Dynamic SIMS ion microscopy imaging of intracellular boron accumulation from carboranyl nucleosides in glioma cells.

BACKGROUND: Selective accumulation of boron-10 isotope in the nuclei of cancer cells is pivotal to the success of Boron Neutron Capture Therapy (BNCT). Sophisticated microanalytical techniques are needed for checking the selectivity and boron delivery characteristics of experimental BNCT drugs. The present study employs a secondary ion mass spectrometry (SIMS) based subcellular isotopic imaging technique of ion microscopy for testing four carboranyl nucleosides. MATERIALS AND METHODS: CDU, HMCDU, CTU, and CFAU were tested for their boron delivery to the nuclear and cytoplasmic compartments of U251 human and F98 rat glioma cells. Quantitative SIMS analysis of boron was carried out in cryogenically prepared cells. RESULTS: For all drugs, the cell cytoplasm revealed significantly higher boron than the nucleus. However, the boron partitioning between the cell nucleus and the nutrient medium indicated 6.4-10.6 times higher boron in the nucleus. CONCLUSION: Carboranyl nucleosides studied here may provide efficient BNCT agents and need further evaluations of their efficacy.

Dynamic secondary ion mass spectrometry analysis of boron from boron neutron capture therapy drugs in co-cultures: single-cell imaging of two different cell types within the same ion microscopy field of imaging.

A co-culture, cryogenic SIMS methodology is presented for the quantitative analysis of cell type-dependent accumulation of boron delivered by BPA-F and BSH, two clinically approved drugs used in boron neutron capture therapy of cancer. T98G human glioblastoma cells were co-cultured with morphologically different normal LLC-PK1 epithelial cells or GM3348 human skin fibroblasts. Our freeze-fracture method of cryogenic sample preparation successfully fractured the different cell types grown together in co-cultures. Quantitative observations revealed an active uptake of boron from BPA-F in both T98G and LLC-PK1 cells but did not show cell type-dependent differences. Accumulation of BSH in all three cell types examined also did not reveal any cell type-dependent differences in co-cultures. As this method relies on the analysis, within the same field of SIMS imaging, of two different cell types that have been maintained under identical conditions of growth, drug exposure, sample preparation, and instrumental analysis, it provides the most effective approach for comparing cell type-specific differences in boron concentrations. The most effective applications of this method will be realized in testing the selectivity of experimental boronated compounds designed to specifically target tumor cells.

Synthesis and biological evaluation of boronated nucleosides for boron neutron capture therapy (BNCT) of cancer.

Several N-3 substituted carboranyl Thd analogs were synthesized. These agents as well as some non-boronated nucleosides were evaluated in phosphoryl transfer assays with recombinant human TK1 and TK2. For some carboranyl thymidine analogs, TK1 phosphorylation rates approached 38% that of thymidine. Their in vitro cytotoxicty appeared to correlate with the TK1 levels in the tested cells. In some cases increased uptake in tumor cell nuclei compared with the surrounding cytoplasm was detected in vitro.

Boron microlocalization in oral mucosal tissue: implications for boron neutron capture therapy.

