2.1.

Mouse Handling and Surgical Procedure

Experiments were conducted in agreement with the European Commission guidelines and directives of the French Research Ministry at University Bordeaux Segalen animal’s facilities. Adult female nude immuno-deficient mice (Swiss Nude, Charles River, laboratories, Lyon, France) were used in this study. For subcutaneous tumor implantation, U87-CMV-lucF cells ($2\times {10}^6\mathrm{cells}/100\mu \mathrm{L}$) were injected on the right posterior leg. For brain tumor implantation, mice (20 to 25 g) were anesthetized with isoflurane (2% in air, Belamont, Nicholas Piramal Limited, London United Kingdom) and placed into a stereotaxic frame support. U87-CMV-lucF tumor cells ($6\times {10}^5/5\mu \mathrm{L}$) were implanted in the brain, 1.5 mm posterior and 2.5 mm lateral to the bregma, 2.5 mm depth from the skull surface.

U87-CMV-lucF was obtained as follows. U87 human glioma cell line was obtained from the American Tissue Culture Collection (ATCC, Manassas, Virginia). Cells were maintained in DMEM (Invitrogen, Carlsbad, California) supplemented with 10% fetal bovine serum. To generate a bioluminescent glioma cell line, U87 cells were first genetically modified for random integration of FRT sites within the genome. A recombinant clone containing several FRT sites was first selected with zeocin and several insertion sites were detected by Southern blot. Then, the U87-FRT line was co-transfected with a pcDNA5-FRT plasmid containing the lucF reporter gene under the control of the constitutive CMV promoter and the pOG44 plasmid encoding for the Flp recombinase and cells were selected with hygromycin (Flp-in system, Invitrogen, Carlsbad, California). The resulting stably transformed cell line U87-CMV-lucF has been amplified and used for all implantations in nude immuno-deficient mice.

2.2.

Nanoparticles Synthesis

For the preparation of a 2 mL dispersion batch of DiR-loaded lipidots, 150 mg of Suppocire® NB (Gatefosse, France), 50 mg of soybean oil (Croda, France), 138 mg of $L\text{-}\alpha$ lecithin (Lipoïd-S75™, Germany), and 800 nmol of DiR (766 μg) (Invitrogen, France) are dissolved in 1 mL of dichloromethane and mixed at 50°C.⁹ After homogenization, the organic solvent is evaporated under reduced pressure, and the lipid phase is crudely mixed with the continuous phase composed of 228 mg of Mirj® 53 (Croda, France), 50 mg of glycerol (Sigma-Aldrich, France) and 1.38 mL of 154 mM NaCl. An oil-in-water nanoemulsion is obtained by ultrasonication of the oil and aqueous phases (VCX750 Ultrasonic processor, 5 min sonication at 25% power). To separate any dye or other compounds not associated with the lipid nanoparticles, lipidots are dialyzed overnight in a large volume ($\approx 800\mathrm{mL}$) of NaCl 154 mM, at room temperature and protected from the light. Samples are sterilized on 0.22 μm filters (Millipore, France), then diluted to the desired concentration, suitable for characterization and in vivo injection. Particle size, polydispersity, and zeta potential are characterized by dynamic light scattering (Zeta Sizer Nano ZS, Malvern Instrument). Absorbance and fluorescence spectra, respectively measured with a Cary 300 scan ultraviolet (UV)-visible spectrophotometer (Varian) and a Perkin-Elmer LS50B fluorimeter, allow the optical characterization of the particles as well as the quantification of dye-loading. Lipid nanodroplets are obtained at a concentration of $4.5\times {10}^{15}\mathrm{particles}/\mathrm{mL}$. They exhibited a diameter of $37\pm 2\mathrm{nm}$, a polydispersity index of $0.12\pm 0.02$, and a zeta potential of $-2.5\pm 2.0\mathrm{mV}$. A mean value of 9 DiR fluorophores per particle core is calculated based on absorbance spectra (180 μM dye, 90% encapsulation yield, absorption maximum 750 nm; emission maximum 770 nm).

2.3.

In Vivo Bioluminescence Imaging

Bioluminescence images of mice were acquired using a NightOWL II LB 983 system equipped with a NC 100 deep-cooled ($-70°\mathrm{C}$) CCD camera (Berthold Technologies, Bad Wildbad, Germany). Mice were injected intra-peritoneal with D-luciferin [Promega, Madison, Wisconsin, 2.9 mg in 100 μL sterile phosphate buffer (PBS)] and were sedated 5 min later with isoflurane (2% in air). Bioluminescence images (2 min integration periods, $4\times 4$ binning) and photos (100 ms exposition) were taken 8 min after the luciferin injection on anesthetized mice. A low light-emitting standard (Glowell, LUX biotechnology, United Kingdom) was placed next to the animal during each image acquisition to provide a constant reference for the resulting images. Grayscale body-surface reference images were collected for superposition of BLI images on anatomical maps. Pseudocolor luminescent images representing the spatial distribution of emitted photons were generated using IndiGO™ 2 software (Berthold Technologies, Bad Wildbad, Germany). BLI analysis was done semi-automatically by first placing a small region of interest (ROI). Within this ROI, the mean light intensity ($\mathrm{Photons}·{\mathrm{s}}^{-1}·{\mathrm{mm}}^{-2}$) was measured.

