JOURNAL
OF SURGICAL
52,631-634
RESEARCH
Adriamycin
(1992)
Enhanced in vitro and in viva Photodynamic Therapy of Mesothelioma PATRICK
Department Presented
at the Annual
of Surgical
Meeting
F. BROPHY, Oncology,
of the Association
M.D., AND STEVEN
Fox Chase
Cancer
for Academic
Center, Surgery,
M. KELLER, Philadelphia, Colorado
M.D. Pennsylvania
Springs,
Colorado,
19111 November
20-23,
1991
the chest. Current therapy including surgery, chemotherapy, and radiation therapy, alone or in combination, has not proven effective in controlling the growth of the tumor in most cases. As a result, long-term survival of patients with mesothelioma is rare [l-6]. Photodynamic therapy (PDT) utilizes a light-activated photosensitizing agent (e.g., Photofrin II) to achieve tumor necrosis via mechanisms that are not fully understood. The effectiveness of PDT on several tumors, including lung, breast, esophagus, and others, has been demonstrated over many years by different investigators [7, 81. The responsiveness of a human malignant mesothelioma cell line (H-MESO-1) to PDT both in vitro and in rho has previously been demonstrated [9, lo]. Other investigators have reported evidence that somechemotherapeutic agents may be active as photosensitizers and that combining PDT with chemotherapy may lead to improved responses [ 11-131. The current investigation was undertaken to determine if combining PDT with Adriamycin, a drug with some activity against mesothelioma and a possible photosensitizer, would be effective in controlling mesothelioma using both in vitro and in uiuo models.
The ability of Adriamycin (AD) to enhance the known in vitro and in vivo tumoricidal effects of photodynamic therapy (PDT) on the H-MESOhuman malignant mesothelioma cell line was investigated. In vitro cytotoxicity was determined by incubating HMESOcells in microtiter plates (2 X 10’ cells/well, 6 wells/group) with the photosensitizer Photofrin II (PF) and varying concentrations of AD (0,2.5,5.0, and 10.0 pg/ml) for 24 hr followed by exposure to gold vapor laser light (GVL) at a fluence of 6000 J/M’. [3H]Thymidine (1 &i) was added to each well 24 hr after treatment. Cells were harvested and counted for thymidine incorporation 24 hr later. PDT alone resulted in a decrease in thymidine incorporation of 23% while the addition of AD to PDT at AD concentrations of 2.5, 5.0, and 10.0 pg/ml resulted in decreases of 62, 85, and 69%, respectively (P = 0.005) as compared to untreated controls. H-MESOtumor bearing nude mice (n = 5) were injected ip with PF (5 mg/kg) and AD (5 mg/kg) 24 hr prior to illumination of the tumor site with GVL (120 J/cm2). Control groups (n = 5) received PDT, AD, and/or GVL alone. Tumor surface area was measured as the product of the greatest perpendicular dimensions every 5 days for 30 days. Administration of PDT without AD resulted in a decrease in tumor surface area of 50% on Day 10 with regrowth of tumor by Day 30 while AD alone with or without GVL had no impact on tumor growth. The addition of AD to PDT resulted in 100% tumor necrosis on Day 5 with no tumor regrowth. The use of PDT led to a significant (P < 0.02) decrease in tumor area while the addition of AD resulted in a more rapid, intense, and prolonged response. The addition of AD to PDT appears to result in greater tumor killing both in vitro and in vivo than does PDT alone. 0 1992 Academic Press, Inc.
