Figure 1.
Reactivity of the NIR-αHLA probe with HCT 116 (human colorectal cancer) cells in vitro and in vivo.
(A) HCT 116 cells were incubated with various NIR-probes, and the in vitro fluorescence signals were specifically detected at wavelengths of 745/800 nm, which were overlaid onto a bright-field image. (B) In vivo fluorescence images of HCT 116 tumor-bearing BRG nude mice. Dose-related effects of the NIR fluorescence intensities after iv injection of various amounts of NIR-αHLA probe can been observed. Fluorescent signal from the NIR-αHLA probe was specifically detected at wavelengths of 720/790 nm. The bright-field image is shown in the top panel, the fluorescent image is shown in the middle panel, and the overlay image is shown in the bottom panel. (C) The time course of the NIR fluorescence intensity of BRG nude mice that had received an iv injection of the NIR-αHLA probe. (D) Fluorescence intensities were quantified using ROIs of equivalent-sized areas from the lower abdominal regions at the indicated time points. Data are presented as the mean ± SD of three individual mice (Student’s t test, *p value of 40 min and 60 min = 0.0390, for 40 min and 24 hr = 0.0151, and for 60 min and 24 hr = 0.0313).
Figure 2.
Proof of concept experiments for the in vivo imaging of human tumors with the NIR-probes.
(A) In vivo fluorescence images of BxPC-3 and HCT 116 tumor-bearing BRG nude mice (left and right flank, respectively) were taken 1, 2, and 9 days after iv injections with various NIR-probes. (B) Ex vivo fluorescence images of the BxPC-3 and HCT 116 xenografts and tissues of recipient BRG nude mice. The bright-field photograph is shown in the left panel, the fluorescence image is shown in the center panel, and the overlay image is shown in the right panel. The fluorescent signal from the NIR-probes were specifically detected at 745/800 nm using an IVIS SpectrumCT.
Figure 3.
Validation of in vivo imaging of human tumors with the NIR-conjugated macromolecule probes.
(A) Bright-field images and fluorescence images of the T-HCT 116 cells (which express tdTomato) and HCT 116 cells in vitro. Fluorescent signal from the orange-red fluorescent protein tdTomato and the NIR-αHLA probe were specifically detected at wavelengths of 535/600 nm and 720/790 nm, respectively. The absence or presence of the NIR-αHLA antibody is indicated as NIR-αHLA (–) or (+), respectively. (B) In vivo fluorescence images of T-HCT 116 and HCT 116 tumor-bearing BRG nude mice. The NIR fluorescence intensity 2 days after iv injection of the NIR-αHLA probe can be observed. Fluorescent signal from tdTomato and NIR-αHLA probe were specifically detected at wavelengths of 535/600 nm and 720/790 nm, respectively, using the Kodak In-Vivo Imaging System FX. The absence or presence of the NIR-αHLA probe is indicated as NIR-αHLA (–) or (+), respectively. The red and yellow arrows indicate engraftment sites of T-HCT 116 cells and HCT 116 cells, respectively. (C) Fluorescent signal of the NIR-conjugated macromolecule probes co-localized with tdTomato in T-HCT 116 cells in tumor-bearing BRG mice. The fluorescent signals at 535/600 nm and 745/800 nm were overlaid (composite) using Living Image software 4.1.3. Li; liver, Sp; spleen. (D) Immunohistochemical staining of dissected tumors; anti-RFP (RFP; left), anti-mouse IgG2a (MIgG2a; center), and anti-CD31 (CD31; right); Enlarged view of boxed area shown below. Arrowheads indicate same position the on serial section. Scale bar, 200 µm.
Figure 4.
Time course of fluorescence intensities in human tumor xenografts after iv injection of NIR-conjugated macromolecule probes.
(A) The fluorescent signal from the tdTomato (left panels) and NIR-probes (right panels) in subcutaneous T-HCT 116 tumor-bearing BRG nude mice (left and right flank, respectively) were detected at 1, 7, and 14 days after iv injection with NIR-probes, at wavelengths of 535/600 nm and 745/800 nm, respectively, using an IVIS SpectrumCT. (B) Fluorescence intensities of the tdTomato signal were quantified using ROIs of equivalent-sized areas from the tumor regions at the indicated time points (Student’s t test, NIR-Isotype probe injected: *p value for 1 day and 14 days = 0.0216 and for 7 days and 14 days = 0.0126; NIR-αHLA probe injected: *p value for 1 day and 7 days = 0.0299). (C) Fluorescence intensities of the NIR-probe signal were quantified using ROIs of equivalent-sized areas from the tumor regions at the indicated time points (Student’s t test, NIR-Isotype probe injected: *p value for 7 days and 14 days = 0.0176; NIR-αHLA probe injected: *p value for 1 day and 14 days = 0.0172, and ***p value for 7 days and 14 days<0.0001). Data were presented as the mean ± SD of four individual tumors (two individual tumors in case of day1-#4 mouse. (D) The signal-to-background ratio (S/B) at 1, 7, and 14 days post-administration of the NIR-probes was calculated using the following formula: S/B = (fluorescence intensity of ROI)/((background intensity) – (fluorescence intensity of ROI)). In each case, the background was derived from equivalently sized areas containing the same number of pixels.
Figure 5.
In vivo imaging of the liver metastasis model with the NIR-αHLA probe.
(A) In vivo fluorescence images of T-HCT 116 tumor-bearing BRG nude mice. The NIR fluorescence intensity 24 hr after iv injection of the NIR-αHLA probe can be observed using an IVIS SpectrumCT. The fluorescent signal from tdTomato and the NIR-αHLA probe were specifically detected at wavelengths of 535/600 nm and 745/800 nm, respectively. Nontransplant indicates that the BRG mouse had not received the T-HCT 116 cell transplant. Li; liver, Pa; pancreas. (B) Immunohistochemical staining of dissected livers; anti-RFP (RFP; left), and anti-mouse IgG2a (MIgG2a; right); Enlarged view of boxed area shown below. Scale bar, 200 µm.
Figure 6.
In vivo imaging with the NIR-αHLA probe detected human tumors in various transplantation models.
(A) In vivo fluorescence images of BRG nude mice that had been implanted with small pieces of LC11-JCK xenograft tumors were taken 48 hr after iv injection with the NIR-αHLA probe or the NIR-Isotype probe (left panels). In laparotomized body ex vivo fluorescence images (center panels) and ex vivo fluorescence images (right panels), the fluorescent signal was specifically detected at wavelengths of 720/790 nm using the Kodak In-Vivo Imaging System FX. GI; gastrointestinal tract, Ki; kidney, Xe; LC11-JCK tumor xenograft, Li; liver. (B) In vivo fluorescence images of BRG nude mice that had received an intravenous (via tail-vein) injection of 1×105 HCT 116 cells were taken 48 hr after iv injection with the NIR-αHLA probe (left panels). Laparotomized body ex vivo fluorescence images (right panels) are also shown. A bright-field image is shown in the top panel, a fluorescence image is shown in the center panel, and the composite image is shown in the right panel. (C) An in vivo fluorescence image of a NOG mouse that had received an intrasplenic injection of 1×106 BxPC-3 cells was taken 48 hr after iv injection with the NIR-αHLA probe (left panels). Laparotomized body ex vivo fluorescence images (right panels) are also shown. The fluorescent signal from the NIR-probes was acquired using the Kodak In-Vivo Imaging System FX.