Biol Cell (1992) 74, 105-108
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© Elsevier, Paris
Original article
Intraceilular boron localization and uptake in cell cultures using imaging secondary ion mass spectrometry (ion microscopy) for neutron capture therapy for cancer Brian D Bennett, Xiaohui Zha, Isabelle Gay, George H. Morrison * Department of Chemistry, Cornell University, Ithaca, N Y 14853, USA (Received and accepted 6 November 1991)
Summary - Quantitative ion microscopy of freeze-fractured, freeze-dried cultured cells is a technique for single cell and subcellular elemental analysis [1 - 3]. This review describes the technique and its usefulnessin determining the uptake and subcellular distribution of the boron from boron neutron capture therapy drugs. SIMS microscopy / boron neutron capture therapy drugs / subcellular
Introduction Boron neutron capture therapy (BNCT) is a cancer treatment that combines selective uptake of boronated drugs into tumor tissue and destruction of that tissue by neutron irradiation [5]. Capture of neutron radiation by l°B gives rise to destructive fission products via the I°B(n,~)TLi reaction. We have applied quantitative ion microscopy to determine the uptake and intracellular distribution of the BNCT drugs CBU-2' (2'-O-(carboran-l-ylmethyl) uridine) and p-boronophenylalanine (BPA). Nucleosides, such as CBU-2', have been developed by BNCT chemists for incorporation into the nuclear material of rapidly proliferating ceils. Selective tumor incorporation is based on a relatively high growth rate compared to normal tissue. High concentration of BNCT drugs in the nucleus is desirable, since the killing effect of the I°B(n,~t)7Li reaction is enhanced two-fold when this localization is achieved. Ion microscopy of cultured cells can readily determine if a boronated nucleoside meets this criterium. The use of BPA is based on its structural similarity to the melanin precursor, tyrosine. As such, BPA is directed against malignant melanocytes, or melanoma. We have tested BPA for its ability to selectively concentrate into the cytoplasmically located melanosome, which is the organelle where melanin is produced. Ion microscopy, with its ability to quantitatively locate boron intracellular deposition at a spatial resolution of 1 ~m, is uniquely suited for testing the design of newly developed BNCT drugs.
Quantitative ion microscopy OM) of cell monolayers Chandra and Morrison recently reviewed our approach to ion microscopy in biology and medicine [4]. We have seen * Correspondence and reprints
that cell cultures prepared by cryo methods result in cells that present the least problems to the ion microscopist when considering artifacttaal ion intensity data. With this in mind, we will briefly describe how freeze-fractured cells can be imaged for quantitative subcellular elemental determinations.
Cryofixation Cryofixation is used to preserve the native intracellular distribution of highly diffusible species. A cell monolayer, grown on a polished silicon wafer, is ~andwiched by placing another wafer on top. Cryofixation is achieved by plunging the sandwich into a freon-22 slush formed at liquid nitrogen temperatures. The sandwich is maintained at liquid nitrogen temperature and opened. This freezefracture produces uncontaminated intracellular space by removing the apical cell surface and adhering growth medium. Often a confluent group of uncontaminated cells are obtained for ion microscopy.
Quantitation Sputter yield and practical ion yield do not vary between the subcellular compartments that are resolvable with the C~/meca 3f. Therefore, correction for local matrix effects are unnecessary [2]. B, K, Na, Ca, and Mg concentrations are determined by an empirical quantitation scheme based on a relative sensitivity factor (RSF) using the matrix element carbon as the reference element [1]. Carbon is used as the reference element since it is homogeneously distributed inside the cell. Calibration standards consisting of spiked homogenated cells are analyzed by ion microscopy and inductively coupled plasma atomic emission spectrometry (ICP-AES). The RSF, given in equation 1, is a correlation between the ion microprobe analyte to carbon ratio (ix~iref) and the analyte concentration (Cx) as determined by inductively coupled plasma atomic emission spectrometric analysis.
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RSFx/re r =
(i#Gf) -
CCxCO -
: w h e r e f~ = isotopic abundance (1)
The RSF's are obtained in microprobe mode using electron multiplier (EM) detection. Quantitative ion microscopic imaging with the CCD camera requires calibration that relates CDD pixel values to EM count rates for each secondary ion of interest. Here, spiked gelatin films are used as calibration standards that provide laterally homogeneous secondary ion micrographs. Once the IM imaging system is calibrated, the RSF's can be used to determine the element concentration at the subcellular level.