MATERIALS
AND
METHODS
H-MESOcells were obtained from Biomeasure, Inc. (Hopkinton, MA). The cells were maintained in vitro by growth in tissue culture flasks using Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in a 5% CO, atmosphere. For in uiuo experiments, tumors were grown in nude mice by subcutaneous implantation of cells onto the backs of the animals. Nude mice used in this study were obtained from the animal colony at our institution and the experiments were approved by the Animal Care Committee. Photofrin II (PF) was obtained from Quadralogics Technologies (Vancouver, British Columbia). The lypholyzed powder was reconstituted with sterile water. Adriamycin (doxorubicin; Adria Laboratories, Dublin, OH) was obtained from the pharmacy of the Fox Chase
INTRODUCTION
Malignant mesothelioma is a rare human tumor that most commonly arises in the pleural cavity. Patients with this disease usually present with extensive local disease and die as a result of progressive disease within 631
All
002%4804/92 $4.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
632
JOURNAL
OF
SURGICAL
RESEARCH:
Cancer Center. Both drugs were diluted to the final concentrations used with tissue culture media or phosphate-buffered saline. To minimize activation of PF by ambient light, minimal lighting conditions were employed. Light at 628 nm, the activating wavelength of PF, was provided by a gold vapor laser (GVL) (Metalaser Technologies, Pleasanton, CA) and transmitted to the cells or tumors via a quartz fiber. Light output was measured with a power meter and maintained at 500 mW. The duration of exposure required to deliver the appropriate light fluence was calculated with the formula: desired joules/laser output in watts = exposure duration in seconds. H-MESOcells were placed into 96-well microtiter plates (2 X lo5 cells/well, 6 wells/group) for 24 hr prior to the addition of test drugs. The concentration of PF used was 0 or 10 pg/ml (final concentration per well). This concentration (10 pg/ml) was chosen to limit cell death from PDT alone [9]. AD was added to wells at final concentrations of 0, 2.5, 5.0, and 10.0 pg/ml. Twenty four hours after addition of the drugs, the plates were exposed to light at fluences of 0 or 6000 J/M’. In vitro cytotoxicity was assessed using a [3H]thymidine incorporation assay. Briefly, 1 &i of radiolabel was added to the cells 24 hr after treatment. Following an additional 24 hr incubation, cells were harvested using a automated cell harvester and counted in a scintillation counter. Results are expressed as percentage incorporation of controls. Groups of tumor-bearing mice (n = 5) were pretreated with combinations of PF and AD (0 or 5 mg/kg) for 24 hr. This was followed by exposure of the tumors to GVL (120 J/cm2) in some groups. The groups are identified as follows: Group l-PF = 0, AD = 0, GVL = 0 (untreated controls); Group 2-PF = 5, AD = 0, GVL = 120 (PDT controls); Group 3-PF = 0, AD = 5, GVL = 0; Group 4-PF = 5, AD = 5, GVL = 0; Group 5-PF = 0, AD = 5, GVL = 120; and Group 6-PF = 5, AD = 5, GVL = 120 (AD PDT group). Two additional control groups (PF = 5, AD = 0, GVL = 0 and PF = 0, AD = 0, GVL = 120) were not included as they have previously been shown to be no different than the untreated controls. Tumor surface area was obtained by calculating the product of the greatest perpendicular dimensions of the tumor as measured with calipers. Measurements were obtained at Day 0 (GVL exposure) and every 5 days for a total of 30 days. Results are expressed as a percentage of change in area from pretreatment measurement. Statistical analysis of the in vitro data was performed using Wilcoxon’s rank-sum two-sample tests. The in vivo data were analyzed using analysis of variance and Mann-Whitney tests. RESULTS
In vitro results of the effects of adriamycin photodynamic therapy (PDT) of H-MESO-
(AD) on cells are
VOL.
52, NO.
Ouahnl
6, JUNE
1992
2.5 @ml
Adriamycin
5.0 ug/ml
10.0 cgml
Concentration
FIG. 1. Effects of the addition of Adriamycin in uitro on treatment with Photofrin II (PFrg/ml) with or without exposure to laser light determined by [3H]thymidine incorporation.
