3.5. In Vivo NIRF Optical Imaging

Generate the tumor xenografts by subcutaneous injection of five million HT-1080 cells suspended in 50 μL of cell culture media and 50 μL of BD Matrigel into the right shoulder of female athymic nude mice (Harlan Laboratories, Livermore, CA, USA) (see Note 9). The mice are subjected to in vivo NIRF imaging studies when the tumor volume reaches 200–300 mm³ (about 2 weeks after inoculation). Monitor the tumor volume every other day using a caliper following the formula V = (witdth)² × length/2.

Set up the IVIS Imaging System 200 Series (PerkinElmer Inc., Alameda, CA, USA). A Cy5.5 filter set is used to acquire the fluorescent signals.

Obtain the fluorescent signals by scanning a tumor-bearing mouse before the injection of Cy5.5-NGR2.

Inject 1.5 nmol of Cy5.5-NGR2 into a tumor-bearing mouse via the tail vein while the mouse is under isoflurane anesthesia.

For the blocking experiment, inject 1.5 nmol of Cy5.5-NGR2 with a non-labeled monomeric NGR peptide (20 mg/kg) into a tumor-bearing mouse via the tail vein.

Scan the animal at 0.5, 1, 2, 3, and 4 h (see Note 10). The identical illumination settings (lamp voltage, filters, f/stop, field of views, binning) are used to acquire all images. All NIRF images are acquired using 1-s exposure time (f/stop = 4). Fluorescence emission images are normalized and reported as photons per second per centimeter squared per steradian (p/s/cm²/sr) (see Note 11).

Representative NIRF images are shown in Fig. 5 after HT-1080 tumor-bearing mice are injected with 1.5 nmol of Cy5.5-NGR2. To determine tumor contrast, mean fluorescence intensities of the tumor area at the right shoulder of the animal and of the normal tissue at the surrounding tissue are calculated using the region-of-interest (ROI) function of the IVIS Living Image 4.4 software (PerkinElmer Inc., Alameda, CA, USA). The fluorescence signal resulting from Cy5.5-NGR2 is clearly visible and the tumor fluorescence intensity reaches maximum at 2 h post-injection (Fig. 6). The NIRF images of HT-1080 tumor-bearing mice at 2 h pi from the non-blocking and blocking group are presented in Fig. 7. Tumor contrast as quantified by the ROI analysis of images shows that the tumor-to-muscle value at 2 h pi is reduced from 2.65 ± 0.13 to 1.05 ± 0.06 (P<0.05) by blocking CD13 receptor with a non-labeled NGR peptide (Fig. 8) (see Note 12).

Time-course fluorescence imaging of subcutaneous HT-1080 tumor-bearing nude mice after intravenous injection of 1.5 nmol of Cy5.5-NGR2. The tumors are clearly visualized as indicated by an arrow from 0.5 to 4 h pi. The fluorescence intensity is recorded as per second per centimeter squared per steradian (p/s/cm²/sr). Reproduced from Ref. 6 with permission from Springer

Quantification and kinetics of in vivo targeting character of Cy5.5-NGR2 in the HT-1080 tumor vs. muscle. The Cy5.5-NGR2 uptake in HT-1080 tumor at various time points is significantly higher than that in muscle. Error bar is calculated as the standard deviation (n = 3). Reproduced from Ref. 6 with permission from Springer

Representative optical imaging (at 2 h pi) of mice bearing HT-1080 tumor on the right shoulder demonstrating blocking of Cy5.5-NGR2 (1.5 nmol) uptake by co-injection with a non-labeled monomeric NGR peptide (20 mg/kg). Reproduced from Ref. 6 with permission from Springer

Fluorescence intensity ratio of tumor to muscle based on the ROI analysis of Cy5.5-NGR2 uptake at 2 h pi in HT-1080 tumors without (non-blocking) or with (blocking) co-injection of a non-labeled monomeric NGR peptide (20 mg/kg). Error bar is calculated as the standard deviation (n = 3). Reproduced from Ref. 6 with permission from Springer