Leukemia Research Vol. 14, No. 3, pp. 209-219, 1990. Printed in Great Britain.
0145-2126/90 $3.00 + .00 Pergamon Press plc
P R E F E R E N T I A L UPTAKE OF BENZOPORPHYRIN D E R I V A T I V E BY LEUKEMIC VERSUS NORMAL CELLS CATRIONA H. M. JAMIESON, WILLIAMN. MCDONALD,* and JULIA G. LEVY Department of Microbiology, University of British Columbia, and *Department of Internal Medicine, St. Paul's Hospital, Vancouver, British Columbia, Canada V6T 1W5 (Received 3 May 1989. Revision accepted 19 August 1989)
Abstract--Benzoporphyrin derivatives (BPDs) are photosensitizers, which fluoresce strongly at 690 nm, and may be candidates for various applications of photodynamic therapy (PDT). Fluorescence-activated cell sorting (FACS) analysis, subsequent to ultraviolet light excitation, revealed pronounced differences in red fluorescence between leukemic cell lines (HL60, K562 and L1210), leukemic clinical isolates, and normal human or murine bone marrow cells incubated with BPD. These observed differences in BPD-mediated fluorescence provide the rationale for sorting leukemic from normal cells via FACS or may constitute a novel method for extracorporeal purging of remission marrow by photodynamic therapy in autologous bone marrow transplantation. Key words: Benzoporphyrin derivative, fluorescence, leukemia, FACS.
INTRODUCTION
available immunotoxins have been employed in an attempt to purge remission marrow of residual leukemic cells. However, the non-specific cytotoxicity of chemotherapeutic agents such as cyclophosphamide and the lack of monoclonal antibodies to well characterized leukemia antigens have hindered these attempts [4-7]. There is clearly a need for a purging agent which may be selectively triggered and which is capable of eradicating leukemic cells rather than normal bone marrow constituents. This need has generated interest in phototoxic amphipathic dyes, some of which have been reported to photosensitize leukemic cells to a greater extent than normal marrow mononuclear cells when activated by exposure to light [8]. The majority of clinical applications of photodynamic therapy (PDT) to date have centered on the use of Photofrin II and its use in the treatment of solid tumors after intravenous administration and exposure to laser light via a fibre optic 24-48 h later. Recently, the development of audio-endoscopes capable of detecting fluorescence emitted by porphyrin accumulated in neoplastic tissue has rekindled interest in porphyrin fluorescence as a potential diagnostic indicator for metastatic tumors. The question remains, however whether porphyrin fluorescence may be used to distinguish non-solid neoplasias such as leukemias from normal cells and if so whether the potential exists for fluorescence-activated cell sorting (FACS) of normal from leukemic cells on the basis of characteristic differences in porphyrin fluorescence.
AUTOLOGOUS bone marrow transplantation (ABMT) involves the removal and cryopreservation of a leukemic patient's marrow during remission followed by reconstitution upon relapse [1]. This technique obviates the need for an HLA-matched donor. Age (over 45 years) and the lack of a histocompatible donor restrict allogeneic bone marrow transplants to 6% of adults with acute myelogenous leukemia (AML) [2]. The lack of a graft versus leukemia response and the risk of reinfusing residual leukemic cells, present but undetected in remission marrow, has weighed against the use of autologous bone marrow transplants [3]. Pharmacological purging techniques and currently Abbreviations: BPD-MA or B-MA, benzoporphyrin derivative monoacid ring A; BPD-MB or B-MB, benzoporphyrin derivative monoacid ring B; BPD-DA or B-DA, benzoporphyrin derivative diacid ring A; BPD-DB or BDB, benzoporphyrin derivative diacid ring B; PDT, photodynamic therapy; DiO, 3,3'-dioctadecyloxacarbocyanine perchlorate; PBL, peripheral blood leukocytes; FACS, fluorescence-activated cell sorting; AML, acute myelogenous leukemia; ABMT, autologous bone marrow transplantation; FCS, fetal calf serum; PBS, phosphate buffered saline; u.v., ultraviolet light; A TCC, American type culture collection; O.D., optical density; DMSO, dimethyl sulfoxide. Correspondence to: Catriona H. M. Jamieson, Department of Microbiology, University of British Columbia, 6174 University Blvd., Vancouver, B.C., Canada V6T lW5.
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Benzoporphyrin derivative (BPD), a longwave absorbing drug with a demonstrated affinity for tumor tissue, is currently under investigation in a number of laboratories [9]. The fluorescent properties of BPD have not been extensively analyzed in vitro or in vivo and the crucial question of whether BPD is taken up preferentially by leukemic cells remains to be addressed. Fluorescence-activated cell sorting (FACS) analysis provides a powerful means of assessing differences in the relative fluorescence intensity between cells. FACS analysis therefore was employed to determine whether substantial differences existed between the uptake of B P D by different leukemic cell lines, leukemic clinical isolates, and normal bone marrow and peripheral blood mononuclear cells. DiO, 3,3'dioctadecyloxacarbocyanine perchlorate (Molecular Probes) is a cationic lipophilic probe which emits green fluorescence when excited by 489 nm and exhibits negligible dye transfer properties. It was used in this study to mark murine leukemic (L1210) cells incubated with BPD to distinguish them from normal mouse bone marrow cells also incubated with BPD subsequent to FACS on the basis of red fluorescence [10].
MATERIALS AND METHODS
Spectrofluorometric analysis Prior to spectrofluorometric analysis, BPD was dissolved in phosphate buffered saline (PBS) at a concentration of 2 ~tg/ml resulting in an absorbance reading of 0.10 O.D. units at 420 nm. Excitation and emission scans were performed using an SLM 500 C spectrofluorometer in a wavelength acquisition mode with an excitation bandpass of 0.5 nm, an emission bandpass of 20 nm, and a step size of 0.5 nm. The emission reference utilized during excitation scans of BPD was 690 nm. The maximal fluorescence excitation peak of BPD was used to determine the maximal fluorescence emission peak during the fluorescence emission scans.
