2.1.

Animals

All animals were used in accordance with the policies and an approved protocol of the Institutional Animal Care and Use Committee at Dartmouth College. Fifteen 6-week-old, male NCR athymic nude mice were obtained from the National Cancer Institute–Frederick Animal Production Program (Frederick, Maryland) and used in this study. There were 12 mice (9 tumor implanted, 3 sham surgery controls) that underwent MRI and PpIX study only, while an additional 3 mice were used for fluorescence staining.

2.2.

Cell Culture and Murine Orthotopic Glioma Model

U251-GFP was used for implantation.¹⁵ The U251-GFP cells were cultured in Dulbecco's Modification of Eagle's Medium (DMEM, Mediatech, Inc., Herndon, Virginia) supplemented with 10% fetal bovine serum (FBS, Mediatech, Inc.) and 1% penicillin-streptomycin (Mediatech, Inc.). The cells were incubated in a humidified environment at 37 °C with 95% air and 5% carbon dioxide. In preparation for injection, the cells were trypsinized, live cell count performed on a hemocytometer with Trypan blue to stain the dead or damaged cells, and then were subsequently suspended in phosphate buffer saline (PBS) for implantation at the appropriate concentration.

The procedure for orthotopic brain tumor implantation has been described previously, but is described briefly here. Mice were anesthetized by interperitoneal (i.p.) injection with ketamine-xylazine (90:10 mg/kg) and a small incision was created on the left side of the head exposing the skull landmarks. A small hole was created 2 mm behind the bregma and 2 mm to the left of the midline using a 1 mm rotary drill. A Hamilton syringe (Hamilton Company, Reno, Nevada) was placed 2-mm deep into the brain tissue and 1×10⁶ cells in 10 μl was injected over a 5 minute period, followed by a slow retraction of the needle to prevent cell leakage. The control mice received the same treatment, but were injected with 10 μl of PBS only. The hole in the skull was closed using bone wax (Ethicon, Inc., Piscataway, New Jersey) and the incision closed with Vetbond (J. A. Webster, Inc., Sterling, Massachusetts). All mice implanted with U251-GFP cells displayed tumor cell growth, although tumor size varied considerably due to the diffuse and infiltrative nature of the cell line as previously reported.^{15, 16} Mice were imaged 10 to 16 days post-implantation when they displayed clinical signs of tumor growth.

2.3.

MRI

All MR experiments were performed on a 7T/21cm magnet (Magnex Scientific, Abingdon, United Kingdom) equipped with an imaging gradient set (Resonance Research Inc, Billerica, Massachusetts), interfaced to a Varian UNITY-INOVA console (Varian Inc., Walnut Creek, California). A Litz coil of 20-mm diameter (Doty Scientific Inc, Columbia, South Carolina) was used in transmit/receive mode. The mice were anesthetized with isoflurane (1 to 1.5 vol.% in 70:30 O₂:N₂) with a nose cone, and an animal torso was placed on a thermostated water circulating heating element at 37 °C for the duration of MR scans to maintain body temperature. Pre- and post-contrast T₁ MR images were acquired with a standard spin echo sequence using the following acquisition parameters: TR = 700 ms, TE = 9 ms, matrix size = 128 × 128, field of view (FOV) = 30 × 30 mm, 2 signal averages, slice thickness = 0.75 mm, number of slices = 20, total acquisition time = 3 min 3 s. For post-contrast T₁, Magnevist (0.2 mmol/kg) was injected i.p. 10 min before acquisition of T₁ MRI. A multiecho, multislice spin echo sequence was used to acquire absolute T₂ MR images with parameters as follows: TR = 3 s, TE = 20 ms, number of echoes = 4, 2 signal averages, matrix size = 128 × 128, FOV = 30 × 30 mm, slice thickness = 0.75 mm, number of slices = 20, total acquisition time = 12 min 55 s. The trace of the diffusion images (D _av) was acquired using the sequence described by Mori and van Zijl¹⁷ with acquisition parameters as follows: TR = 1.5 s, TE = 55 ms, matrix size = 128×64, FOV = 30×30 mm, 2 signal averages, slice thickness = 0.75 mm, number of slices = 15 to 20, three b-values = 0, 496.6 and 1056.7 s/mm², total acquisition time = 9 min 41 s. The Aedes routine ( http://aedes.uku.fi) performed under a MATLAB platform (Mathworks Inc, Bolder, Colorado) was used to compute both absolute T₂ and D _av images from the acquired data sets.

