hours, IR-783 was completely cleared from all vital organs examined. B, a representative mouse bearing orthotopic ARCaP_M human prostate tumor was imaged after IR-783 10 nmol/20 g i.v. injection at 0.5, 24, 48, 72, and 96 hours. Note dye uptake and retention seen in an ARCaP_M orthotopic tumor. C, a representative mouse bearing a s.c. ARCaP_M tumor subjected to NIR imaging after IR-783 i.p. injection at a dose of 10 nmol/20 g. The left panel shows the retention of IR-783 in the tumor 24 hours after dye administration in whole body in vivo imaging. The right panel shows the ex vivo imaging of surgically dissected tissues, which confirmed the uptake and retention of IR-783 in a surgically dissected ARCaP_M tumor. Top row from left, liver, lung, and heart; bottom row from left, spleen, kidneys, and tumor. Tumor tissue displayed strong signals in both in vivo and ex vivo imaging. D, a standard curve was constructed based on the fluorescence emission intensity of IR-783 at 820 nm (see Materials and Methods) with the dye added to a PBS solution at concentrations of 0.5, 1, 2, 4, 8, 16, and 32 μmol/L. The correlation coefficient between the fluorescence emission intensity and concentration of IR-783 was estimated to be r = 0.9991 (left). The apparent dye concentration (μg/g) in organs and tumor was calculated based on the standard curve established above (right). The apparent dye concentration is defined here by the light emission intensity at 820 nm, which could include the parental IR-783 and its metabolites. Data are expressed as average ± SEM of three determinations.