FA CS analysis Cell lines. Cell lines were maintained in phenol-red free Dulbecco's Modified Eagle's (DME) media supplemented with 10% fetal calf serum (FCS) in a 10% CO2 incubator at 37°C and were subcultured according to American Type Culture Collections (ATCC) specifications. Cell lines analyzed included: L1210, a mouse lymphocytic leukemia HL60, a human acute promyelocytic leukemia, and K562, a human chronic myelogenous leukemic cell line. Patient samples. Mononuclear cells were extracted from leukemic clinical isolates, normal human bone marrow and human peripheral blood (collected in heparinized tubes) over Ficoll-Hypaque (Pharmacia) density gradients. Briefly, whole blood was diluted by a factor of two in PBS and layered over 3 ml of Ficoll-Hypaque. The mononuclear band, containing all mononuclear white blood cells, was collected subsequent to a 10 min centrifugation period at 1500rpm. Cells were washed three times in PBS to
remove Ficoll-Hypaque. Mononuclear cells were analyzed fresh or subsequent to cryopreservation in DME media containing 10% dimethyl sulfoxide (DMSO) and 40% FCS. Vials containing cryopreserved cells were warmed quickly in a 37°C water bath and rinsed with ethanol. Cryopreserved cells were then diluted by a factor of ten in PBS and washed three times in PBS to remove DMSO. Mouse bone marrow and spleen preparation. Bone marrow was extracted from 6-8-week old DBA/2 female mice which were euthanized by CO2. The femur and tibia were cleaned of muscle using sterile forceps and scissors and transferred to sterile PBS where bone marrow was flushed from the bones and aspirated with a 25 gauge needle to form a single cell suspension. Bone marrow cells were centrifuged and washed in sterile PBS and viability counts performed. Spleens were removed asceptically from the same mice used for bone marrow extraction and passed through a wire mesh to create a single cell suspension. Bone marrow or spleen cells were cryopreserved or analyzed fresh. FACS parameters. Before FACS analysis, cells were incubated in the dark in a 10% CO2, 37°C incubator with BPD-monoacid ring A (BPD-MA), BPD-monoacid ring B (BPD-MB), BPD-diacid ring A (BPD-DA), BPD-diacid ring B (BPD-DB), or DiO for 30 min in phenol red-free DME media in the absence of FCS. Cells were washed in PBS to remove excess photosensitizer, centrifuged at 1100 rpm and resuspended in phenol red-free DME. The excitation wavelengths employed for FACS analysis involving BPD were 351.1-363.8 nm (ultraviolet light) and 488 nm (visible light) and a 590 nm emission (longpass) filter was utilized to detect BPD (red) fluorescence. The excitation wavelength used for cells incubated with DiO was 488 nm and a 530 nm emission filter (515-545 nm) was used to detect DiO (green) fluorescence. Both dual and quantitative single parameter FACS analyses were performed using a Becton-Dickinson FACS 440 flow cytometer equipped with both a visible light laser and an ultraviolet (u.v.) light laser.
RESULTS
Spectrofluorometric analysis Spectrofluorometric excitation and emission scans revealed that B P D ( B P D - D A ) (Fig. 1) is maximally excited by 420 nm (Fig. 2) and excited to a lesser extent by 356 nm (u.v.), resulting in a major fluorescence emission peak at 690 nm (Fig. 3). The monoacids are excited to fluoresce to a greater extent by ultraviolet light (data not shown).
Fluorescence comparison of four structural analogues of BPD In order to determine whether the four structural analogues of B P D differed in their fluorescent properties in the presence of cells, HL60 cells were incubated with 5, 10, or 20 t~g/ml B P D - M A , BPD-MB, B P D - D A , or B P D - D B for 24 h. Fluorescence emitted by cells subsequent to either u.v. or visible light excitation was measured via quantitative FACS analysis and demonstrated that although negligible
Preferential uptake of benzoporphyrinderivative by leukemicversus normal cells H3C~._
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211
studies correlate well with observed differences in photosensitizer activity of the four analogues. In a number of cell lines studied, the monoacid forms of BPD were significantly more cytotoxic than the diacid forms, and these differences were related to selective uptake [12, 13].
H3¢ RI= Rz= C02Me
R3= (CH2)2COzMe or (CH2)2CO2H
FIG. 1. Structure of BPD. Four structural analogues of BPD have been synthesized: BPD-MA, BPD-MB, BPDDA, and BPD-DB. The monoacids differ from the diacids at the R3 position in that in the monoacids one of the carboxylic acid groups has been esterified to form a methyl ester. Ring A or B refers to the benzene derivative on the left (ring A) or the right (ring B) of the porphyrin ring.
differences in mean red fluorescence emitted existed at 488 nm (Fig. 4) when excited by u.v. light, cells incubated with B P D - M A fluoresced to the greatest extent followed by those incubated with BPD-MB, B P D - D A , and B P D - D B respectively (Fig. 5). These
t
300
FA CS analysis o f leukemic cell lines versus normal bone marrow incubated with B P D Therefore, B P D - M A was used in subsequent FACS analyses to determine if substantive differences existed between normal and leukemic cells with regard to BPD uptake as judged by mean reflective fluorescence intensity. Normal human bone marrow mononuclear cells incubated with B P D - M A for 30 min fluoresced substantially less in the red spectrum subsequent to u.v. light excitation than human leukemic cell lines (HL60 and K562) or mouse leukemic cells (L1210) incubated under the same conditions (Fig. 6). Similarly, normal mouse bone marrow or spleen cells cells fluoresced to a lesser extent than murine leukemic cells (L1210) (Fig. 7).
10.0
400 500 excitation wavelength scan (nm) FIG. 2. Spectrofluorometric analysis of BPD in PBS-excitation wavelength scan. The emission reference employed was 690 nm. A slit width of 0.5 nm was used and fluorescence excitation was scanned between 300 and 600 nm.
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CATRIONAH. M..[AMIESONet al.
212
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500
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600
700
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scan
800
(nm)
FIG. 3. Spectrofluorometric analysis of BPD in PBS--emission wavelength scan. The excitation reference employed was 420 nm, which corresponded to the Soret peak of the porphyrin, and fluorescence emission was scanned between 500 and 800 nm.
