2.1.

Vector Construction

The HIV-derived recombinant lentivirus vector expressing iRFP was made by replacing the deoxyribose nucleic acid (DNA) of EGFP in the lentiviral vector pRRLSIN.cPPT.PGK-GFP.WPRE (referred to as pRRL, kindly provided by Dr. Trono, University of Geneva, Switzerland) by the iRFP DNA from the pShuttle-CMV-iRFP plasmid (a gift of Dr. Verkhusha, Addgene plasmid # 31856). To this end, iRFP DNA was excised with BglII and SalI, gel purified and ligated into BamHI/SalI-digested pRRL resulting in pRRL/iRFP construct [Fig. 1(a)].

Fig. 1

(a) Schematic diagram of pRRL/iRFP construct obtained by swapping deoxyribose nucleic acid (DNA) of EGFP in pRRLSIN.cPPT.PGK-GFP.WPRE vector by iRFP DNA. The expression of the transgene is under the control of the human phosphoglycerate kinase promoter (PGK). The central polypurine tract (cPPT) and the posttranscriptional Woodchuck hepatitis virus (WPRE) are located upstream and downstream of the transgene, respectively. RSV, rous sarcoma virus; SD, splice donor; SA, splice acceptor; Gag, deleted gag region; RRE, rev-responsive element; LTR, long terminal repeat; EGFP, enhanced green fluorescent protein; iRFP, infrared fluorescent protein. (b) Phase contrast and iRFP-based fluorescence of tumor cells transduced with pRRL/iRFP and observed with the Cy5.5 filter at excitation/emission of $660/710\mathrm{nm}$ (magnification $400\times$). (c and d) Sequential images and the deduced growth curve after subcutaneous injection of iRFP-expressing U87 MG cells. The color bar indicates the total fluorescence radiant efficiency.

2.2.

Cells and Lentiviral Transduction

The human embryonic kidney cells HEK293T, the glioblastoma U87 MG and the melanoma IGR37 cells were cultured in Dulbecco's modified Eagle medium (Cambrex, Verviers, Belgium). The osteosarcoma U2OS cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 (Cambrex). All media were supplemented with 10% fetal calf serum and antibiotics penicillin and streptomycin (Eurobio, Les Ullis, France). Lentiviral particles were produced according to Mathieu et al.¹⁴

2.3.

Animals and Tumor Implantations

Four- to six-week old male athymic Naval Medical Research Institute nude mice (Harlan, Gannat, France) were maintained in a barrier facility on high efficiency particules arresting (HEPA)-filtered racks and fed with autoclaved alfalfa-free rodent diet to avoid digestive tract autofluorescence.¹⁵ Animal experiments were conducted in accordance with the European Directive EEC/2010/63, under the supervision of authorized investigators. Subcutaneous (s.c) and intracranial (i.c) injections of U87 MG glioblastoma cells were performed as described earlier^16,17 using $1\times {10}^5$ cells (s.c) or $5\times {10}^5$ cells (i.c), respectively. For the experimental metastases, we injected $1.5\times {10}^6$ melanoma cells IGR37 through the tail veins. For intratibial xenografts, mice were anesthetized and $1\times {10}^5$ U2OS cells were injected into the tibia with a needle introduced at the top of the tibial metaphysics. Immunohistochemistry of melanoma tumors was performed according to Ref. 18, using S100, HBM-45, and Melan-A as markers.

2.4.

In Vivo Imaging and Analyses

Unless otherwise indicated, animals were subjected to NIR imaging every 3 to 4 days with a Pearl^® Impulse Small Animal Imaging System Li-Cor^® Biosciences (LI-COR Biosciences GmbH, Bad Homburg, Germany). Excitation and emission wavelengths were fixed at 690 and 710 nm, respectively. The fluorescence signal was overlaid with the white channel light-emitting diode [(LED) light] and detected by a cooled CCD camera of the imager. During the in vivo imaging process, mice were kept on the imaging bed by isoflurane inhalation (Abbott Laboratories, Rungis, France) and warmed at 37°C. In some experiments, organs or tumor deposits were retrieved with the minimum surrounding tissue from mice sacrificed by cervical dislocation and subjected to ex vivo imaging. Excised tissues were then placed on the stage and their fluorescence captured as indicated above. Image analysis was performed according to Nakayama et al.¹⁹ In brief, regions of interest (ROIs) were targeted on iRFP fluorescent sites and circles with the minimum size were drawn around lesions on each image. The pixel density in the selected ROI was quantitated using the software for image analysis provided with the Pearl^® Imager. The fluorescence signal on the plots was normalized to the background and presented as arbitrary units (a.u.) according to Filonov et al.¹¹

2.5.