to H-MESOcells = 0 pg/ml, PF+ = 10 (GVLor GVL+) as
shown in Fig. 1. For each concentration of AD used there were four treatment groups. These groups were designed to demonstrate any individual effects of the agents involved in PDT [Photofrin II (PF) and GVL light] in addition to the combined effects. Untreated cells (AD = 0 pg/ml, PF-/GVL-) were used as controls and the results of [3H]thymidine incorporation by these cells was set at 100%. All results are expressed as percentage thymidine incorporation as compared to this group unless otherwise specified. For AD concentration of 0 pg/ml, the addition of PF(PF+/GVL-) resulted in a slight increase (6%) in thymidine uptake while exposure to GVL(PF-/GVL+) resulted in a 7% decrease in radiolabel incorporation. Cells treated by PDT(PF+/GVL+) in the absence of AD showed a 23% decrease in thymidine incorporation. These differences in radiolabel uptake were not statistically significant (P > 0.05). The addition of AD to cells caused statistically significant (P < 0.01) decreases in radiolabel incorporation at all concentrations tested. Specifically, at AD concentration of 2.5, 5.0, and 10.0 pg/ml decreases of thymidine uptake of 42, 26, and 34% were seen, respectively. The increase of AD concentration from 2.5 to 5.0 ag/ml also resulted in a significant decrease (P = 0.04) in radiolabel uptake but the increase from 5.0 to 10.0 pg/ml did not result in a significant change. When PF was added to cells at the above concentrations of AD(PF+/GVL-), the results were mixed. At AD = 2.5 pg/ml thymidine uptake was increased by 49% (P = 0.005) as compared with cells at that concentration of AD alone. At 5.0 and 10.0 pg/ml concentrations of AD, the radiolabel incorporation was decreased by 27% (P = 0.008) and 6% (P > 0.05), respectively, when PF was added to the cells. Exposure of cells treated with AD concentrations of 2.5,5.0, and 10.0 pg/ml to GVL(PF-/GVL+) resulted in
BROPHY
AND
KELLER:
633 . PDT group (Group 6), 100% tumor necrosis occurred by Day 5. No tumor regrowth occurred during the observation period. Analysis of all groups revealed statistically significant differences (P < 0.02) between groups receiving PDT (Groups 2 and 6) and those that did not while differences between Groups 2 and 6 were significant at Day 5 but not at the later observation points.
ADRIAMYCIN
250% 200% 150%
k% 50 100% 8
ENHANCED
PDT
DISCUSSION
50% 0% -50% -100%
Days
Post Treatment
FIG. 2. Response of subcutaneous ment with combinations of Adriamycin, expressed as a percentage change from for description of groups.
tumors in nude mice to treatPhotofrin II, and laser light tumor area on Day 0. See text
an approximately 50% decrease in thymidine incorporation as compared to similarly treated cells without AD (P < 0.02). Only at an AD concentration of 5.0 pg/ml did the addition of GVL result in a statistically significant decrease (P = 0.005) in radiolabel uptake when compared to cells treated with similar concentrations of AD alone. Increasing the AD concentration from 2.5 to 5.0 to 10.0 pg/ml did not result in any significant changes in thymidine uptake. Photodynamic therapy (PF+/GVL+) of H-MESOcells treated with AD concentrations of 2.5,5.0, and 10.0 pg/ml resulted in significant (P = 0.005) decreases in radiolabel uptake of 62, 85, and 69%, respectively, as compared to the untreated controls. This represents enhancement of the PDT effect of cells without AD of 39% (P = O.Ol), 62% (P = O.OOS), and46% (P = O.OOS), respectively. For each concentration of AD the addition of PDT resulted in significantly decreased thymidine incorporation (P = 0.008). Additional decreases of 20% at AD = 2.5 pg/ml, 59% at AD = 5.0 @g/ml, and 25% at AD = 10.0 pg/ml were seen as compared to cells at these AD concentrations alone. A significant change (P = 0.04) in radiolabel incorporation was seen when the AD concentration of PDT-treated cells increased from 2.5 to 5.0 pug/ml but not with further increases in AD concentration. The results of treatment of subcutaneous tumors in nude mice are shown in Fig. 2. Groups 1, 3, 4, and 5 showed progressive growth with tumor area nearly doubling by Day 15. There was no statistically significant differences between these groups. The PDT controls (Group 2) showed a slight decrease in tumor area at Day 5 and a 50% decrease in tumor area at Day 10. Tumor regrowth occurred at Day 30 in this group after having remained stable during the interim period. In the AD