The results shown in these figures illustrate representative data from individual experiments which have been repeated a n u m b e r of times. HL60, a human cell line representing promyelocytic leukemia was chosen for these purposes because its size more closely resembles the normal cell types found in blood and bone marrow, and therefore would minimize differences in fluorescence contributed by size. K562 is a human chronic myelogenous leukemic cell line, composed predominantly of blasts. L1210 is a T-cell leukemia of D B A / 2 mice and was chosen because this tumor line is being used in continuing research in bone marrow purging in an animal model.
Comparison of BPD uptake by leukemic and normal bone marrow To ensure that the observed differences in uptake of B P D - M A between leukemic cells and normal leu-
kocytes were not associated solely with leukemic cell lines; acute myelogenous leukemia clinical isolates and normal h u m a n peripheral blood or bone marrow mononuclear cells were subjected to FACS analysis subsequent to incubation with B P D - M A . Clear differences were recorded between normal peripheral blood or bone marrow and A M L mononuclear cells in terms of B P D - M A - m e d i a t e d fluorescence, although these differences were not as pronounced as those observed with leukemic cell lines. Significant differences in fluorescence between normal and leukemic cells were not observed when cells were incubated with BPD-DA (Student's t-test 0.05 < p ~< 0.1). Representative results are shown in Fig. 8, and a s u m m a r y of all data is given in Table 1. To ensure that differences in fluorescence between normal and leukemic cells were not solely a consequence of differences in size, cell size, as measured
Preferential uptake of benzoporphyrin derivative by leukemic versus normal cells FAC$ Analvsis of BPD-MA, -MB, -DA, -DB (488 nm} 102
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BPD 488 nm excitation. HL60 cells were incubated for 24 h with 0 (control), 5, 10, or 20 ~tg/ml BPD-MA, BPDMB, BPD-DA, or BPD-DB. The excitation wavelength used for FACS analysis was 488 nm. A 550 nm longpass filter was used to detect red fluorescence characteristic of BPD. As in all subsequent experiments involving FACS analysis, 10,240 cells were analyzed per sample.
FACS ANALYSIS
OF BPD-MA,
- MB, - DA, - DB (uv)
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FIG. 5. FACS analysis of the four structural analogues of BPD--u.v. excitation. HL60 cells were incubated as mentioned previously, however, u.v. light (351.1363.8 nm) was used as the excitation wavelength.
213
214
CATRIONAH. M. JAMIESONet al. 120 • "o c
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L1210 + BPD-MA
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FIG. 6. Comparison of BPD uptake by normal bone marrow versus leukemic cell lines. Duplicate samples of 1 x 106 cryopreserved human monuclear cells extracted from bone marrow were incubated for 30 min with 0 (control) or 10 ~tg/ml BPD-MA, as were the same number of HL60, K562, and L1210 cells. Mean red fluorescence emitted by these cell populations was compared via FACS analysis subsequent to u.v. excitation and BPD fluoresence detected with a 590 nm longpass filter as in all subsequent FACS analyses. Background refers to the level of fluorescence emitted by cells incubated in the absence of the drug.
T A B L E 1.
FACS ANALYSIS O F CELLS I N C U B A T E D WITH BPD-MA: M E A N F L U O R E S C E N C E -- B A C K G R O U N D
5 ~tg/ml -+ S.E. Normal cells murine bone marrow spleen cells human PBL bone marrow Leukemic cells murine L1210 human K562 HL60 clinical samples AML CML
10 ~tg/ml --_ S.E.
No. of samples
13.75 --+5.44 9.79 --- 1.47
2 2
10.15 -+ 1.47
12.53 -+ 1.61" 5.23 + 1.50
10 7
70.74 -+ 13.72
122.19 +- 6.67
10
43.43 + 1.41
53.21 +- 13.12 86.30 +- 28.45
3 4
29.18 -+ 0.20
22.98 -+ 0.27 37.28 -+ 3.32
2 10
* Mean fluorescence of human PBL incubated with 10 ktg/ml BPD-MA was compared to that of CML cells by the Student's t-test (p ~< 0.0005), to AML cells (p < 0.016), to HL60 ceils (p ~
~8
~
FIG. 10. Dual parameter FACS analysis of L1210 and mouse spleen cells. L1210 cells (1 x 106) incubated, as described above, with 10 ~tg/ml BPD-MA and 100 ~tg/ml DiO were mixed immediately before FACS analysis with 1 x 106 mouse spleen cells incubated with 10 ~tg/ml BPDMA. Total events refers to the number of cells analyzed (10,240). Cells were analyzed for DiO (green) fluorescence (y-axis) using a 530 nm filter subsequent to 488 nm excitation and also analyzed for BPD fluorescence, subsequent to u.v. excitation, but utilizing a 590 nm longpass filter.
in red fluorescence subsequent to u.v. excitation. Cells were sorted into ten fractions and each fraction was reanalyzed for green (DiO) fluorescence via 488 nm excitation and the use of a 530 nm filter
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2
3
4
5
6
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8
9
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FIG. 11. Sorting of L1210 cells from mouse spleen cells on the basis of differences in BPD fluorescence. The ceils described above were sorted into ten fractions with approximately 1,000 cells per fraction on the basis of differences in BPD (red) fluorescence and reanalyzed for DiO (green) fluorescence (y-axis) using a 530 nm filter. The graph represents two experiments. There were significant differences in green fluorescence between fractions 1 and 10 as judged by the Student's t-test (p = 0.003).
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to determine the extent of contamination of each fraction by leukemic cells, the argument being that only the strongly red fluorescing cells (i.e. L1210) should also show strong green fluorescence since only they were loaded with DiO (Fig. 11). Because only L1210 cells were incubated with DiO and DiO exhibits negligible dye transfer properties, green fluorescence should only be emitted by leukemic cells and so a way of distinguishing normal from leukemic cells in the normal sorted pool is provided. Pronounced differences existed between the extent of green fluorescence in fraction 10 compared to fraction 1 suggestive of a large proportion of leukemic cells in fraction 10 compared to fraction 1. Fluorescent microscopic examination of cytospins of fractions 1 and 10 revealed that no green fluorescing cells existed in fraction 1, whereas fraction 10 was composed solely of bright green fluorescing cells subsequent to blue light excitation (data not shown). Obviously, this preliminary experiment did not establish protocols for purging procedures but does indicate a justification for further work on this model.
