2.1.

Cell Line and Animal Model

All studies were conducted in compliance with Washington University Animal Welfare Committee’s requirements for the care and use of laboratory animals in research. U87-GL-GFP PDE7B H217Q cells²² were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, penicillin ($100\text{units}/\mathrm{mL}$), and streptomycin ($100\mu \mathrm{g}/\mathrm{mL}$), all obtained from Gibco (Life Technologies, New York). Tumors were initiated by stereotactic injection of $5\times {10}^4\text{cells}$ in $2\text{-}\mu \mathrm{L}$ phosphate buffered saline (PBS) in the brain (relative to bregma 0.5 mm anterior, 2.2 lateral, 0.6 to 0.65 mm ventral) into Athymic NCr-nu/nu female mice aged 7 to 13 weeks (Charles River Laboratories). Sham control mice were injected with $2\mu \mathrm{L}$ of PBS. Mice were maintained on a low-fluorescence chow diet.

2.2.

Bioluminescence Imaging

For BLI of living animals, mice were injected intraperitoneally with $150\mu \mathrm{g}/\mathrm{g}$ d-luciferin (Gold Biotechnology, Missouri) in PBS, anesthetized with 2.5% isoflurane, and imaged with a charge-coupled device camera-basedBLI system (IVIS 50 Perkin Elmer, Massachusetts); exposure time 1 to 60 s, binning 4 to 8, field of view 12, $f/\text{stop}1$, and open filter. Radiance is displayed as $\text{photons}/\mathrm{s}/{\mathrm{cm}}^2/\mathrm{sr}$.

2.3.

Fluorescence Molecular Tomography

Mice were anesthetized with 2% isoflurane and injected intravenously with either $60\mu \mathrm{M}$ ICG (Cardiogreen, Sigma-Aldrich, Missouri) or Cypate-cyclo(D-Cys-Gly-Arg–Asp-Ser-Pro-Cys)-Lys-OH (LS301) in $100\text{-}\mu \mathrm{L}$ PBS or Alexa Fluor™ 680 Conjugate (ThermoFisher, Massachusetts). 3-D tumor images were obtained using the FMT 4000 system (PerkinElmer, Inc., Massachusetts) with $1.5\mathrm{mm}\times 1.5\mathrm{mm}$ source density. Reconstruction and image analyses were performed using TrueQuant™ software (PerkinElmer, Inc., Massachusetts). Fluorescence quantification of fluorophores was based on concentration-dependent calibration using the calibration phantom provided with the system. Rectangular prism ROIs were drawn manually around tumors of 3-D reconstructed images, using the topographic projections, stereotactic coordinates, and bioluminescence images for guidance. Quantification of data is given as mean fluorophore concentration in regions of interest, omitting voxels with arbitrary low signal ($<{10}^{-9}\mathrm{nM}$). Background subtracted quantification was obtained by subtracting the mean concentration in the preinjection data from postinjection data.

2.4.

Longitudinal Imaging

The U87GL cells ($n=8$) or PBS for sham controls ($n=5$) were initiated by stereotactic cortical injection.BLI was used to track tumor progression. Mice were then imaged weekly by BLI and FMT with successive injections of ICG and LS301. Typically, images were acquired preinjection and at 0.5, 1, 4, and 24 h, following each injection using the 790-nm channel of the FMT for up to 7 weeks postcortical injection.

2.5.