2.4.

Ex vivo Frozen Tissue Fluorescence Imaging and Histology

Post-MR imaging, the mice were administered 100 mg/kg ALA dissolved in PBS by i.p. injection. Two hours post-ALA injection, the mice were sacrificed; the brains were extracted and sectioned in the coronal plane into four pieces, ensuring that one slice was made directly through the needle track to optimize tumor detection. The sections were laid cut face down on a glass slide and were imaged on a fluorescence plate scanner (Typhoon 9410, GE Healthcare Life Sciences) for both PpIX (633-nm excitation, 650-nm LP emission) and GFP (488-nm excitation, 526-nm BP emission) fluorescence. Following fluorescence imaging the brain slices were sent to pathology for routine H&E staining on 4 μm tissue slices. Routinely, the surface slice was kept for each tissue section and subsequent slices were taken every 100 μm throughout the entirety of the tissue. The tissue between each H&E slice was discarded at the time of preparation.

Additional fluorescence imaging on frozen sections was performed in three mice to verify compromised vasculature and blood-brain barrier breakdown as a mode of ALA delivery. In addition to the ALA administered 2 h prior to sacrifice, the mice were i.v. injected with the vascular marker Hoechst 33258 (15 mg/kg in PBS) via the tail vein 1 min prior to sacrifice. The brains were removed, submerged in Tissue Tek® Optimum Cutting Temperature medium, and flash frozen in a mixture of methylbutane and dry ice. The frozen brain samples were stored short term at −20 °C and long term at −80 °C. Ten micrometer thick frozen tissue sections were made on a cryotome (CM 1850, Leica Biosystems). Two consecutive slices were made every 100 μm throughout the entirety of the brain sample. The first slice was used for fluorescence imaging and the second slice was sent for routine H&E staining to confirm tumor location. Fluorescence imaging was performed on a Nikon DIAPHOT-TMD inverted fluorescence microscope with a QColor3 CCD camera and QCapture Suite imaging software (QImaging, Surrey, BC, Canada). GFP (ex: 470 to 490 nm, em: 520 to 560 nm; DM510, BIE filter cube, Nikon, Garden City, New York), PpIX (ex: 445 nm, em: 685 to 715 nm; 560DCXR, C36848; Chroma Technology Corp., Rockingham, Vermont), and Hoechst 33258 (ex: 360 to 370 nm, em: 425 to 475, HQ450/50m dichroic; Chroma Technology Corp.) were imaged in the same field of view for fluorescence correlation. The GFP fluorescence overlaps with Hoechst 33258 fluorescence in the 520 to 560 nm range; however, the Hoechst 33258 fluorescence could be separated using the shorter wavelength filter cube.

2.5.

Contrast Analysis

Corresponding regions of interest (ROIs) for the fluorescence and MR images were produced to compare the contrast between image types. ROIs were created using ImageJ (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, http://rsb.info.nih.gov/ij/, 1997 to 2005) from the GFP-fluorescence images and were copied to the PpIX images and the correlating MR image slices. The tumor location was confirmed in the H&E slices, and the ROIs were divided into two groups based on the GFP fluorescence: H&E positive (H&E+) and negative (H&E−) ROIs. The image contrast was determined using the following formula:

3.1.