DISCUSSION When BPD analogues are excited to fluoresce in the red spectrum by u.v. light (351.1-363.8 nm) and 420 nm, a powerful signal is emitted. Use of these parameters in the Becton-Dickinson FACS 440 flow cytometer has enabled us to distinguish differential uptake of these dyes by malignant versus normal hemopoietic cells in both murine and human systems. The most extensive work involving selective uptake of malignant hemopoietic cells by photosensitizers is that of Dr Sieber and his associates working with merocyanine 540 [3, 8]. These investigators have shown selective uptake and phototoxic killing of malignant cells in bone marrow samples of patients with advanced lymphomas. Similarly, Edelson and colleagues have shown that a light activated psoralen, known as 8-MOP can be used to treat, extracorporeally, the blood of patients with cutaneous T-cell lymphoma with concomitant activation with u.v. light [13]. BPD-MA and its analogues are photosensitizers derived from hematoporphyrin which show promise as agents for the photodynamic therapy of solid tumors, in that they accumulate somewhat selectively in tumor tissue, are readily activated by light at 690 nm to cause singlet oxygen release, and have been shown to be effective in eradicating tumors in a murine model [11, 12]. The present work constitutes a preliminary study to evaluate the capacity of BPD to accumulate selectively in leukemic cells, with the
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CATRIONA H. M. JAMIESON et al.
long term goal of testing its potential in selective killing of malignant hemopoietic cells. When u.v. light excitation was used to compare uptake of the four structural analogues of B P D : B P D MA, BPD-MB, B P D - D A , and B P D - D B , HL60 cells incubated with B P D - M A clearly fluoresced to the greatest extent. This observation correlates with earlier studies which indicated that B P D - M A was a more potent photosensitizer than the other structural analogues of BPD, when the analogues were tested for phototoxicity with a variety of cell lines [11, 12]. We have examined, not only leukemic cell lines, but also both normal bone marrow and peripheral blood, and leukemic cells from a variety of donors and patients. The results (summarized in Table 1) attest to our statement that leukemic cells selectively take up three to six fold more B P D - M A than do normal cells derived from bone marrow, spleen and peripheral blood. The results from one FACS analysis to another are surprisingly consistent. The fact that the second fluorescent dye used in these experiments, DiO, does not show this selective accumulation (since leukemic and normal cells took up essentially the same amount of the dye), attests to the selective nature of B P D - M A rather than to inherent differences in malignant versus normal cells. The use of dual labelling with B P D - M A and DiO of leukemic cells provides some preliminary evidence that the differences in BPD-mediated red fluorescence exhibited between normal and leukemic cells may be utilized in a novel form of purging remission marrow of residual leukemic cells via FACS. However, these results are very preliminary. The observations presented here provided a base for ongoing research on the possible application of this apparent selectivity in BPD uptake by leukemic as opposed to normal hemopoietic cells. Clearly, experiments must be performed to determine whether selective uptake translates into selective photoxic killing of malignant versus normal stem cells. It will be necessary to establish that this selective difference applies to self-renewing cells as well as other more mature hemopoietic populations. An in v i v o reconstitution model is currently being developed in which sorted populations of murine bone marrow cells mixed with L1210 cells will be used to reconstitute lethally irradiated D B A / 2 mice. These studies should reveal not only the presence of stem cells in the " n o r m a l " population but also the extent of contamination of the stem cell containing fraction with tumor cells, since between 1 and 10 L1210 cells will cause lethal ascites in recipients. At the same time, the potential use of BPD as an in vitro bone marrow purging agent through selective photosensitization will be followed in both the murine
model, and with human cells with the aid of in vitro colony assays (both C F U - G M and long term stem cell culture). Preliminary studies indicate that the LDs0 of progenitors from C G L patient samples is lower (in B P D - M A dosage) than that for normal C F U - G M progenitors (ongoing research). This study provides preliminary evidence that substantial differences exist in the uptake of BPDMA by leukemic versus normal cells and lays the ground work for developing a novel extra-corporeal purging protocol for autologous bone marrow transplantation. The mechanism of differential uptake has yet to be elucidated and is currently under investigation in this laboratory. Acknowledgements--This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (5-80268). CHMJ is the recipient of a B.C. Science Council GREAT Scholarship. The authors gratefully acknowledge the expert advice and technical assistance provided by Denise MacDougal, Lindsay Eltis, Dr Frank Tufaro and Dr Anna Richter.
REFERENCES 1. Atzpodien J., Gulati S. C. & Clarkson B. (1986) Comparison of the cytotoxic effects of Merocyanine 540 on leukemic cells and normal human bone marrow. Cancer Res. 46, 4892. 2. Singer C. R. J., Linch D. C., Brown S. G., Hughes E. R. & Goldstone A. H. (1988) Differential phthalocyanine photosensitisation of acute myeloblastic leukemia progentior cells: a potential purging technique for autologous bone marrow transplantation. Br. J. Hemat. 68, 417. 3. Sieber F., Rao S. & Rowley S. D. (1986) Dye-mediated photolysis of human neuroblastoma cells: Implications for autologous bone marrow transplantation. Blood 68, 32. 4. Gale R. P. & Foon K. A. (1987) Therapy of acute myelogenous leukemia. Semin. Hemat. 24, 40. 5. Champlin R. E., Goldman J. M. & Gale R. P. (1988) Bone marrow transplantation in chronic myelogenous leukemia. Semin. Hemat. 25, 74. 6. Laurent G., Kuhlen E., Casella P., Carrat X., Caragon P., Poncelot P., Corrall S., Rigat F. & Jansen F. K. (1986) Determination of sensitivity of fresh leukemia cells to immunotoxins. Cancer Res. 46, 2289. 7. Tulpaz M., Kantarjian H. M., Kusroch R. & Gutterman J. (1988) Therapy of chronic myelogenous leukemia: chemotherapy and interferons. Semin. Hemat. 25, 62. 8. Sieber F. (1987) Elimination of residual tumor cells from autologous bone marrow grafts by dye-mediated photolysis: Preclinical data. Photochem. Photobiol. 46, 71. 9. Richter A. M., Kelly B., Chow J., Liu D., Towers G., Dolphin D. & Levy J. G. (1987) Preliminary studies on a more effective phototoxic agent than hematoporphyrin derivative. J. natn. Cancer Inst. 79, 27. 10. Honig M. G. (1986) Fluorescent carbocyanine dyes
Preferential uptake of benzoporphyrin derivative by leukemic versus normal cells allow living neurons of identified origin to be studied in long-term cultures. J. cell. Biol. 103, 171. 11. Jamieson C., Dolphin D., Durand R., Levy J. G. (1989) Differential uptake of benzoporphyrin derivative by normal versus leukemic cells. In Proc. SPIE Conference, Los Angeles, Jan. 19-20. 1065, 152. 12. Richter A., Cerruti-Sola S., Sternberg E. D. & Dolphin D. Biodistribution of a tritiated benzoporphyrin derivation (3H-BPD-MA), a new potent photosensitizer in normal and tumor bearing mice. J. Photochern. Photobiol. (in press).
