Microchemical Analysis of Boron in Rat Brain Tumor:
A Spectromicroscopy Study with MEPHISTO
Gelsomina De Stasio
Institut de Physique Appliquée, Ecole Polytechnique Fédérale, PH-Ecublens,
CH-1015 Lausanne, Switzerland
and
Istituto di Struttura della Materia del CNR, Via Enrico Fermi 38,
I-00044 Frascati, Roma, Italy
Abstract. The boron microscopic distribution in brain tissue sections is an extremely
important issue for the success of boron neutron capture therapy for cancer. We
present the first results of a new approach to assess this distribution and its
homogeneity: photoelectron spectromicroscopy with synchrotron radiation enabled us
to detect boron in rat brain tissue specimens with a lateral resolution of 0.2 µm and a
detection sensitivity of a few ppm. These experiments were performed by the newly
commissioned MEPHISTO spectromicroscope (from the French acronym
“Microscope à Emission de Photoélectrons par Illumination Synchrotronique de Type
Onduleur”, or “Photoelectron Emission Microscope by Synchrotron Undulator
Illumination”). The specimens were brain tissue sections from two rats injected with
BPA (borono-phenylalanine) or BSH (dodecahydro-dodecaborate). We found boron
only in cancer-related structures, and with a higher concentration in the BPA-treated
specimens than in the BSH-case. These results are in good agreement with the
quantitative results obtained by inductively coupled plasma atomic emission
spectroscopy.
1 Introduction
One of the fundamental issues for the success of boron neutron capture therapies (BNCT)
[1] is the availability of analytical techniques to study the microscopic boron distribution in
tissues [2]. In fact, techniques like the widely used Inductively Coupled Plasma Atomic
Emission Spectroscopy (ICP-AES) offer no spatial resolution [3, 4]. We present a feasibility test to solve this fundamental problem, based on synchrotron radiation photoelectron
spectromicroscopy [5, 6]. This approach is an alternative to electron energy loss spectroscopy combined with transmission electron microscopy (EELS-TEM) [2], immunohistochemical methods [7], boron neutron capture autoradiography [8], or SIMS [9] also used for
the same purpose.
Spectromicroscopy does not require ultra-thin tissue sections (<60 nm for EELS-TEM)
and can analyze tissue sections 5-7 µm thick. It can detect trace concentrations of boron of
the order of a few ppm. EELS-TEM can detect the presence of boron only if its local
concentration is higher than 100 ppm [10].
On the other hand, EELS-TEM has a spatial resolution of the order of a few Å. At
present, we are limited to 200 nm; at the end of the commissioning period of our spectromicroscope MEPHISTO we should be able to reach the 50 nm level. MEPHISTO's
resolution is, and will always be, much worse than that of EELS-TEM, but still better than
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G. De Stasio
other techniques based on optical microscopy such as neutron capture autoradiography or
immunohistological methods, or with respect to SIMS [9].
Compared to immunohistochemical or neutron capture radiography techniques,
spectromicroscopy with MEPHISTO can detect any element present in the specimen, with
no need for staining or labeling. The elements are not required to have a high neutron
absorption cross section to be detectable, and the analysis is not limited by the availability
of the proper enzyme to stain specific biological targets, with a limited number of elements
for staining and limited histological parts detectable.
Another convenient characteristic of spectromicroscopy is its dynamic range: the field
of view of the micrographs obtained with MEPHISTO may be quickly varied between 5 µm
and 500 µm, enabling the microchemical analysis of a single cells or the whole region of
tissue surrounding it.
One of the main limitations of spectromicroscopy when compared to the other
techniques is that it requires a synchrotron source of photons.
In the present work we used MEPHISTO to analyze the boron distribution in specimens
of brain tissues of two rats with experimental cerebral tumors, previously injected with 10B
enriched BPA (borono-phenylalanine) or BSH (dodecahydro-dodecaborate) two hours prior
to sacrifice. We observed strong spectroscopic signal from boron in the experimental
glioma of the BPA and BSH injected rats, and we determined the boron spatial distribution
with a lateral resolution of 0.2 µm.
One critical point in BNCT is the boron distribution, which is required to be
concentrated selectively in cancer cells. The results presented here, obtained for the BPAand BSH-treated cases, revealed indeed that boron is localized in the specimen structures
that are related to cancer. By contrast, no boron signal was observed outside these
structures.
