Multimodal evaluation of 19F-BPA internalization in pancreatic cancer cells for boron capture and proton therapy potential applications

Published:January 06, 2022DOI:


      • 19F-BPA is significantly internalized in PANC-1 cells.
      • At 120 ppm of 11B 19F-BPA has an internalization ratio equal to 0.5.
      • 19F-MRS can detect and quantify the therapeutic 19F-BPA doses useful in BNCT/PBFT.
      • 19F-BPA is a promising BNCT/PBFT carrier and tracer for the treatment of pancreatic cancer.



      One of the obstacles to the application of Boron Neutron Capture Therapy (BNCT) and Proton Boron Fusion Therapy (PBFT) concerns the measurement of borated carriers' biodistribution. The objective of the present study was to evaluate the in vitro internalization of the 19F-labelled p-boronophenylalanine (19F-BPA) in the human cancer pancreatic cell line (PANC-1) for the potential application of BNCT and PBFT in pancreatic cancer. The 19F-BPA carrier has the advantage that its bio-distribution may be monitored in vivo using 19F-Nuclear Magnetic Resonance (19F NMR).

      Materials and methods

      The 19F-BPA internalization in PANC-1 cells was evaluated using three independent techniques on cellular samples left in contact with growing medium enriched with 13.6 mM 19F-BPA corresponding to a 11B concentration of 120 ppm: neutron autoradiography, which quantifies boron; liquid chromatography hyphenated to tandem mass spectrometry and UV-Diode Array Detection (UV-DAD), which quantifies 19F-BPA molecule; and 19F NMR spectroscopy, which detects fluorine nuclei.


      Our studies suggested that 19F-BPA is internalized by PANC-1 cells. The three methods provided consistent results of about 50% internalization fraction at 120 ppm of 11B. Small variations (less than 15%) in internalization fraction are mainly dependent on the proliferation state of the cells.


      The ability of 19F NMR spectroscopy to study 19F-BPA internalization was validated by well-established independent techniques. The multimodal approach we used suggests 19F-BPA as a promising BNCT/PBFT carrier for the treatment of pancreatic cancer. Since the quantification is performed at doses useful for BNCT/PBFT, 19F NMR can be envisaged to monitor 19F-BPA bio-distribution during the therapy.


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        • Sereti E.
        • Karagianellou T.
        • Kotsoni I.
        • Magouliotis D.
        • Kamposioras K.
        • Ulukaya E.
        • et al.
        Patient Derived Xenografts (PDX) for personalized treatment of pancreatic cancer: Emerging allies in the war on a devastating cancer?.
        J Proteomics. 2018; 188: 107-118
        • Rawla P.
        • Sunkara T.
        • Gaduputi V.
        Epidemiology of Pancreatic Cancer: Global Trends, Etiology and Risk Factors.
        World J Oncol. 2019; 10: 10-27
        • Seshacharyulu P.
        • Baine M.J.
        • Souchek J.J.
        • Menning M.
        • Kaur S.
        • Yan Y.
        • et al.
        Biological determinants of radioresistance and their remediation in pancreatic cancer.
        Biochim Biophys Acta, Rev Cancer. 2017; 1868: 69-92
        • Dell’Oro M.
        • Short M.
        • Wilson P.
        • Bezak E.
        Clinical Limitations of Photon, Proton and Carbon Ion Therapy in Pancreatic Cancer.
        Cancers (Basel). 2020; 12: 163
        • Barth R.F.
        • Coderre J.A.
        • Vicente M.G.H.
        • Blue T.E.
        Boron Neutron Capture Therapy of Cancer: Current Status and Future Prospects.
        Clin Cancer Res. 2005; 11: 3987-4002
        • Moss R.L.
        Critical review with an optimistic outlook, on Boron Neutron Capture Therapy (BNCT).
        Appl Radiat Isotop. 2014; 88: 2-11
        • Barth R.F.
        Boron neutron capture therapy at the crossroads: challenges and opportunities.
        Appl Radiat Isot. 2009; 67: S3-S6
        • Savolainen S.
        • Kortesniemi M.
        • Timonen M.
        • Reijonen V.
        • Kuusela L.
        • Uusi-Simola J.
        • et al.
        Boron neutron capture therapy (BNCT) in Finland: technological and physical prospects after 20 years of experiences.
        Phys Med. 2013; 29: 233-248
      1. Porcari P, Capuani S, Pastore FS. Novel Pharmacological and Magnetic Resonance Strategies to Enhance Boron Neutron Capture Therapy (BNCT) Efficacy in the Clinical Treatment of Malignant Glioma, in Management of CNS Tumors. 2011; 267. doi: 10.5772/24756.

