Duane R. Smith

Peer Reviewed: A Subcellular Imaging by Dynamic SIMS Ion Microscopy.

By imaging isotopes, the transport of ions, molecules, and therapeutic drugs can be studies in single cells.

Boron-Containing Nucleosides as Potential Delivery Agents for Neutron Capture Therapy of Brain Tumors

The purpose of the present study was to evaluate both in vitro and in vivo a series of boron-containing nucleosides that potentially could be used as delivery agents for neutron capture therapy. The rationale for their synthesis was based on the fact that proliferating neoplastic cells have increased requirements for nucleic acid precursors, and, therefore, they should preferentially localize in the tumor. A series of 3-carboranlyalkyl thymidine analogs has been synthesized and a subset, designated N4, N5, and N7, and the corresponding 3-dihydroxypropyl derivatives, designated N4–2OH, N5–2OH, and N7–2OH, have been selected for evaluation. Using these compounds as substrates for recombinant human thymidine kinase-1 and the mitochondrial isoenzyme thymidine kinase-2, the highest phosphorylation levels relative to thymidine were seen with N5 and the corresponding dihydroxypropyl analog N5–2OH. In contrast, N4, N4-OH, N7, and N7-OH had substantially lower phosphorylation levels. To compare compounds with high and low thymidine kinase-1 substrate activity, N5 and N7 and the corresponding dihydroxypropyl derivatives were selected for evaluation of their cellular toxicity, uptake and retention by the F98 rat glioma, human MRA melanoma, and murine L929 cell lines, all of which are thymidine kinase-1(+), and a mutant L929 cell line that is thymidine kinase-1(−). N5–2OH was the least toxic (IC50, 43–70 μm), and N7 and N7–2OH were the most toxic (IC50, 18–49 μm). The highest boron uptake was seen with N7–2OH by the MRA 27 melanoma and L929 wild-type (wt) cell lines. The highest retention was seen with L929 (wt) cells, and this ranged from 29% for N5–2OH to 46% for N7. Based on the in vitro toxicity and uptake data, N5–2OH was selected for in vivo biodistribution studies either in rats bearing intracerebral implants of the F98 glioma or in mice bearing either s.c. or intracerebral implants of L929 (wt) tumors. At 2.5 hours after convection-enhanced delivery, the boron values for the F98 glioma and normal brain were 16.2 ± 2.3 and 2.2 μg/g, respectively, and the tumor to brain ratio was 8.5. Boron values at 4 hours after convection-enhanced delivery of N5–2OH to mice bearing intracerebral implants of L929 (wt) or L929 thymidine kinase-1(−) tumors were 39.8 ± 10.8 and 12.4 ± 1.6 μg/g, respectively, and the corresponding normal brain values were 4.4 and 1.6 μg/g, thereby indicating that there was selective retention by the thymidine kinase-1(+) tumors. Based on these favorable in vitro and in vivo data, neutron capture therapy studies will be initiated using N5–2OH in combination with two non-cell cycle dependent boron delivery agents, boronophenylalanine and sodium borocaptate.

Quantitative Subcellular Secondary Ion Mass Spectrometry (SIMS) Imaging of Boron-10 and Boron-11 Isotopes in the Same Cell Delivered by Two Combined BNCT Drugs: In Vitro Studies on Human Glioblastoma T98G Cells

Abstract Chandra, S., Lorey, D. R., II and Smith, D. R. Quantitative Subcellular Secondary Ion Mass Spectrometry (SIMS) Imaging of Boron-10 and Boron-11 Isotopes in the Same Cell Delivered by Two Combined BNCT Drugs: In Vitro Studies on Human Glioblastoma T98G Cells. Radiat. Res. 157, 700–710 (2002). Ion microscopy was used for subcellular quantitative imaging of the isotopes 10B and 11B in the same cell to evaluate boron delivery using a mixture of two neutron capture therapy drugs, p-boronophenylalanine-fructose (BPA-F) and sodium borocaptate (BSH). The application of 10B-labeled BPA-F and 11B-labeled BSH allowed independent imaging of both 10B and 11B in the same cell using a CAMECA IMS-3f ion microscope. Mixed-drug treatments were compared to single-drug exposures given under identical conditions. 10BPA-F delivered 10B heterogeneously to T98G human glioblastoma cells, with a significantly reduced concentration in an organelle-rich perinuclear region. The intracellular distribution of 11B from 11BSH contrasted with that of the 10B from 10BPA-F, with 11B distributed nearly homogeneously throughout cells. The subcellular distributions of 10B and 11B were sustained in mixed-drug treatments and resembled their localizations after the single-drug treatments. In both single- and mixed-drug treatments, cellular levels of 10B from 10BPA-F nearly doubled between 1 h and 6 h, with a 3:1 intracellular to nutrient medium partitioning, while cellular levels of 11BSH remained essentially unchanged. The net effect of the combined treatment with 10BPA-F and 11BSH was an additive delivery of boron to cells. This study introduces a novel approach for checking potential synergistic, antagonistic or simple additive delivery of two mixed boronated compounds in cellular/subcellular compartments.