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13. Yemul S., Berger C., Estabrook A., Suarez S., Edelson R. & Bayley H. (1987) Selective killing of T lymphocytes by phototoxic liposomes. Proc. natn. Acad. Sci. U.S.A. 84, 246. 14. Edelson R. (1987) Treatment of cutaneous T-cell lymphoma by extracorporal photochemotherapy: preliminary results. New Engl. J. Med. 316, 297. 15. Gulliya K. S., Fay J., Dowben R. M., Berkholder S. & Matthews J. L. (1988) Elimination of leukemic cells by laser photodynamic therapy. Cancer Chemother. Pharmac. 22, 211.
0145-2126/90 $3.00 + .00 Pergamon Press plc
P R E F E R E N T I A L UPTAKE OF BENZOPORPHYRIN D E R I V A T I V E BY LEUKEMIC VERSUS NORMAL CELLS CATRIONA H. M. JAMIESON, WILLIAMN. MCDONALD,* and JULIA G. LEVY Department of Microbiology, University of British Columbia, and *Department of Internal Medicine, St. Paul's Hospital, Vancouver, British Columbia, Canada V6T 1W5 (Received 3 May 1989. Revision accepted 19 August 1989)
Abstract--Benzoporphyrin derivatives (BPDs) are photosensitizers, which fluoresce strongly at 690 nm, and may be candidates for various applications of photodynamic therapy (PDT). Fluorescence-activated cell sorting (FACS) analysis, subsequent to ultraviolet light excitation, revealed pronounced differences in red fluorescence between leukemic cell lines (HL60, K562 and L1210), leukemic clinical isolates, and normal human or murine bone marrow cells incubated with BPD. These observed differences in BPD-mediated fluorescence provide the rationale for sorting leukemic from normal cells via FACS or may constitute a novel method for extracorporeal purging of remission marrow by photodynamic therapy in autologous bone marrow transplantation. Key words: Benzoporphyrin derivative, fluorescence, leukemia, FACS.
INTRODUCTION
available immunotoxins have been employed in an attempt to purge remission marrow of residual leukemic cells. However, the non-specific cytotoxicity of chemotherapeutic agents such as cyclophosphamide and the lack of monoclonal antibodies to well characterized leukemia antigens have hindered these attempts [4-7]. There is clearly a need for a purging agent which may be selectively triggered and which is capable of eradicating leukemic cells rather than normal bone marrow constituents. This need has generated interest in phototoxic amphipathic dyes, some of which have been reported to photosensitize leukemic cells to a greater extent than normal marrow mononuclear cells when activated by exposure to light [8]. The majority of clinical applications of photodynamic therapy (PDT) to date have centered on the use of Photofrin II and its use in the treatment of solid tumors after intravenous administration and exposure to laser light via a fibre optic 24-48 h later. Recently, the development of audio-endoscopes capable of detecting fluorescence emitted by porphyrin accumulated in neoplastic tissue has rekindled interest in porphyrin fluorescence as a potential diagnostic indicator for metastatic tumors. The question remains, however whether porphyrin fluorescence may be used to distinguish non-solid neoplasias such as leukemias from normal cells and if so whether the potential exists for fluorescence-activated cell sorting (FACS) of normal from leukemic cells on the basis of characteristic differences in porphyrin fluorescence.
AUTOLOGOUS bone marrow transplantation (ABMT) involves the removal and cryopreservation of a leukemic patient's marrow during remission followed by reconstitution upon relapse [1]. This technique obviates the need for an HLA-matched donor. Age (over 45 years) and the lack of a histocompatible donor restrict allogeneic bone marrow transplants to 6% of adults with acute myelogenous leukemia (AML) [2]. The lack of a graft versus leukemia response and the risk of reinfusing residual leukemic cells, present but undetected in remission marrow, has weighed against the use of autologous bone marrow transplants [3]. Pharmacological purging techniques and currently Abbreviations: BPD-MA or B-MA, benzoporphyrin derivative monoacid ring A; BPD-MB or B-MB, benzoporphyrin derivative monoacid ring B; BPD-DA or B-DA, benzoporphyrin derivative diacid ring A; BPD-DB or BDB, benzoporphyrin derivative diacid ring B; PDT, photodynamic therapy; DiO, 3,3'-dioctadecyloxacarbocyanine perchlorate; PBL, peripheral blood leukocytes; FACS, fluorescence-activated cell sorting; AML, acute myelogenous leukemia; ABMT, autologous bone marrow transplantation; FCS, fetal calf serum; PBS, phosphate buffered saline; u.v., ultraviolet light; A TCC, American type culture collection; O.D., optical density; DMSO, dimethyl sulfoxide. Correspondence to: Catriona H. M. Jamieson, Department of Microbiology, University of British Columbia, 6174 University Blvd., Vancouver, B.C., Canada V6T lW5.
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CATRIONAH. M. JAMIESONet al.
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Benzoporphyrin derivative (BPD), a longwave absorbing drug with a demonstrated affinity for tumor tissue, is currently under investigation in a number of laboratories [9]. The fluorescent properties of BPD have not been extensively analyzed in vitro or in vivo and the crucial question of whether BPD is taken up preferentially by leukemic cells remains to be addressed. Fluorescence-activated cell sorting (FACS) analysis provides a powerful means of assessing differences in the relative fluorescence intensity between cells. FACS analysis therefore was employed to determine whether substantial differences existed between the uptake of B P D by different leukemic cell lines, leukemic clinical isolates, and normal bone marrow and peripheral blood mononuclear cells. DiO, 3,3'dioctadecyloxacarbocyanine perchlorate (Molecular Probes) is a cationic lipophilic probe which emits green fluorescence when excited by 489 nm and exhibits negligible dye transfer properties. It was used in this study to mark murine leukemic (L1210) cells incubated with BPD to distinguish them from normal mouse bone marrow cells also incubated with BPD subsequent to FACS on the basis of red fluorescence [10].