Application of ion microscopy to boron microanalysis of BNCT drugs Boron neutron capture therapy is a two component cancer treatment that combines mB and thermal neutrons. l°B is selectively targeted at tumor tissue and upon irradiation with thermal neutrons generates lethal fission products from the l°B(n,~)TLi reaction: l°B + nth ~ [liB]---' 4He + 7Li + 2.79 Mev (6070) 4He + 7Li + 0.48 MeV ~- + 2.31 Mev (94070) Selective irradiation of tumor combined with selective uptake of l°B should yield high LET radiation leading to specific destruction of tumor cells without significant damage to normal tissue [5]. CBU-2" CBU-2' (2'-O-(carboran-l-yl-methyl) uridine) (fig 1) is a boronated nucleoside designed to cross the plasma membrane rapidly, become trapped within the cell after transformation to the nucleotide, and then incorporate into the DNA of rapidly proliferating tumor cells [5, 6]. The BNCT killing effect is increased two-fold if a nuclear localization is obtained. The ion micrograph in figure 2 shows that the boron from CBU-2' localizes in the cytoplasm of F-98 glioma cells. The cytoplasmic compartment contains about 63°70 of the total intracellular boron. Quantitative comparison
Fig 2. F98 glioma cells treated with 50 ~g/ml 2'-0-(0carboran-l-yl-methyl) uridine (CBU-2') for 6 h. Top = calcium, bottom = boron. The calcium ion image shows a physiologically normal cytoplasmic localization. The nucleus is easily distinguished by the low calcium content. Boron from CBU-2' concentrates in the cytoplasm preferentially. Bar = 20 tzm.
O
HO
I°i
HO o - -
CH ]~10H10
Fig 1. CBU-2' (2'-0-(carboran-l-yl-methyl) uridine).
of nuclear and cytoplasm reveals that concentrations high enough for effective BNCT exist in both compartments. Boron levels in F-98 glioma cells after treatment with 50 ~zg/ml CBU-2' for 6 h are 109 p p m and 195 p p m for the nuclear and cytoplasmic compartments, respectively. The cytoplasmic localization of this nucleoside is consistent with incorporation into RNA. But one must note that the nuclear concentration is significant, thus there may be incorporation into both DNA and RNA. CBU-2' incorporation into nucleotides remains unproven. We intend to determine if CBU-2' uptake is dependent on RNA synthesis utilizing actinomycin D, which is a specific inhibitor of RNA synthesis. A variety of nucleosides, nucleotides, and oligonucleotides are becoming available for testing [7]. Ion microscopy can readily determine if any of these experimental nucleic acid precursors localize within the cell nucleus.
Intracellular boron localization by ion microscopy
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Fractionation study of melanoma cells treated with B P A in vitro show that most o f the b o r o n is in the soluble frac-
tion and that the level in the m e l a n o s o m e fraction is higher than any other particulate fraction [10]. Thus, b o r o n from B P A m a y be incorporated into melanin. But, this incorporation is an insignificant portion o f the total intracellular b o r o n and p r o b a b l y is not the cause o f selective uptake. Coderrer et al [8] have shown that b o r o n (from BPA) selectively concentrates in m e l a n o m a in vivo, but is rapidly eliminated. Thus, incorporation into melanin is unlikely. Coderre et al [8] propose that selective B P A uptake into melanoma is due to increased amino acid transport relative to normal tissue. Ion microscopic analysis shows that b o r o n f r o m B P A does not localize in the cytoplasm o f m e l a n o m a cells, but has a h o m o g e n e o u s distribution (fig 3). Indeed, b o r o n from B P A does not concentrate into the cytoplasm the site o f the melanosomal matrix. If incorporation into melanin occurs, it is at a low level and thus cannot account for the m e l a n o m a uptake mechanism seen in vivo. Unfortunately, we do not know the catabolic fate of BPA. Thus, the distribution o f b o r o n may not adequately describe the fate o f BPA. However, ion microscopic observation reveals what killing effect can be expected in BNCT, since the true b o r o n c o n c e n t r a t i o n and localization is determined.
