2.1.

Cell Culture

Two cell lines were used in the studies presented; the 9L rat gliosarcoma cell line transfected with green fluorescent protein (9L-GFP) and the human glioma cell line U251. The 9L-GFP cell line was a generous gift from Alexi Bogdanov.¹⁸ The cell lines were cultured in Dulbecco’s modification of eagle’s medium (Cellgro, Mediatech, Herndon, Virginia), supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, Georgia) and 1% penicillin/streptomycin from a stock solution of 10,000 IU penicillin and $\mathrm{10,000}\mu \mathrm{g}∕\mathrm{ml}$ streptomycin (Mediatech, Herndon, Virginia), and were kept incubated at $37°\mathrm{C}$ in a 95% air and 5% carbon dioxide humidified environment.

2.2.

Murine Glioma Model

Both cell lines (9L-GFP and U251) were used for intracranial implantation into male athymic nude mice about $6\text{weeks}$ of age. Mice were anesthetized using ketamine/xylazine in a $90:10\text{-}\mathrm{mg}∕\mathrm{kg}$ ratio, and their body temperature was maintained during anesthesia via a heating pad. A small incision was made in the scalp exposing the top of the skull so that the landmarks on the brain were visible. A Dremel drill was used to make a $1\text{-}\mathrm{mm}$ hole in the skull, located $2\mathrm{mm}$ in front of the bregma and $2\mathrm{mm}$ to the left of the midline, after which the needle was inserted $2\mathrm{mm}$ deep into the brain via guidance from a sterotactic frame. A total of $1\times {10}^6$ cells in $10\mu \mathrm{l}$ of phosphate buffered saline (PBS) were injected over $5\min$ using a Hamilton syringe. After the injection, the needle was slowly retracted, the skull was cleaned to ensure cells were not deposited outside the brain, and bone wax was used to cover the hole drilled in the skull. Finally, the incision in the scalp was closed with a small amount of Vetbond Tissue Adhesive (J.A. Webster, Inc., Sterling, Massachusetts). Mice were examined daily following surgery to ensure proper healing of the scalp. Control mice were implanted with $10\text{-}\mu \mathrm{l}$ PBS without cells to allow similar surgical procedures to be performed on all mice.

2.3.

Single Channel Fluorescence Spectroscopy System

A schematic of the single channel spectroscopy system can be seen in Fig. 1a . The system consisted of a $250\text{-}\mathrm{mW}$ , $635\text{-}\mathrm{nm}$ diode laser (Power Technology, Incorporated) for excitation collimated onto the chin of the mouse. The light transmitted through the mouse head was collected through a second collimator, and passed through a $650\text{-}\mathrm{nm}$ LP filter prior to spectrally resolved detection through a spectrometer ( $1200\text{-}\mathrm{l}∕\mathrm{mm}$ grating, SpectraPro 300, Acton Research, Acton, Massachusetts) and onto a cooled charge-coupled device (CCD) camera (Spec-10:400BR/XTE, Princeton Instruments, Acton, Massachusetts). Data from the camera were captured and transferred using commercial software (Winspec, Acton Research). The spectrometer was centered at $705\mathrm{nm}$ to collect the PpIX fluorescence emission peak, and at $615\mathrm{nm}$ to collect the transmitted intensity from the $635\text{-}\mathrm{nm}$ laser. A mouse holder consisted of a bed in a light tight box with holes $180\text{-}\mathrm{deg}$ apart from one another to hold the collimators, for collection of spectroscopy data [Figs. 1b and 1c].

Fig. 1

(a) A schematic of the single channel spectroscopy system is shown containing a $635\text{-}\mathrm{nm}$ laser, a spectrometer, a $650\text{-}\mathrm{nm}$ LP filter, and a computer. In (b), a photograph of the system with the light tight box open is shown. In (c), a sample mouse is shown in the holder in position for measurement.

2.4.