The effectiveness of PDT on H-MESOcells in vitro has previously been shown to be dependent on both PF concentration and light fluence [9]. PF and light parameters utilized in this study were chosen to allow identification of additional AD effect against a background of minimal PDT. Several significant relationships of AD, PF, and GVL treatment of mesothelioma cells in vitro were demonstrated. Incubation of H-MESOcells with AD alone led to significant decreases in [3H]thymidine incorporation at all drug concentrations. However, a characteristic dose response was not seen. An interesting observation in this study is the ability of PF to stimulate thymidine uptake in the cells when they are not subsequently exposed to light. This effect was seen in a previous study [9] but in the current study was most pronounced when AD was added at the 2.5 pg/ml concentration. At the higher concentrations of AD, addition of PF led to decreases in radiolabel uptake but only at the AD of 5.0 pg/ml was this significantly different. The explanation of this phenomenon is not known but could include the presence of a limiting growth factor in the heterogeneous PF mixture or of a factor that competes with or counteracts the effects of AD at the lower doses but is lost as the AD dose increases. Exposure of AD-treated cells to GVL resulted in decreases in thymidine uptake as compared to cells exposed to light in the absence of AD. However, it was only with AD at the 5.0 pg/ml concentration that the decrease achieved significance. This suggests that AD alone does have some inherent photosensitizing ability but that it may not be inducible beyond a certain level regardless of the concentration. The combination of AD and PDT resulted in enhanced in vitro cytotoxicity as measured by thymidine incorporation. The dose response is similar to that seen in cells treated with AD alone and is most pronounced at the 5.0 pg/ml concentration of AD. At that dose the effect appears to be synergistic but at the other doses this is not clear. A possible explanation for this variability in the effect of PDT may be that pretreatment with AD may interfere with pathways needed for the expression of the PDT effect. The combination of AD and PDT resulted in a complete and sustained necrosis of subcutaneous tumors in nude mice. Standard PDT resulted in a decrease in tumor area but it was not sustained nor complete. Treat-
634
JOURNAL
OF
SURGICAL
RESEARCH:
ment with AD alone as well as other combinations of AD with PF and GVL did not have any effect on tumor growth. The time to maximal response in the two PDT groups was different and this suggests an increase in the photodynamic effect related to the presence of AD. The prolonged response to the combination of AD and PDT is most likely related to elimination of tumor cells. Continued observation of this group to 90 days post-treatment resulted in tumor recurrence in only two animals (data not shown). An explanation of the observed effect probably involves complex interactions within the tumor including vascular injury and hypoxia as well as the intracellular effects of activated PF and AD. In summary, AD appears to enhance the effectiveness of PDT against H-MESOmalignant mesothelioma cells both in vitro and in uivo. In addition, AD appears to have inherent photosensitizing abilities that are not dose dependent. Lastly, PF may have protective or stimulatory properties when not activated. Widespread application of PDT in cancer therapy has been limited by several factors such as dose standardization of both drugs and light and the complexity of the light delivery systems available. These factors are being slowly resolved and should lead to an increased use of this treatment modality [7, 81. With further refinement of PDT in the management of human cancers, combinations of PDT with chemotherapy may provide improved responses in a select group of tumors such as mesothelioma that are not currently controlled by standard forms of therapy.
VOL.
Antman, K. H., and Corson, J. M. Benign and malignant mesothelioma. Clin. Chest Med. 6: 127, 1985.
pleural
6, JUNE
1992
2.
Martini, N., McCormack, P. M., Bains, Burt, M. E., and Hilaris, B. S. Pleural Thoruc. Surg. 43: 113, 1987.
3.
Antman, K. H., Blum, R. H., Greenberger, J. S., Flowerdew, G., Skarin, A. T., and Canellos, G. P. Multimodality therapy for malignant mesothelioma based on a study of natural history. Am. J. Med. 68: 356,198O.
4.
Elmes, P. C., and Simpson, M. J. C. The clinical thelioma. Q. J. Med. 45: 427, 1976.
5.
Law, M. R., Gregor, A., Hodson, M. E., Bloom, H. J. G., and Turner-Warick, M. Malignant mesothelioma of the pleura: A study of 52 treated and 64 untreated patients. Thorax 39: 255, 1984.
6.
Alberts, A. S., Falleson, G., Goedhals, L., Vorobiof, D. A., and VanDerMerwe, C. A. Malignant mesothelioma: A disease unaffected by current therapeutic maneuvers. J. Clin. Oncol. 6: 527, 1988.
7.
Doughtery, min. Surg.
8.
Kessel, Raton:
9.
