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Photodynamic Treatment of Normal Endothelial Cells or Glioma Cells In Vitro Marie-Thdr~se Foultier, Ph.D., Thierry Patrice, M.D., Sergio Yactayo, M.D., Youenn Lajat, M.D., and Frangois Resche, M.D. Laser Department and Department of Neurosurgery, H6pital Laennec, Nantes, France
Foultier M-T, Patrice T, Yactayo S, Lajat Y, Resche F. Photodynamic treatment of normal endothelial cells or glioma cells in vitro. Surg Neurol 1992;37:83-8.
Photodynamic therapy is based on the interaction of a sensitizer (hematoporphyrin derivative) selectively retained by tumor cells, which becomes toxic after light exposure. We studied the influence of exogenous prostaglandins and indomethacin on photodynamic therapy of normal human endothelial cells and glioma cells. Although differing in origin and kinetic properties, endothelial cells exhibited photodynamic therapy sensitivity quite comparable to that of C6 cells. However, in contrast to studies performed using radiotherapy, exogenous prostaglandins decreased rather than protected the surviving fraction of both cell types treated by photodynamic therapy. Indomethacin, a potent inhibitor of endogenous prostaglandin synthesis, increased the surviving fraction of C6 glioma cells but not that of endothelial cells. Exogenous or endogenous prostaglandins seem to influence in vitro photodynamic therapy in a different way than does radiotherapy. KEY WORDS: Photodynamic therapy; Hematoporphyrin derivative; Glioma cells; Endothelial cells; Prostaglandins
Photodynamic therapy (PDT) mediated by hematoporphyrin derivative (HPD) is now undergoing phase I clinical trials in various medical fields, including neurosurgery [6,21]. A complete review of P D T applications in neurosurgery has been done by Kaye et al [19]. The efficiency of P D T has been related to the preferential localization of a sensitizer (HPD) in neoplastic tissues in vivo [12,13]. This photosensitizer is then able to destroy tissue selectively when activated by light of an appropriate wavelength by producing an excited and toxic form of oxygen (singlet oxygen lo2 [341. HowAddre.~s reprint requests to: Thierry Patrice, M.D., Laser Department/Laboratoire de Physiologie, Facultd de Pharmacie, 1 Rue G Veil, 44035 Nantes Cddex, France. Received September l 1, 1989; accepted July 15, 1991.
~) 1992 by Elsevier Science Publishing Co., Inc.
ever, in vivo, several authors have pointed out that not only tumor tissue but also the neovasculature of tumors can be damaged early [2,28,29]. Anoxia then supposedly contributes to tumor growth control. However, neither the mechanism of endothelial cell sensitivity to PDT nor the parameters influencing such sensitivity have, to our knowledge, been investigated. One aim of our study was to evaluate whether vessel occlusions occurring after H P D - P D T could be accounted for by a special sensitivity of endothelial cells to PDT. Verification of this point is essential to determine whether further experimental P D T studies should be focused on vessel endothelium or on cancer tissue itself. A second aim of our work was to study the sensitivity of endothelial cells (slowly dividing and known to secrete large amounts o f prostaglandins) in the presence or absence of exogenous prostaglandin E (PGE) or indomethacin, a potent inhibitor of endogenous prostaglandin synthesis. These drugs influence the cyclic adenosine monophosphate (cAMP) content of cells and have been shown to decrease cell sensitivity to radiotherapy [16,25] and to interfere with the rate of cell replication [31]. All our experiments on endothelial cells have been performed in comparison with murine C6 glioma cells that strongly differ from endothelial cells in their origin and kinetics but are of similar cell size (a parameter that influences cell photosensitivity [3] and adhesiveness). C6 cells routinely used in experiments concerning neurosurgical oncology were included in this work as a reference to evaluate endothelial cell sensitivity to PDT.
Materials and Methods
Chemicals The PGEI synthetic analogue misoprostol was kindly provided by Searle Research Development (Skokie, Ill.). Misoprostol purity was greater than 99%, as determined by high-pressure chromatography. The shelf life ofmisoprostol was 6 to 12 months at 0°C. Indomethacin was obtained from Sigma Chemicals (U.K.). 0090-3019/92/$5.00
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Hematoporphyrin derivative was prepared according to the method of Gregorie et al [14] and Lipson et al [22] from hematoporphyrin hydrochloride (Roussel, Basel, Switzerland). The p H of the ready-to-use solution was adjusted to 7.2 to 7.4 by the addition of hydrochloric acid solution and made isotonic by the addition of sodium chloride. The final 5-mg/mL solution was sterilized by filtration through a Millipore filter and kept in darkness until used.
at room temperature and shaking. PGEI was dissolved at the final concentration in RPMI 1640 without the addition of FCS or phenol. Indomethacin was added to cells 24 hours before laser irradiation or immediately after, at a final concentration of 10 -4 to 10 ~' M, and for some experiments of 10 4 to 10 -r M. Incubation times were determined from literature data [35] but checked in our own experimental conditions during broad preliminary screening (data not shown).
