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

Abstract

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.

Measure of carbon and nitrogen stable isotope ratios in cultured cells

“…The 13 C/ 12 C isotope ratios were obtained by two methods: By dividing the counts of 13 C 14 N Ϫ by the simultaneously recorded counts of 12 C 14 N Ϫ , and by dividing the counts for 13 C Ϫ by the simultaneously recorded counts for 12 C Ϫ . The precision of these ratios is essentially a function of the secondary ion currents for 12 C 15 N Ϫ , 13 C 14 N Ϫ , or 13 C Ϫ , which were all considerably larger than 1000 s Ϫ1 (3% precision). Data analysis was performed with an SGI O2 workstation using Prophet statistical software (NCRR, NIH).…”

“…Each dish contained a 5-mm ϫ 5-mm Si chip, to which cells readily attached (Montco Silicon Technologies, Inc., Spring City, PA; cut to size at Microsystems Technology Laboratories, MIT, Cambridge, MA). The cells were then grown to 80% confluence, and the chips with the attached cells were transferred to dishes with fresh, prewarmed media supplemented with 1 mM of either 13 C glycine (99%) or 15 N glycine (98%) (Cambridge Isotope Laboratories Inc., Andover, MA). No glycine was added to the control medium (DME/F12 ϩ 10% FBS).…”

“…Images of the distribution of 15 N and other isotopes have been provided by secondary ion mass spectrometry [3][4][5][6][7][8][9][10][11][12][13][14]. Although nitrogen alone does not produce a secondary ion, the secondary cyanide ion, CN Ϫ , primarily released from protein-rich regions of biological tissue, provides anatomically recognizable images.…”

“…The evaluation of enrichment in 15 N is based upon the measure of its increase above its natural, terrestrial abundance, calculated from the 15 N/ 14 N isotope ratios. Because it is necessary to measure nitrogen from the secondary ion CN Ϫ , the mass resolution must be sufficient to allow the separation of the mass 27 isobars, 12 C 15 N and 13 C 14 N. Hindie et al [15] have reported in elegant experiments the measure of 12 C 15 N/ 12 C 14 N, 13 C 14 N/ 12 C 14 N, and 13 C 15 N/ 12 C 14 N isotope ratios in cultured cells using a CAMECA IMS 3F. The 12 C 15 N/ 12 C 14 N isotope ratio has also been measured in pollen grains [16].…”

“…Here, we report the use of MIMS to measure-in parallel-the 12 C Ϫ , 13 C Ϫ , 12 C 14 N Ϫ , and 12 C 15 N Ϫ ion currents in subcellular volumes of cells that had been cultured in normal medium or in media supplemented with the amino acid glycine labeled with either 13 C or 15 N. The results demonstrate that, for the first time, 13 C/ 12 C and 15 N/ 14 N isotope ratios can be directly measured at a subcellular scale. The capability of this new generation of instruments to directly measure stable isotope ratios lends itself to a wide range of applications in biomedical research.…”