resistance to photodynamic therapy in radiation induced fibrosarcoma-1 and chinese hamster...

Photochemistry and Photobiology Vol. 54, No. 2, pp. 307-312, 1991 Printed in Great Britain. All rights reserved

0031-8655191 $03 .oO+O.OO Copyright 0 1991 Pergamon Press plc

RESEARCH NOTE

RESISTANCE TO PHOTODYNAMIC THERAPY IN RADIATION INDUCED FIBROSARCOMA-1 AND

CHINESE HAMSTER OVARY-MULTI-DRUG RESISTANT CELLS in vitro

GURMIT sINGH',3, BRIAN c. WILSON''4*, SHEILA M. sHARKEY3, GEORGE P. BROWMAN''2 and PAULA DESCHAMPS3

'Hamilton Regional Cancer Centre and McMaster University Departments of ZMedicine, 3Pathology and 4Radiology, Hamilton, Ontario, Canada L8V 1C3

(Received 29 October 1990; accepted 1 February 1991)

Abstract-A degree of resistance to photodynamic therapy (PDT) has been induced in radiation- induced fibrosarcoma-1 (RIF-1) tumor cells by repeated photodynamic treatment with Photofrin (4 or 18 h incubation) in vitro to the 0.1-1% survival level, followed by regrowth from single surviving colonies. The resistance is shown as increased cell survival in the strain designated RIF-gA, compared to the wild-type RIF-1 cells, when exposed to increasing Photofrin concentration for 18 h incubation and fixed light exposure. No difference was found between RIF-1 and RIF-8A in the uptake of Photofrin per unit cell volume at 18 h incubation.

Resistance to PDT was also observed in Chinese hamster ovary-multi-drug resistant (CHO-MDR) cells compared to the wild-type CHO cells, possibly associated with decreased cellular concentration of Photofrin in the former. By contrast, the PDT-resistant RIF-8A cells did not show any cross- resistance to Adriamycin, nor was there any significant drug concentration difference between RIF-1 and RIFdA. These findings suggest that different mechanisms are responsible for PDT-induced resistance and multi-drug resistance.

INTRODUCTION

Photodynamic therapy (PDT)?, based on photoactivation of the photosensitizer Photofrin (Quadra- logic Technologies, Vancouver, BC, Canada), is currently undergoing clinical trials for a variety of solid tumors (Marcus, 1990; Dougherty, 1988; Dougherty et al., 1990). There are also numerous pre-clinical studies in progress with new photosensitizers with improved biochemical, pharmaco- logical or photobiological properties (as reviewed by Morgan and Skalkos, 1990). In spite of the considerable body of clinical and preclinical experience with this form of treatment, the basic mechanisms of tumor cell and tumor tissue destruction are only partially understood (Gomer et al . , 1990; Hender- son, 1990a,b). Studies of PDT mechanisms are com- plex because, in vitro, the subcellular targets for PDT damage depend on both the photosensitizer used and the treatment conditions, and the tumor response in vivo may have contributions from both

*To whom correspondence should be addressed, at: Dr. Brian C. Wilson, Hamilton Regional Cancer Centre, 711 Concession Street, Hamilton, Ontario, L8V 1C3. [Tel. 416-387-9495; Fax 416-575-6330]

TAbbreviations: CHO, Chinese hamster ovary; CTAB, cetyltrimethylammoniumbromide; FCM, flow cytometry; MDR, multi-drug resistant; PDT, photodynamic therapy; RIF, radiation induced fibrosarcoma.

direct tumor cell phototoxicity and indirect tumor cell kill due to damage to the vasculature of the tumor tissue or tumor bed (Henderson and Fingar, 1987; Henderson, 1990a,b; Selman et al., 1984).