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,α)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.

Pulsed laser assisted epitaxy of GexSi1−x alloys on Si 〈100〉

The epitaxial quality of GexSi1−x films grown on Si〈100〉 by a novel laser‐assisted technique has been investigated for compositions in the range x≤0.37 and thicknesses between 50 and 500 nm. Epitaxy is induced during electron beam deposition of GexSi1−x films on Si substrates by pulses of an excimer laser operating at 308 nm with 30 ns pulse duration. Good epitaxial growth is obtained for 50 nm films for x≤0.04 on chemically cleaned Si(100) surfaces even in the presence of substantial fluorine coverage. Using a predeposition/crystallization of a 50 nm pure‐Si buffer layer, good epitaxy is then obtained in 50 nm films for x=0.05–0.13. At x=0.07, defect‐free alloys have been grown up to thicknesses of 0.5 μm. However, for films with compositions above 19 at.% Ge, dislocations at a nearly constant density of ≊10 μm/μm2 are observed. This uniform density suggests a breakdown mechanism other than simple strain relaxation.

A Rat Model of Oral Mucosal Response to Boron Neutron Capture Irradiation

Oral mucositis is a frequent side effect during conventional radiotherapy for advanced head and neck tumours. While acute radiation damage to the oral mucosa is not a problem for the majority of glioma patients involved in the current boron neutron capture therapy (BNCT) clinical trial at BNL, a subset of patients with tumours located towards the front of the brain are presenting with transient acute effects in the oral mucosa. This suggests that the oral mucosa could become a potential dose limiting tissue in future treatment protocols involving higher doses and larger irradiation field sizes. The objective of the present study was to evaluate dose-effect relationships and estimate relative biological effectiveness (RBE) and compound biological effectiveness (CBE) factors for this critical normal tissue. The model selected for study was rat ventral tongue mucosa. Ventral tongue offers the most readily accessible area of representative multi-layered oral epithelium. It can be locally irradiated (and readily scored), greatly reducing the radiation burden to the head.

Ion Microscopy Imaging of Boron from p-Boronophenylalanine in Surgically Acquired Samples of Human Brain Tumor Tissue

Using ion microscopy1 we are able to quantitatively image physiologically relevant isotopes, including 10B, in cancerous and contiguous normal tissues with cellular to subcellular resolution and with detection limits in the low-ppm to ppb range. Over the past several years we have been using this technique to analyze tissue sections from animal brain tumor models,1, 2, 3 as well as specimens prepared from tissue cultures,4,5 to develop appropriate cryogenic sampling procedures to be applied to the in situ analyses of 10B in human brain tumor specimens. An ion microscopy imaging study is presented which demonstrates the localization of 10B from the fructose complex of p-boronophenylalanine (BPA-F) in tumor cells from a patient with glioblastoma multi-forme. Patient A was infused intravenously for one hour to deliver 170mg BPA/kg body weight. The craniotomy commenced one-half hour after the end of the infusion. Tissue specimens from patient A were frozen on dry ice, stored at -80 °C, and at a later date cryosectioned and freeze-dried for ion microscopy analysis. The boron concentration in tumor cells, calculated from the grayscale intensity of ion images, was found to have a dynamic distribution with an average approximately 3.5 times higher than the 10B levels in the normal brain and blood. Ion images are shown to illustrate the complexities of cryogenic sample preparation for the analysis of 10B in human brain tumor tissues at the microscopic level.