MATERIALS AND METHODS
Spectrofluorometric analysis Prior to spectrofluorometric analysis, BPD was dissolved in phosphate buffered saline (PBS) at a concentration of 2 ~tg/ml resulting in an absorbance reading of 0.10 O.D. units at 420 nm. Excitation and emission scans were performed using an SLM 500 C spectrofluorometer in a wavelength acquisition mode with an excitation bandpass of 0.5 nm, an emission bandpass of 20 nm, and a step size of 0.5 nm. The emission reference utilized during excitation scans of BPD was 690 nm. The maximal fluorescence excitation peak of BPD was used to determine the maximal fluorescence emission peak during the fluorescence emission scans.
FA CS analysis Cell lines. Cell lines were maintained in phenol-red free Dulbecco's Modified Eagle's (DME) media supplemented with 10% fetal calf serum (FCS) in a 10% CO2 incubator at 37°C and were subcultured according to American Type Culture Collections (ATCC) specifications. Cell lines analyzed included: L1210, a mouse lymphocytic leukemia HL60, a human acute promyelocytic leukemia, and K562, a human chronic myelogenous leukemic cell line. Patient samples. Mononuclear cells were extracted from leukemic clinical isolates, normal human bone marrow and human peripheral blood (collected in heparinized tubes) over Ficoll-Hypaque (Pharmacia) density gradients. Briefly, whole blood was diluted by a factor of two in PBS and layered over 3 ml of Ficoll-Hypaque. The mononuclear band, containing all mononuclear white blood cells, was collected subsequent to a 10 min centrifugation period at 1500rpm. Cells were washed three times in PBS to
remove Ficoll-Hypaque. Mononuclear cells were analyzed fresh or subsequent to cryopreservation in DME media containing 10% dimethyl sulfoxide (DMSO) and 40% FCS. Vials containing cryopreserved cells were warmed quickly in a 37°C water bath and rinsed with ethanol. Cryopreserved cells were then diluted by a factor of ten in PBS and washed three times in PBS to remove DMSO. Mouse bone marrow and spleen preparation. Bone marrow was extracted from 6-8-week old DBA/2 female mice which were euthanized by CO2. The femur and tibia were cleaned of muscle using sterile forceps and scissors and transferred to sterile PBS where bone marrow was flushed from the bones and aspirated with a 25 gauge needle to form a single cell suspension. Bone marrow cells were centrifuged and washed in sterile PBS and viability counts performed. Spleens were removed asceptically from the same mice used for bone marrow extraction and passed through a wire mesh to create a single cell suspension. Bone marrow or spleen cells were cryopreserved or analyzed fresh. FACS parameters. Before FACS analysis, cells were incubated in the dark in a 10% CO2, 37°C incubator with BPD-monoacid ring A (BPD-MA), BPD-monoacid ring B (BPD-MB), BPD-diacid ring A (BPD-DA), BPD-diacid ring B (BPD-DB), or DiO for 30 min in phenol red-free DME media in the absence of FCS. Cells were washed in PBS to remove excess photosensitizer, centrifuged at 1100 rpm and resuspended in phenol red-free DME. The excitation wavelengths employed for FACS analysis involving BPD were 351.1-363.8 nm (ultraviolet light) and 488 nm (visible light) and a 590 nm emission (longpass) filter was utilized to detect BPD (red) fluorescence. The excitation wavelength used for cells incubated with DiO was 488 nm and a 530 nm emission filter (515-545 nm) was used to detect DiO (green) fluorescence. Both dual and quantitative single parameter FACS analyses were performed using a Becton-Dickinson FACS 440 flow cytometer equipped with both a visible light laser and an ultraviolet (u.v.) light laser.
RESULTS
Spectrofluorometric analysis Spectrofluorometric excitation and emission scans revealed that B P D ( B P D - D A ) (Fig. 1) is maximally excited by 420 nm (Fig. 2) and excited to a lesser extent by 356 nm (u.v.), resulting in a major fluorescence emission peak at 690 nm (Fig. 3). The monoacids are excited to fluoresce to a greater extent by ultraviolet light (data not shown).
Fluorescence comparison of four structural analogues of BPD In order to determine whether the four structural analogues of B P D differed in their fluorescent properties in the presence of cells, HL60 cells were incubated with 5, 10, or 20 t~g/ml B P D - M A , BPD-MB, B P D - D A , or B P D - D B for 24 h. Fluorescence emitted by cells subsequent to either u.v. or visible light excitation was measured via quantitative FACS analysis and demonstrated that although negligible
Preferential uptake of benzoporphyrinderivative by leukemicversus normal cells H3C~._
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211
studies correlate well with observed differences in photosensitizer activity of the four analogues. In a number of cell lines studied, the monoacid forms of BPD were significantly more cytotoxic than the diacid forms, and these differences were related to selective uptake [12, 13].
H3¢ RI= Rz= C02Me
R3= (CH2)2COzMe or (CH2)2CO2H
FIG. 1. Structure of BPD. Four structural analogues of BPD have been synthesized: BPD-MA, BPD-MB, BPDDA, and BPD-DB. The monoacids differ from the diacids at the R3 position in that in the monoacids one of the carboxylic acid groups has been esterified to form a methyl ester. Ring A or B refers to the benzene derivative on the left (ring A) or the right (ring B) of the porphyrin ring.
differences in mean red fluorescence emitted existed at 488 nm (Fig. 4) when excited by u.v. light, cells incubated with B P D - M A fluoresced to the greatest extent followed by those incubated with BPD-MB, B P D - D A , and B P D - D B respectively (Fig. 5). These
t
300
FA CS analysis o f leukemic cell lines versus normal bone marrow incubated with B P D Therefore, B P D - M A was used in subsequent FACS analyses to determine if substantive differences existed between normal and leukemic cells with regard to BPD uptake as judged by mean reflective fluorescence intensity. Normal human bone marrow mononuclear cells incubated with B P D - M A for 30 min fluoresced substantially less in the red spectrum subsequent to u.v. light excitation than human leukemic cell lines (HL60 and K562) or mouse leukemic cells (L1210) incubated under the same conditions (Fig. 6). Similarly, normal mouse bone marrow or spleen cells cells fluoresced to a lesser extent than murine leukemic cells (L1210) (Fig. 7).