Historically, induction of resistance to treatment has been a fruitful approach to the study of mechanisms of action of many antineoplastic drugs and physical agents such as hyperthermia, as reviewed by Morrow and Coman (1988). By analogy, it is hypothesized that comparative studies using PDT- sensitive cells and resistant cells derived from survivors of PDT treatment will help clarify the critical targets for PDT killing in vitro. Comparison of the photosensitivity of tumors in vivo grown from cells with different PDT sensitivities in vitro may also allow differentiation between direct and indirect tumor cell killing in vivo, because indirect effects would be expected to operate independently of the intrinsic tumor cell sensitivity. This paper reports initial findings on the induction of PDT resistance in the radiation induced fibrosarcoma cell line RIF- 1 by repeated PDT treatment in vitro and re-growth from single surviving colonies. It will be shown that a degree of PDT resistance can be induced in this way, consistent with recent reports from other groups (Luna, et al., 1990; Rucker et al., 1990). The RIF model was selected because it may be grown both in vitro as a monolayer culture and sub- cutaneously in vivo in mice (Twentyman et al.,

307

308 GURMIT SINGH et d.

1980), and has been well characterized in PDT studies with Photofrin. In particular, clonogenic assays of tumor cells removed after various times in situ following in vivo PDT treatment have been used to investigate indirect tumoricidal mechanisms (Hen- derson et al., 1985; Henderson, 1990a,b), and we propose that such experiments may be repeated for PDT-sensitive cells and resistance-induced cells to further clarify the relative contributions of direct and indirect mechanisms.

In addition in this study, the cross-resistance to Photofrin-PDT of Chinese hamster ovary (CHO) cells and CHO-multi-drug resistant cells (CHO- MDR) has been measured, as a complementary approach to the study of PDT resistance. This is also of potential clinical interest in considering PDT treatment of tumors which are multi-drug resistant. The wild-type PDT-sensitive RIF cells and the PDT- resistant RIF cells have also been assayed for Adria- mycin sensitivity. The possibility that reduced photosensitizer or drug uptake is the cause of cellular resistance has been examined by measuring the concentration of Photofrin and Adriamycin in each of the RIF-sensitive, RIF-resistant, CHO, and CHO-MDR cells. The results suggest different mechanisms of resistance between PDT induction and multi-drug induction.

Few systematic studies of cellular resistance to photodynamic therapy have been reported to date, and these fall into two categories: those involving induction of PDT resistance by light or dark exposure to photosensitizers, and those which have assessed the PDT sensitivity of cells showing multi-drug resistance (MDR) or resistance to hyperthermia.

For the former, Luna et a f . (1990) have induced Photofrin-PDT resistance in RIF cells by a sequence of 10 cycles of Photofrin-PDT treatment in vitro, which parallels the study reported here. The findings are essentially consistent with our RIF results, and a detailed comparison will be made in the discussion.

No decrease in Photofrin-PDT sensitivity was found by Gomer et a f . (1990) in Chinese hamster fibroblasts or RIF cells having significant resistance to hyperthermia, for either short (1 h) or long (16 h) photosensitizer incubation. There was also no alteration of photosensitizer uptake. Thus, although the possible sub-cellular targets for PDT and hyper- thermic toxicity and the types of induced damage may be similar, this study suggests that the mechanisms of cytotoxicity are different for the two modalities, so that they are not cross-resistant. Absence of hyperthermia cross-resistance was observed also in PDT-resistant RIF cells, even though various stress proteins, which are associated with thermotolerance, were induced by PDT treatment.

As regards chemotoxic cross-resistance in PDT- resistance cells, Fisher and Gomer (1990) found decreasing sensitivity to Adriamycin in the 24 h

period following Photofrin-PDT treatment of V79 Chinese hamster cells, the effect being most pro- nounced in those cells surviving severe PDT treatment (high cell killing). The effect was not associated with altered Adriamycin concentration in the cells.

In other MDR cell lines, Giannotti et al. (1990) recently reported no difference in PDT sensitivity with sulfonated aluminum phthalocyanine between parental and MDR+ human erythroleukemic cells, while Marchesini et al . (1990) found a 1.4-fold Phot- ofrin-PDT resistance under specific treatment conditions in Adriamycin-resistant human MCF-7 cells. Mitchell et af. (1988) also observed a degree of porphyrin-PDT resistance and impaired porphyrin accumulation in CHO-MDR cells but not in an MDR human breast cancer line. These various preliminary studies indicate a range of possible approaches to the use of PDT resistance as a tool in the clarification of PDT mechanisms.