2.4.

Fluorescent Diffuse Optical Tomography

Mice were injected intravenously with 200 μL of lipid nanoparticle suspension in PBS isotonic to blood (100 μM DiR; $5\times {10}^{14}$ particles). Before and at different times after injection, mice were anesthetized (2% isoflurane in air) and positioned on a dedicated holder. For brain tumor detection, mouse head is gently maintained between the bottom glass plates, and a small, upper glass plate positioned on the mouse head. For a subcutaneous tumor, the free space mode was used.¹¹ The holder is placed in the dark chamber of the fDOT prototype. The instrument consists of a laser source (690 nm, 21 mW, Powertechnology) mounted on two motorized translation stages and a CCD cooling ($-30°\mathrm{C}$) camera (Hamamatsu Orca AG, Massy, France). The laser is placed under the animal for transillumination. Typically, the excitation source describes a regular $11\times 11$, 2 mm spaced grid ($2\times 2{\mathrm{cm}}^2$ field of view) under the examined sample. For each source position, the CCD camera focused onto the upper glass window, records at first the transmitted (excitation) images. Then a RG9 high-pass filter (cut off at 720 nm, Schott, Mainz, Germany) is inserted, and the fluorescence (emission) images are acquired. The exposure time is adapted for each source position in order to take advantage of the entire dynamic range of the camera, even in highly heterogeneous regions of the animal. The transmitted images lead to the reconstruction of an optical heterogeneity map; it depicts compounds of the intrinsic tissues heterogeneity and the border effects due to the complex shape of the turbid medium.

The second step is the reconstruction of the fluorescence yield from the fluorescent images using this heterogeneity map. Convergence of the algorithm is achieved within 15 iterations by using a classical iterative ART algorithm with a relaxation parameter of 0.1.¹²

2.5.

Fluorescence Reflectance Imaging (2D-FRI)

The fDOT prototype could also be used for FRI using a light emitting diode (LED) ring at 660 nm (Hamamatsu) as excitation source. Near-infrared fluorescence images (exposure time 1 s) were acquired through the RG9 filter by the charge coupled device (CCD) camera.

2.6.

Magnetic Resonance Imaging

Animals were sedated with isoflurane (2% in air) and maintained in the fDOT double plate holder. The animal was positioned in prone position into the MRI scanner (1.5 Tesla, Achieva Philips Healthcare). A 23-mm surface receiver coil was positioned above the head mouse hind leg to provide sufficient signal-to-noise ratio.T1-weighted ($\mathrm{TR}/\mathrm{TE}=650/7.6\mathrm{ms}$) and T2-weighted ($\mathrm{TR}/\mathrm{TE}=2220/80\mathrm{ms}$) sequences were performed in coronal view, with a 0.31 mm in plane resolution, to localize and to measure the size of the tumors. Slice thickness of both, MR and reconstructed three-dimensional (3-D) fluorescent images, were identical (1 mm).

3.1.

Time Course of DiR-Lipidots Fluorescence in Mouse-Bearing Subcutaneous Tumor

U87-CMV-lucF cells were injected to generate subcutaneous tumors in immuno-deficient mice ($n=6$). Tumors were left growing for about two months, taking care that diameter never exceeded 10 mm. Tumors were then assayed for lucF expression by BLI to attest the presence of viable U87-CMV-lucF cells within the tumor [Fig. 1(a)]. Mice were quickly transferred from BLI system to fDOT machine and mice auto-fluorescence was determined by 2D-FRI [Fig. 1(b)] and fDOT using the free space mode [Fig. 1(g)]. The 3-D fluorescent maps computed using the fDOT software provide a stack of 18 fluorescent slices with 1 mm of thickness, from ventral ($z=0$) to dorsal ($z=17$). As shown on Fig. 1(g), no fluorescent signal was found within the 3-D reconstruction maps.

