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Experimental aspects of in vitro and in vivo photochemotherapy
Biochimie, 68 (1986) 923-926 © Soci6t6de Chimie biologique/Elsevier.Paris
923
Experimental aspects of in vitro and in vivo photochemotherapy Thierry PATRICE I, Vincent PRALORAN 2, Marie-Fran¢oise LE BODIC 3 and Louis LE BODIC I I D~partement Laser, H6pital G. et R. La6nnec, B.P. 1005, Nantes, and 2Laboratoire d'Hdmatologie and 3Laboratoire d'Anatomie Pathologique A et B, CHR de Nantes, Nantes, France (Received 6-2-1986, accepted 26-3-1986)
S u m m a r y - The selectivity of bt vitro photodynamic reactions and the in vivo effects induced by PRT, whether the irradiation is applied interstitially or externally, still remains unclear, hi vitro studies were performed using leukemic cell lines and syngeneic normal hemopoietic progenitors. For these, cells incubated with hematoporphyrin derivative (HPD) and non-incubated cells were irradiated with an argon laser. Data were obtained as the count of cell colonies found after a 7-day incubation period on semi-solid collagen gel medium. In vivo studies employed the HT 29 tumor model grafted into nude mice. Both animals injected with HPD and non-infected controls were irradiated with a dye laser pumped by an argon laser (Coherent) using a 400 tzm optic fiber located either at a distance of 65 mm from the skin or inserted into the tumor. The temperature increase occurring during PRT was measured using non-absorbing thermocoupies. ht vitro, after HPD treatment and argon irradiation leukemic cells showed a greater phototoxicity ( > 2 logl0) than did the normal cells (0.25 logl0), hz vivo, when the heat rise is very similar ( < 4°C) in both the tissues irradiated externally and those irradiated interstitially after HPD injection, histological examination of these did not reveal any quantitative differences (90°7o of tumor mass). These results are discussed.
hematoporphyrin derivative I hi vitro photocylotoxicity / L 1210, P 388, HT 29 / argon laser / dye laser
R~sum6 - Aspects exp~rimentaux de la photochimioth~rapie in vitro et in vivo. La s61ectivit~ des rdactions photochimiqttes in vitro, les efrets anatomo-pathologiques indttits in vivo par la r6action photochimique, lors d'une irradiation soit externe soit interstitielle demettrent real connus. Les ~tudes in vitro ont ~td tnendes sttr des modules celhtlaires lettcdntiques et sttr des prog~nitettrs hdntopo't~tiqttes syngdniqttes. Dans ce grottpe d"expdriences, l'irradiation des celhdes sottmises ott 11011~t tree incubation de deux hettres avec H P D est r6alis6e en utilisant le laser gt l'argon ionis& Les rdsuitats sont obtenus par comptage de colonies celhtlaires aprbs sept jours d'incubation sur ttn miliett demi-solide de gel de collagkne. Les 6tudes in vivo ont reposd sttr le tnodble tttntoral H T 29, addnome colique humain greff~ sur la sottris nttde. L "irradiation pratiqu6e gt 632 nm (Dyelaser pomp6 par laser argon ionis6 Coherent©) a 6t~ r~alis6e soit ?t l'aide d'une fibre 400 tzm maintentte gt 65 m m de la peatt (irradiation externe) soit par bnplantation de la fibre optiqtte. Des ntesures d'dldvation thermiqlte par therntocouple non absorbant flnplantd ont ~t6 r6alis6es. In vitro, notts avons ptt ntettre en 6vidence ttne phototoxicit6 de phts de 2 log~o sur les celhdes leuc6tniqttes trait~es par H P D et laser, mais seulement de 0,25 Ioglo sttr des celhdes prog6nitrices normales. In vivo, alors que les ~16vations thermiqttes sont comparables avec les dettx m~thodes d'irradiation, hlfdriettres ?t 4°C, l'~tttde anatomo-pathologique ne met pas en dvidence de diffdrences quantitatives quant ?t la ndcrose tissulaire htduite par laser + HPD. Ces r~sttltats sont discutds. h6matoporphyrine ddriv~e I photoc.vtotoxicit~in vitro ! L 1210, P 388, H T 291 laser argon I laser h colorants
924
T. Patrice et al.
Introduction Photodynamic reactions mediated by hematoporphyrin derivative have been applied for many years in experimental cancer therapy, but many problems still remain, even regarding such fundamental points as selectivity. The preferential uptake o f the sensitizer by neoplastic tissues was first demonstrated for solid tumors using 3H- or 14Clabelled H P D [1]. More recently, Andreoni et al. [2] demonstrated that P R T produced greater cytotoxicity in virus transformed thyroid cells than in normal cells. In this preliminary in vitro study, a new colony-forming assay [3] was used in which no temperature rise was involved at any moment. It was thus possible to assess the efficacy o f P R T on LI210 cells, as well as the selectivity of this treatment on normal syngeneic cells. The effect of PRT treatment #z vivo is also unclear as regards the role o f laser-induced hyperthermia or the efficiency o f interstitial or external laser delivery. With our H T 29 tumor model, we attempted to clarify these two points.
