(CANCER RESEARCH 56. 4302-43(16. Oclubcr I, 19961

Advances in Brief

Ion Microscopy Imaging of 10Bfrom /7-Boronophenylalanine in a Brain TumorModel for Boron Neutron Capture Therapy1

Duane R. Smith, Subhash Chandra, Jeffrey A. Coderre, and George H. Morrison2

Depurimeli! of Chemistry. Baker Laboratory, Cornell Universii\. Ithaca. New York ¡4853¡D.K. S.. S. C., G. H. M.I. ami Medical De/Hirlmenl. Brookhaven National iMhorattiry.Upton. New York 11973 ¡J.A. C.¡

Abstract

Boron neutron capture therapy (BNCT), a binary treatment modalitythat can potentially irradiate tumor tissue within cellular dimensions, iscritically dependent on the preferential delivery of '"IS to individual

neoplastic cells. In this study, ion microscopy was used to quantitativelyevaluate the selectivity of p-boronophenylalanine-fructose (BPA-F) in therat 9L gliosarcoma brain tumor model. With a spatial resolution of —¿�0.5

/

QUANTITATIVE ION IMAGING FOR NEUTRON CAPTURE THERAPY

of each rat. resulting in a locally expanding cerebral tumor. The BNL strain of the9L gliosurcomu originated from an .V-nitrosomethylurea-induced neoplasm in a

Fischer 344 rat (18). Details of the intracranial tumor implantation have beenpublished ( 16. 19). BPA-F treatment and tissue acquisition were performed on the

14th day after inoculation, when the tumors were approximately 4 mm in diameter,a size proportionate to that of most human glioblastoma multiforme tumors whenfirst diagnosed. Single i.p. injections of BPA-F (trial 1, 1200 mg/kg body weight.

four rats: trial 2. 600 mg/kg body weight, eight rats) were administered 2 h priorto euthanasia to approach the maximum accumulation in the time-dependentdistribution of "'B in tumor tissue (20). One rat. which did not receive an injection

of BPA-F. was treated as a control to check for any detectable naturally occurring"'B in the brain and tumor tissue. Rats were anesthetized by single i.p. injection of

a mixture of ketamine (140 mg/kg body weight) and xylazine (28 mg/kg bodyweight) prior to tissue acquisition. These animal studies were reviewed andapproved by the Institutional Animal Care and Use Committee of the Brkhaven

National Laboratory.Sample Preparation. Small pieces of brain tissue from rats were cryogen-

ically prepared. Most specimens were plunged in liquid isopentane cooled to-150°C in liquid nitrogen and sectioned at -20°C in a Reichert HistoSTAT

cryostat microtome. Sections. 4 /urn thick, were taken in series to correlate thetissue morphology (via optical microscopy) with the '"B distribution (via ion

microscopy). Sections prepared for optical microscopy were placed on cleanglass microscope slides and stained with H&E. Those sections prepared for ionmicroscopy were mounted on high-purity indium substrates (21). freeze dried,

and coated with 400 A Au/Pd alloy.An alternate, more rigorous cryogenic procedure provided an independent

confirmation of the preservation of the in vivo "'B distributions. The tissue

specimens (pieces approximately 1 mm3) prepared in this manner wereplunged and frozen in propane slush ( —¿�180°C)and maintained in liquidnitrogen. Cryosections (0.5-0.75 /¿mthick) were obtained at - 140°Cusing a

Reichert Ultracut S/FC S Cryoultramicrotome. mounted on indium substrates,

freeze dried, and coated with Au/Pd prior to ion microscopy analysis.Ion Microscopy Imaging. A CAMECA IMS-3C ion microscope, a direct-

imaging secondary ion mass spectrometer ( 14). was used in this study. Allsamples were analyzed in the positive secondary ion imaging mode using amass-filtered O,+ primary ion beam accelerated to 5.5 keV with a nominal

beam current of 120 nA and an approximate beam diameter of 50 ¡an. Formost analyses, the primary ion beam was raster scanned over 250 X 250-/nmsquare regions. The ion microscope was operated using the I50-/j.m transferoptics in conjunction with a 60-ju.m contrast aperture to produce ion imageswith a spatial resolution of ~0.5 /LUTI.The energy window of the mass

spectrometer was centered and set to the maximum value of 130 eV. Secondaryion images were generated on a single-microchannel plate/phosphor screen

image detection assembly. The gain of the microchannel plate was set to 70%of maximum. Images were recorded directly from the image detection assembly using a Photometries. Ltd.. CH220 charged-coupled device liquid-cooledcamera head equipped with a Thomson-CSF TH7882 CDA charged-coupled

device camera and digitized to 14 bits/pixel by a Photometries camera controller. For each region analyzed, sequential ion images from WK+. 2*Na+.10B+, I2C+. 24Mg+, 40Ca+, and ' "ln+ were acquired with integration times of