Fig. 1

Optical imaging of a subcutaneous tumor. (a) Bioluminescence image of a mouse bearing a U87-CMV-lucF tumor. Next to the mouse, a low light-emitting standard is placed. (b), (c), (d) fluorescence reflectance imaging of the same mouse before (b); 24 h (c), and 120 h (d) after systemic injection of DiR-lipidots. White bar represents 9 mm (e) position of regions of interest (ROI) for quantification by FRI (circle). Square represents the scanning zone for fDOT. (f) Evolution of the fluorescent level after DiR-lipidots intravenous injection in the 3 different ROI (ROI 1 is an area on the back of the mouse; ROI 2 is on the head and ROI 3 is on the subcutaneous tumor as illustrated in panel E). (g) and (h): Results of fDOT reconstructions before (g) and 24 h after (h) DiR-lipidots injection. $Z$ cross-sections (1 mm thickness) are presented in the same color scale from $z=0$ (ventral) to $z=17$ (dorsal). fDOT images were acquired using the free space mode. Images are representative of $n=\mathrm{six}\mathrm{mice}$.

Mice are allowed to wake up from isoflurane anesthesia and 10 min later injected intravenously by 200 μL of DiR-lipidots solution. As shown on Fig. 1(c) and 1(d), the fluorescence level as determined by 2D-FRI, rapidly increases in the whole body of the animal and in the tumor. A maximum of fluorescence was observed 4 h after injection, and then 2D-FRI signal slowly decreased [Fig. 1(f)]. The fluorescence level in the ROI located on the subcutaneous tumors was more intense and decreased more slowly than in ROIs placed on other areas of the mouse [Fig. 1(e)]. The difference in fluorescence levels between inside and outside the tumor was maximum 8 h post-injection, [Fig. 1(f)]. The fluorescent signal of DiR fluorophore remained detected in the tumor up to seven days after injection. A fDOT acquisition was performed 24 h post-injection [Fig. 1(h)] using the free space mode with 3-D reconstruction maps. The signal of fluorescence found on slices $z=9$ to $z=13$ was consistent with an accumulation of DiR fluorophore into the tumor.

3.2.

Multimodal Detection of Brain Tumors Using BLI/fDOT/MRI

After stereotaxic injection of U87-CMV-lucF cells into the brain (right parietal area), tumor growth was followed every week by BLI. The BLI signal was used to determine the optimal time for DiR-lipidots injection. Small tumors exhibiting BLI signal lower than $\mathrm{50,000}\text{photons}·{\mathrm{s}}^{-1}·{\mathrm{mm}}^{-2}$ were found not detectable using fDOT and the current MRI setup (data not shown).

Mice used for multi-imaging sequence exhibited a BLI signal from the brain tumor above $\mathrm{200,000}\text{photons}·{\mathrm{s}}^{-1}·{\mathrm{mm}}^{-2}$. Mice were first imaged by BLI [Fig. 2(a)] and immediately submitted to a fDOT acquisition [Fig. 2(c)]. The 3-D fluorescent maps computed using the fDOT software provide a stack of 16 fluorescent slices with 1 mm of thickness, from ventral ($z=0$) to dorsal ($z=15$). As shown in Fig. 2(c) substantial amounts of fluorescence were detected before DiR-lipidots injection. After a fDOT acquisition, mice are allowed to rescue from anesthesia (10 min) and are then injected intravenously with the DiR-lipidots solution. MRI and fDOT were performed 8 h after injection, sequentially and using the double plates contention support [Fig. 2(b)].

Fig. 2

Imaging of a brain tumor. (a) Bioluminescence image of a mouse bearing an intracerebral U87-CMV-lucF tumor. (b) Picture of the mouse in the fDOT double-plate contention holder. Upper glass plate is limited to the mouse’s head. (c) and (d) Results of fDOT reconstructions before (c) and 8 h (d) after DiR-lipidots injection. $Z$ cross-sections (1 mm thickness) are presented in the same color scale from $z=0$ (ventral) to $z=15$ (dorsal). (e): T2-weighted MR images of the same mouse maintained in the fDOT double-plate contention holder. $Z=12$ to $z=14$ cross-sections (1 mm thickness corresponding with the fDOT frame) showing the brain tumor localization. Slices ($z=0$ to $z=9$) corresponding with the outside of the brain. Images are representative of $n=\mathrm{six}\mathrm{mice}$.

As shown on Fig. 2(d), the 3-D fluorescent maps reveal fluorescent hot spots on slices $z=12$, $z=13$, and $z=14$. The MRI images are fitted on the fDOT thickness to provide the same stack of slices (1 mm of thickness). fDOT and MRI images from the $z$-stack were compared $z$-slice to $z$-slice. As shown in Fig. 2(e), slices $z=12$, $z=13$, and $z=14$ exhibiting hot fluorescent spots fit with MRI slices corresponding to brain tumors location. A substantial amount of fluorescent signal was also found outside the brain in slices ($z=0$ to $z=9$) corresponding to the buccal area. Alternatively, fluorescence distribution of the DiR dye could be provided as a 3-D representation as illustrated in Fig. 3.

Fig. 3

3-D imaging of fDOT reconstruction after DiR-lipidots injection of a mouse bearing an intracerebral U87-CMV-lucF tumor. Image representative of the same batch of animals than Fig. 2 ($n=6$).