2 Sample Preparation
The specimens were brain tissue sections from rats that had been injected with 4 x 104
cancer cells (C6 glioma cell line # CCL107) twelve days before the experiment. These cells
were injected in the striatum at a depth of 5.5 mm. The rats were intraperitoneally injected
with 150 mg of BPA in 2 ml of isotonic solution or 150 mg of BSH suspended in 3 ml of
isotonic solution. Two hours after the injection, the rats were sacrificed by decapitation and
the brains extracted. The brain tissues were cryofixed by quick immersion in isopentane
kept at liquid nitrogen temperature; this quick freezing prevents the formation of large ice
crystals and the consequent microscopic damage to the tissue structures. Then, the tissue
was cut in 5 µm thick sections with a cryostat. The morphology of individual sections was
examined after immunohistochemical staining and tumoral tissue identified.
Normal brain or tumoral cryocut specimens were put on silicon wafer substrates, and
then ashed with a cold plasma (150 °C, Plasma-Processor 300E, Techn. Plasma GmbH,
München) in the presence of oxygen for • 24 hours. The ashing removes most of the
tissues’ carbon; the absence of boron from substrate areas indicates that the ashing process
does not promote boron diffusion or spread through the specimen [11].
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3 Experimental Techniques
3.1 Spectromicroscopy
The specific experimental technique used for microchemical analysis was the photoelectron spectromicroscopy approach known as x-ray secondary electron (emission)
microscopy [5], using our newly commissioned MEPHISTO system. This technique uses
monochromatized x-ray photons emitted by a synchrotron source, in our case the 6-m
Toroidal Grating Monochromator (TGM) beamline of the Aladdin ring at the Wisconsin
Synchrotron Radiation Center. The photons stimulated the emission of primary and mostly
secondary photoelectrons, through intermediate energy-loss steps.
Intensity vs photon energy spectra taken in this way (total yield) correspond to the x-ray
optical absorption coefficient. [12] The secondary electrons emitted by the sample
interacted with an electron-optical magnifying system [5] yielding images of the
specimen’s geometric features and spectra of microscopic areas. The electron optics are
basically composed by a cathode lens (the sample is kept at -20 kV, the objective lens'
intermediate element is kept at <20 kV), an aperture (70, 40 or 25 µm pinhole) in the back
focal plane of the objective lens, and intermediate lens, a projective lens, a series of two
microchannel plates to intensify the electron magnified image produced by the optical
system and a phosphor screen to convert this electron image into a visible image, which is
finally collected by a video acquisition system.
Extensive tests were systematically performed to assess the possible presence of
problems such as radiation damage or sample charging, with negative results. Specifically,
samples exposed for hours to the monochromatized x-ray beam (whose size was of the
order of 0.3 x 0.3 mm2) did not exhibit any detectable evidence of damage – such as
spectral or morphological changes. Substantial damage was observed instead after several
minutes of exposure to a much more intense unmonochromatized beam (which was never
used in our present experiments).
3.2 ICP-AES
ICP-AES, allows to perform quantitative chemical analysis on liquid samples. An aerosol
of the sample solution and argon is heated up to a cold-plasma temperature (8000 °C) by
an electromagnetic field. At this temperature, each element emits a specific near-ultraviolet
light wavelength, that can be accurately identified.
Measuring the emission intensity at each wavelength, and comparing it to a standard
reference, make it possible to determine the concentration of each element (boron, in the
present case) in the solution. The sensitivity reaches 1 ppb, evidently with no spatial
resolution: this, in fact, is a space-averaged technique. The conceptual background of the
ICP-AES technique can be found, for example, in Refs. 13 and 14.
Samples for ICP-AES analysis were not ashed. For these experiments tissue samples
were taken from the tumor volumes or the contra-lateral hemispheres, for comparison, from
both rats treated with BSH and BPA. After quick freezing in isopentane, the tissue portions
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G. De Stasio
were weighted, then added to 2 ml HNO3 and sonicated until the solution appeared
homogeneous.
Thirteen replicates were taken for each ICP-AES measurement, and the blank HNO3
was repeatedly tested to rule out the presence of boron after each experiment on different
parts of the rat brain.
4 Results and Discussion
Figures 1-7 show typical sets of experimental data, extracted from a much larger body of
results. Specifically, Fig. 1 presents a photoelectron microimage taken at low magnification, and Fig. 2 presents a set of spectra from the correspondingly labeled microscopic
areas of Fig. 1.