0.3 s, 0.3 s, 2 min, 2 min, I min. I min. and 0.3 s, respectively. High-mass

resolution analyses confirmed that the mass interferences at the studied masseswere negligible (22). The '"B+ signal was below the detection limit of the ion

microscope (2Cimages and assuming 85% water content.

Results and Discussion

An optical image of cryosectioned tumor and brain tissue andsequential ion images taken from an adjacent 4-j^m-thick cryosection

show part of an interface running diagonally between a main tumormass and the normal rat brain (Fig. 1). In the optical image (Fig. la),a portion of the main tumor mass (7~) is visible to the left of the

interface (arrows) and is identified by the densely packed, dark-

stained nuclei. The cytoplasmic volumes of the tumor cells are small,and cellular outlines are ill defined. Spongiform circular spaces, signsof vasogenic edema, are evident along the tumor-brain interface. The

normal brain (NB) to the right of the interface is apparently free fromtumor infiltration. The circle superimposed over the interface in Fig.1«is scaled to 150 jam in diameter and correlates with the region onthe adjacent cryosection of tissue analyzed with the ion microscope(Fig. 1, b-d). Regions such as this were chosen for ion microscopy

analysis to simultaneously sample the main tumor mass and thenormal brain within the same field of view. The change in brightnessof the ion image (Fig. \b) quantitatively reflects the difference inconcentration of "'B between the main tumor mass (T) and normal

brain (NB) and coincides with the interface (arrows). Note that themagnification of the '"B ion microscopy image is higher than that ofthe optical image in Fig. la. This allowed recording of several "'Bimages along the interface to confirm the partitioning of "'B between

the main tumor mass and the normal brain. These observations indicate that IOB from BPA-F preferentially accumulated in the maintumor mass. At the subcellular scale, "'B concentrations appear equal

in the cytoplasmic and nuclear compartments, and the distribution of'°Bis relatively homogeneous.

The mass spectrometer of the ion microscope enables physiologically relevant species (39K, 23Na, 24Mg. and 4

QUANTITATIVE ION IMAGING FOR NEUTRON CAPTURE THERAPY

Fig. 1. Interface between the main tumor mass of the 9L rat gliosarcoma and the normal brain of a BPA-F-treated male Fiseher 344 rat. Adjacent 4-^tm-thick cryosections wereused for optical microscopic and ion microscopic imaging, a. optical image of a H&E-stained cryosection on glass, b-il. sequential ion images showing the distribution of the isotopesIOB(b). ll>K(f), and 4"Ca (

QUANTITATIVE ION IMAGING FOR NEUTRON CAPTURE THERAPY

•¿�v

Fig. 2. "'B distribution in clusters of neoplastia cells invading the normal rat brain beyond the main lumor mass. The images in a and b and c and d were from adjacent 4-jim-lhickcryosections. a and c. optical images of H&E-siained cryoseclions on glass from different rats, b and tl. respective "'B ion images from freeze-dried cryosections. 7",main tumor mass;

NB. normal brain; A. artery. Black arrows in a, cross-sections of a few of the neoplastic clusters invading the normal brain beyond the periphery {dotted line} of the tumor. Arrow inh. neoplaslic cluster (see circle in a} showing a significantly higher uptake of '"B than the surrounding normal brain. In r. several perivascular infiltrating neoplastic cells (arrows)are visible along with the cross-section of an artery. In d. the perivascular infiltrating neoplastic cells («rrmv.v)show a significant uptake of IOBin comparison to the walls of the arteryand the normal hrain. fiar* in n and h-tf, UK)and 50 /Am. respectively. The images were processed for publication quality.