Keller, S. M., Taylor, D. D., and Weese, J. L. In uitro killing human malignant mesothelioma by photodynamic therapy. Surg. Res. 48: 337, 1990.
T. J. Photodynamic Oncol. 5: 6, 1989.
D. Photodynamic CRC Press, 1990.
Therapy
therapy.
of
M. S., Kaiser, mesothelioma.
aspects
L. R., Ann.
of meso-
New
approaches.
Se-
Neopkxstic
Disease.
Boca of J.
10.
Feins, R. H., Hilf, R., Ross, H., and Gibson, therapy for human malignant mesothelioma J. Surg. Res. 49: 311, 1990.
11.
Mang, T. S., Khan, S., and Crean, D. H. Pre-clinical and clinical evaluation of chemotherapeutic agents with Photofrin II in the management of cutaneous metastases of breast cancer. Photothem. Photobiol. 51: 85S, 1990. [Abstract]
12.
Edell, E. S., and Cortese, D. A. Combined effects of hematoporphyrin derivative phototherapy and adriamycin in a murine tumor model. Lasers Surg. Med. 8: 413, 1988.
13.
Cowled, P. A., MacKenzie, L., and Forbes, photodynamic therapy with hematoporphyrin cocorticoids. Cancer Lett. 29: 107, 1985.
REFERENCES 1.
52, NO.
S. L. Photodynamic in the nude mouse.
I. J. Potentiation of derivatives by glu-
OF SURGICAL
52,631-634
RESEARCH
Adriamycin
(1992)
Enhanced in vitro and in viva Photodynamic Therapy of Mesothelioma PATRICK
Department Presented
at the Annual
of Surgical
Meeting
F. BROPHY, Oncology,
of the Association
M.D., AND STEVEN
Fox Chase
Cancer
for Academic
Center, Surgery,
M. KELLER, Philadelphia, Colorado
M.D. Pennsylvania
Springs,
Colorado,
19111 November
20-23,
1991
the chest. Current therapy including surgery, chemotherapy, and radiation therapy, alone or in combination, has not proven effective in controlling the growth of the tumor in most cases. As a result, long-term survival of patients with mesothelioma is rare [l-6]. Photodynamic therapy (PDT) utilizes a light-activated photosensitizing agent (e.g., Photofrin II) to achieve tumor necrosis via mechanisms that are not fully understood. The effectiveness of PDT on several tumors, including lung, breast, esophagus, and others, has been demonstrated over many years by different investigators [7, 81. The responsiveness of a human malignant mesothelioma cell line (H-MESO-1) to PDT both in vitro and in rho has previously been demonstrated [9, lo]. Other investigators have reported evidence that somechemotherapeutic agents may be active as photosensitizers and that combining PDT with chemotherapy may lead to improved responses [ 11-131. The current investigation was undertaken to determine if combining PDT with Adriamycin, a drug with some activity against mesothelioma and a possible photosensitizer, would be effective in controlling mesothelioma using both in vitro and in uiuo models.
The ability of Adriamycin (AD) to enhance the known in vitro and in vivo tumoricidal effects of photodynamic therapy (PDT) on the H-MESOhuman malignant mesothelioma cell line was investigated. In vitro cytotoxicity was determined by incubating HMESOcells in microtiter plates (2 X 10’ cells/well, 6 wells/group) with the photosensitizer Photofrin II (PF) and varying concentrations of AD (0,2.5,5.0, and 10.0 pg/ml) for 24 hr followed by exposure to gold vapor laser light (GVL) at a fluence of 6000 J/M’. [3H]Thymidine (1 &i) was added to each well 24 hr after treatment. Cells were harvested and counted for thymidine incorporation 24 hr later. PDT alone resulted in a decrease in thymidine incorporation of 23% while the addition of AD to PDT at AD concentrations of 2.5, 5.0, and 10.0 pg/ml resulted in decreases of 62, 85, and 69%, respectively (P = 0.005) as compared to untreated controls. H-MESOtumor bearing nude mice (n = 5) were injected ip with PF (5 mg/kg) and AD (5 mg/kg) 24 hr prior to illumination of the tumor site with GVL (120 J/cm2). Control groups (n = 5) received PDT, AD, and/or GVL alone. Tumor surface area was measured as the product of the greatest perpendicular dimensions every 5 days for 30 days. Administration of PDT without AD resulted in a decrease in tumor surface area of 50% on Day 10 with regrowth of tumor by Day 30 while AD alone with or without GVL had no impact on tumor growth. The addition of AD to PDT resulted in 100% tumor necrosis on Day 5 with no tumor regrowth. The use of PDT led to a significant (P < 0.02) decrease in tumor area while the addition of AD resulted in a more rapid, intense, and prolonged response. The addition of AD to PDT appears to result in greater tumor killing both in vitro and in vivo than does PDT alone. 0 1992 Academic Press, Inc.