        • Barth R.F.
        • Mi P.
        • Yang W.
        Boron delivery agents for neutron capture therapy of cancer.
        Cancer Commun. 2018; 38: 35
        • Capuani S.
        • Gili T.
        • Bozzali M.
        • Russo S.
        • Porcari P.
        • Cametti C.
        • et al.
        L-DOPA Preloading Increases the Uptake of Borophenylalanine in C6 Glioma Rat Model: A New Strategy to Improve BNCT Efficacy.
        Int J Radiat Oncol Biol Phys. 2008; 72: 562-567
        • Malouff T.D.
        • Seneviratne D.S.
        • Ebner D.K.
        • Stross W.C.
        • Waddle M.R.
        • Trifiletti D.M.
        • et al.
        Boron Neutron Capture Therapy: A Review of Clinical Applications.
        Front Oncol. 2021; 11
        • Capuani S.
        • Gili T.
        • Bozzali M.
        • Russo S.
        • Porcari P.
        • Cametti C.
        • et al.
        Boronophenylalanine uptake in C6 glioma model is dramatically increased by L-DOPA preloading.
        Appl Radiat Isot. 2009; 67: S34-S36
        • Wongthai P.
        • Hagiwara K.
        • Miyoshi Y.
        • Wiriyasermkul P.
        • Wei L.
        • Ohgaki R.
        • et al.
        Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATBo,+, LAT1 and LAT2.
        Cancer Sci. 2015; 106: 279-286
        • Nakagawa Y.
        • Pooh K.
        • Kobayashi T.
        • Kageji T.
        • Uyama S.
        • Matsumura A.
        • et al.
        Clinical review of the Japanese experience with boron neutron capture therapy and a proposed strategy using epithermal neutron beams.
        J Neurooncol. 2003; 62: 87-99
        • Sköld K.
        • Stenstam B.H.
        • Diaz A.Z.
        • Giusti V.
        • Pellettieri L.
        • Hopewell J.W.
        Boron Neutron Capture Therapy for glioblastoma multiforme: Advantage of prolonged infusion of BPA-f.
        Acta Neurol Scand. 2010; 122: 58-62
        • Wang L.-W.
        • Chen Y.-W.
        • Ho C.-Y.
        • Hsueh Liu Y.-W.
        • Chou F.-I.
        • Liu Y.-H.
        • et al.
        Fractionated BNCT for locally recurrent head and neck cancer: experience from a phase I/II clinical trial at Tsing Hua Open-Pool Reactor.
        Appl Radiat Isot. 2014; 88: 23-27
        • Wang L.-W.
        • Liu Y.-W.
        • Chou F.-I.
        • Jiang S.-H.
        Clinical trials for treating recurrent head and neck cancer with boron neutron capture therapy using the Tsing-Hua Open Pool Reactor.
        Cancer Commun. 2018; 38: 37
        • Jung J.Y.
        • Lu B.
        • Yoon D.K.
        • Hong K.J.
        • Jang H.
        • Liu C.
        • et al.
        Therapy region monitoring based on PET using 478keV single prompt gamma ray during BNCT: A Monte Carlo simulation study.
        Phys Med. 2016; 32: 562-567
        • Jung J.-Y.
        • Yoon D.-K.
        • Barraclough B.
        • Lee H.C.
        • Suh T.S.
        • Lu B.o.
        Comparison between proton boron fusion therapy (PBFT) and boron neutron capture therapy (BNCT): a Monte Carlo study.
        Oncotarget. 2017; 8: 39774-39781
        • Cirrone G.A.P.
        • Manti L.
        • Margarone D.
        • Petringa G.
        • Giuffrida L.
        • Minopoli A.
        • et al.
        First experimental proof of Proton Boron Capture Therapy (PBCT) to enhance protontherapy effectiveness.
        Sci Rep. 2018; 8
        • Hanaoka K.
        • Watabe T.
        • Naka S.
        • Kanai Y.
        • Ikeda H.
        • Horitsugi G.
