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A Brain Tumor Molecular Imaging Strategy Using A New Triple-Modality MRI-Photoacoustic-Raman Nanoparticle

The vexing difficulty in delineating brain tumor margins represents a major obstacle toward better outcome of brain tumor patients. Current imaging methods are often limited by inadequate sensitivity, specificity, and spatial resolution. Here we show that a unique triple-modality Magnetic resonance imaging - Photoacoustic imaging – surface enhanced Raman scattering (SERS) nanoparticle (MPR) can accurately help delineate the margins of brain tumors in living mice both pre- and intra-operatively. The MPRs were detected by all three modalities with at least picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to specific MPR accumulation and retention by the tumors, allowing for non-invasive tumor delineation by all three modalities through the intact skull. Raman imaging allowed guidance of intra-operative tumor resection, and histological correlation validated that Raman imaging is accurately delineating brain tumor margins. This novel triple-modality nanoparticle approach holds promise to enable more accurate brain tumor imaging and resection.


A. Supplementary Data Serum stability of Photoacoustic signal
To verify the stability of the optical absorbance of the MPRs in serum, MPRs were added to 50% mouse serum / 50% PBS (total volume 1 mL, MPR concentration 13 pM). We monitored the optical absorbance of the solution at 532 nm at multiple time points for 22 h (Supplementary Figure 1).
Control solution included 50% serum only. The optical absorbance of the control serum vial had increased slightly over the 22 h, likely due to the evaporation of water from the vial, leading to higher concentration of the serum. This effect has likely also occurred in the vial containing the MPRs in serum. However, subtracting the "serum only" absorbance from the "MPR in serum" absorbance shows a stable and consistent absorbance curve with a standard deviation from the mean average absorbance of 9.5%. Figure 1. Optical serum stability of MPRs. The MPRs showed a high level of optical stability when exposed to serum. Over the course of 22 h, the optical absorbance of a vial containing 50% serum only (blue curve) and a vial containing MPRs with 50% serum (red curve) were monitored. The blue and red curves show a slight increase of absorbance over time, an effect which is likely due to the evaporation of water from the vials, thereby increasing the concentration of serum in the vial. The green curve represents the subtraction of the "serum only" curve from the "MPR in serum" curve.

Stability of the hydrodynamic size during serum incubation
To validate the stability of the MPRs in serum, we measured the MPR's hydrodynamic size distribution using a dynamic light scattering instrument (Zetasizer, Malvern). The MPRs were added to 50% mouse serum / 50% PBS at a concentration of 0.068 nM and incubated at 37 °C for 24 h. The sample was analyzed at 0 h, 1 h, 2 h, 4 h, 6 h and 24 h post-incubation by dynamic light scattering.
Over the course of 24 h, we noticed a slight increase in the hydrodynamic radius and the polydispersity index (PDI) of the MPRs (peaking at 1 h post-incubation with serum), which is attributed to association with blood proteins. In addition, a small peak was observed near 4,500 nm, attributed to aggregation; however, this peak was less than 5% of the total intensity for all measurements. Hence, we concluded that the MPRs are stable in serum (Supplementary Fig. 2).

Supplementary Figure 2. Stability of the MPR hydrodynamic size during serum incubation.
MPRs were incubated with 50% mouse serum / 50% PBS for 24 h at 37 o C. Hydrodynamic measurements of the samples taken with a dynamic light scattering instrument indicated that the particles' hydrodynamic size is stable over the course of 24 h of serum incubation.  We further explored the correlation between the MRI signal-concentration response curve to the Photoacoustic signal-concentration response curve, to the Raman signal-concentration response curve in the phantom experiment (Supplementary Figure 4). For this purpose, we plotted 3 graphs:

vivo)
We then explored the correlation between the MRI signal-concentration response curve to the