Cells and Cell Culture
PDT experiments. Hematoporphyrin derivative was added to the medium at a final concentration of 0.5, 0.75, and 1/zg/mL. In a first series o f experiments, the influence of phenol red was studied. This strongly colored substance is routinely added to cell media as a p H indicator (10 rag/L). However, as phenol red is tightly bound to cell estrogen receptors [11], it was considered to have absorbed the b l u e - g r e e n laser light, particularly if cell washing was not feasible before P D T (because of the presence of PGE~ or indomethacin). Light illumination was performed using an all-linesemitting argon laser (SP.770, Spectra Physics, Palo Alto, Calif.) after a 2-hour incubation time (allowing a sufficient intracellular concentration for PDT experiments [1]). The beam was transmitted through a silicasilicon step index optical fiber with a 400-~zm-diameter core, giving a divergence of 8 °. Power (3 W) was calibrated using a T R G energy meter (Palo Alto, Calif.) to obtain 50 J/cm 2 energy density. Distance from the end of the optical fiber to the bottom of the well was 60 mm, giving a spot diameter of 16 mm (equal to the diameter of the well).
Endothelial cells were obtained from human umbilical cord vein and identified according to Jaffe et al [18]. Briefly, the cord was severed from the placenta soon after birth and placed in sterile cord buffer (0.14 M NaCl, 0.004 M KCI, 0.011 M glucose, 0.001 M phosphate, p H 7.4) at 4°C until processing (maximum delay 24 hours). After cannulation, the umbilical vein was perfused with 80 mL of cord buffer before being infused with 10 mL o f 0.2% collagenase (CLS I Worthington, Intermed, France) in cord buffer. After a 12-minute incubation, endothelial cells and collagenase solution were flushed out with 30 mL of cord buffer containing bovine serum albumin (0.4 mg/mL). The effluent was collected with 10 mL of RPMI 1640 medium. Cells were centrifuged at 2000 rpm for 10 minutes before seeding in culture medium in 25-cm 2 culture flasks (Falcon®). Cells were allowed to grow to confluence (6 days) in RPMI 1640 supplemented with 20% fetal calf serum (FCS) and glutamine (2 mM). C6 murine glioma cells [4] (ATCC CCL 107), obtained at 41 to 51 culturing passages from Flow Laboratories (Puteaux, France), were maintained in RPMI 1640 supplemented with 20% FCS and 2 mM glutamine. C6 cells were trypsinized (trypsin 0.025%, 0.2% EDTA) and aliquoted in two flasks every 4 days when confluenced.
Methods The different cell types were aliquoted after trypsinization (trypsin 0.05%, EDTA 0.02%) in 24-well plates (0.5 mL/well) at a cell concentration of 5.104/mL (as measured by a hemocytometer) in RPMI 1640 with or without phenol red according to the experiments, and containing 20% FCS, antibiotics, and antifungics. Twenty-four hours later, after a change of medium, the different chemicals were added. PGEI was stored as an ethanol stock solution of 1 mg/mL and frozen. The ready-to-use solution in e t h a n o l - - p H 7.4 buffer (NaH2PO4, Na2HPO4 : 1/7), at a concentration of 100 lzg/mL--was stored at 20°C until used. It was added to cells 3 hours before laser irradiation, after soft warming
Controls. Controls receiving either H P D alone, laser alone, or neither were obtained for both cell types. The influence of PGE1 or indomethacin alone has also been checked for both our cell types. Generally, each experimental condition was replicated four times. Estimation of cell death. Immediately after laser irradiation, cells were returned to the incubator for 24 hours. Cell survival was calculated by the trypan blue exclusion test according to a modification of the method by Philips [24]. At the time of counting, the medium was released and cells were washed, trypsinized (4 minutes) with 0.1 mL trypsin/EDTA solution, and resuspended in RPMI 1640 with FCS. Trypan blue in phosphate-buffered saline solution was then added to give a final dye concentration of 0.18%. Before addition of trypan blue, cell suspension was checked by optically inverted microscopy. Trypan-blue-excluded cells were counted as alive. Results are expressed as a percentage of control cells receiving no treatment of any kind.