a region of high "'B concentration, which corresponds to the larger The '"B ion image intensity from largest neoplastic cluster in Fig. 2d

neoplastic cluster visible on the lower inside edge of the circle in Fig.2a. Fig. 2c shows the optical image of a H&E-stained, 4-^m-thick

cryosection showing perivascular infiltrating neoplastic cells (arrows), the cross-section of a small artery (/4), and the contiguousnormal brain (M?) from a rat treated with 600 mg BPA-F/kg bodyweight. The ion image (Fig. 2d) of the adjacent 4-/Mm-thick cryosection ofthat in Fig. 2c shows the distribution of IOBin the perivascular

infiltrating cells (arrows), the artery (A), and the normal brain (/Vß).

arises from two cells. The wall of the artery contains ~ 1.7 times more10B than the surrounding normal brain.

The ion microscopic images of Fig. 2 demonstrate that a significantdifference in "'B concentration exists between the small clusters of

neoplastic cells invading the contiguous normal brain beyond themain tumor mass. However, the quantity of IOB was lower than that

observed in the tissues composing and immediately surrounding themain tumor mass of the 9L gliosarcoma. The wet weight concentra-

4305

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QUANTITATIVE ION IMAGINO FOR NEUTRON CAPTURE THERAPY

tion of "'B in the neoplastic clusters was 47 ±15 /xg/g tissue versus

16 ±8 ju,g/g tissue in the contiguous normal brain (n = 12 observa

tions from four rats).An interesting pattern of IOBaccumulation in the tumor and normal

brain tissue emerges in this study. The estimated "'B concentration in

the main tumor mass is approximately twice that of the neoplasticclusters infiltrating the normal brain (99 versus 47 /xg/g). Similarly,the concentration of IOB ¡nthe normal brain tissue adjacent to the

main tumor mass is approximately 1.5 times higher than in the normalbrain tissue surrounding the neoplastic clusters (27 versus 16 jxg/g).These differences may be related to the integrity of the BBB, whichwas found to be disrupted around the main tumor mass of the 9Lgliosarcoma at 14 days after tumor implantation in Fischer 344 rats(25). Note that the present study was also conducted in the samemodel at 14 days after tumor implantation. The disruption of the BBBaround the main tumor mass would increase the amount of BPA-F

present in the extracellular fluid, thus increasing tissue concentrationsof "'B. From these observations, it is also clear that BPA-F is capableof crossing the intact BBB, because 10B is distributed in the normal

brain far from the main tumor mass.The results of this study clearly indicate that BPA-F does selec

tively deliver "'B to the 9L gliosarcoma at the cellular level, produc

ing a substantial difference in boron concentration between neoplasticcells and the contiguous normal rat brain. The data are both encouraging and supportive of the use of BPA-F in the ongoing clinical trials

of BNCT of human glioblastoma multiforme. Because the tumor cellsinvading the normal brain parenchyma are responsible for regrowth ofthe tumor after surgical intervention, the observation of the reducedIOBcontent within the neoplastic cells invading the contiguous normal

rat brain beyond the main tumor mass must be considered carefully byBNCT researchers. We have observed a similar pattern of distributionof '"B from BPA-F in the F98 rat glioma model of human glioblastoma multiforme.6

The present study demonstrates an ion microscopic approach tostudying boron localization in physiologically relevant regions of thebrain associated with tumor infiltration. We have been able to establish a much-needed baseline measurement of performance of a clin

ically approved BNCT drug within the short dimensions of the LET ofthe boron neutron capture reaction. With this baseline, we are nowable to compare the performance of other potential BNCT drugs. Themethods described herein are currently being used to study the distribution pattern of BPA-F in human glioblastoma multiforme.

Acknowledgments

We thank V. L. Smith for critically reading the manuscript: P. L. Micca andM. M. Nawrocky for technical assistance in handling of the rats; J. Wilson forassistance in sample preparation for ion microscopy; and A. D. Chanana. D. D.Joel, and D. N. Slatkin for comments throughout this study.

6 D. R. Smith. W. Yang, L. Liu. J. H. Rotaru. R. F. Barth. and G. H. Morrison,

unpublished results.

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1996;56:4302-4306. Cancer Res Duane R. Smith, Subhash Chandra, Jeffrey A. Coderre, et al. a Brain Tumor Model for Boron Neutron Capture Therapy

-Boronophenylalanine inpB from 10Ion Microscopy Imaging of

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