Dynamic triple-modality imaging
In order to determine the kinetics of the MPRs in all three modalities, we prepared additional eGFP + U87MG orthotopic tumor-bearing mice (n = 4 for MRI and n = 4 for Raman imaging. The data for Photoacoustic imaging is derived from the same mice (n = 4) described in Figure Fig. 11). This behavior can be explained by the fact that Raman imaging, unlike MRI or Photoacoustic imaging, is integrating the signal from all tissues under the surface into a 2D image. This includes the skin and healthy brain tissue in which the MPRs circulate but are not retained.
Supplementary Figure 11. MPR in vivo signal kinetics. Unlike the MRI (top) and Photoacoustic (middle) kinetic curves, the Raman (bottom) kinetic curve showed an initial peak at 0.5 h, followed by a decrease to a plateau at 1.5 h. This effect is presumably due to initial nonspecific circulation of MPRs in superficial layers, before clearance of MPRs from the blood-stream has occurred. Raman is most sensitive to superficial layers such as the skin because of the proximity of the lens to the skin surface of the animal. Error bars represent s.e.m. . At 24 h post-injection, brains were excised and cryosections (10 µm slice thickness) were stained with hematoxylin and eosin (H&E) and subsequently analyzed with a scanning electron microscope (SEM). Adjacent sections were stained with Olig2-specific immunohistochemistry (center image). (a) H&E stain of the brain showing a tumor in the right hemisphere (boxed region). (b) An SEM scan of this H&E section was then acquired to sample high magnification SEM images from precise locations of the tumor and surrounding brain parenchyma (exact sample locations indicated by '+' signs). (c) Higher magnification SEM images taken from five different locations in the brain were acquired (overlay of secondary electron SEM on backscattered electron SEM, MPRs shown as white dots). Location 1 and 5 represent normal brain tissue as confirmed by IHC, whereas locations 2-4 represent zones of infiltrating tumor margin. Note that many clusters of MPRs were found not only in the bulk tumor but indeed also in the diffusely infiltrating margins of the tumor. Scale bar in (a) equals 2.5 mm; scale bar in all SEM images equals 500 nm. . At 24 h post-injection, brains were excised and cryosections were analyzed with Raman microscopy (500 µm slice thickness) and Olig2specific immunofluorescent staining of an adjacent section (with DAPI counterstain; 10 µm slice thickness). The Raman signal (red) represents the location of the MPRs, the Olig-2 signal (green) the presence of tumor cells and DAPI (blue) the presence of cellular nuclei of both tumor and surrounding brain (Note that certain differences in co-localization between Raman signal and Olig2/DAPI staining are expected as these images are not taken from the exact same section and volume averaging in the Raman image due to the larger slice thickness can occur). Left column: A finger-like tumor protrusion is shown at the edge of the brain tumor. As demonstrated by the good co-localization of the Raman and Olig-2 signal, MPRs accumulated in and were therefore able to outline such a finger-like protrusion. Right column: An isolated satellite metastasis away from the main tumor is also depicted by Raman imaging (note the good co-localization of red and green signal). Data is representative of three random slices through each of the four tumors.

Intra-operative Photoacoustic imaging
In addition to the Raman intra-operative experiment (Fig. 5), we have demonstrated that Photoacoustic imaging can further be used to complement Raman imaging in intra-operative guidance of tumor resection. A mouse bearing a glioblastoma tumor (primary human xenograft) was injected with MPRs (150 µl, 16 nM). 24 h post-injection, the mouse was perfused with PBS, sacrificed and the brain excised and embedded in agarose. Photoacoustic images were acquired before and after partial tumor resection (Supplementary Fig. 14). An absence of Photoacoustic signal in the resected portion of the tumor was observed, while a residual Photoacoustic signal was observed from the nonresected tumor. This experiment provides preliminary demonstration that the Photoacoustic tomographic information further complements the two-dimensional Raman image, as it provides the surgeon with depth-information on the area that should be resected.
Supplementary Figure 14. Intra-operative Photoacoustic imaging. A mouse bearing a glioblastoma tumor (primary human xenograft) was injected with MPRs (150 µl, 16 nM). After 24 h, the brain was perfused with PBS, excised, and embedded in an agarose gel. Coronal Photoacoustic images were acquired before (left image) and after (right image) partial tumor resection. An absence of Photoacoustic signal in the resected portion of the tumor was observed, while residual Photoacoustic signal (arrow-head) was observed in the area of the non-resected tumor. Note that the increased grayscale ultrasound signal to the right of the resected cavity is likely due the surgical manipulation, an effect that is commonly observed during surgery. Photoacoustic images (color scale from 0 to max) were overlaid on conventional ultrasound images (gray), which outline the gross anatomy of the mouse brain.