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no effect on endothelial or C6 cell survival (data not shown).
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Figure 1. Hematoporphyrin derivative-photodynamic treatment of C6 glioma cells (1) or endothelial cells (2). Results are the mean percentage of four separate experiments guntreated control cells = 100%~. Cells were incubated with RPMI 1640 containing phenol red [] or not ~ . Phenol red 10 mg/L increased significantly (p < 0.01) the surviring fraction of both cell types,
Figures 2 and 3 show the effect o f 5 or 10/*g o f misoprostol per milliliter when added to culture medium 1 hour before H P D . The percentage of surviving C6 or endothelial cells was significantly (p < 0.01) decreased in the presence o f misoprostol. PGEI influence on P D T was found for all H P D doses used. C6 cells incubated with misoprostol but not subjected to P D T were significantly (p < 0.001) more affected than endothelial cells in similar conditions as compared with controls. This inhibition of cancer cell growth by misoprostol alone was confirmed by sophisticated flow cytometry D N A analysis [23].
Effect of Indomethacin on P D T Results
Effect of H P D - P D T on Endothelial or C6 Glioma Cells A close relationship was found between cell death anti the H P D concentration used for 50 J/cm 2. C6 and endothelial cells exhibited a highly comparable sensitivity to H P D - P D T . Figure 1, which shows photodynamic experiments performed in the presence or absence o f phenol red in culture medium, indicates a significantly greater sensitivity o f both cell types when irradiation was done in the absence o f phenol red (p < 0.01). Hematoporphyrin derivative alone or laser alone had
While indomethacin alone had only a slight stimulating effect on C6 cells [23], indomethacin added 24 hours before laser irradiation gave a 30Uc improvement of C6 cell survival as compared with controls. This effect was found for 10 • and 10 ~ M indomethacin concentrations and for both 0.75 and 1 tzg/mL H P D doses (Figure 4). In contrast, 10 ~' M or 10 : M indomethacin had no effect on the surviving fraction o f endothelial cells when added before the addition of 0 . 7 5 / , g / m L H P D . H o w ever, when indomethacin was added to endothelial cells before a 1-tzg/ml H P D dose, it decreased (p < 0.01) the surviving fraction o f endothelial cells regardless o f the dose used (Figure 5). Indomethacin had no effect when
Figure 2. Surviving fraction of endothelial cells after photodynamic treatment. Cells were incubated with 0.5, 0.75, and 1 tzg/mL HPD after preincubation (1 hour) with misoprosto/ (ffA) (i) 5 tzg/mL or (2) 10 tzg/mL or uithout (~). Laser irradiation (25 J/cm2~ u'as performed after 2-hour incubation with H P D and misoprostol. The effect of mi.~oprostol alone on endothelial celk is represented b3, the separate bar ¢ffJ?.
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Figure 4. Surviving fraction of C6 glioma cells incubated with indomethacin for 24 hours before H P D addition and photodynamic therapy. Indomethacin doses ranged between 10 -7 and 10 ~ M, HPD concentration was 0.75 tzg/mL ( ~ ) or I ~g/mL (~). The laser dose was 50 J/cm ~, Indomethacin increased significantly the surviving fraction of C6 cells regardless of the H P D dose.
Figure 6. Surviving fraction of endothelial cells after hematoporphyrin addition (0.75 or 1 ~g/mL) and laser irradiation (50 J/cm:). Indomethatin I0 ~'. 10 5 or 10 4 M was added to cells immediately after laser irradiation. In these conditions, indomethacin had no influence on HPDPDT.
added to C6 or endothelial cells immediately after PDT (Figures 6 and 7).
ble for the in vivo tumor-growth inhibition. To investigate this last point, it seemed of interest to us to determine whether endothelial cells (one of the essential determinants of tumor blood flow) exhibit special photosensitivity to PDT as compared with routinely cultured cancer cells. Endothelial cells from human umbilical cord are often used in experiments in physiology [9]. As these cells retain their capacity to divide until confluence is reached [17], they may be considered to mimick as closely as possible endothelial cells of tumor neovasculature. Endothelial cells and C6 glioma cells, although of
Discussion Photodynamic therapy is now under phase I clinical trials in various medical fields. It has been shown to act both on solid glial tumors [2,7] and cultured glioma cells [13,18]. However, the mechanism of tumorgrowth inhibition remains unclear. Several authors have suggested that vascular occlusions occurring after P D T and evidenced experimentally [2,28] could be responsi-
Figure 5. Surviving fraction of endothelial cells incubated with indomethacin and HPD in similar conditions to those described in Figure 4. When the H P D dose was 1 ~*g/mL, indomethacin decreased the sum,iving fraction," however, there was no effect with the 0.75-HPD dose ([]).