Focused Ultrasound

Sonication was performed as described previously.^23,24 A preclinical FUS system (VIFU 2000, Alpinion US Inc., Washington) was used for FUS sonication. The FUS transducer has a center frequency of 1.5 MHz, focal depth of 60 mm, and aperture of 60 mm. The transducer was attached to a water balloon, which was filled with degassed water to provide acoustic coupling. The water balloon was immersed in a degassed-water container. The bottom of the water container had a window sealed with an almost acoustically and optically transparent membrane. The container was placed on the mouse head and coupled with degassed ultrasound gel. The FUS transducer has a circular central opening of 38 mm in diameter. A B-mode imaging probe (L8-17, Alpinion, Seoul, South Korea) was inserted into the opening and aligned with the FUS focal plane. The pressure amplitude of the FUS transducer was calibrated using a needle hydrophone (Onda, California) in a degassed water tank before the in vivo experiment. The reported pressure amplitudes were the measured peak negative pressure calculated using the hydrophone measured pressure values attenuating by 18% to correct for mouse skull attenuation. The lateral and axial full-width-at-half maximum pressures of the beam were 6.04 and 0.62 mm, respectively. The FUS transducer was attached to a 3-D positioning system (Velmex, Lachine, Quebec, Canada). In this study, the tumor was targeted with the assistance of a grid. The grid was positioned in the water container on top of the skull with the crossing point in alignment with the tumor in reference to the BLI image and the stereotaxic coordinates of the tumor injection. The B-mode imaging probe was used to scan through the grid and form an image of the grid. Then, the crossing point of the grid was then identified. The depth of FUS was adjusted to be 0.65 mm to the skull by measuring the distance using the B-mode imaging. Five adjacent points (four located at the corner of a square with one in the middle) were targeted to cover the whole tumor. Freshly diluted microbubble suspension ($30\mu \mathrm{L}$) was administered through a bolus injection via the tail vein before FUS sonication. Immediately after injection (5 s), pulsed FUS (center frequency: 1.5 MHz; ultrasound pressure: 0.85 MPa; pulse length: 6.7 ms; pulse repetition frequency: 5 Hz; duration: 1 min each point) was applied. LS301 or ICG was injected after each treatment.

2.6.

Histology

Mouse brains were harvested and frozen in optimal cutting temperature embedding medium (Fisher Healthcare, Texas) and stored at $-20°\mathrm{C}$. Fluorescence and brightfield images of $10\mu \mathrm{m}$ sections were obtained by epifluorescence microscopy at $4\times$ and $20\times$ magnifications. Fluorescence images from LS301 were acquired before fixation and immunohistochemistry. Slides were stained with a monoclonal antibody to murine p-ANXA2 (Santa Cruz Biotechnology, Inc., Texas) at a 1:250 dilution, followed by secondary staining with a donkey anti-mouse Alexa Fluor 594 conjugate (ThermoFisher, Massachusetts) at a 1:1000 dilution.

2.7.

Data Analysis and Statistics

Statistical significance was measured by a two-tailed Student’s $t$-test using GraphPad Prism software (GraphPad, California). Comparison of longitudinal data included the Holm–Sidak correction for multiple comparisons. All values are means and error bars are standard deviations. *$P<0.05$, **$P<0.01$, ***$P<0.001$.

3.1.

Comparison of ICG and LS301 FMT Images in Tumor-Bearing and Sham-Treated Mice

Previous reports demonstrated a high tumor-to-background fluorescence 24 h after intravenous injection in GBM models.^11,12 Recently, we showed that LS301 also provides excellent tumor contrast 24 h postintravenous injection in mice.^14–16 Given these findings, we compared LS301 and ICG uptake in brain tumor-bearing mice at 24 h postinjection. We validated the uptake pattern by time-course imaging of the imaging agents up to 24 h and comparing the mean tumor ROI fluorescence in tumor-bearing mice to naïve and sham-treated controls. While enhancement of LS301 uptake relative to these controls is observed at 24 h postinjection at week 7 post-tumor implantation, accumulation of ICG in tumors is not statistically significant relative to controls at any time point recorded. LS301 reveals focal fluorescence in tumor tissue 24 h after LS301 injection at weeks 1, 4, and 7 (Fig. 2). Epifluorescence microscopy of brain tumor sections harvested at 72 h postinjection of LS301 at 7 weeks postimplantation shows the presence of GFP signal [Fig. 2(m)] and NIR fluorescence from LS301 [Fig. 2(n)] from the tumor in the cerebral cortex. Subsequently, immunohistochemistry of p-ANXA2 expression demonstrates excellent agreement between areas of LS301 accumulation and p-ANXA2 expression in the tumor [Fig. 2(o)].