Clinical studies of the treatment of glioma and cutaneous melanoma using boron neutron capture therapy (BNCT) are currently taking place in the USA, Europe and Japan. New BNCT clinical facilities are under construction in Finland, Sweden, England and California. The observation of transient acute effects in the oral mucosa of a number of glioma patients involved in the American clinical trials, suggests that radiation damage of the oral mucosa could be a potential complication in future BNCT clinical protocols, involving higher doses and larger irradiation field sizes. The present investigation is the first to use a high resolution surface analytical technique to relate the microdistribution of boron-10 (10B) in the oral mucosa to the biological effectiveness of the 10B(n,alpha)7Li neutron capture reaction in this tissue. The two boron delivery agents used clinically in Europe/Japan and the USA, borocaptate sodium (BSH) and p-boronophenylalanine (BPA), respectively, were evaluated using a rat ventral tongue model. 10B concentrations in various regions of the tongue mucosa were estimated using ion microscopy. In the epithelium, levels of 10B were appreciably lower after the administration of BSH than was the case after BPA. The epithelium:blood 10B partition ratios were 0.2:1 and 1:1 for BSH and BPA respectively. The 10B content of the lamina propria was higher than that measured in the epithelium for both BSH and BPA. The difference was most marked for BSH, where 10B levels were a factor of six higher in the lamina propria than in the epithelium. The concentration of 10B was also measured in blood vessel walls where relatively low levels of accumulation of BSH, as compared with BPA, was demonstrated in blood vessel endothelial cells and muscle. Vessel wall:blood 10B partition ratios were 0.3:1 and 0.9:1 for BSH and BPA respectively. Evaluation of tongue mucosal response (ulceration) to BNC irradiation indicated a considerably reduced radiation sensitivity using BSH as the boron delivery agent relative to BPA. The compound biological effectiveness (CBE) factor for BSH was estimated at 0.29 +/- 0.02. This compares with a previously published CBE factor for BPA of 4.87 +/- 0.16. It was concluded that variations in the microdistribution profile of 10B, using the two boron delivery agents, had a significant effect on the response of oral mucosa to BNC irradiation. From a clinical perspective, based on the findings of the present study, it is probable that potential radiation-induced oral mucositis will be restricted to BNCT protocols involving BPA. However, a thorough high resolution analysis of 10B microdistribution in human oral mucosal tissue, using a technique such as ion microscopy, is a prerequisite for the use of experimentally derived CBE factors in clinical BNCT.

Imaging of total calcium in urediospore germlings of Uromyces by ion microscopy.

Calcium has been implicated in growth and appressorium formation of urediospore germlings of the bean rust fungus, Uromyces appendiculatus. Using ion microscopy, a mass spectrometry-based imaging technique, intracellular stores of calcium were analyzed by direct imaging of total calcium in frozen freeze-dried germlings. Calcium concentration was calculated by ratioing and spatially registering (40)Ca to (12)C signals. Intracellular distributions of total potassium, sodium, magnesium, and carbon were similarly imaged in the same germlings for a direct comparison of their localizations to total calcium. Calcium was remarkably heterogeneous with highest concentrations (2 to 10 mM) in the mid-region of the germling between the nuclei and the apex. A similar distribution of Ca(2+) (assessed using Fluo-3) was also noted sequestered in organelles in live germlings. Distributions of remaining elements (K, Na, Mg, and C) were mostly homogeneous throughout the cytoplasm and nuclei of the fungal cell. The K/Na ratio ranged from 17 to 31.

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies.

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK1 cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144. 15-37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K+, Na+ and Ca2+). Structurally damaged cells revealed lower K+ and higher Na+ and Ca2+ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.

Evaluation of 26Mg stable isotope as an in vivo tracer of magnesium transport for SIMS ion microscopy imaging studies.

Isotopic detection with high sensitivity, one of the most important features of ion microscopy, allows the in vivo application of stable isotopes as tracers for unravelling smaller tissue structures implicated with transport capabilities. The evaluation of the mass interferences associated with a particular mass of secondary ion signals is a necessity for tracer studies with stable isotopes. We have tested the feasibility of 26Mg stable isotope as a tracer of magnesium transport in the killifish. The fish were given a single intraperitoneal injection of 3 mumol 26MgCl2.6H2O (99.5% 26Mg enrichment), and the renal distribution of 26Mg was examined in frozen freeze-dried cryosections with ion microscopy. High-mass resolution analyses were performed to evaluate the purity of positive secondary ion signals of the nominal masses 24, 25 and 26 in order to assess the purity of 24Mg, 25Mg and 26Mg signals, respectively, in kidneys of control and 26Mg-injected fish. In kidneys of control fish, the purities of 24Mg, 25Mg and 26Mg signals were approximately 97%, 82% and 90%, respectively. In fish that were injected with 26Mg stable isotope, an enhancement of 26Mg+ secondary ion signals was observed with signal purity reaching 95%. These observations indicate that 26Mg can be used successfully as a tracer of magnesium transport in animal models. To uncover the distribution of tracer 26Mg from the naturally abundant background of this isotope, a pixel-by-pixel digital subtraction is applied to the raw ion microscopy mass 26 image.