B. Supplementary Discussion
As many disease states do not exhibit an intrinsic Photoacoustic contrast, it is necessary to administer a Photoacoustic contrast agent. The nanoparticle used here with its 60 nm diameter gold core was shown to exhibit the highest Photoacoustic signal reported to our knowledge (e.g., 4,500-fold higher than carbon nanotubes 1 and ~ten-fold higher than most gold nanorods 2 ). While other Photoacoustic contrast agents may exhibit lower absolute Photoacoustic signal, some were reported to have peak absorption in the near infra-red 2-5 , where tissue background signal is reduced. However, targeting them to tumors has been proven to be highly challenging 2-6 . In fact, apart from this current work, we are not aware of any report where nanoparticles were shown to accumulate in deep tumors and produce a detectable Photoacoustic signal. Hence, while the peak Photoacoustic wavelength of the MPRs is in theory not optimal for tissue penetration, their very high optical absorbance at this wavelength compensates for this fact to some degree (see Supplementary Figure 15, showing 123fold higher absorbance of MPR nanoparticles than gold nanorods (Nanorodz™; Nanopartz Inc., 10 nm diameter, 38 nm length; peak absorbance at 780 nm) at 532 nm and 2.5-fold higher absorbance than gold nanorods at 780 nm). While the exact depth of penetration achievable using the MPRs is not fully known in vivo, this parameter could be evaluated in future studies.
Another potential imaging modality that can be leveraged for identifying tumor margins is fluorescence imaging. Compared to Raman or Photoacoustic imaging, fluorescence imaging suffers from a lower sensitivity on the order of 0.5 nM; however, it could be further improved using multispectral imaging analysis 7 . On a per-molar concentration basis, the MPRs exhibit a higher sensitivity compared to fluorescence agents. However, on a per-mass basis, fluorescent dyes may show better sensitivity than Raman imaging, partially due to the relatively large size of the MPRs.
Nanoparticles also carry some unique advantages such as a very high EPR effect (i.e., accumulation in tumors) compared to small fluorescent dyes.
High grade gliomas, particularly WHO Grade IV glioma (glioblastoma multiforme), are characteristically diffuse, grossly hemorrhagic and necrotic. Therefore, establishing a correspondence between pre-operative and intra-operative tumor delineation represents a significant challenge in neuro-oncology and is one that may be overcome with a multimodal imaging agent such as the one described here. A second significant difficulty in tumor resection is the frequent poor definition of the tumor margin, caused by finger-like protrusions of tumor into the adjacent brain parenchyma. Thus, invasive cells may be found to follow small blood vessels (perivascular cuffing), and in myelinated fiber tracts in which cells show intrafascicular, perifascicular, and interfibrillary migration. The detection of such small tumor extensions requires the highest possible sensitivity and resolution.
While most studies examining the effect of intra-operative image guidance on survival used MRI and found an associated improved survival due to the more complete resection 8,9 , the resolution of clinical MRI does not resolve such small structures. This could represent a reason why the majority of studies using currently available intra-operative imaging methods report a significant increase in length of survival, but are still not able to achieve long-term survival in high-grade gliomas. In our study we have observed that such finger-like protrusions were also detectable by Raman imaging, indicating that this approach may allow more complete resection in these critical areas. Although we have demonstrated the feasibility of the MPR approach in both the U87MG model as well as in an infiltrating human primary xenograft model, a caveat remains that neither of these two models completely recapitulate human brain tumor pathology and thus eventual clinical studies will be needed for further validation.