The micrograph corresponds to an ashed section of cryocut brain cancer tissue. We
note that most of the image exhibits a reticulate pattern of fine corrugations with limited
vertical protrusion. There are, however, more protruding, elongated structures (appearing
as clear areas in the image, corresponding to higher photoelectron yield). The analysis of
the specimens indicates that these elongated structures are typical of the cancer tissue,
since none was observed in non-cancer areas.
Fig. 1 . An X-ray secondary pho-toelectron
emission micrograph taken with the
MEPHISTO system on a section of
extracranially grown rat brain cancer tissue,
after ashing. The rat had been treated
intraperitoneally with BPA.
Fig. 2. X-ray secondary photoelectron
emission spectra taken with MEPHISTO
in the labeled areas of Fig. 1. Note that the
B signal is confined to the elongated
bright areas.
The spectra of Fig. 2 indicate the presence of three absorption structures, due to the
elements boron, sulfur and phosphorous, of which the last two are naturally present in the
Microchemical Analysis of Boron in Rat Brain Tumor
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brain tissue. It is evident from Fig. 2 that extreme care must be used in distinguishing from
each other the boron and phosphorous signals, which fall in the same spectral region.
The key point in discriminating between P and B was the analysis of control specimens
extracted from normal tissue areas. Figure 3 shows one of the spectra obtained on this
normal tissue: it is quite clear that the P and S signals are still present, whereas no B signal
can be observed. This conclusion is supported by an analysis of the first and second
derivatives of the spectra (data not shown). Quite to the contrast, the absence of the B
signal corroborates the identification of the corresponding spectral structure in Fig. 2 as
due, indeed, to this element.
Having solved this crucial issue, we can now analyze some interesting points in Figs. 1
and 2. The relative intensity of the B-related spectral feature at 190 eV is not constant from
place to place in the examined areas. On the contrary, it disappears in some points and is
rather weak in others, indicating a strongly inhomogeneous distribution of boron.
Fig. 3. Spectrum from a non-cancer control specimen: note the absence of the B signal.
More specifically, we found very strong B signal in areas corresponding to the elongated
and protruding structures in the cancer specimens -- see, for example, curves (a), (b), (d)
and (g) in Fig. 2. Outside the elongated structures, on the reticulate tissue, no B signal was
observed (curves (e) and (f)). These specific results are representative of a much larger
body of consistent data, including about 100 spectra and 20 images acquired on 3 different
sections of the same BPA treated tumor.
The strong localization of boron in the elongated structures is indicated by the results of
Figs. 4 and 5, which illustrate a “zoom” test. Figure 4 shows micrographs taken in the same
specimen area with increasing magnification; the area is centered around point (g) in Fig. 1.
Spectra were taken in microscopic regions of the higher-magnification images of Fig. 4 with
the following two relevant results.
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G. De Stasio
Fig. 4. Three different images centered at point (g) in Fig. 1, taken with increasing magnification.
Fig. 5. Spectra taken from the areas labeled as (a) and (b) in Fig. 4 and (g) in Fig. 1, but much smaller
than for the spectrum (g) in Fig. 2. Note that the relative boron intensity is higher in curve (a) and
lower in curve (b) than in the spectrum (g) of Fig. 2, indicating that this element is not
homogeneously distributed within the elongated structures.
First of all, a fine analysis of the reticulate pattern regions close to, but outside, the
elongated protruding structures always failed to produce any evidence of boron. Second,
spectra taken in different areas within the elongated structures give different relative
intensities of the B signal. An example is shown in Fig. 5; the spectra (a) and (b) were taken
at the same point as region (g) in Fig. 2a, but from two much smaller regions
(approximately 7 x 7 µm2, whereas the acquisition area (g) was approximately 12 x 20
µm2): the relative B intensity in curve (a) is higher than in the (g) spectrum of Fig. 2, and
lower in the curve (b).
These results demonstrate that (1) boron is strongly localized and confined to the
cancer-related elongated protruding structures; (2) the distribution of boron within such
structures is not homogeneous. This confirms the need of a high-spatial-resolution technique
for microchemical analysis in this type of problems.
At the present time it is not clear to what cellular/tissue structures the reticulate pattern
and the elongated protruding structures of Figs. 1 and 4 correspond. It is tempting to
Microchemical Analysis of Boron in Rat Brain Tumor
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speculate that the bright reticulate edges might be delimiting individual cells (5-20 mm size)
and could represent the extracellular space.