Ion Microscopy Imaging of Boron in Animal Tissue Sections for Neutron Capture Therapy

Ion microscopy imaging1 of animal tissue sections enables us to evaluate the in situ distribution of physiologically relevant elements, including the isotope 10B, with cellular to subcellular resolution and with detection limits ranging from the low-ppm to ppb levels. An overview of three ion microscopy imaging studies is presented. The first two address the issue of the localization of 10B from BPA-F in the mucosal and glandular tissues associated with the oral cavity. Of concern is the possibility that BNCT-related radiation damage in soft tissues of the head and neck may be dose-limiting. The third study explores the time dependence of BPA-F boron delivery by intravenous (iv) infusion. This study was undertaken in direct response to our previous ion microscopy imaging studies1,2 which show a fixed, 2/1 partitioning of 10B from BPA-F between tumor cells in the main tumor mass and tumor cells invading the normal brain.

Abstract 4028: Analysis and recovery of functionally defined single immune cell clones through opto-electro-positioning technology

Cancer patients mount protective innate and adaptive immune responses against tumor cells. Such responses are able to control or even eliminate tumor burden if harnessed properly. Identifying clonal adaptive immune cells targeting tumor cells with designated function is extremely valuable as these cells are able to either fight the tumor directly or provide tumor targeting immune receptors to engineer other effector cells like autologous CD8 T cells or NK cells. The process of isolating cancer specific immune cells and their receptors, however, remains technically challenging and labor intensive. Opto-Electro-Positioning (OEP) technology employs a light actuated dielectrophoretic force to maneuver live cells in their culture environment. We have combined OEP with a nanofluidic cell culture system to produce an integrated microfluidic platform that enables multi-dimensional investigation of lymphocyte specificity and function. The system is able to 1) deterministically capture designated lymphocytes, 2) actively maintain and observe colony growth, and 3) conduct cytokine release assays, all in a single enclosed microfluidic chip. The new platform is suitable for high throughput screening to identify and harvest antigen specific and/or functionally defined T cells. We have successfully applied it to isolate primary human T cells targeting either EBV/Tetanus toxin antigens or tumor associated antigens such as MART-1 through tetramer staining or a proliferation assay. The system is also exceptionally efficient for investigating immune cells from clinical specimens typically of low cellularity. From tumor biopsies, we are able to capture tumor infiltrating T cells, stimulate proliferation, investigate their TNF-α secretion on chip, and obtain full transcriptomic analysis after export. Overall, our data suggest the OEP nano-fluidic environment provides a powerful high throughput screening tool to identify rare lymphocytes against tumor associate antigens or neo-antigens and to investigate precious clinical samples for biomarker discovery. Citation Format: Xiaohua Wang, Yelena Bronevetsky, Kristin Beaumont, Guido Stadler, Xiaoyan R. Bao, Duane Smith, Peter Beemiller, Kevin Chapman. Analysis and recovery of functionally defined single immune cell clones through opto-electro-positioning technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4028. doi:10.1158/1538-7445.AM2017-4028

In Vitro Exposure of Human T98G Glioblastoma Cells to Mixed BNCT Drugs

The selective targeting with sufficient quantities of 10B atoms to the tumor cells is critical for Boron Neutron Capture Therapy (BNCT) of cancer. At present there are two BNCT drugs, p-boronophenylalanine (BPA) and sodium borocaptate (BSH), approved for use in clinical trials. Since both BPA and BSH may deliver boron to tumor cells by different mechanisms, the concept of mixing the two compounds may have the merit of providing a unique approach for attaining a higher net accumulation of boron atoms in tumor cells than would otherwise be attained by an individual compound. Clinical applications of this mixed-drug approach would require that both drugs provide 10B atoms. For laboratory research purposes, however, one can use a mixture such as 10B labeled 10BPA and 11B labeled 11BSH and an isotopic imaging technique of ion microscopy for studying intracellular accumulations of both 10B and 11B in the same cell. Ion microscopy, based on secondary ion mass spectrometry (SIMS),1,2 is capable of quantitatively imaging the subcellular distribution of both 10B and 11B atoms delivered by two different BNCT agents in the same cell since the technique distinguishes isotopes based on their mass-to-charge (m/z) ratio. The exposure of cells to two BNCT compounds mixed together may result in the enhancement of boron uptake by tumor cells. In the present study, by using 10B labeled p-boronophenylalanine-fructose (10BPA-F) and 11B labeled sodium borocaptate (11BSH) as a mixed-drug model we were able to image the subcellular distribution of both 10B and 11B atoms independently in the same cell with ion microscopy. Such independent imaging of boron atoms from two mixed-drugs in the same cell introduces a novel approach for checking the boron delivery characteristic of two BNCT compounds combined together.