Regions of Interest

The H&E slices were used to guide the location of the tumors in the GFP fluorescence images for the initial ROI determination. There were 36 ROIs that displayed GFP fluorescence (GFP+). It was readily noted that within these 36 ROIs there were distinct regions that displayed GFP fluorescence (i.e., U251-GFP cells) in the thick tissue sections used for fluorescence imaging that did not appear in the 4 μm surface H&E sections. Therefore, the paraffin embedded tissue blocks were further sectioned by taking a 4 μm slice every 100 μm for the entirety of the tissue. Although this increased the number of correlating GFP fluorescence and tumor H&E positive ROIs, there were still many that could not be correlated with H&E stained tissues. Therefore, the GFP+ ROIs were further divided into two categories: H&E positive (H&E+) and H&E negative (H&E−); although the latter group is designated H&E−, it is believed that the GFP fluorescence indicates the presence of tumor. There were 36 GFP + ROIs noted in 9 mice, with 14 of those ROIs H&E+ and 22 H&E negative (H&E−). Twenty-four ROIs were created for three sham surgery control mice.

Figure 1 demonstrates a complete set of images for one representative mouse in the study. The regions of interest within the brain are denoted with arrows. Note that there are three H&E + ROIs in this particular mouse. The corresponding ROIs in the GFP and PpIX fluorescence images are shown in Figs. 1b, 1c, respectively, while the MR images are shown in Figs. 1d, 1e, 1f, 1g, 1h, 1i, 1j.

Fig. 1

The diffusely growing U251-GFP tumor images are shown using ex vivo fluorescence, in vivo MR imaging, and ex vivo H&E staining. (a) H&E staining of a mouse brain implanted with U251-GFP shows three regions (black arrows – dotted, dashed, and solid) of diffusely growing glioma cells. H&E was used as the gold standard for tumor detection. (b) GFP fluorescence from thick tissue slices was used to determine the ROIs for fluorescence and MR imaging contrast analysis. The three ROIs are circled with dotted, dashed, and solid lines corresponding to the ROIs indicated in (a). (c) PpIX fluorescence of the same tissue slice was analyzed. The following MR imaging scans were also analyzed for tumor contrast: (d) T1W without gadolinium contrast [dotted, dashed, and solid circles correspond to GFP fluorescence ROIs indicated in (b)], (e) T1W with gadolinium, (f) T1W contrast difference, (g) T2, (h) absolute T2, (i) diffusion, and (j) D _av image. The ROIs created in the MR images corresponding to fluorescence and H&E images are shown in the T1W without gadolinium image (d).

3.2.

PpIX Fluorescence and MRI Contrast Comparison

All ROIs were analyzed for PpIX fluorescence, and contrast in the following MR images: T1W, T1W+Gd, T1WCD, T2, T2 map, diffusion, and D _av map. The resultant image contrast values are presented in box and whisker plots in Fig. 2. When considering the GFP + ROIs [Fig. 2b] the PpIX fluorescence, T1W+Gd, and T2 Map are all statistically significant from the control ROIs [Fig. 2a] at p < 0.05. For the H&E + ROIs, the same three imaging types produce statistically significant results as compared to the control (p < 0.05). However, when considering only the H&E − ROIs, the PpIX and T2 Map are statistically significant from the control group (p < 0.05). The diffusion and D _av map did not show any statistical significance in any of the ROI groups.

Fig. 2

The comparison of MRI and fluorescence contrast (where contrast = [ROI] / [Contralateral ROI]) of the control mice (a), and the GFP+ ROIs (b) show that the T1W+Gd, absolute T2, and PpIX fluorescence are all significant (black asterisks) from the controls. The GFP+ ROIs are further broken down into groups of H&E + ROIs (c) and H&E − ROIs (d) to illustrate the large variation between these two groups. The H&E positive ROIs have the same statistically significant groups as all tumor bearing mice, but the H&E negative ROIs are only significant for PpIX fluorescence and T2 map.

