Biol Cell (1992) 74, 105-108
105
© 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.