PII S0360-3016(99)00172-8 BIOLOGY CONTRIBUTION BORONATED PROTOPORPHYRIN (BOPP): LOCALIZATION IN LYSOSOMES OF THE HUMAN GLIOMA CELL LINE SF-767 WITH UPTAKE MODULATED BY LIPOPROTEIN LEVELS DANIEL E. CALLAHAN,PH.D.,* TRUDY M. FORTE,PH.D.*, S. M. JAVED AFZAL,PH.D.,* DENNIS F. DEEN,PH.D., ‡ STEPHEN B. KAHL,PH.D., † KATHLEEN A. BJORNSTAD, B.S.,* WILLIAM F. BAUER,PH.D., § AND ELEANOR A. BLAKELY,PH.D.* *Life Sciences Division, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA; † Department of Pharmaceutical Chemistry, and ‡ Brain Tumor Research Center, University of California, San Francisco, CA; and § National Center for BNCT Measurement and Development, Idaho National Engineering and Environmental Laboratory EG&G Idaho, Inc., Idaho Falls, ID Purpose: Boronated protoporphyrin (BOPP) is a candidate for use in both boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of glioblastoma multiforme (GBM). Our objectives are to identify factors that influence the uptake and retention of BOPP in vitro and to determine BOPP distribution in a human glioma cell line in vitro. This information will aid the development of compounds and treatment strategies that increase the effectiveness of BNCT therapy for GBM. Methods and Materials: The amount, distribution pattern, and site of internalization of BOPP were assessed using fluorescence microscopy. Living human glioma (SF-767) cells were imaged after a 24-h exposure to BOPP (20 –135.6 g/ml, normal serum). Dose-dependent uptake of BOPP was determined using both fluorescence microscopy of individual living cells and inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of cell pellets. Lysosome- or mitochondria-specific fluorescent probes were used to identify the cellular compartment containing BOPP. Two human fibroblast cell lines, AG-1522 (LDL receptor-positive) and GM019- 15C (LDL receptor-deficient), were used to investigate LDL receptor-dependent BOPP uptake. The dependence of BOPP uptake on lipoproteins in the media was determined by exposing each of the three cell types to BOPP in medium containing either normal (NS) or lipoprotein deficient serum (LPDS). Results: BOPP accumulated in the lysosomes of human glioma cells in vitro, and not in the mitochondria, as reported for C6 rat glioma cells in vitro. BOPP uptake was concentration-dependent and was also dependent on the amount of lipoproteins in the medium. Over the range of incubation concentrations studied and at the single exposure duration time point investigated (24 h), all cells retained a similar amount of BOPP. At the lowest incubation concentration (20 g/ml, NS), the amount of boron retained was near 10 9 atoms per cell (15 g B/g cells). Lysosomes containing high concentrations of BOPP were randomly distributed throughout the cytoplasm; however, larger lysosomes containing BOPP were concentrated around the cell nucleus. Little or no BOPP accumulated in the cell nucleus. At incubation concentrations of 20 and 40 g/ml (24-h time point), BOPP uptake in SF-767 cells was reduced in LPDS compared with NS (66% reduction). A similar result was observed for normal human fibroblasts (AG-1522 cells, 40 g/ml, 24 h). At 40 g/ml, in both NS and LPDS at 24 h, BOPP accumulation in LDL receptor-deficient human fibroblasts (GM019-15C cells) was reduced relative to AG-1522 cells. BOPP accumulation in GM019-15C cells (40 g/ml, 24 h) was not affected by serum lipoprotein levels. Conclusion: In cell culture, BOPP is taken up by human glioma cells via the LDL pathway and is compartmen- talized into cellular lysosomes. Knowledge of this mechanism of BOPP uptake and retention will be important in attempts to modify toxicity and efficacy of this drug. © 1999 Elsevier Science Inc. BOPP, Porphyrins, Boron neutron capture therapy, Brain tumors, LDL receptor. INTRODUCTION Selective tumor uptake of boronated protoporphyrin (BOPP) has been demonstrated in mice bearing an im- planted rat C6 intracerebral glioma (1). BOPP (2,4-Bis- (,-dihydroxyethyl) deuteroporphyrin IX tetrakiscarbo- rane carboxylate ester; C 46 H 77 B 40 N 4 O 12 Na 2 ) is a porphyrin Reprint requests to: Dr. E. A. Blakely, Ernest Orlando Lawrence Berkeley National Laboratory, Life Sciences Division, 1 Cyclotron Road, MS 70A/1118, Berkeley, CA 94720. Tel: (510) 486-6595; Fax: (510) 486-4475; E-mail: EABlakely@lbl.gov This work was supported by the Director, Office of Energy Research, Office of Laboratory Policy and Infrastructure Manage- ment, of the U.S. Department of Energy under contract No. DE- ACO3-76SF00098 and by NIH HL18574 (Dr. Forte), NIH CA- 13525 (Dr. Deen), and NIH CA-37961 (Dr. Kahl). Presented in part at The Proton Therapy Cooperative Group Meeting PTCOG XXIV, April 24 –26, 1996, Detroit, MI; The Seventh International Symposium on Neutron Capture Therapy for Cancer, September 4 –7, 1996, Zurich, Switzerland; and The Eighth International Symposium on Neutron Capture Therapy for Cancer, September 13–18, 1998, La Jolla, CA. Accepted for publication 28 April 1999. Int. J. Radiation Oncology Biol. Phys., Vol. 45, No. 3, pp. 761–771, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/99/$–see front matter 761