ROC curves were created for the GFP+ and H&E+ ROIs [Figs. 3a, 3b, respectively] for PpIX fluorescence, T1W+Gd and T2 Image. The receiver operating characteristic (ROC) curves demonstrate the relationship between the false positive fraction and the true positive fraction based on the image contrast, as compared to the contrast in the control mice. PpIX fluorescence image contrast displays the highest sensitivity and specificity of the three imaging types in both GFP+ and H&E + ROI groups, with perfect scoring for the H&E + ROIs. T1W+Gd image contrast displays higher sensitivity in the H&E + ROI group than that in the GFP+ ROI group and has similar specificities. The T2 map exhibits similar sensitivity and specificity in both GFP+ and H&E + ROI groups.

Fig. 3

The ROC curves for the GFP+ ROIs (left) and the H&E + ROIs (right) show that PpIX fluorescence has the highest specificity and sensitivity, perfect in the H&E + ROIs case, as compared to the other imaging techniques. The T1W+Gd image contrast is significantly better in the H&E + case, while the T2 map image contrast is relatively similar in both the GFP+ and H&E + ROIs. The AUC for each of the image-ROI combination is summarized in Table 1.

The area under the curve (AUC) for each ROC curve image type is summarized in Table 1 and describes how accurately the tumor can be differentiated from the control; where an area of 1 indicates 100% sensitivity and specificity, while an area of 0.5 is indicative of 50% sensitivity and specificity, or random guessing. The AUCs for the H&E + ROI group are all higher than the AUCs for the GFP+ ROI group, although the individual imaging contrast types all show the same trends with PpIX fluorescence > absolute T2 > T1W+Gd. The H&E − ROI group shows a high level of false positives for the PpIX fluorescence and absolute T2, while the T1W+Gd is close to the control group.

Table 1

Diagnostic test results for the three ROI analysis groups: All ROIs, H&E + ROIs and H&E − ROIs.

A complete summary of diagnostic tests is provided in Table 1. The results of the diagnostic tests demonstrate the same basic trends as seen for the AUC. PpIX image contrast is consistently higher in sensitivity, NPV, and diagnostic accuracy for all three ROI groups. T1W+Gd image contrast also shows perfect specificity and PPV for all three ROI groups; however, T1W+Gd has lower NPV and diagnostic accuracy values than PpIX. In fact, T1W+Gd has lower sensitivity, NPV, and diagnostic accuracy values than T2 Map.

Correlations between PpIX fluorescence and both T1WCD and T2 maps were identified. Pearson's correlation coefficients were 0.79 (p < 1 × 10⁻⁸) and 0.74 (p < 0.003) for PpIX fluorescence and T1W+Gd image contrast for the GFP+ and the H&E + ROIs, respectively, demonstrating highly significant positive linear correlation between PpIX fluorescence and gadolinium enhancement. The Pearson's correlation coefficient for PpIX fluorescence and absolute T2 image was 0.34 (p < 0.05) for the GFP+ ROI group, indicating a weak positive correlation. No correlation was found between PpIX fluorescence and absolute T2 image in the H&E + ROI group, and no significance was found between PpIX fluorescence and any MR image type in the H&E − ROI group.

3.3.

Ex vivo Fluorescence Imaging of Frozen Tissue

Ex vivo fluorescence imaging of frozen brain tissue sections displayed co-localized regions of GFP, Hoechst 33258, and PpIX fluorescence (Fig. 4). The GFP fluorescence channel had some bleed through of the Hoechst 33258 due to the overlapping spectra in the 480 to 560 nm region. Therefore, the GFP and Hoechst 33258 fluorescence images were overlaid and regions of overlap were indicated to distinguish Hoechst 33258. The PpIX fluorescence corresponded well with both the tumor cells (GFP fluorescence) and with perfusing vasculature (Hoechst 33258 fluorescence), but was not observed elsewhere in the brain.

Fig. 4

Ex vivo fluorescence imaging of frozen tissue sections confirms the co-localization of GFP (green, left image), perfusing blood vessels (Hoechst, blue, left image), and PpIX fluorescence (red, right image). Due to the nature of the filter, some Hoechst fluorescence leaked into the GFP channel; therefore, these regions are marked with white arrows and indicate areas that contain fluorescence from both the green and blue fluorescence channels and can be attributed to perfusive blood vessels. Scale bar = 200 μm.