Fig. 2

Comparison of (a)–(c), (g)–(i) ICG and (d)–(f), (j)–(l) LS301 uptake in tumor-bearing mice imaged at 24 h postinjection on weeks 1, 4, and 7 after tumor initiation. (a)–(f) Coronal projections of images with the corresponding axial projection images in (g)–(l) viewed head first for improved visualization. Epifluorescence microscopy of U87-GL PDE7B H217Q tumors harvested at 72 h postinjection of LS301 at 7 weeks post-tumor implantation. (m) GFP fluorescence of tumors (green). (n) NIR fluorescence of brain tissue (red). (o) p-ANXA2 stain of the tumor (magenta).

Further analysis of the data suggests that LS301 fluorescence may not completely co-localize with the tumor tissue. To account for nonspecific fluorescence in the tumor ROI, naïve and sham-treated controls were injected with ICG and LS301. Following the experimental workflow shown in Fig. 1, naïve mice received stereotaxic cortical injections of PBS and were imaged weekly up to 7 weeks to match data from the tumor cohort. Our result shows a clear difference in the distribution of LS301 at 24 h postinjection compared to sham-treated mice (Fig. 3). The sham-treated mice have low and diffuse LS301 fluorescence in the ROI similar to tumor implantation site, whereas tumor-bearing mice display a focal fluorescence distribution. This is best visualized in the axial view [Figs. 3(j)–3(l)]. However, the mean fluorescence in the ROI for both tumor and sham was not statistically distinguishable in the early time points until 7 weeks postimplantation, reflecting the poor penetration of LS301 into the brain until tumor degrades the BBB.

Fig. 3

Comparison of FMT images of tumor ROIs in tumor-bearing and sham-treated mice. Representative images of (a)–(c), (g)–(i)  sham-treated and (d)–(f), (j)–(l) tumor-bearing mice imaged at 24 h post-LS301 injection on weeks 1, 4, and 7 after tumor initiation or sham injection. (a)–(f) Coronal projections of images with the corresponding axial projection images in (g)–(l).

3.2.

Quantification of Bioluminescence and Tumor ROI Fluorescence

Longitudinal bioluminescence quantification demonstrates a log-linear growth of the tumors [Fig. 4(a)] but LS301 fluorescence at 24 h postinjection remains stable from weeks 1 to 6 and then begins to separate from the sham control at week 7 [Fig. 4(b)]. A statistically significant difference in LS301 uptake is only achieved at 7 weeks postimplantation between the naïve and tumor-bearing cohort. In addition, a clear difference between LS301 fluorescence in the sham-treated and tumor-bearing cohorts is only apparent at week 7, although the diffuse nature of background LS301 fluorescence across different animals prevented the attainment of statistical significance after correcting for multiple comparisons ($p=0.058$). Comparing LS301 fluorescence at 24 h postinjection as a function of bioluminescence signal shows a weak linear correlation [$r^2=0.2797$, $p<0.0001$, Fig. 4(d)], which could be attributed to a combination of factors that include LS301 penetrance in tumor, limited access to tumor tissue in the brain, and differences in the imaging strategies. In all cases, the mean ROI fluorescence for ICG at 24 h postinjection was close to zero, with no significant difference between sham-treated and tumor-bearing mice [Fig. 4(c)].

Fig. 4

Quantification of tumor fluorescence and bioluminescence. (a) Longitudinal quantification of tumor bioluminescence. (b) Longitudinal quantification of LS301 mean fluorescence at 24 h postinjection in naïve, sham-treated, and tumor-bearing mice. (c) Longitudinal quantification of ICG mean fluorescence at 24 h postinjection in naïve ($n=5$), sham-treated ($n=5$), and tumor-bearing mice ($n=8$). (d) Longitudinal quantification of LS301 mean fluorescence as a function of BLI counts. BLI data are shown on log scale for ease of viewing.

3.3.