Identification of Mg-transporting renal tubules and cells by ion microscopy imaging of stable isotopes.

Sites of renal Mg transport were identified in seawater killifish (Fundulus heteroclitus) using a Cameca model IMS-3f ion microscope. Killifish were given an intraperitoneal injection of the stable isotope 26Mg (99.5% enrichment) to stimulate and trace renal Mg excretion. We identified two sites of 26Mg transport in frozen freeze-dried cryosections of kidney: the proximal tubule, known to secrete Mg, and the collecting duct, heretofore not known to handle Mg. In epithelial cells of the proximal tubule, the punctate distribution of injected 26Mg suggests transcytotic excretion of Mg in bound form. In collecting ducts, a subpopulation of Mg/Ca-rich cells was identified with high accumulations of injected 26Mg. Here, the punctate distribution of 26Mg decreased from the apical to the basal region of the cells, revealing a transcytotic gradient of apparently bound Mg. Since proximal tubules of fish are implicated with Mg secretion, Mg/Ca-rich cells in the collecting duct may reabsorb Mg, thereby providing the usual two-step of renal regulation, now also for Mg.

Ion microscopy imaging of 10B from p-boronophenylalanine in a brain tumor model for boron neutron capture therapy.

Boron neutron capture therapy (BNCT), a binary treatment modality that can potentially irradiate tumor tissue within cellular dimensions, is critically dependent on the preferential delivery of 10B to individual neoplastic cells. In this study, ion microscopy was used to quantitatively evaluate the selectivity of p-boronophenylalanine-fructose (BPA-F) in the rat 9L gliosarcoma brain tumor model. With a spatial resolution of approximately 0.5 microm, ion microscopy images show that BPA-F delivers 3.5 times more boron to the main tumor mass [99 +/- 36 microg/g tissue (mean +/- SD)] than to the contiguous normal brain (27 +/- 12 microg/g tissue). A similar, but lower, accumulation was observed away from the main tumor mass in small clusters of neoplastic cells (47 +/- 15 microg/g tissue) invading the surrounding brain (16 +/- 8 microg/g tissue). These findings establish for the first time the selectivity of BPA-F to the neoplastic cells invading the normal brain and provide a much-needed baseline measurement of the distribution of a clinically approved BNCT drug. Given the propensity for malignant brain tumors to infiltrate the surrounding normal brain, these observations have particular significance for clinical trials of BNCT for human glioblastoma multiforme using the drug BPA-F.

Ion microscopic imaging of calcium during 1,25-dihydroxyvitamin D-mediated intestinal absorption.

A combination of ion microscopic and conventional radionuclide techniques was employed to investigate the temporal-spatial dynamics of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated intestinal calcium (Ca) absorption. At varying times following the administration of a single intravenous dose of 1,25(OH)2D3 to vitamin D-deficient chicks, transepithelial transport and tissue retention of Ca were quantitated in vivo, using the ligated duodenal loop technique and 47Ca as the tracer. The localization of Ca in the intestinal tissue during absorption was monitored by ion microscopy, using the stable Ca isotope, 44Ca, as the absorbed species. There was little transepithelial absorption of Ca in the vitamin D-deficient animals despite a substantial tissue accumulation of luminally derived Ca, the latter localizing predominantly in the brush border region of the enterocyte, as shown by the 44Ca-ion microscopic images. The early (30 min-1 h) response to 1,25(OH)2D3 was an increased tissue uptake of luminal 47Ca, which also primarily associated with the brush border region, again as shown by ion microscopy. At 2-4 h after the 1,25(OH)2D3 dose, there was a progressive redistribution of Ca from the brush border region throughout the cytoplasm and into the lamina propria. At 8-16 h, 47Ca absorption was maximal and 44Ca was sparsely distributed in the intestinal tissue. 47Ca absorption gradually declined and reached pre-dose levels by 72 h. At this time, tissue 44Ca was again largely limited to the brush border region. These results provide support for the multiple actions of 1,25(OH)2D3 on the intestinal Ca absorption process. The ion microscopic images provided unique information on the specific time-dependent changes in the tissue localization of Ca during the process of its intestinal absorption as affected by 1,25(OH)2D3.