Acknowledgments
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This work was supported by DOE DE-FG02-91ER61138 grant to GHM and a NSF Graduate Fellowship to IG. The Cornell NIH/NSF Developmental Resource for Biophysical Imaging and Optoelectronics was used for cell culture work. CBU-2' was a kind gift from Rolf F Barth, Albert H Soloway, and Abul K Anisuzzaman from Ohio State University.
• References
Fig 3. [NIe4,D-PheT]-~-MSH induced M-3 malignant melanoma cells treated with 100 ~zg/ml L-BPA for 6 h. Melanogenesis was induced in a culture of the M-3 clone of Cloudman $91 mouse malignant melanoma (ATCC CCL.53.1) cells with 6 × 10 -5 M [NIe4,D-PheT]-~-MSH [I 1] for 18 h [12]. The hormone containing medium was removed and replaced with BPA treatment medium. Top = calcium, bottom = boron. Three cells are seen, which demonstrate relatively low calcium in the nucleus. The corresponding ~°B ion image shows a homogeneous intracellular distribution. Bar = 10/~m.
BPA
p - B o r o n o p h e n y l a l a n i n e (BPA) is taken up selectively into malignant m e l a n o m a in vivo [8]. The mechanism o f this selective uptake has not been fully elucidated. B P A is t h o u g h t to be a false precursor o f melanin pigment, since it is an analog o f tyrosine, a true substrate [5]. If B P A is an effective melanin precursor it should be incorporated into m e l a n o m a cells that are actively synthesizing melanin. Further, this incorporation should be localized in the melanosome, the site o f melanin biosynthesis [9].
1 Ausserer WA, Ling YC, Chandra S, Morrison GH (1989) Quantitative imaging of boron, calcium, magnesium, potassium, and sodium distributions in cultured cells with ion microscopy. Anal Chem 61, 2690-2695 2 Chandra S, Ausserer WA, Morrison GH (1987) Evaluation of matrix effects in ion microscopic analysis of freezefractured, freeze-dried cultured cells. J Microsc (Oxf) 148, 223-239 3 Ausserer WA, Chandra S, Morrison GH (1989) Morphological and elemental integrity of freeze-fractured, freeze-dried cultured cells during ion microscopic analysis. J Microsc (Oxf) 154, 39-57 4 Chandra S and Morrison GH (1988) Ion microscopy in biology and medicine. In: Methods in Enzymology 158, 157-179 5 Barth RF, Soloway AH, Fairchild RG (1990) Boron neutron capture therapy of cancer. Cancer Res 50, 1061 - 1070 6 Soloway AH, Anisuzzman AKM, Liu L, Alam F, Barth RF (1991) Carboranyl precursors o f nucleic acids-potential DNA probes for BNCT. In: Neutron Capture Therapy f o r Cancer (Allen BJ, ed) Plenum Publishing Corp, NY, NY, (in press) 7 Neutron Capture Therapy f o r Cancer (Allen BJ, ed) Plenum Publishing Corp, NY, NY, (in press) 8 Coderre JA, Glass JD, Fairchild RG, Roy U, Cohen S, Fand I (1987) Selective targeting of boronophenylalanine to melanoma in BALB/c mice for neutron capture therapy. Cancer Res 47, 6377-6383 9 Seiji M, Shimao K, Birbeck MSC, Fitzpatrick TB Subcellular localization of melanin biosythesis. (1963) Ann N Y A c a d Sci 100, 497-533
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10 Tsuji M, Ichihashi M, Mishima Y (1983) Selective affinity of ~째B-para-boronophenylalanine-HCL to malignant melanoma: for thermal neutron capture therapy. Jpn J Dermatol 93,773-778 11 Sawyer TK, Sanfilippo P J, Hruby V J, Engel MH, Heward CB, Burnett JB, Hadley ME (1980) 4-Norleucine,7-D-
phenylalanine-a-melanocyte-stimulating hormone: A highly potent ~-melanotropin with ultralong biological activity. Proc Natl Acad Sci USA 77, 5754-5758 12 Pawlek J, Wong G, Sansone M, Morowitz J (1973) Molecular controls in mammalian pigmentation. Yale J Biol Med 46, 430-443