MATERIALS
AND
METHODS
H-MESOcells were obtained from Biomeasure, Inc. (Hopkinton, MA). The cells were maintained in vitro by growth in tissue culture flasks using Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in a 5% CO, atmosphere. For in uiuo experiments, tumors were grown in nude mice by subcutaneous implantation of cells onto the backs of the animals. Nude mice used in this study were obtained from the animal colony at our institution and the experiments were approved by the Animal Care Committee. Photofrin II (PF) was obtained from Quadralogics Technologies (Vancouver, British Columbia). The lypholyzed powder was reconstituted with sterile water. Adriamycin (doxorubicin; Adria Laboratories, Dublin, OH) was obtained from the pharmacy of the Fox Chase
INTRODUCTION
Malignant mesothelioma is a rare human tumor that most commonly arises in the pleural cavity. Patients with this disease usually present with extensive local disease and die as a result of progressive disease within 631
All
002%4804/92 $4.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
632
JOURNAL
OF
SURGICAL
RESEARCH:
Cancer Center. Both drugs were diluted to the final concentrations used with tissue culture media or phosphate-buffered saline. To minimize activation of PF by ambient light, minimal lighting conditions were employed. Light at 628 nm, the activating wavelength of PF, was provided by a gold vapor laser (GVL) (Metalaser Technologies, Pleasanton, CA) and transmitted to the cells or tumors via a quartz fiber. Light output was measured with a power meter and maintained at 500 mW. The duration of exposure required to deliver the appropriate light fluence was calculated with the formula: desired joules/laser output in watts = exposure duration in seconds. H-MESOcells were placed into 96-well microtiter plates (2 X lo5 cells/well, 6 wells/group) for 24 hr prior to the addition of test drugs. The concentration of PF used was 0 or 10 pg/ml (final concentration per well). This concentration (10 pg/ml) was chosen to limit cell death from PDT alone [9]. AD was added to wells at final concentrations of 0, 2.5, 5.0, and 10.0 pg/ml. Twenty four hours after addition of the drugs, the plates were exposed to light at fluences of 0 or 6000 J/M’. In vitro cytotoxicity was assessed using a [3H]thymidine incorporation assay. Briefly, 1 &i of radiolabel was added to the cells 24 hr after treatment. Following an additional 24 hr incubation, cells were harvested using a automated cell harvester and counted in a scintillation counter. Results are expressed as percentage incorporation of controls. Groups of tumor-bearing mice (n = 5) were pretreated with combinations of PF and AD (0 or 5 mg/kg) for 24 hr. This was followed by exposure of the tumors to GVL (120 J/cm2) in some groups. The groups are identified as follows: Group l-PF = 0, AD = 0, GVL = 0 (untreated controls); Group 2-PF = 5, AD = 0, GVL = 120 (PDT controls); Group 3-PF = 0, AD = 5, GVL = 0; Group 4-PF = 5, AD = 5, GVL = 0; Group 5-PF = 0, AD = 5, GVL = 120; and Group 6-PF = 5, AD = 5, GVL = 120 (AD PDT group). Two additional control groups (PF = 5, AD = 0, GVL = 0 and PF = 0, AD = 0, GVL = 120) were not included as they have previously been shown to be no different than the untreated controls. Tumor surface area was obtained by calculating the product of the greatest perpendicular dimensions of the tumor as measured with calipers. Measurements were obtained at Day 0 (GVL exposure) and every 5 days for a total of 30 days. Results are expressed as a percentage of change in area from pretreatment measurement. Statistical analysis of the in vitro data was performed using Wilcoxon’s rank-sum two-sample tests. The in vivo data were analyzed using analysis of variance and Mann-Whitney tests. RESULTS
In vitro results of the effects of adriamycin photodynamic therapy (PDT) of H-MESO-
(AD) on cells are
VOL.