MATERIALS AND METHODS

Cells. RIF-1 cells were obtained originally from Dr. B. Henderson, Roswell Park Memorial Institute, Buffalo, NY. Chinese hamster ovary and CHO-MDR cells were obtained from Dr. V. Ling, Ontario Cancer Institute, Toronto, Ontario. The CHO-MDR line, containing the P- glycoprotein gene (CHRC5) was derived from the AUX B1 parent line as described by Ling and Thompson (1974). The RIF and CHO cells were grown as monolayers in a- MEM medium (Gibco, Mississauga, Canada), plus deoxy- ribonucleosides and ribonucleosides, supplemented with 10% fetal calf serum, penicillin and streptomycin in 50 mL culture flasks. Cells were maintained at 37°C and 5% C02/95% air in a humid environment.

Incubation and treatment. The protocol for each treatment of cells was as follows. Preconfluent cells were removed from the culture flasks using 10% trypsin, centrifuged at 1000 rpm for 5 min and resuspended in medium. The cells were then transferred to culture plates and allowed to adhere for 4 h. At this time, the medium was removed and replaced with fresh medium alone, as control, or with the same volume of medium containing the desired concentration of either Photofrin I1 (QLT, Vancouver, BC) or Adriamycin (Adria Laboratories of Canada Ltd., Mississauga, Ontario). After incubation for a selected period, the medium containing drug was removed and replaced with fresh medium. For PDT treatments the plates were then irradiated to a known light fluence. Triplicate plates were used for each drug and/or light dose. Treated and control cells were allowed to grow undisturbed for 5 days. At this time the cells were either assayed for survival or, for the resistance-induction procedure in RIF cells (see below), single surviving colonies were harvested manually from the culture plates, and separate cultures were initiated from each individual col- ony harvested. For the survival assay, the plates were stained with methylene blue, and colonies of >20 cells were counted.

Irradiation procedure. For the PDT treatments, culture plates containing the cells which had been incubated with or without Photofrin were placed on a 100 cm x 50 cm light diffusing surface illuminated by a bank of fluorescent tubes (Philips type TL/83), filtered with red acetate filters (Roscolux, No. 19, Rosco, CA), to give wide-band illumi- nation above 585 nm. The energy fluence rate was 9.2 W m-* in the wavelength band 623-634 nm, representing 12% of the total filtered output. Irradiation for 5 min

Research Note 309

Table 1. Cyclca of PDT treatmcnt to inducc resistance in RIF- 1 cells. Light fluencc of 2.7 x 10' J m-* was used in all cases

Photofrin Cycle No. concentration ()*g/mL) Incubation timc (h)

1 2 1s 18 3,4 2s 18

5 SO 4 5 60 4 7 8 100 4

-+4A

+UA

resulted in an incident energy fluence of 2.7 x lo1 J m-2 in this photoactivation band. Except for this controlled light irradiation, all procedures were carried out in mini- mal ambient lighting after the addition of photosensitizer to the cells.

Protocol for inducing resistance in RIF cells. The RIF cells were subjected to a series of Photofrin incubation and light irradiation treatments as described above in order to obtain PDT-resistant strains. At the end of each treatment cycle, new cultures were started from single surviving colonies; harvested colonies were not mixed at any stage. The data to be presented below are for the selected variant designated 8A, and an intermediate variant 4A. The cycle of treatments used to derive these is shown in Table 1 . The 4A cells were thus derived by 4 cycles of treatment and regrowth, each treatment being at the fixed light dose of 2.7 X 10' J m-* and 18 h incubations at Photofrin concentrations of either 15 or 25 pg/mL. The further 4 cycles of treatment and regrowth used to obtain the UA cells were carried out with 4 h incubations at 50, 60, 100 and 100 pg/mL. This change in incubation time was intended to produce a more stable resistance, based on work of other investigators ( C . Gomer et al., personal communication). Each cycle of treatment was aimed at achieving survival levels in the 0.1-1'4 range, based on survival assays performed in the previous cycle. The increasing Photofrin concentration through the cycles indicates decreasing sensitivity to treatment at each cycle, representing stepwise increases in resistance. Data are only presented below for 8A (and, in one case, also for 4A). Multiple cultures from single surviving colonies were grown and tested at each selection cycle. The 8A cells showed the greatest reduction in Photofrin-PDT sensitivity compared with the parent cells, and, therefore, represent the most resistant variant to date.