Spectroscopy Data Collect and Processing

At each time point, the mouse was placed in the mouse holder with the collimators in contact with the head. Fluorescence emission data was collected with the spectrometer centered at $705\mathrm{nm}$ , and the exposure time adjusted to obtain signals in the linear range of the spectrometers with maximal signal-to-noise level. The light was passed through the $650\text{-}\mathrm{nm}$ LP filter to collect the PpIX fluorescence emission prior to detection by the spectrometer [Fig. 2a ]. Prior to any movement of the mouse, transmittance data were also collected, where the spectrometer was centered at $615\mathrm{nm}$ to allow detection of the $635\text{-}\mathrm{nm}$ laser intensity through the $650\text{-}\mathrm{nm}$ LP filter, and the exposure time was again adjusted to ensure detection in the linear range of the spectrometers [Fig. 2b].

Fig. 2

In (a), the PpIX fluorescence emission data are plotted for a sample mouse. In (b), the transmittance spectrum from the $635\text{-}\mathrm{nm}$ laser through the head of a sample mouse is shown. In (c), the PpIX fluorescence data are spectrally fitted to a reference PpIX spectrum from tissue-simulating phantom data. In (d), the deconvolved fluorescence and nonspecific background signals are shown.

The raw spectral excitation and emission data were postprocessed by a two-step process involving spectral fitting and then normalization. PpIX fluorescence data were collected using a liquid tissue simulating phantom composed of 1% Intralipid to simulate scattering, $1\text{-}\mathrm{mg}∕\mathrm{L}$ India ink to simulate absorption, $1\text{-}\mu \mathrm{g}∕\mathrm{mL}$ PpIX in dimethyl sulfoxide to simulate in vivo PpIX concentrations, and 5% Tween 20 to decrease aggregation of PpIX, a hydrophobic molecule, in solution. The spectral shapes of PpIX obtained from these tissue-simulating phantoms were normalized to one and used for spectral fitting of in vivo PpIX data. The fluorescence data were spectrally fitted using a MatLab program to perform a linear least squares fit to the PpIX phantom data, so that the nonspecific background signal could be deconvolved from the PpIX fluorescence signal [Figs. 2c and 2d], as it had a distinctly different spectral shape. The area under the deconvolved PpIX fluorescence curve was then calculated and reported as a single number. Both the fluorescence emission data and the transmittance data were normalized to counts/second to account for differences in exposure time. Then the integrated fluorescence intensity was normalized to the integrated transmitted laser intensity to account for positional differences between measurements of a single mouse, as well as variations in optical properties,¹⁴ resulting in the fluorescence-to-transmittance ratio.

2.5.

Protoporphyrin IX Detection of Brain Tumors

At $10\text{to}14\text{days}$ after tumor implantation, the mice were imaged via a large bore Philips 3T Magnetic Resonance Imaging (MRI) Achieva system, using a modified rodent body coil insert.¹⁹ A plastic insert was used in the rodent coil to raise the mouse into the isocenter of the magnetic field [Figs. 3a and 3b ]. Intracranial tumors were identified using T1 turbo spin echo (TSE) images with and without Gadolinium enhancement ( $1\text{-}\mu \mathrm{l}∕\mathrm{mg}$ body weight) [Fig. 3c], as well as T2 TSE imaging sequences. T1 TSE and T2 TSE images were also collected prior to sacrifice for in vivo and ex vivo tumor comparison.

Fig. 3

In (a), the rodent coil inside the 3T MRI is shown, and in (b) an example mouse in the rodent coil with IP catheter for Gd-DTPA injection is shown. In (c), a sample set of T1 coronal turbo spin echo contrast enhanced (TSE-CE) images are shown for a 9L-GFP tumor.

