Quantitative subcellular imaging of boron compounds in individual mitotic and interphase human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

Abstract

Boron measurements at subcellular scale are essential in boron neutron capture therapy (BNCT) of cancer as the nuclear localization of boron-10 atoms can enhance the effectiveness of killing individual tumour cells. Since tumours contain a heterogeneous population of cells in interphase as well as in the M phase (mitotic division) of the cell cycle, it is important to evaluate the subcellular distribution of boron in both phases. In this work, the secondary ion mass spectrometry (SIMS) based imaging technique of ion microscopy was used to quantitatively image boron from two BNCT agents, clinically used p-boronophenylalanine (BPA) and 3-[4-(o-carboran-1-yl)butyl]thymidine (N4), in mitotic metaphase and interphase human glioblastoma T98G cells. N4 belongs to a class of experimental BNCT agents, designated 3-carboranyl thymidine analogues (3CTAs), which presumably accumulate selectively in cancer cells due to a process referred to as kinase-mediated trapping (KMT). The cells were exposed to BPA for 1 h and N4 for 2 h. A CAMECA IMS-3f SIMS ion microscope instrument capable of producing isotopic images with 500 nm spatial resolution was used in the study. Observations were made in cryogenically prepared fast frozen, and freeze-fractured, freeze-dried cells. Three discernible subcellular regions were studied: the nucleus, a characteristic mitochondria-rich perinuclear cytoplasmic region, and the remaining cytoplasm in interphase T98G cells. In metaphase cells, the chromosomes and the cytoplasm were studied for boron localization. Intracellular concentrations of potassium and sodium also were measured in each cell in which the subcellular boron concentrations were imaged. Since the healthy cells maintain a K/Na ratio of approximately 10 due to the presence of Na-K-ATPase in the plasma membrane of mammalian cells, these measurements provided validation for cryogenic sample preparation and indicated the analysis healthy, well preserved cells. The BPA-treated interphase cells revealed significantly lower concentrations of boron in the perinuclear mitochondria-rich cytoplasmic region as compared to the remaining cytoplasm and the nucleus, which were not significantly different from each other. In contrast, the BPA-treated metaphase cells revealed significantly lower concentration of boron in their chromosomes than cytoplasm. In addition, the cytoplasm of metaphase cells contained significantly less boron than the cytoplasm of interphase cells. These observations provide valuable information on the reduced uptake of boron from BPA in mitotic cells for BPA-mediated BNCT. SIMS observations on N4 revealed that boron was distributed throughout the interphase and mitotic cells, including the chromosomes. The presence of boron in chromosomes of metaphase cells treated with N4 is indicative of a possible incorporation of this thymidine analogue into DNA. The 3-D SIMS imaging approach for the analysis of mitotic cells shown in this work should be equally feasible to the evaluation of other BNCT agents.