Statistical Analyses

Data were collected from the three independent experiments. Animal implantation experiments were performed with 3 to 4 mice per each group of glioblastoma, osteosarcoma, or melanoma tumor models and results were expressed as a standard error of the mean (SEM).

3.1.

Visualization of iRFP-Expressing Tumor Xenografts in Mice

Tumor cells expressing iRFP were produced as described and analyzed for the NIR fluorescence before inoculation to animals [Fig. 1(b)]. As shown in Fig. 1(c), fluorescence of iRFP-expressing U87 MG glioma cells,²⁰ not only clearly distinguishes the tumor from the host but also allows a real-time monitoring of subcutaneous tumor growth, without any additive [Fig. 1(d)]. By subcutaneously inoculating increasing numbers of iRFP-labeled cells, we found that the minimal number of detectable cells by our NIR imaging device is 50,000 cells (data not shown). Filonov et al. advocated the use of iRFP for imaging of deep organs such as the liver.¹¹ We, therefore, wanted to ask whether iRFP would uncover intraosseous tumors using intracranial and intratibial inoculated iRFP-expressing tumor cells. As shown in Fig. 2(A), the growth of an intracranial implant of iRFP-expressing U87 MG cells can be easily detected and monitored in real-time in vivo [Fig. 2(A)] and ex vivo, after the brain of this mouse has been retrieved [Fig. 2(Aa)]. The deduced growth curve shows a good correlation with the images [Fig. 2(Ab)].

Fig. 2

(A) Serial whole-body images of mice harboring intracranial tumors derived from U87 MG cells. Shown also is a retrieved brain from mice at day 33 (a) and the deduced growth curve from A. The color bar indicates the total fluorescence radiant efficiency. (B) Intratibial tumors of U2OS cells and images of retrieved tibias from mice at days 43 (a) and 70 (b). The color bar indicates the total fluorescence radiant efficiency. The deduced growth curve of U2OS growth is shown in (c).

Intratibial growth of U2OS cells is shown in Fig. 2(B). Note that the presence of additional NIR fluorescent spots on the abdomen of mice between days 43 and 51, which could be due to traces of alfalfa in this animal food;¹⁵ a problem that we subsequently solved. To further analyze the intrabone tumor progression of iRFP-expressing U2OS cells, two animals from a parallel series and corresponding to those of days 43 and 70 were sacrificed and their tibias retrieved. As shown in Fig. 2(Ba), the tumor that initially localized to the head of the bone at day 43, progressively invaded the rest of the organ a week later (at day 51), eventually leading to fracture of the bone and spreading over surrounding soft tissue at day 70 [Fig. 2(Bb)]. Therefore, the iRFP-emitted fluorescence crosses the bone barrier, thus allowing us to reliably estimate tumor burden without sacrificing the animal [see also Fig. 2(Bc)].

3.2.

Visualization of Metastasis

These data being acquired, we then asked whether iRFP-based imaging could be applied to the study of metastatic spread. For this purpose, we used melanoma cells IGR37, originating from a metastatic spread of the primary human melanoma cells IGR39.²¹ As shown in Fig. 3(A), injection of iRFP-expressing IGR37 cells in the tail vein resulted in a tumor deposit in the liver [see m1 series and after dissection of the animal Fig. 3(Aa)], consistent with a previous work showing the liver to be a potential niche for melanoma metastasis.²² Surprisingly, injection of the same cells to two other mice led to tumors that appeared, not in the liver, but in the space between omoplates [Fig. 3(B), see m2 series]. Dissection of the animal revealed the presence of well-defined tumors that strongly adhered to the cervical vertebrae (data not shown) but which were positive for melanoma immunological markers such as S100, HBM-45, and Melan-A [Fig. 3(C)]. These data show that whole-body NIR imaging with iRFP uncovers different metastatic targets for melanomas.

Fig. 3

Melanoma-derived metastatic lesions were formed after tail vein injection of IGR37 cells to three series of mice. One series developed a liver metastasis (A, m1 series) and the others developed a dorsal tumor (B, only the m2 series is shown). The color bar indicates the total fluorescence radiant efficiency. (C) The tumor from the mouse m2 was retrieved and subjected to hematoxylin/eosin (HE) and immunohistochemistry using S100, HBM-45 and Melan-A markers (magnification $50\times$).