Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular-scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

Authors:   S. Chandra,T. Ahmad,R. Barth,G. Kabalka
Journal: Journal of Microscopy
https://doi.org/10.1111/jmi.12126
Abstract: Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron-10 (10B) atoms to individual tumour cells. Cell killing results from the 10B (n, $alpha$)7Li neutron capture and fission reactions that occur if a sufficient number of 10B atoms are localized in the tumour cells. Intranuclear 10B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of 10B atoms reflects both bound and free pools of boron in individual tumour cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular-scale resolution by clinically applicable techniques such as positron emission tomography and magnetic resonance imaging. In this study, a secondary ion mass spectrometry based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high-grade gliomas, recurrent tumours of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumour cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols. Lay Description: The effectiveness of boron neutron capture therapy (BNCT) of cancer depends on the selective incorporation of about 20 microgram/gram boron-10 (10B) atoms in tumor cells. The cell killing is enhanced via the neutron capture reaction if the 10B atoms are localized in nuclei of tumor cells. The net content of 10B atoms reflects both bound and free pools of boron in tumor cells. The assessment of these pools of boron, delivered by a BNCT drug, currently cannot be made at subcellular scale resolution by clinically applicable techniques of Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI). BNCT is currently under development for the treatment of brain tumors, especially the high grade gliomas such as glioblastoma multiforme. BNCT is potentially capable of killing the infiltrating (spreading) individual and clusters of glioblastoma cells in the normal brain. In this study, a highly specialized secondary ion mass spectrometry (SIMS) based instrument (CAMECA IMS-3F SIMS Ion Microscope) capable of imaging boron atoms in single cells at 500 nanometer spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high grade gliomas, recurrent tumors of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumor cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with the extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine amino acid in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols. {textcopyright} 2014 Royal Microscopical Society.