        • et al.
        FBPA PET in boron neutron capture therapy for cancer: prediction of (10)B concentration in the tumor and normal tissue in a rat xenograft model.
        EJNMMI Res. 2014; 4
        • Ishiwata K.
        4-Borono-2-18F-fluoro-l-phenylalanine PET for boron neutron capture therapy-oriented diagnosis: overview of a quarter century of research.
        Ann Nucl Med. 2019; 33: 223-236
        • Tirotta I.
        • Dichiarante V.
        • Pigliacelli C.
        • Cavallo G.
        • Terraneo G.
        • Bombelli F.B.
        • et al.
        19F Magnetic Resonance Imaging (MRI): From Design of Materials to Clinical Applications.
        Chem Rev. 2015; 115: 1106-1129
        • Porcari P.
        • Capuani S.
        • D’Amore E.
        • Lecce M.
        • La Bella A.
        • Fasano F.
        • et al.
        In vivo 19F MR imaging and spectroscopy for the BNCT optimization.
        Appl Radiat Isot. 2009; 67: S365-S368
        • Porcari P.
        • Capuani S.
        • D'Amore E.
        • Lecce M.
        • La Bella A.
        • Fasano F.
        • et al.
        In vivo 19F MRI and 19F MRS of 19F-labelled boronophenylalanine–fructose complex on a C6 rat glioma model to optimize boron neutron capture therapy (BNCT).
        Phys Med Biol. 2008; 53: 6979-6989
        • Wittig A.
        • Michel J.
        • Moss R.L.
        • Stecher-Rasmussen F.
        • Arlinghaus H.F.
        • Bendel P.
        • et al.
        Boron analysis and boron imaging in biological materials for Boron Neutron Capture Therapy (BNCT).
        Crit Rev Oncol Hematol. 2008; 68: 66-90
        • Martano G.
        • Delmotte N.
        • Kiefer P.
        • Christen P.
        • Kentner D.
        • Bumann D.
        • et al.
        Fast sampling method for mammalian cell metabolic analyses using liquid chromatography-mass spectrometry.
        Nat Protoc. 2015; 10: 1-11
        • Johnson C.H.
        • Ivanisevic J.
        • Siuzdak G.
        Metabolomics: beyond biomarkers and towards mechanisms.
        Nat Rev Mol Cell Biol. 2016; 17: 451-459
        • Kabalka G.W.
        • Reddy N.K.
        • Wang L.
        • Malladi R.R.
        Synthesis of 4-borono-2-fluorophenylalanine.
        Org Prep Proced Int. 2000; 32: 290-293
        • Ferrari C.
        • Bakeine J.
        • Ballarini F.
        • Boninella A.
        • Bortolussi S.
        • Bruschi P.
        • et al.
        In Vitro and In Vivo Studies of Boron Neutron Capture Therapy: Boron Uptake/Washout and Cell Death.
        Radiat Res. 2011; 175: 452-462
        • Bi H.
        • Krausz K.W.
        • Manna S.K.
        • Li F.
        • Johnson C.H.
        • Gonzalez F.J.
        Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells.
        Anal Bioanal Chem. 2013; 405: 5279-5289
        • Luo X.
        • Gu X.
        • Li L.
        Development of a simple and efficient method of harvesting and lysing adherent mammalian cells for chemical isotope labeling LC-MS-based cellular metabolomics.
        Anal Chim Acta. 2018; 1037: 97-106
        • Postuma I.
        • Bortolussi S.
        • Protti N.
        • Ballarini F.
        • Bruschi P.
        • Ciani L.
        • et al.
        An improved neutron autoradiography set-up for 10B concentration measurements in biological samples.
        Rep Pract Oncol Radiother. 2016; 21: 123-128
        • Bortolussi S.
        • Bakeine J.G.
        • Ballarini F.
        • Bruschi P.
        • Gadan M.A.