MRI -additional details
For phantom experiments, 50 µL of serial MPR dilutions in MES buffer were placed in customized 384 well plates. For in vivo experiments, animals were first anesthetized in a knockdown box with 3% isoflurane. The animals were then placed on a custom-designed MRI-compatible cradle in a 3 cm inner diameter in house quadrature birdcage radio frequency transmit/receive coil and the tube was connected to a ventilator with 1.3-1.5% isoflurane. A fiber-optic temperature probe and respiratory sensor were placed adjacent to the abdomen of the animal. The coil with the animal was inserted to the iso-center of the magnet. Heated air was pumped into the bore to maintain the body temperature of the animals at physiological levels (34-38 °C).
For quantification of tumor enhancement on MRI, the contrast-to-noise ratio (CNR) was used 10 . An axial slice through the middle of the tumor was chosen in the software Osirix. On this slice, an ROI was drawn outlining the margins of the tumor. A second ROI of the same size was placed over brain tissue of the contralateral hemisphere, which did not contain visible tumor. A third ROI was placed outside of the skin near the skull to record image noise. The CNR was then calculated using the following formula: (signal tumor -signal normal brain )/standard deviation of noise 10 .

Raman Imaging -additional details
For Raman microscopy, frozen sections were placed on quartz slides (Ted Pella, Inc.) and air-dried. A greater slice thickness (50 µm or 500 µm, depending on the purpose of the experiment) than for immunohistochemistry was chosen in order to increase Raman signal intensity. A 5x or 12x lens was used, and Raman spectral maps and correlating white light images were acquired using the Renishaw Streamline function. Spectra were analyzed by least squares analysis using Wire 2.0 Software (Renishaw).

Photoacoustic Imaging -additional details
A precision xyz-stage (U500, Aerotech Inc.) with a minimum step size of 1 µm was used to move the transducer and the fiber ring along a planar 2D trajectory. At every position, the acquired signal was averaged over 2 to 4 laser pulses. The time of arrival and the intensity of the laser pulses were recorded using a silicon photodiode (DET10A, Thorlabs). This information was used to synchronize the acquisition and compensate for pulse-to-pulse variations in laser intensity. The analog Photoacoustic signals were amplified using a 30 dB preamplifier (5676/115VAC, Panametrics-Olympus NDT) and digitized using an oscilloscope (Infiniium 54825A, Agilent). The Photoacoustic and ultrasound images were reconstructed as follows: the a-scan from each position of the transducer was band pass filtered with 100% fractional bandwidth, compensated for laser intensity variations and envelope detected. The a-scans were then combined to reconstruct a 3D intensity image of the target.
No further post-processing was done on the images. The ultrasound images were acquired using a 5 or 25 MHz transducer.

Photoacoustic-MRI and Raman-MRI co-registration and 3D visualization
Since the Photoacoustic image is intrinsically three-dimensional, we co-registered it to the MRI image and rendered their overlay in 3D. A conventional ultrasound image was taken with the same transducer that was used to acquire the Photoacoustic image, thereby producing perfectly coregistered Photoacoustic and ultrasound images. The ultrasound image highlighted the mouse brain anatomy such as skull curvature, which then allowed us to manually find the exact rigid-body alignment needed to register the ultrasound image to the MRI image. We then applied the same rigidbody alignment to the Photoacoustic image. The 3D rendering of the two images was done in the commercial software Amira(Visage Imaging, Inc.). The Raman images were acquired using precise topographical landmarks of the mouse anatomy. The Raman image with its known dimensions and matrix size and the MRI data were then manually co-registered based on these landmarks and the known size and position of the acquired images.