Ion microscopy evidence that La3+ releases Ca2+ from Golgi complex in LLC-PK1 cells.

The effect of La3+ on LLC-PK1 cells was investigated by ion microscopy, a mass spectrometry-based technique with a spatial resolution of approximately 0.5 micron. Cells were incubated with LaCl3 for 10 min. (1 mM) or 30 min (0.1 mM), and intracellular calcium distributions were measured with a Cameca IMS-3f ion microscope in cryogenically prepared cells. Compared with control cells, La3+ reduced total calcium in the Golgi complex by > 100 microM in both treatments, whereas other cellular regions, such as the nucleus and cytoplasm, remained largely unchanged. These two treatments were repeated on cells that were preincubated with 1 mM ouabain. The presence of ouabain in the medium increased the loss of calcium from the Golgi by about fourfold compared with the treatments without ouabain. The La3+ effect, therefore, was amplified by ouabain-induced Na+ loading, indicating a possible involvement of a Na+/La3+ exchanger. La3+ was detected within cells and its influx was facilitated by Na+ loading. These results suggest that La3+ may affect cellular calcium homeostasis by actions other than as a simple Ca2+ antagonist.

Golgi apparatus is involved in intracellular Ca2+ regulation in epithelial LLC-PK1 cells.

Several lines of evidence suggest that the Golgi apparatus is involved in Ca2+ regulation in renal epithelial LLC-PK1 cells. Laser scanning confocal microscopy (LSCM) was employed to establish that a prominent perinuclear region is occupied mainly by the Golgi apparatus in this cell line. LSCM measurements in individual cells with the ionized Ca2+ indicator calcium green revealed that stimulation of LLC-PK1 cells with arginine vasopressin (AVP) resulted in the elevation of ionized Ca2+ levels. However, the vasopressin-induced rise in ionized Ca2+ was attenuated if the Golgi apparatus was disassembled by pretreating the cells with brefeldin A (BFA). Subcellular measurements of total Ca2+ with ion microscopy in cryogenically prepared cells indicated that 1) within 1 min of AVP treatment significant quantities of sequestered Ca2+ were released from the perinuclear Golgi region and 2) the BFA treatment reduced the total Ca2+ stored in the Golgi region. These observations indicate that the Golgi apparatus is sensitive to hormonal stimulation and may play important roles in intracellular Ca2+ regulation in LLC-PK1 cells.

Parameter control, characterization, and optimization in the fabrication of optical fiber near-field probes.

Tip diameter and transmission efficiency of a visible-wavelength near-field optic probe determine both the lateral spatial resolution and experimental utility of the near-field scanning optical microscope. The commonly used tip fabrication technique, laser-heated pulling of fused-silica optical fiber followed by aperture formation through aluminization, is a complex process governed by a large number of parameters. An extensive study of the pulling parameter space has revealed a time-dependent functionality between the various pulling parameters dominated by a photon-based heating mechanism. The photon-based heat source results in a temperature and viscosity dependence that is a complex function of time and fiber diameter. Changing the taper of the optical probe can affect transmission efficiency by an order of magnitude or more.

Subcellular localization of p-boronophenylalanine-delivered boron-10 in the rat 9L gliosarcoma: cryogenic preparation in vitro and in vivo.

A well-characterized in vitro cryogenic preparation for ion microscopic isotope imaging, which minimizes redistribution of diffusible species, was used to determine the distribution of boron in GS-9L gliosarcoma cells incubated with the boron neutron capture therapy agent, p-boronophenylalanine (BPA). At the subcellular level, boron from BPA distributes relatively homogeneously within the glioma cell. Boron from BPA was eliminated rapidly, indicating that most is unbound. Thus a large pool of boron is susceptible to diffusion artifact. Removal of this artifact increases the degree of confidence in microdosimetric results inferred from the homogeneous subcellular distribution. The ion microscopic imaging of boron in subcutaneous tumors cryofixed in situ was achieved in rats treated with BPA. Boron signals from BPA were adequate to image microdistributions at the 1-micron resolution level. As in the in vitro case, boron did not localize discretely at the subcellular level. However, boron heterogeneity was seen at the tissue level. Physiologically valid cellular potassium and sodium levels were seen, which demonstrates minimized redistribution artifact. Future tissue studies designed to correlate ion microscopic boron images to microscopic structure are feasible using cryogenic sample preparation and ion microscopy.