10.0
400 500 excitation wavelength scan (nm) FIG. 2. Spectrofluorometric analysis of BPD in PBS-excitation wavelength scan. The emission reference employed was 690 nm. A slit width of 0.5 nm was used and fluorescence excitation was scanned between 300 and 600 nm.
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CATRIONAH. M..[AMIESONet al.
212
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500
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600
700
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800
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FIG. 3. Spectrofluorometric analysis of BPD in PBS--emission wavelength scan. The excitation reference employed was 420 nm, which corresponded to the Soret peak of the porphyrin, and fluorescence emission was scanned between 500 and 800 nm.
The results shown in these figures illustrate representative data from individual experiments which have been repeated a n u m b e r of times. HL60, a human cell line representing promyelocytic leukemia was chosen for these purposes because its size more closely resembles the normal cell types found in blood and bone marrow, and therefore would minimize differences in fluorescence contributed by size. K562 is a human chronic myelogenous leukemic cell line, composed predominantly of blasts. L1210 is a T-cell leukemia of D B A / 2 mice and was chosen because this tumor line is being used in continuing research in bone marrow purging in an animal model.
Comparison of BPD uptake by leukemic and normal bone marrow To ensure that the observed differences in uptake of B P D - M A between leukemic cells and normal leu-
kocytes were not associated solely with leukemic cell lines; acute myelogenous leukemia clinical isolates and normal h u m a n peripheral blood or bone marrow mononuclear cells were subjected to FACS analysis subsequent to incubation with B P D - M A . Clear differences were recorded between normal peripheral blood or bone marrow and A M L mononuclear cells in terms of B P D - M A - m e d i a t e d fluorescence, although these differences were not as pronounced as those observed with leukemic cell lines. Significant differences in fluorescence between normal and leukemic cells were not observed when cells were incubated with BPD-DA (Student's t-test 0.05 < p ~< 0.1). Representative results are shown in Fig. 8, and a s u m m a r y of all data is given in Table 1. To ensure that differences in fluorescence between normal and leukemic cells were not solely a consequence of differences in size, cell size, as measured
Preferential uptake of benzoporphyrin derivative by leukemic versus normal cells FAC$ Analvsis of BPD-MA, -MB, -DA, -DB (488 nm} 102
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BPD 488 nm excitation. HL60 cells were incubated for 24 h with 0 (control), 5, 10, or 20 ~tg/ml BPD-MA, BPDMB, BPD-DA, or BPD-DB. The excitation wavelength used for FACS analysis was 488 nm. A 550 nm longpass filter was used to detect red fluorescence characteristic of BPD. As in all subsequent experiments involving FACS analysis, 10,240 cells were analyzed per sample.
FACS ANALYSIS
OF BPD-MA,
- MB, - DA, - DB (uv)
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FIG. 5. FACS analysis of the four structural analogues of BPD--u.v. excitation. HL60 cells were incubated as mentioned previously, however, u.v. light (351.1363.8 nm) was used as the excitation wavelength.
213
214
CATRIONAH. M. JAMIESONet al. 120 • "o c
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FIG. 6. Comparison of BPD uptake by normal bone marrow versus leukemic cell lines. Duplicate samples of 1 x 106 cryopreserved human monuclear cells extracted from bone marrow were incubated for 30 min with 0 (control) or 10 ~tg/ml BPD-MA, as were the same number of HL60, K562, and L1210 cells. Mean red fluorescence emitted by these cell populations was compared via FACS analysis subsequent to u.v. excitation and BPD fluoresence detected with a 590 nm longpass filter as in all subsequent FACS analyses. Background refers to the level of fluorescence emitted by cells incubated in the absence of the drug.
T A B L E 1.
FACS ANALYSIS O F CELLS I N C U B A T E D WITH BPD-MA: M E A N F L U O R E S C E N C E -- B A C K G R O U N D
5 ~tg/ml -+ S.E. Normal cells murine bone marrow spleen cells human PBL bone marrow Leukemic cells murine L1210 human K562 HL60 clinical samples AML CML
10 ~tg/ml --_ S.E.
No. of samples
13.75 --+5.44 9.79 --- 1.47
2 2
10.15 -+ 1.47
12.53 -+ 1.61" 5.23 + 1.50
10 7
70.74 -+ 13.72
122.19 +- 6.67
10
43.43 + 1.41
53.21 +- 13.12 86.30 +- 28.45
3 4
29.18 -+ 0.20
22.98 -+ 0.27 37.28 -+ 3.32
2 10
* Mean fluorescence of human PBL incubated with 10 ktg/ml BPD-MA was compared to that of CML cells by the Student's t-test (p ~< 0.0005), to AML cells (p < 0.016), to HL60 ceils (p ~
~8
~
FIG. 10. Dual parameter FACS analysis of L1210 and mouse spleen cells. L1210 cells (1 x 106) incubated, as described above, with 10 ~tg/ml BPD-MA and 100 ~tg/ml DiO were mixed immediately before FACS analysis with 1 x 106 mouse spleen cells incubated with 10 ~tg/ml BPDMA. Total events refers to the number of cells analyzed (10,240). Cells were analyzed for DiO (green) fluorescence (y-axis) using a 530 nm filter subsequent to 488 nm excitation and also analyzed for BPD fluorescence, subsequent to u.v. excitation, but utilizing a 590 nm longpass filter.
in red fluorescence subsequent to u.v. excitation. Cells were sorted into ten fractions and each fraction was reanalyzed for green (DiO) fluorescence via 488 nm excitation and the use of a 530 nm filter
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2
3
4
5
6
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8
9
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FIG. 11. Sorting of L1210 cells from mouse spleen cells on the basis of differences in BPD fluorescence. The ceils described above were sorted into ten fractions with approximately 1,000 cells per fraction on the basis of differences in BPD (red) fluorescence and reanalyzed for DiO (green) fluorescence (y-axis) using a 530 nm filter. The graph represents two experiments. There were significant differences in green fluorescence between fractions 1 and 10 as judged by the Student's t-test (p = 0.003).