Histology
Brain tissue was embedded in optimal cutting temperature material (O.C.T.; Sakura) and snap-frozen in liquid nitrogen. Frozen sections (10 µm slice thickness) were obtained using a cryotome (Leica).
For Raman microscopy, 50 µm or 500 µm sections were cut immediately adjacent to a slice used for immunohistochemistry.
Slides were air-dried for 2 h at RT, then fixed in cold acetone for 5 min and air dried for 1 h. Slides

Statistical Methods
Correlation of signal increase with increasing concentration in vitro was tested with mixed effects linear regression of log 10 signal ratio to baseline on log 10 concentration, imaging method, and their interaction, with individual phantom area as the random factor. Correlation of signal increase with increasing concentration in living mice, and differences in rate of signal increase between imaging methods in living mice were tested with mixed effects linear regression of log 10 signal ratio to baseline on log 10 concentration, imaging method, and their interaction, with mouse as the random factor. The MPR tumor targeting experiments were analyzed via one-sided t-test, where MRI and Photoacoustic post-injection measurements were compared against a ratio of 1 with a one-sided one-sample t-test, and Raman post-injection measurements were compared against an average noise value of 0.001 with a one-sided one-sample t-test. The MPR tumor targeting kinetics study initial increase was tested with one-sided student's t-test. The signals were then re-expressed as log of ratio to pre-injection baseline. Differences between imaging methods in time course of signal from 30-120 min were tested with mixed effects linear regression of log 10 signal ratio to baseline on log 10 concentration, imaging method, and their interaction, with mouse as the random factor.

Mouse arrangement in the Photoacoustic system
Female nude mice were used for all the Photoacoustic studies. The mice that were scanned in the Photoacoustic system were fully anesthetized using isoflurane delivered through a nose-cone. Prior to the Photoacoustic scan, the head of the mouse was slightly tilted to expose the right lobe to the imaging system. The head was then covered by Gonak gel (Akorn) and covered by a saran-wrap water bath. The ultrasonic transducer in the water bath was therefore acoustically coupled to the mouse brain tissues. This setup allowed the ultrasonic transducer to move freely in 3D while applying only minimal physical pressure on the mouse head (Supplementary Figure 16).
Supplementary Figure 16: Photoacoustic imaging instrument. A tunable pulsed laser (Nd:YAG laser) illuminated the subject through a fiber optic ring light. The Photoacoustic signals produced by the sample were acquired using either a 5 MHz or 25 MHz focused transducer. A precision xyz-stage was used to move the transducer and the fiber ring along a planar 2D trajectory. The time of arrival and the intensity of the laser pulses were recorded using a silicon photodiode. This information was used to synchronize the acquisition and compensate for pulse-to-pulse variations in laser intensity. The analog Photoacoustic signal was amplified using a 30 dB preamplifier and digitized using an oscilloscope.

Stereotactic brain tumor implantation
All aspects of experimental manipulation were in strict accord with guidelines from the National

Institute of Health and have been approved by members of the Stanford Institutional Animal Care and
Use Committee (IACUC). Ten week old female nude mice (Charles River), or five week old male SCID mice (Taconic Farms, Inc.), respectively, were anesthetized using 2.0% isoflurane and positioned in a Benchmark® (Leica) stereotactic instrument (Supplementary Figure 17).

Supplementary Figure 17. Stereotactic brain tumor implantation.
Mice also received a subcutaneous injection of 0.1 mL of a 1:10 dilution of 0.3 mg/mL buprenorphine as additional anesthesia. The top of the animal's head was cleaned with 70% ethanol and betadine.
Ophthalmic ointment was applied, a linear skin incision was made over the bregma, and 3% hydrogen peroxide was applied to the periost with a cotton swab. A 27G needle was then used to drill a burrhole into the skull 0.5 mm anterior and 2 mm lateral to the bregma. A 10 µL gas-tight syringe (Hamilton) was then used to inject 2 µL of the eGFP + U87MG-cell suspension or TS543 human glioblastoma cells

Optical and Raman photobleaching studies
We tested the optical stability of the MPRs under increasing durations of light exposure (photobleaching). For assessment of photobleaching by the Photoacoustic laser, the sample was continuously exposed over 30 min (532 nm, power density of 8 mJ/cm 2 , 10 Hz repetition rate) which were the wavelength and power settings used in our animal experiments.
For assessment of photobleaching by the Raman laser, a 20 µL sample of MPRs in MES buffer was placed in a cell-counting slide. The sample was then continuously exposed to the Raman excitation laser (785 nm) and the Raman signal was recorded every 30 seconds.