Photofrin and Adriamycin uptake. The uptake of Photo- frin or Adriamycin in each of the RIF-1 parent, RIF-gA, CHO and CHO-MDR cells was measured by fluorescence flow cytometry and by spectrofluorometry. Drug exposures were carried out on exponentially growing cells which had been trypsinized, counted, resuspended in a- MEM medium, plated out and allowed to adhere, as in the survival assays. All drug exposures and subsequent procedures were carried out in the dark.

A11 four cell strains were exposed to Photofrin for 18 h, after which time the medium containing drug was removed, the cells trypsinized, resuspended in 10 mL medium, split into two aliquots and centrifuged (5 min, 37°C 1000 rpm). The cell pellets were then resuspended in either 2 mL PBS for flow cytometry or in 2 mL 0.2N NaOH in lo/" CTAB (cetyltrimethylammoniumbromide, Aldrich Chemical Co., Milwaukee, W1) for those cells destined for fluorometry. Cells in NaOHCTAB were then placed in a sonicating ice bath for 30 min and centrifuged at 2000 rpm for 10 min. The supernatant was placed into cuvettes, as was the fractionated cell pellet after resuspension in 2 mL NaOHiCTAB.

Exponentially growing cells were also exposed to various concentrations of Adriamycin for 3 h. After removal of the medium containing drug and trypsinization, cells were kept cold (4°C) throughout the remaining procedures. Cells were treated as in the protocol for Photofrin uptake measurements, except that the fluorometer solvent was 0.3N HCI/50% ethanol for both the cell extract and the fractionated membrane component resuspension (Bachur et al., 1970).

Fluorometry measurements of the cell extracts and fractionated membrane components were done on a Perkin- Elmer LS-5 Luminescence Spectrometer using an exci- tation wavelength of 488 nm and emission measurements at 575 nm, which corresponded to the wavelengths used in the flow cytometry (FCM) analysis. The Photofrin and Adriamycin results discussed below are based on FCM. In order to validate these, the uptake of either Photofrin or Adriamycin in each of the cell types was measured as a function of drug concentration by both FCM and fluorometry. Good correlation was found between the two methods; e.g. with Photofrin in RIF-1 cells the FCM and fluorometry measurements of drug concentration were proportional, with a linear regression correlation of R = 0.88. It should be pointed out that in the case of Photofrin, selective accumulation of differently-fluorescing porphyrin fractions could complicate the interpretation of the uptake data.

In the FCM, a Coulter Electronics Epics Profile I1 flow cytometer was used. Prior to analysis, cells were passed through a 37 km nylon filter in order to obtain single cell suspensions. Forward scatter measurements were used as an estimate of cell volume for each cell type so that relative fluorescence per unit cell volume could be reported.

RESULTS

Induced PDT resistance in RIF cells

Figure l(a) shows representative clonogenic survival curves for the parent, 4A and 8A RIF cells, as a function of the Photofrin concentration for 18 h incubation and fixed light fluence. The 100% survival in each case was the average of three different control conditions: no Photofrin, no light; 15 p.g/mL Photofrin, no light; no Photofrin, 2.7 x lo3 J m-2 light. There was no significant difference found between these three controls.

The 4A strain showed an intermediate degree of resistance between the parent and 8A cells, and illustrates the progressive and incremental change in the clonogenic response obtained with each cycle of treatment and regrowth. At the highest Photofrin levels studied and 2.7 x lo3 J m-' light dose, there was approximately a 2-log difference in cell kill in the parent RIF cells compared with the 8A cells. The survival curves at fixed Photofrin dose (15 p.g/mL) and varying light dose (obtained by varying the treatment time) showed similar general behav- iour, but a systematic study over a range of Photo- frin and light doses has not been performed.