        • Protti N.
        • et al.
        Boron uptake measurements in a rat model for Boron Neutron Capture Therapy of lung tumours.
        Appl Radiat Isot. 2011; 69: 394-398
        • Portu A.
        • Postuma I.
        • Gadan M.A.
        • Saint Martin G.
        • Olivera M.S.
        • Altieri S.
        • et al.
        Inter-comparison of boron concentration measurements at INFN-University of Pavia (Italy) and CNEA (Argentina).
        Appl Radiat Isot. 2015; 105: 35-39
        • Kur-Kowalska K.
        • Przybyt M.
        • Miller E.
        The study of phenylboronic acid optical properties towards creation of a glucose sensor.
        Biotechnol Food Sci. 2014; 78: 101-110
        • Panov V.
        • Salomon Y.
        • Kabalka G.W.
        • Bendel P.
        Uptake and washout of borocaptate sodium and borono-phenylalanine in cultured melanoma cells: a multi-nuclear NMR study.
        Radiat Res. 2000; 154: 104-112[0104:uawobs];2
        • Porcari P.
        • Capuani S.
        • Campanella R.
        • Bella A.L.
        • Migneco L.M.
        • Maraviglia B.
        Multi-nuclear MRS and 19F MRI of 19F-labelled and 10B-enriched p-boronophenylalanine-fructose complex to optimize boron neutron capture therapy: phantom studies at high magnetic fields.
        Phys Med Biol. 2006; 51: 3141-3154
        • Vähätalo J.
        • Tuominen J.
        • Kokkonen J.
        • Kriz O.
        • Karonen S.L.
        • Kallio M.
        Trace impurities identified by high performance liquid chromatography/electrospray mass spectrometry in two different synthetic batches of 4–boronophenylalanne.
        Rapid Commun Mass Spectrom. 1998; 12: 1118-1122<1118::AID-RCM289>3.0.CO;2-1
        • Gibson C.R.
        • Staubus A.E.
        • Barth R.F.
        • Yang W.
        • Kleinholz N.M.
        • Jones R.B.
        • et al.
        Electrospray ionization mass spectrometry coupled to reversed-phase ion-pair high-performance liquid chromatography for quantitation of sodium borocaptate and application to pharmacokinetic analysis.
        Anal Chem. 2002; 74: 2394-2399
        • Mauri P.L.
        • Basilico F.
        • Pietta P.G.
        • Pasini E.
        • Monti D.
        • Sauerwein W.
        New approach for the detection of BSH and its metabolites using capillary electrophoresis and electrospray ionization mass spectrometry. J. Chromatogr. B. Analyt. Technol. Biomed.
        Life Sci. 2003; 788: 9-16
        • Shichi Y.
        • Sasaki N.
        • Michishita M.
        • Hasegawa F.
        • Matsuda Y.
        • Arai T.
        • et al.
        Enhanced morphological and functional differences of pancreatic cancer with epithelial or mesenchymal characteristics in 3D culture.
        Sci Rep. 2019; 9
        • Gradiz R.
        • Silva H.C.
        • Carvalho L.
        • Botelho M.F.
        • Mota-Pinto A.
        MIA PaCa-2 and PANC-1 – pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors.
        Sci Rep. 2016; 6: 21648
        • Watanabe M.
        • Sheriff S.
        • Lewis K.B.
        • Cho J.
        • Tinch S.L.
        • Balasubramaniam A.
        • et al.
        Metabolic Profiling Comparison of Human Pancreatic Ductal Epithelial Cells and Three Pancreatic Cancer Cell Lines using NMR Based Metabonomics.
        J Mol Biomark Diagn. 2012; 3: S3-002
        • Holzgrabe U.
        Quantitative NMR spectroscopy in pharmaceutical Applications.
        Prog Nucl Magn Reson Spectrosc. 2010; 57: 229-240