Signal linearity phantom study
To test the linearity of the Photoacoustic signal as a function of MPR concentration, we used an agarose phantom with no scattering or absorbing additives. MPRs at increasing concentrations were mixed with warm liquid agarose (n = 3 samples for each concentration) forming MPR solutions at 0, 1. 2, 2.4, 4.9, 9.8, 19, 39, 78, 156 2D ROIs encompassing the inclusions were placed within a slice through the middle of the inclusion using the software Osirix. The signal intensity expressed in arbitrary units for each of the three modalities represents the mean for each of the three mice.

Quantitative Raman spectral analysis
The direct classical least squares (DCLS) method was used in this work to perform quantitative analysis of Raman spectroscopy 12,13 . DCLS finds the linear combination of spectra from the MPR Raman signal contained in the area of interest (brain) that most closely matches the Raman spectrum

Scanning Transmission Electron Microscopy of tissue samples
Tissue samples from both healthy brain and tumor were fixed in a solution of 4% paraformaldehyde in 0.1 M sodium cacodylate buffer. Samples were then stained with 1% osmium tetroxide in water at 4ºC for 2 h. After 2 h, the tissue samples were rinsed with deionized water and stained with 1% uranyl acetate at 4ºC overnight. Samples were then dehydrated in progressively higher concentrations of ethanol at 4ºC; 50%, 70%, and 95%. The tissue samples were then allowed to gradually warm to RT.
Samples were further dehydrated 2x in 100% ethanol and 3x in propylene oxide. Samples were then embedded in Embed 812 epoxy resin (EMSdiasum). Samples were placed in 1:1 solution of Embed 812:propylene oxide for 1 h at RT. Samples were then placed in 2:1 solution of Embed 812:propylene oxide overnight. Finally samples were placed in 100% Embed 812 for 1 h before being placed in molds and cured overnight at 60ºC. Thin sections (150 nm) were then cut from the tissue samples using an Ultracut S microtome (Leica) and placed on 200 mesh bare copper grids. The sections were examined without coverslip using a Tecnai G2 X-Twin (FEI) scanning transmission electron microscope (STEM) operating at 120 kV in scanning mode.

Scanning Electron Microscopy of tissue samples
Images of histology slides of brain tissue stained with hematoxylin and eosin were acquired using a DM 2000 light microscope (Leica). Images were taken and stitched together to create a large composite image of the entire brain section. The histology slides were then coated with a thin AuPd film to improve conductivity and placed in a XHR scanning electron microscope (SEM) (Magellan).
The SEM was operated at 15 kV with a probe current of 50 pA. Both secondary electron and backscattered electron images were collected. Backscattered electron imaging was utilized to locate the MPRs in the brain tissue.

Inductively coupled plasma atomic emission spectroscopy (ICP-AES)
An aliquot of 385 µL of 3.86 nM MPR suspension was washed 2x in water and then pelleted by centrifugation. The pellet was re-suspended in 2 mL of 10 M of sodium hydroxide and sonicated for 10 min in a sonication bath at RT. This was followed by slow addition of 70% HNO 3 until the pH became acidic. The volume of the resulted mixture was brought to 10 mL and analyzed for the presence of Gd +3 ions using an IRIS Advantage/1000 Radial ICAP Spectrometer (Thermo Scientific).

Raman imaging
The phantom was based on a 1% agarose mixed with India Ink to mimic tissue absorption (to make the final agarose-solution at optical absorbance of 1.0 cm -1 ) and Liposyn 0.5% to mimic tissue scattering. The agarose liquid was left to solidify in a plastic container that was slightly tilted. As the agarose solidified, small inclusions of ~3 mm diameter and 1-2 mm in depth were made using a small pipette tip. MPRs mixed in liquid agarose-solution at 1 nM concentration were embedded in the small inclusions. As the inclusions solidified, the plastic container was positioned on a flat surface and an additional layer of agarose-solution was poured on the phantom to seal it.