Prior to ALA administration, the mice were placed in the single channel spectroscopy mouse holder and their background PpIX fluorescence was measured while they were under inhaled isofluorane anesthesia. The mice were then administered $100\text{-}\mathrm{mg}∕\mathrm{kg}$ ALA dissolved in PBS by intraperitonal (IP) injection. Two hours after ALA administration, the PpIX fluorescence was again measured using the single channel spectroscopy system. The mice were then sacrificed, their brains extracted, and placed back into the spectroscopy system for ex vivo measurement of the tumor in situ. Following collection of all spectroscopy measurements, the brain was sectioned in a coronal plane and imaged on a fluorescence plate scanner (Typhoon 9410, GE Healthcare Life Sciences) with the cut faces of the brain facing the imaging plane of the scanner. The tissue slices were imaged for PpIX fluorescence using a $633\text{-}\mathrm{nm}$ excitation laser and a $650\text{-}\mathrm{nm}$ LP emission filter, and when appropriate, then scanned for GFP fluorescence using a $488\text{-}\mathrm{nm}$ laser with a $526\text{-}\mathrm{nm}$ SP emission filter. Following fluorescence imaging, the brain slices were sent to pathology for routine hematoxylin and eosin (H&E) staining.

2.6.

Red Light Time Course Photobleaching

Four control mice, nine 9L-GFP tumor-bearing mice, and seven U251 tumor-bearing mice were compared in this study. In each group the tumors were implanted, identified via MRI, and the PpIX fluorescence of each mouse was then measured using the single channel spectroscopy system as described previously. Following the measurement $2\mathrm{h}$ after the administration of ALA, the $635\text{-}\mathrm{nm}$ laser was left running and subsequent measurements were collected after 1, 2, 4, 8, 16, 24, and $32\min$ to allow for photobleaching of the skin PpIX fluorescence signal.

2.7.

Statistical Analysis

Differences between all groups were established through box and whisker plots and unpaired two-tailed students-t tests to establish $p$ -values for comparison of all groups of interest. All box and whisker plots shown illustrate the median as the center line and the interquartile range as the enclosed box showing 75% of the data. The upper whisker represents $\mathrm{Q}3+1.5(\mathrm{Q}3-\mathrm{Q}1)$ and the lower whisker represents $\mathrm{Q}1-1.5(\mathrm{Q}3-\mathrm{Q}1)$ , where Q is the quartile. Each open circle represents the fluorescence-to-transmittance ratio of one mouse.

The ability to quantify PpIX fluorescence spectroscopy as a method to detect tumors was assessed by receiver operator characteristic (ROC) analysis. The values for sensitivity and specificity were estimated based on the number of true positives, false positives, true negatives, and false negatives, where tumor status was confirmed through MRI and histology. Sensitivity was calculated as the number of true positive (TP) cases divided by the number of true positive and false negative (FN) cases $[\text{sensitivity}=\mathrm{TP}∕(\mathrm{TP}+\mathrm{FN})]$ . Specificity was calculated as the number of true negative cases (TN) divided by the number of true negative and false positive (FP) cases $[\text{specificity}=\mathrm{TN}∕(\mathrm{TN}+\mathrm{FP})]$ .²⁰

ROC curves were constructed using different intensity threshold levels on the PpIX fluorescence-to-transmittance ratio collected from the control, 9L-GFP, and U251 tumor-bearing mice. As the intensity threshold was changed, the true positive fraction (TPF) and false positive fraction (FPF) were calculated, where TPF is synonymous with sensitivity and FPF is representative of 1 minus the specificity of the tumor detection modality. The area under the curve (AUC) was calculated from the ROC curves and used as a direct measure of sensitivity and specificity of the spectroscopy system for tumor detection. An AUC of 1 would indicate that the tumor detection modality had 100% sensitivity and specificity of detection, while an AUC of 0.5 would indicate there was only 50% sensitivity and specificity, equivalent to random guessing of tumor status.

3.1.