52, NO.
Ouahnl
6, JUNE
1992
2.5 @ml
Adriamycin
5.0 ug/ml
10.0 cgml
Concentration
FIG. 1. Effects of the addition of Adriamycin in uitro on treatment with Photofrin II (PFrg/ml) with or without exposure to laser light determined by [3H]thymidine incorporation.
to H-MESOcells = 0 pg/ml, PF+ = 10 (GVLor GVL+) as
shown in Fig. 1. For each concentration of AD used there were four treatment groups. These groups were designed to demonstrate any individual effects of the agents involved in PDT [Photofrin II (PF) and GVL light] in addition to the combined effects. Untreated cells (AD = 0 pg/ml, PF-/GVL-) were used as controls and the results of [3H]thymidine incorporation by these cells was set at 100%. All results are expressed as percentage thymidine incorporation as compared to this group unless otherwise specified. For AD concentration of 0 pg/ml, the addition of PF(PF+/GVL-) resulted in a slight increase (6%) in thymidine uptake while exposure to GVL(PF-/GVL+) resulted in a 7% decrease in radiolabel incorporation. Cells treated by PDT(PF+/GVL+) in the absence of AD showed a 23% decrease in thymidine incorporation. These differences in radiolabel uptake were not statistically significant (P > 0.05). The addition of AD to cells caused statistically significant (P < 0.01) decreases in radiolabel incorporation at all concentrations tested. Specifically, at AD concentration of 2.5, 5.0, and 10.0 pg/ml decreases of thymidine uptake of 42, 26, and 34% were seen, respectively. The increase of AD concentration from 2.5 to 5.0 ag/ml also resulted in a significant decrease (P = 0.04) in radiolabel uptake but the increase from 5.0 to 10.0 pg/ml did not result in a significant change. When PF was added to cells at the above concentrations of AD(PF+/GVL-), the results were mixed. At AD = 2.5 pg/ml thymidine uptake was increased by 49% (P = 0.005) as compared with cells at that concentration of AD alone. At 5.0 and 10.0 pg/ml concentrations of AD, the radiolabel incorporation was decreased by 27% (P = 0.008) and 6% (P > 0.05), respectively, when PF was added to the cells. Exposure of cells treated with AD concentrations of 2.5,5.0, and 10.0 pg/ml to GVL(PF-/GVL+) resulted in
BROPHY
AND
KELLER:
633 . PDT group (Group 6), 100% tumor necrosis occurred by Day 5. No tumor regrowth occurred during the observation period. Analysis of all groups revealed statistically significant differences (P < 0.02) between groups receiving PDT (Groups 2 and 6) and those that did not while differences between Groups 2 and 6 were significant at Day 5 but not at the later observation points.
ADRIAMYCIN
250% 200% 150%
k% 50 100% 8
ENHANCED
PDT
DISCUSSION
50% 0% -50% -100%
Days
Post Treatment
FIG. 2. Response of subcutaneous ment with combinations of Adriamycin, expressed as a percentage change from for description of groups.