Imaging of total intracellular calcium and calcium influx and efflux in individual resting and stimulated tumor mast cells using ion microscopy.

Ion microscopy was employed to investigate intracellular total calcium concentrations and calcium influx, and efflux in resting and antigen-stimulated tumor mast cells (RBL-2H3 cells). The nucleus, a perinuclear region which included the Golgi apparatus (Golgi region), and the remaining cytoplasm were spatially resolved with the Cameca IMS-3f ion microscope in cryogenically prepared cells. In resting cells the nucleus contained about 0.60 mM, the Golgi region about 1.2 mM, and the remaining cytoplasm about 1.0 mM total calcium. Antigen stimulation of rat basophilic leukemia cells resulted in a significant loading of calcium in all three cellular compartments. Antigen stimulation in the absence of extracellular calcium resulted in a significant loss of total calcium from all three intracellular compartments. Influx and efflux of calcium were measured simultaneously in resting and stimulated cells by using stable 44Ca in the extracellular solution, and by imaging mass 40 to determine the native intracellular calcium (40Ca) and mass 44 to localize the 44Ca that entered the cell from extracellular solution. After a 10-min incubation, 0.240 fmol of the total calcium per cell had been replaced with 44Ca, which amounts to about 33% of the total cell calcium. If antigen was present during this incubation there was an additional loss of 0.229 fmol of 40Ca and an added gain of 0.476 fmol of 44Ca per cell, which corresponds to a net increase in total intracellular calcium of 0.247 fmol.

Ion microscopy in biology.

Ion microscopy, a mass spectrometry based isotopic imaging technique, is uniquely suited for ion transport related problems in biological systems. Due to its high sensitivity, it can image the transport and distribution of both major and minor elements (isotopes) at subcellular resolutions. The images of major elements such as K, Na, CI, etc., can be viewed directly and recorded in real-time from the microchannel plate-fluorescent screen detector of the instrument. The low concentration physiologically important elements, such as Ca, need about one minute of integration for good quality imaging. The isotopic imaging capability of ion microscopy provides a unique approach for the use of stable isotopes as tracers. In this way, one can image both the endogenous and the transported isotopes independently. Strict cryogenic sample preparations are essential for ion transport studies. Correlative imaging of the same cell with laser scanning confocal microscopy and ion microscopy can positively identify smaller cytoplasmic compartments such as the Golgi apparatus in calcium images. We have identified the Golgi apparatus as a calcium storing organelle. Another unique application of ion microscopy is the imaging of boron from boronated drugs used in Boron Neutron Capture Therapy (BNCT) of cancer. Ion microscopy is capable of rapid screening of potential drugs for BNCT. This critical information is essential for the fundamental understanding of BNCT. Ion microscopy is now at the stage where it can provide previously unattainable answers to important biomedical questions.

Quantitative imaging of a radiotherapeutic drug, Na2B12H11SH, at subcellular resolution in tissue cultures using ion microscopy.

The effectiveness of boron neutron capture therapy is predicted to be dependent not only on the amount of boron taken up by the target cells but also on the intracellular distribution of boron. Using the isotopic imaging technique ion microscopy, we have quantitatively determined uptake and intracellular distribution of Na2B12H11SH, a promising boron drug for boron neutron capture therapy, in four human cell lines: U87 glioblastoma cells, HeLa epithelioid carcinoma cells, GM 2408b mutant skin fibroblasts, and GM 3348b skin fibroblasts. The boron uptake of all four cell lines, after exposure to 100-500 micrograms/ml Na2B12H11SH, increased as the dosages were increased but showed a tendency toward saturation. Boron was more concentrated in the cytoplasm than in the nucleus but was not strongly localized within cells. There were no significant differences in boron uptake among the four cell lines. A retention experiment identified at least two different intracellular boron pools, and cells lost greater than 60% of intracellular boron within 1 h upon changing to Na2B12H11SH-free medium, indicating a largely low affinity binding.