Protoporphyrin IX Fluorescence Detection of Brain Tumors

At $18\text{to}22\text{days}$ following tumor implantation, the mice were measured in the single channel spectroscopy system prior to ALA administration, $2\mathrm{h}$ after ALA administration and ex vivo following brain extraction. As can be seen in Fig. 4a, prior to the administration of ALA the mean PpIX fluorescence of the control mice, 9L-GFP, and U251 tumor-bearing mice were very similar. However, $2\mathrm{h}$ after the administration of ALA, there was a significant difference between the means of the control group and the 9L-GFP tumor-bearing group $(p\text{-}\text{value}=0.016)$ , as well as between the means of the control group and the U251 tumor-bearing group $(p\text{-}\text{value}=0.004)$ . The mean PpIX fluorescence of the 9L-GFP and U251 groups were similar $2\mathrm{h}$ after ALA administration; however, the variance of the 9L-GFP group was large compared to that of the U251 group [Fig. 4b]. Following sacrifice, ex vivo measurements were collected on the bulk brain tissue. As can be seen in Fig. 4c, the mean fluorescence of the 9L-GFP group was slightly higher than that of the control group, while the mean fluorescence of the U251 group was significantly higher than either the control group $(p\text{-}\text{value}<0.0001)$ or the 9L-GFP group $(p\text{-}\text{value}=0.021)$ .

Fig. 4

The PpIX fluorescence-to-transmittance signal ratio (F/T ratio); (a) prior to ALA administration in vivo, (b) $2\mathrm{h}$ after ALA administration in vivo, and (c) $2\mathrm{h}$ after ALA administration, measured ex vivo on the extracted whole brain.

3.2.

Protoporphyrin IX Tumor Tissue Production Heterogeneity

PpIX production following ALA administration was heterogeneous between mice, as can be seen in Fig. 5, which shows three sample 9L-GFP tumor-bearing mice. For each mouse the same brain section is shown in a PpIX fluorescence image, a GFP fluorescence image, and an H&E stained image. Approximately the same section can be viewed in vivo via the T1 turbo spin echo (TSE) Gadolinium contrast enhanced MR image. Figure 5 illustrates that while some of the 9L-GFP mice showed PpIX production in the bulk tumor, as can be seen in Fig. 5a, most of the 9L-GFP mice only had PpIX fluorescence on the periphery of the tumor tissue and virtually no increase in PpIX fluorescence in the bulk tumor tissue over the surrounding normal brain tissue [Figs. 5b and 5c]. The pattern of tumor tissue PpIX production in the 9L-GFP gliosarcoma model was very different from that seen in the U251 glioma model. Figure 6 shows that the PpIX production in the U251 tumors was confined to the bulk tumor when compared to the H&E stained section in all three examples. It can also be seen that the T1 TSE Gadolinium-enhanced MR images and T2 TSE MR images allowed for in vivo visualization of approximately the same tumor tissue section shown ex vivo.

Fig. 5

Images of mouse brain tissue with 9L-GFP tumors shown with ex vivo GFP (first row), PpIX (second row), and H&E (third row) of the same sections of the brain. Also, in the bottom row, in vivo T1 turbo spin echo contrast enhanced (TSE-CE) MRI of approximately the same section. In (a), the PpIX fluorescence can be seen in the bulk tumor as compared to the H&E stained image and the GFP fluorescence image. In (b) and (c), the PpIX fluorescence was observed only at the periphery of the tumor tissue, not in the bulk tumor as can be seen by comparing the PpIX fluorescence image with the corresponding H&E stained images and GFP fluorescence images.

Fig. 6

Images of mouse brain tissue with U251 tumors shown with ex vivo PpIX fluorescence images (first row) and corresponding H&E stained images (second row) of the same sections of the brain. In vivo T1 turbo spin echo contrast enhanced (TSE-CE) MRI (third row) and T2 TSE MR images (fourth row) of approximately the same section shown in ex vivo images. In (a), (b), and (c), all mice exhibit increased PpIX fluorescence corresponding to the bulk tumor area as can be seen by comparing the PpIX fluorescence images with the corresponding H&E stained images.

3.3.