The protruding structures, on the other hand, may originate from tumor specific blood
vessels. Neovascularization (or angiogenesis), in fact, is an important component of tumor
growth. But these structures could as well possibly originate from higher density areas
characteristic of the tumor tissue, which "resist" ashing more than the surrounding areas,
and are therefore thicker after ashing. Further investigation and histochemical analysis will
be necessary to univocally identify such structures.
We note that however these protruding structures are interpreted, our technique could be
detecting relative tumor tissue vs normal tissue B uptake, or vascular structure vs tumor cell
uptake. In this latter case the spectromicroscopical information retrieved would also be
valuable. If we are detecting boron in blood vessels, it means that BNCT would destroy
them, possibly leading to life threatening hemorrhages in the brain.
Fig. 6. MEPHISTO micrograph of a section of
intracranial rat brain cancer tissue, after
ashing. The rat had been treated
intraperitoneally with BSH.
Fig. 7. Spectra taken in the labeled areas of
Fig. 6. Note that the B signal is also in this
case confined to the elongated bright areas,
but it’s corresponding signal is much lower
than the one found treating the rat with BPA
(spectra of Fig. 2).
Figures 6 and 7 show spectromicroscopy results similar to those of Figs. 1 and 2,
obtained on tumor tissue from a rat treated with BSH. Note that boron is barely visible in
the spectra of Fig. 7. First and second derivative analysis of these spectra confirms that the
spectral feature at 190 eV can indeed be overlapped to the corresponding signal of Fig. 2,
only much less intense. Also in this case, boron, when detectable, is confined to the
protruding cancer-related structures, and absent from the reticulate tissue. In this BSH-case
too, the control sample obtained from the contra-lateral (with respect to cancer) hemisphere
did not show any boron signal.
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G. De Stasio
As already mentioned, we also performed ICP-AES analysis of boron, with no spatial
resolution, of tissue samples extracted from the same two rats used for spectromicroscopy
experiments.
Table 1. ICP-AES results on boron concentration in the various rat brain tissue parts. The results for
B concentration are normalized to the tissue volume (10-50 mg). The errors shown are the standard
deviation calculated on the 13 different measurements performed on each sample.
Sample from
B - compound injected
Boron in tissue(ppm)
HNO3
BSH
BSH
BPA
BPA
0.0 ± 0.3
1.3 ± 0.5
1.9 ± 0.8
0.7 ± 0.3
5.7 ± 0.3
normal brain tissue
intracranial tumor
normal brain tissue
extra-cranial tumor
Table 1 shows the results of the quantitative ICP-AES analysis. Note, in particular, that
the BPA-treated rat tumor tissue contains about 6 ppm boron, whereas the cancer tissue
from the BSH treated rat contains about 2 ppm boron. This of course is an average
concentration overall the entire tissue volume examined (between 10 and 50 mg of tissue in
the various different cases).
A comparison of the results obtained by a space-resolved and a space-integrated
technique is not in general feasible. Nevertheless, we note that we detected more boron in
the BPA- than in the BSH-treated case, both with the space-resolved spectromicroscopy and
with the space-integrated ICP-AES technique.
5 Conclusions
In summary, our preliminary results demonstrate the feasibility of a spectromicroscopic
analysis of boron distribution in brain tumor tissues. We concur with the opinion expressed
by Gabel [2,7], Setiawan [15], and many other authors, that high spatial resolution is
essential for the complete analysis of boron in tissues, in view of cancer therapy -- in
particular as far as the assessment of spatial distribution is concerned for a complete
destruction of cancer cells. Our approach provides a satisfactory and practical method to
answer to this need.
Acknowledgments
Work supported by the Fonds National Suisse de la Recherche Scientifique, by the Istituto
di Struttura della Materia del CNR and by the EPFL. We are indebted to Giorgio
Margaritondo and Paolo Perfetti for their constant support, to Brian P. Tonner for his help
and encouragement during the spectromicroscopy experiments, and for the construction of
the electron optics for MEPHISTO. We thank Erwin Van Meir, Marie-France Hamou, C.
Jayet, B. Ess, Gian Francesco Lorusso, Jose Redondo, Delio Mercanti and Maria Teresa
Ciotti for rat treatments and sample preparation, Didier Perret and Benjamin Gilbert for
performing the ICP-AES experiments. We also thank Mario Capozi, Sandro Rinaldi, and
Microchemical Analysis of Boron in Rat Brain Tumor
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Mary Severson, Tom Nelson and the entire staff of the Wisconsin Synchrotron Radiation
Center (a national facility supported by the NSF) for their expert professional help. We are
grateful to Borje Larsson for his encouragement and advise.
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