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Figure 7. Surviring fraction of HPD-treated C6 cellJ (as described in Figure 6) receiving indomethacin immedMtely after laaer irradiation. In these conditions, indomethacin had no influence on HPD-PDT. 100
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Photodynamic Treatment
very different kinetics (thus reproducing to some extent the in vivo situation) and of different origin, were of very similar size, as measured by flow cytometry in endothelial and C6 co-culture (data not shown). This parameter has been shown to be of importance for cell sensitivity to P D T [2]. However, endothelial cells exhibited a similar photosensitivity to that of C6 cells, which could explain why microvasculature damage occurs after P D T [2,28,29]. Nonetheless, endothelial cell sensitivity was not greater than that ofglioma cancer cells, suggesting that the control of tumor growth in vivo was only partially due to tumor anoxia resulting from destruction of endothelium. Furthermore, cell doubling time seemed to have little influence on cell photosensitivity, at least in our experimental conditions. Photosensitivity of endothelial cells as compared with C6 glioma cells was not consistent with observations in radiotherapy [5]: rapidly dividing cells (C6 doubling time 16 hours) were no more sensitive than slowly dividing endothelial cells (doubling time 68 hours). The presence of phenol red, although noted only rarely in the Materials and Methods sections of papers on in vitro PDT, is not without effect on survival curves of C6 or endothelial cells. This point required checking in our experiments, since cell washing could not be performed when there was drug addition (ie, prostaglandins) in culture media. Prostaglandins have been shown to protect cells as well as tissues from damage induced by radiotherapy [16,25,33] in the course of peptic ulcer disease [26,30]. One possible mechanism for this is that the increased cAMP level induced by PGEI incubation may be inversely related to cell radiosensitivity [16]. Thus, it could be of interest to protect endothelial cells in vivo from PDT in order to maintain efficient blood flow after PDT. Unfortunately, in our in vitro experiments, the PGE, synthetic analogue misoprostol (5 or 10/.tg/mL) failed to protect endothelial cells from damage induced by H P D - P D T . The in vivo situation is now under investigation in our laboratory. Similar results were obtained for C6 cells. However, in control groups it was noted that prostaglandin alone was able to decrease (proportionately to the dose) the number of C6 cells counted after the trypan-blue exclusion test, whereas its influence in reducing the number of endothelial cells was only slight. These results were confirmed by sophisticated flow-cytometry analysis [23]. Such inhibition of cancer cells after incubation with prostaglandin has been previously described [28], particularly for glioma cells [35], but to our knowledge the lack of responsiveness o f endothelial cells to prostaglandin has never been reported.
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These results were not totally opposed to those obtained by Farrell et al [10], who showed that ibuprofen (a nonsteroidal antiinflammatory agent) treatment of rats implanted with C6 glioma cells reduced the size of tumors, suggesting that prostaglandin-associated angiogenesis could be of importance for tumor growth. Furthermore, the results of Farrell et al in vitro concerning C6 cell line were in full agreement with ours. lndomethacin, a powerful inhibitor of endogenous prostaglandin synthesis [32], increased the percentage of surviving C6 cells after P D T but decreased this percentage for endothelial cells incubated with 1 /,g/mL H P D or had no effect with lower H P D doses. These dissociated effects imply that cell response to PDT differs according to the type of cell and perhaps to the severity of cell injury induced. Finally, the effect of indomethacin on C6 cells is another factor contradicting the hypothesis developed in radiotherapy studies that P D T efficiency would be better after a decrease in cell prostaglandin content. Moreover, indomethacin seems to influence PDT in a different way compared with glucocorticoids [8]. All of this suggests that prostaglandins do not have the same influence on cell death induced after P D T as they do after radiotherapy. They apparently have no influence on cell repair, since indomethacin seems to act only when added to cells before PDT.
Conclusion Endothelial cells and C6 glioma cells exhibit very similar sensitivity to PDT, suggesting that another mechanism other than special endothelial cell photosensitivity must account for the microvasculature damage observed in vivo. It is of importance to protect endothelial cells from PDT in order to permit the use of PDT in the course of combined treatments that often require sufficient blood flow. Unfortunately, PGEI analogue has failed to provide such protection. Research for such protectors should remain an essential aim for the future of PDT.
This study was supported by grant CRE INSERM N. 87 9013 and ARC No. 6727. We also wish to thank the Notre Dame de Graces Clinic fi)r umbilical cord supply and Dr. J. Gray fi~r English translation.
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