In order to test the repeatability of these results, six separate PDT survival experiments were carried out with the parental RIF and 8A cells run in paral- lel under identical conditions. Although there was considerable experiment-to-experiment variation in the RIF-1 and RIF-8A survival curves, resistance

310 GURMIT SINCH et al.

Photofrln II [rplml] Photofrln I1 [rg/ml]

Figure 1. Survival curves for Photofrin PDT or Adriamy- cin treatment of RIF and CHO cells. (a) Clonogenic survival curves for RIF-1 (parental cells) (0); PDT-resistance induced RIF-4A (A) and RIFdA (0). Cells were incubated with 10, 15, 20 or 25 pg mL-' Photofrin I1 for 18 h and exposed to 2.7 x lo3 J m+. Each point is the average of triplicate plates and error bars indicate the standard error. (b) Survival of CHO- wild-type cells (0) and CHO-MDR cells (0) to same conditions as above. J indicates survival below 0.001. (c) Survival of RIF-1 and RIF3A to Adriamycin. (d) Survival of CHO and CHO- MDR to Adriamycin. 1 indicates survival below 0.01.

of the 8A strain was consistently observed in every experiment. The average ratio, Dlo(RIF-8A)/ D,,(RIF-1) for 2.7 X lo3 J m-2 of light dose in six independent experiments was 1.8 * 0.4.

PDT and M D R cross-resistance

Figure l(a-d) shows both the Photofrin-PDT and the Adriamycin survival curves for the RIF, RIF- 8A, CHO and CHO-MDR cells. As seen in Fig. l(b), the CHO-MDR cells did exhibit a degree of PDT resistance. The difference in the PDT sensitivity of CHO and CHO-MDR cells appears primar- ily as an increase in the width of the shoulder in the survival curves, rather than as a change in the final slope. As regards Adriamycin response, the CHO-MDR cells showed a very high degree of resistance, as expected. There was, however, no significant difference in the Adriamycin sensitivity between the RIF and 8A cells. That is, multi-drug resistance appeared to confer a degree of PDT resistance, but PDT resistance did not result in resistance to Adriamycin in these cell lines.

Drug uptake

Comparisons of the relative Photofrin uptake between RIF-1, RIF-8A, CHO and CHO-MDR are

I RIF RIFdA CHO 0 CHO-MDR I

Figure 2. Photofrin uptake per unit cell volume in RIF and CHO cells at 18 h incubation determined by fluorescence flow cytometry. Three independent experiments were conducted with each cell type and the error bars indicate the resulting standard error. At 10 and 20 pg/mL the Photofrin uptake difference between CHO and CHO-

MDR is significant at the P = 0.05 level.

shown in Fig. 2. These measurements show that the relative fluorescence per unit cell volume was not significantly different between the RIF-1 and RIF- 8A cells. The ratio of cellular fluorescence over the range of concentration measurements was RIF- 1:RIF-gA = 1.01 2 0.05. However a significant difference was observed between the CHO and CHO-MDR cells; Photofrin uptake ratio between

Flow cytometry has been successfully used in measuring intracellular Adriamycin concentration in CHO-MDR cells when the integrated fluorescence >530 nm was measured (Luk et al . , 1989). Those authors reported that the uptake ratio of CH0:CHO-MDR was approximately 1 O : l . We have observed a high variability in these measurements but we have not found any significant difference in Adriamycin uptake curves between RIF-1 and RIF-8A.

CH0:CHO-MDR = 1.70 2 0.09.

DISCUSSION

The primary goal of this work was to derive PDT- resistant RIF cells for future use as a tool in the study of PDT mechanisms of action in vitro and in vivo. This has been achieved, but the degree of resistance is not large, so work on further enhancing the resistance is in progress. In addition, the results suggest a number of other observations.