217
to determine the extent of contamination of each fraction by leukemic cells, the argument being that only the strongly red fluorescing cells (i.e. L1210) should also show strong green fluorescence since only they were loaded with DiO (Fig. 11). Because only L1210 cells were incubated with DiO and DiO exhibits negligible dye transfer properties, green fluorescence should only be emitted by leukemic cells and so a way of distinguishing normal from leukemic cells in the normal sorted pool is provided. Pronounced differences existed between the extent of green fluorescence in fraction 10 compared to fraction 1 suggestive of a large proportion of leukemic cells in fraction 10 compared to fraction 1. Fluorescent microscopic examination of cytospins of fractions 1 and 10 revealed that no green fluorescing cells existed in fraction 1, whereas fraction 10 was composed solely of bright green fluorescing cells subsequent to blue light excitation (data not shown). Obviously, this preliminary experiment did not establish protocols for purging procedures but does indicate a justification for further work on this model.
DISCUSSION When BPD analogues are excited to fluoresce in the red spectrum by u.v. light (351.1-363.8 nm) and 420 nm, a powerful signal is emitted. Use of these parameters in the Becton-Dickinson FACS 440 flow cytometer has enabled us to distinguish differential uptake of these dyes by malignant versus normal hemopoietic cells in both murine and human systems. The most extensive work involving selective uptake of malignant hemopoietic cells by photosensitizers is that of Dr Sieber and his associates working with merocyanine 540 [3, 8]. These investigators have shown selective uptake and phototoxic killing of malignant cells in bone marrow samples of patients with advanced lymphomas. Similarly, Edelson and colleagues have shown that a light activated psoralen, known as 8-MOP can be used to treat, extracorporeally, the blood of patients with cutaneous T-cell lymphoma with concomitant activation with u.v. light [13]. BPD-MA and its analogues are photosensitizers derived from hematoporphyrin which show promise as agents for the photodynamic therapy of solid tumors, in that they accumulate somewhat selectively in tumor tissue, are readily activated by light at 690 nm to cause singlet oxygen release, and have been shown to be effective in eradicating tumors in a murine model [11, 12]. The present work constitutes a preliminary study to evaluate the capacity of BPD to accumulate selectively in leukemic cells, with the
218
CATRIONA H. M. JAMIESON et al.
long term goal of testing its potential in selective killing of malignant hemopoietic cells. When u.v. light excitation was used to compare uptake of the four structural analogues of B P D : B P D MA, BPD-MB, B P D - D A , and B P D - D B , HL60 cells incubated with B P D - M A clearly fluoresced to the greatest extent. This observation correlates with earlier studies which indicated that B P D - M A was a more potent photosensitizer than the other structural analogues of BPD, when the analogues were tested for phototoxicity with a variety of cell lines [11, 12]. We have examined, not only leukemic cell lines, but also both normal bone marrow and peripheral blood, and leukemic cells from a variety of donors and patients. The results (summarized in Table 1) attest to our statement that leukemic cells selectively take up three to six fold more B P D - M A than do normal cells derived from bone marrow, spleen and peripheral blood. The results from one FACS analysis to another are surprisingly consistent. The fact that the second fluorescent dye used in these experiments, DiO, does not show this selective accumulation (since leukemic and normal cells took up essentially the same amount of the dye), attests to the selective nature of B P D - M A rather than to inherent differences in malignant versus normal cells. The use of dual labelling with B P D - M A and DiO of leukemic cells provides some preliminary evidence that the differences in BPD-mediated red fluorescence exhibited between normal and leukemic cells may be utilized in a novel form of purging remission marrow of residual leukemic cells via FACS. However, these results are very preliminary. The observations presented here provided a base for ongoing research on the possible application of this apparent selectivity in BPD uptake by leukemic as opposed to normal hemopoietic cells. Clearly, experiments must be performed to determine whether selective uptake translates into selective photoxic killing of malignant versus normal stem cells. It will be necessary to establish that this selective difference applies to self-renewing cells as well as other more mature hemopoietic populations. An in v i v o reconstitution model is currently being developed in which sorted populations of murine bone marrow cells mixed with L1210 cells will be used to reconstitute lethally irradiated D B A / 2 mice. These studies should reveal not only the presence of stem cells in the " n o r m a l " population but also the extent of contamination of the stem cell containing fraction with tumor cells, since between 1 and 10 L1210 cells will cause lethal ascites in recipients. At the same time, the potential use of BPD as an in vitro bone marrow purging agent through selective photosensitization will be followed in both the murine
model, and with human cells with the aid of in vitro colony assays (both C F U - G M and long term stem cell culture). Preliminary studies indicate that the LDs0 of progenitors from C G L patient samples is lower (in B P D - M A dosage) than that for normal C F U - G M progenitors (ongoing research). This study provides preliminary evidence that substantial differences exist in the uptake of BPDMA by leukemic versus normal cells and lays the ground work for developing a novel extra-corporeal purging protocol for autologous bone marrow transplantation. The mechanism of differential uptake has yet to be elucidated and is currently under investigation in this laboratory. Acknowledgements--This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (5-80268). CHMJ is the recipient of a B.C. Science Council GREAT Scholarship. The authors gratefully acknowledge the expert advice and technical assistance provided by Denise MacDougal, Lindsay Eltis, Dr Frank Tufaro and Dr Anna Richter.
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13. Yemul S., Berger C., Estabrook A., Suarez S., Edelson R. & Bayley H. (1987) Selective killing of T lymphocytes by phototoxic liposomes. Proc. natn. Acad. Sci. U.S.A. 84, 246. 14. Edelson R. (1987) Treatment of cutaneous T-cell lymphoma by extracorporal photochemotherapy: preliminary results. New Engl. J. Med. 316, 297. 15. Gulliya K. S., Fay J., Dowben R. M., Berkholder S. & Matthews J. L. (1988) Elimination of leukemic cells by laser photodynamic therapy. Cancer Chemother. Pharmac. 22, 211.