Red Light Time Course Photobleaching

Due to high PpIX production in skin tissue, selective photobleaching of skin PpIX fluorescence should improve noninvasive tumor tissue detection. The mean PpIX fluorescence measured $2\mathrm{h}$ after ALA administration showed a different relationship between the control group and the two tumor-bearing groups than that seen by ex vivo PpIX measurements. Specifically, the $2\text{-}\mathrm{h}$ in vivo measurements showed the 9L-GFP group with slightly higher mean PpIX fluorescence than the U251 group (mean PpIX fluorescence: $\text{control}=25.02$ , $9\mathrm{L}\text{-}\mathrm{GFP}=43.20$ , $\mathrm{U}251=41.50$ ), as can be seen in Fig. 4b, while the ex vivo measurements illustrated that the mean PpIX fluorescence of the U251 group was more than twice that of the 9L-GFP group (mean PpIX fluorescence: $\text{control}=0.85$ , $9\mathrm{L}\text{-}\mathrm{GFP}=3.3$ , $\mathrm{U}251=6.9$ ) as reported in Fig. 4c. The time course photobleaching measurements were examined to determine if the red light photobleaching facilitated in vivo visualization of the same PpIX fluorescence contrast pattern seen in ex vivo measurements.

A similar relationship to that seen $2\mathrm{h}$ after the administration of ALA was seen after 1, 2, and $4\min$ of red light photobleaching, where the mean PpIX fluorescence of the 9L-GFP group was slightly higher than that of the U251 group. The measurement obtained after $8\min$ of red light photobleaching showed the mean PpIX fluorescence of the U251 group was higher than in the 9L-GFP group, which was the same relationship that was seen in the ex vivo measurements. Box and whisker plots that showed the individual mice in each group as well as the interquartile range can be seen in Fig. 7 for the mice after 1, 2, 4, and $8\min$ of red light photobleaching. The measured PpIX fluorescence of the 9L-GFP group was considerably reduced following $8\min$ of red light photobleaching, the mean of which was lower than the U251 group (mean PpIX fluorescence: $\text{control}=19.92$ , $9\mathrm{L}\text{-}\mathrm{GFP}=27.24$ , $\mathrm{U}251=33.38$ ).

Fig. 7

The PpIX fluorescence-to-transmittance ratio (F/T ratio) of the mice in the control group and each tumor-bearing group after (a) $1\min$ , (b) $2\min$ , (c) $4\min$ , and (d) $8\min$ of red light photobleaching. The mean of the U251 group was higher than the mean of the 9L-GFP group for the first time following ALA administration in the photobleaching time course after $8\min$ of red light photobleaching.

3.4.

Receiver Operator Characteristic Analysis

Detection of tumor-bearing animals over nontumor-bearing control animals via ALA-induced PpIX fluorescence spectroscopy before and after red light photobleaching was assessed by ROC analysis. ROC curves were plotted for each tumor type relative to the control animals $2\mathrm{h}$ after ALA administration, but prior to any photobleaching [Fig. 8a ] and after $8\min$ of red light photobleaching [Fig. 8b]. Two hours after ALA administration, prior to any photobleaching, the U251 tumor-bearing group could be detected with higher sensitivity and specificity than the 9L-GFP tumor-bearing group, compared to the control group (U251 $\mathrm{AUC}=0.94$ , 9L-GFP $\mathrm{AUC}=0.72$ ). Following the $8\min$ of red light photobleaching, detection sensitivity and specificity were decreased in both tumor-bearing groups over the control group; however, the decrease was more significant in the 9L-GFP tumor-bearing group than in the U251 tumor-bearing group. This decrease in detection sensitivity could be attributed to increased overlap between the PpIX fluorescence-to-transmittance ratio of the tumor-bearing group and the control group (U251 $\mathrm{AUC}=0.92$ , 9L-GFP $\mathrm{AUC}=0.69$ ).

Fig. 8

Receiver operator characteristic (ROC) curve for PpIX fluorescence-to-transmittance ratio of U251 tumor-bearing group versus control nontumor-bearing group and 9L-GFP tumor-bearing group versus control group. The area under the curve (AUC) was calculated as a metric to compare tumor detection sensitivity and specificity for the two tumor types over control, nontumor-bearing animals. The ROC curve for PpIX fluorescence tumor detection (a) $2\mathrm{h}$ after ALA administration where the U251 $\mathrm{AUC}=0.94$ and the 9L-GFP $\mathrm{AUC}=0.72$ , and (b) after $8\min$ of red light photobleaching where the U251 $\mathrm{AUC}=0.92$ and the 9L-GFP $\mathrm{AUC}=0.69$ .