tumors in nude mice to treatPhotofrin II, and laser light tumor area on Day 0. See text
an approximately 50% decrease in thymidine incorporation as compared to similarly treated cells without AD (P < 0.02). Only at an AD concentration of 5.0 pg/ml did the addition of GVL result in a statistically significant decrease (P = 0.005) in radiolabel uptake when compared to cells treated with similar concentrations of AD alone. Increasing the AD concentration from 2.5 to 5.0 to 10.0 pg/ml did not result in any significant changes in thymidine uptake. Photodynamic therapy (PF+/GVL+) of H-MESOcells treated with AD concentrations of 2.5,5.0, and 10.0 pg/ml resulted in significant (P = 0.005) decreases in radiolabel uptake of 62, 85, and 69%, respectively, as compared to the untreated controls. This represents enhancement of the PDT effect of cells without AD of 39% (P = O.Ol), 62% (P = O.OOS), and46% (P = O.OOS), respectively. For each concentration of AD the addition of PDT resulted in significantly decreased thymidine incorporation (P = 0.008). Additional decreases of 20% at AD = 2.5 pg/ml, 59% at AD = 5.0 @g/ml, and 25% at AD = 10.0 pg/ml were seen as compared to cells at these AD concentrations alone. A significant change (P = 0.04) in radiolabel incorporation was seen when the AD concentration of PDT-treated cells increased from 2.5 to 5.0 pug/ml but not with further increases in AD concentration. The results of treatment of subcutaneous tumors in nude mice are shown in Fig. 2. Groups 1, 3, 4, and 5 showed progressive growth with tumor area nearly doubling by Day 15. There was no statistically significant differences between these groups. The PDT controls (Group 2) showed a slight decrease in tumor area at Day 5 and a 50% decrease in tumor area at Day 10. Tumor regrowth occurred at Day 30 in this group after having remained stable during the interim period. In the AD
The effectiveness of PDT on H-MESOcells in vitro has previously been shown to be dependent on both PF concentration and light fluence [9]. PF and light parameters utilized in this study were chosen to allow identification of additional AD effect against a background of minimal PDT. Several significant relationships of AD, PF, and GVL treatment of mesothelioma cells in vitro were demonstrated. Incubation of H-MESOcells with AD alone led to significant decreases in [3H]thymidine incorporation at all drug concentrations. However, a characteristic dose response was not seen. An interesting observation in this study is the ability of PF to stimulate thymidine uptake in the cells when they are not subsequently exposed to light. This effect was seen in a previous study [9] but in the current study was most pronounced when AD was added at the 2.5 pg/ml concentration. At the higher concentrations of AD, addition of PF led to decreases in radiolabel uptake but only at the AD of 5.0 pg/ml was this significantly different. The explanation of this phenomenon is not known but could include the presence of a limiting growth factor in the heterogeneous PF mixture or of a factor that competes with or counteracts the effects of AD at the lower doses but is lost as the AD dose increases. Exposure of AD-treated cells to GVL resulted in decreases in thymidine uptake as compared to cells exposed to light in the absence of AD. However, it was only with AD at the 5.0 pg/ml concentration that the decrease achieved significance. This suggests that AD alone does have some inherent photosensitizing ability but that it may not be inducible beyond a certain level regardless of the concentration. The combination of AD and PDT resulted in enhanced in vitro cytotoxicity as measured by thymidine incorporation. The dose response is similar to that seen in cells treated with AD alone and is most pronounced at the 5.0 pg/ml concentration of AD. At that dose the effect appears to be synergistic but at the other doses this is not clear. A possible explanation for this variability in the effect of PDT may be that pretreatment with AD may interfere with pathways needed for the expression of the PDT effect. The combination of AD and PDT resulted in a complete and sustained necrosis of subcutaneous tumors in nude mice. Standard PDT resulted in a decrease in tumor area but it was not sustained nor complete. Treat-
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ment with AD alone as well as other combinations of AD with PF and GVL did not have any effect on tumor growth. The time to maximal response in the two PDT groups was different and this suggests an increase in the photodynamic effect related to the presence of AD. The prolonged response to the combination of AD and PDT is most likely related to elimination of tumor cells. Continued observation of this group to 90 days post-treatment resulted in tumor recurrence in only two animals (data not shown). An explanation of the observed effect probably involves complex interactions within the tumor including vascular injury and hypoxia as well as the intracellular effects of activated PF and AD. In summary, AD appears to enhance the effectiveness of PDT against H-MESOmalignant mesothelioma cells both in vitro and in uivo. In addition, AD appears to have inherent photosensitizing abilities that are not dose dependent. Lastly, PF may have protective or stimulatory properties when not activated. Widespread application of PDT in cancer therapy has been limited by several factors such as dose standardization of both drugs and light and the complexity of the light delivery systems available. These factors are being slowly resolved and should lead to an increased use of this treatment modality [7, 81. With further refinement of PDT in the management of human cancers, combinations of PDT with chemotherapy may provide improved responses in a select group of tumors such as mesothelioma that are not currently controlled by standard forms of therapy.
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