Focused Ultrasound Delivery of LS301 and ICG to Brain Tumors

The difficulty in delivering contrast agents across an intact BBB contributes to the inability to implement imaging protocols for the early detection of brain cancer. FUS with microbubbles has been shown to transiently disrupt the BBB.²⁷ To explore this phenomenon, we injected LS301 into mice 4 weeks after tumor initiation when the BBB is intact, followed by FUS treatment and imaging 24 h post-LS301 injection. Four days later, those same mice were again treated with FUS, injected with ICG, and imaged 24 h later. Figure 5(g) shows the focus of the FUS treatment, which was based on BLI imaging and the stereotaxic coordinates of the tumor injection. Quantification of the mean tumor ROI fluorescence for mice injected with LS301 or ICG is shown in Fig. 5(h). Significance testing between groups with and without FUS treatment was carried out using a Student’s $t$-test with Welch’s correction.²⁸ We found that FUS enhances the uptake of LS301 compared with mice the non-FUS-treated mice [Figs. 5(a)–5(f)]. Even with FUS, ICG still clears rapidly from the tumor region and was not visible by 24 h, confirming that tumor-targeting molecular probes confer higher uptake and retention in brain tumors if BBB permeation is feasible.

Fig. 5

FUS delivery of LS301 and ICG at 4 weeks post-tumor initiation. (a), (d) Coronal and axial FMT projections of LS301 fluorescence at 24-h postinjection. (b), (e) Coronal and axial FMT projections of LS301 fluorescence at 24 h postinjection and FUS sonication. (c), (f) Coronal and axial FMT projections of ICG fluorescence at 24 h postinjection and FUS sonication. (g) B-mode ultrasound image showing focus of ultrasound treatment. (h) Quantification of tumor ROI fluorescence with and without FUS treatment.

3.4.

Comparison of LS301 and Alexa Fluor 680 Transferrin

A previous report showed that a transferrin-imaging agent is able to target gliomas selectively.²¹ In this study, we compared the distribution of the reported glioma-targeting agent, AF-Tf and LS301. The differences in their imaging spectral windows (670/690 to 740 nm for AF-Tf and $780/>805\mathrm{nm}$) allowed us to differentiate the spectral signatures of both agents with the dual excitation and emission features of the FMT system. To ensure that the imaging agents had access to the tumors, we coinjected a solution of LS301 (6 nmol) and AF-Tf (2 nmol) in PBS in a subset of the tumor-bearing mice to compensate for differences in the fluorescence quantum yields of Alexa Fluor (0.36) and cypate dye ($\sim 0.1$) used to prepare AF-Tf and LS301, respectively.^19,29 We imaged for up to 72 h at 7 weeks post-tumor implantation, when the BBB is compromised. Fluorescence was observed in the tumor ROIs as well as the surrounding areas that encompass the top 3 to 4 mm of the brain, which we used as background [Figs. 6(a)–6(p)]. Time course imaging and comparison of LS301 with AF-Tf show strong signals in the tumor ROI for the molecular probes. The faster clearance of LS301 from nontumor tissue compared to AF-Tf enhances the tumor-to-background contrast. Quantitative analysis of the data shows that the high background AF-Tf fluorescence made it difficult to distinguish the tumor from surrounding tissue at the 24- and 48-h time points [Fig. 6(r)]. This yields an apparent difference in the tumor-to-background ratio between LS301 and AF-Tf at 24 h, with a statistically significant difference at 48 h postinjection ($p<0.05$), which is in agreement with the ex vivo results [Figs. 6(d), 6(h), 6(l), and 6(p)]. AF-Tf appears to highlight the brain vasculature given the tortuous shape of the signal. This pattern of AF-Tf distribution can be attributed to the high expression of transferrin receptors in the brain capillary endothelium.³⁰

Fig. 6

In vivo and ex vivo images of LS301 (6-nmol injection) and Alexa Fluor 680 transferrin (2 nmol injection). (a)–(d) Coronal projection of LS301 fluorescence in the tumor ROI and surrounding area. (e)–(h) Coronal projection of Alexa Fluor 680 transferrin fluorescence in the tumor ROI and surrounding area. (i)–(l) Axial projection of LS301 fluorescence in the tumor ROI and surrounding area. (m)–(p) Coronal projection of Alexa Fluor 680 transferrin fluorescence in the tumor ROI and surrounding area. (q) Quantification of tumor ROI mean fluorescence. (r) Tumor-to-background ratio.