It appears that the Photofrin-PDT resistance seen in the RIF-8A cells is not the result of reduced photosensitizer uptake. This implies mechanism(s) of resistance different from classical MDR or pleio- tropic resistance, such as altered sub-cellular tar- getting of photosensitizer, photosensitizer detox- ification, enhanced repair, or scavaging of the cytotoxic photoproducts (Kessel, 1989). The fact that the slope of the cell survival curves appears to

Research Note 31 1

be different between RIF-1 and RIF-8A may be relevant in this regard [Fig. l(a)]. By contrast, the Photofrin-PDT resistance seen in CHO-MDR cells [Fig. l(b)] is accompanied by reduced Photofrin concentration compared with the wild-type CHO cells. Similar observations were made by Mitchell et al. (1988) also in CHO-MDR cells and by Mar- chesini et al. (1990) in MCF-7 human breast carcinoma cells. It is interesting to note that Photofrin is anionic, whereas enhanced drug efflux in multi- drug resistance generally relates to cationic drugs. The observation is consistent with the interpretation that P-glycoprotein is at least partly effective in altering Photofrin transport. However, it should be noted that the CHO-MDR cell line used here was derived from multiple drug treatments, not by trans- fection of the mdr, gene, so that other mechanisms may contribute to the multi-drug resistance. Never- theless, these two observations taken together, i.e. uptake/efflux-independent PDT resistance in RIF- 8A and efflux-associated PDT resistance in CHO- MDR, are consistent with our finding that there was no difference in Adriamycin sensitivity between the RIF wild type and RIF-8A strains. If there had been P-glycoprotein amplification in the 8A cells, one would expect the RIF-8A cells to be less Adria- mycin sensitive than the RIF-1 cells. However, direct quantitation of P-glycoprotein in RIF and RIF-8A will be required to confirm this explanation. The Adriamycin fluorescence measurements in RIF- 1 and RIF-8A also did not indicate any significant difference in drug concentration.

The only work which is directly comparable with this study is that of Luna et al. (1990). These investigators induced PDT resistance de novo in RIF-1 cells, The induction protocol involved 10 cycles of Photofrin-light treatment (1 h or 16 h incubation with photosensitizer) and regrowth of survivors. Regrowth following each cycle was not from single colonies, as in our study, but rather the final resistant strains were selected after characterizing a number of clones at the end of the 10 cycle series. The degree of resistance in the 16 h strains, which are likely most comparable to the RIF-8A cells here, was in the range 1.2-1.4 for surviving fractions from 0.005 to 0.1, as measured by the light-dose ratio at fixed Photofrin concentration (25 pglmL). This is somewhat lower than, but of the same order as our 8A resistance, measured by the drug-dose ratio. Luna et al. also found, as we did, that, although the Photofrin uptake per cell was higher in their resistant strains, the uptake expressed per unit protein mass was not significantly different from the wild- type RIF cells.

Luna et al. (1990) have reported reduced in vitro and in vivo growth rates of their PDT-resistant cells compared with the wild-type cells, and preliminary work with RIF-8A supports this finding. We also have preliminary evidence of cross-resistance of RIF-8A to PDT with the photosensitizer aluminum

chlorosulfonated phthalocyanine, whereas Gianotti et al. (1990) have reported no ALSPC cross-resistance in MDR cells. These observations will be the subject of future detailed reports.

In conclusion, we have demonstrated that resistance to PDT in vitro can be achieved. Furthermore, cross-resistance to PDT was also observed in CHO- MDR cells, but PDT-induced resistant RIF-8A cells were not resistant to the chemotherapeutic agent Adriamycin, suggesting different mechanisms of resistance. The fact that the PDT-induced resistance did not appear to be the result of altered photosensitizer transport suggests that the PDT-resistance may be due to factors such as altered sub-cellular localization of photosensitizer, photochemical modi- fication or repair of photodamage. This gives the possibility of making comparative measurements between the PDT-sensitive and PDT resistance vari- ants to investigate specific mechanisms of photodynamic action in vitro, for example by correlating PDT cell killing and binding of photosensitizer to targets such as mitochondria. This will be the subject of future work, as will the assessment of differ- ences in the in vivo photodynamic sensitivities of tumors grown from the RIF and RIF-8A cells.

Acknowledgements-This work was supported by the Hamilton Civic Hospitals and the Ontario Laser and Light- wave Research Centre. Photofrin was kindly supplied by Quadralogic Technologies Inc., Vancouver, B.C. Our thanks are also due to Drs. C. Gomer and A. Rainbow for helpful discussions and to Drs. B. Henderson, Roswell Park Memorial Institute, Buffalo and V. Ling, University of Toronto, for supplying cell lines.

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