Materials and melhods In vitro studies An argon ion (multiwavelength) laser was used which is strongly absorbed by HPD. The laser beam was transmitted by an index step silica-silicon optic fiber of core diameter 400/.zm, cooled by a stream of CO2. The tip of the fiber was maintained at 65 mm from the target, and an energy of 50-150 J/cm 2was delivered, the quantity depending on the exposure time. 25-cm 2 flasks each containing 5 ml of 5× 105 cells/ml were irradiated directly. Two kinds of cells were used: LI210 cells (lymphocytic leukemia induced by methylcholanthrene in DBA2 mice) and normal progenitors obtained by flushing out marrow cells from the femurs of syngeneic Fj mice. In both cases the cells were seeded on collagen gel medium after irradiation. The plating efficiency was established after a 7-dayincubation period. HPD was prepared according to the method of Gregorie and Lipson. In vivo studies A human colonic carcinoma type tumor HT 29 was grafted into albino nude mice by bilateral injection of crushed tumors. 5 mg/kg of HPD was injected intravenously 24 h before irradiation. The laser source was a CR 99 dye laser pumped by an Innova 90 (Coherent R) argon laser emmitting 200 mW at the fiber tip at 632 nm. For external irradiation (14 tumors) the fiber tip was maintained at a distance of 65 mm from the mouse's skin. For interstitial irradiation (12 tumors) the tip was embedded in the tumor at a depth of 5 mm. Temperature measurements were made with non-absorbing thermocouples
from Lelt (Lorient, France). Histopathological examinations were performed on mice killed 48 h after laser irradiation. Control groups each made up of I0 animals with tumors were used for each experiment.
Results In vitro studies The results are summarized in Table I. There was an absence o f toxicity of H P D in the concentration range used for the incubations. Laser irradiation on its own produced a dose-dependent alteration o f plating efficiency. A highly cytotoxic effect of combined H P D and laser treatment was evident with leukemic clonogenic cells, which was dependent on both H P D concentration and laser dose. H P D at a concentration o f equal to or greater than 20 t~g/ml PlUs laser irradiation of equal or greater than 50 J / c m 2 always reduced plating efficiency to 1.8 lOgl0 or 3 lOgl0 under optimal conditions. Normal progenitor cells under the same conditions showed no decrease o f plating efficiency either with H P D alone (max. H P D concentration = 30 ~g/ml) or with laser alone (50 or 100 J/cm2). However, combined H P D plus argon laser treatment (50 J / c m 2) decreased the plating efficiency to 0.25 logl0.
Table 1. Selective photodynamic cytotoxicity of leukemic cells (LI210) compared to normal hemopoietic progenitors (CFU-GM). Normal cells Leukemiccells (CFU-GM, day 7) (LI210),x 103/ml × I05/ml Controls
113.0 +__15 (100%)
Treated cells 20tzg/ml, 50 J/cruZ73.5 _+0.7 64.5 +0.7 20t~g/ml, 100 J/cm 2 31 _+8 27.5+7.7
64 + 5 (100%)
1.2+0.3 (1.8%) 0.7+0.1 (---1% o)
I n vivo s t u d i e s Temperature measurements (Figs. I, 2) using an intrarectal reference thermocouple taken either during external or interstitial irradiation indicated a heat increase of less than 4°C during the first
Experimental aspects of in vitro and in vivo pl~otochemotherapy
AT"
EXTERNAL
d 65 me P 26 O mw
4
P 2 mm .
I
1
925
J
l
I
.
.
.
.
i
3
.
.
.
!
4
t~~.~
2.8 "C .
.
.
I
5
I
6
I
7
8
1
time iaia
Fig. 1. Heat increase during external PRT irradiation (optic fiber-skin surface distance 65 mm). Cu~'ves from top to bottom: intratumoral thermocouple depth 2 ram; intratumoral thermocouple depth 4 mm (after skin incision); intratumoral thermocouple depth 4 mm (without skin incision). The increase in skin temperature was 2.8°C.
I
Z~T
d O,F,/thermo couple (mm) I
1
2
5
8
~T
4,5
4
3
1
maximale (*C)
1
10 0,5
EMBEDDED FIBER
]~
P200 mw
•
I I
•
I 2
time
rain
Fig. 2. Heat increase during interstitial PRT irradiation. Curve a (top): optic fiber-thermocouple distance 2.5 mm. Curve b (bottom): optic fiber-thermocouple distance 5 mm.
926
T. Patrice et al.
10 min of exposure to laser irradiation. In the case of interstitial irradiation the initial temperature increase gradient was very steep, while during the external irradiation the increase in skin temperature was 2.8°C. Dye laser irradiation 24 h after H P D injection induced a decrease in growth rate as compared to controls ( + 1.4 v s + 3.7 relative units, respectively), whereas tumors irradiated by laser alone showed an increase in growth rate as compared to controls ( + 5.7 vs + 3.7 relative units). While no macroscopic change was evident immediately after external irradiation following H P D injection, histological examination revealed that definite skin necrosis had occurred by the time o f sacrifice. Mice that had only received H P D injections showed rare areas o f scanty punctiform necrosis with persistence of more than 90°7o o f the tumor mass as described earlier [4]. Laser-irradiated mice showed no differences as compared with control mice. Tumors treated with laser plus H P D showed necrosis, affecting more than 90070 of the tumor mass. No quantitative differences were detectable between externally irradiated or interstially irradiated mice.
medium, apart from being more physiological than agar, avoids any hyperthermia. In our #1 vivo experiments the heat increase was never above 4°C. The results are in agreement with those o f Gomer et aL [6] but are underestimated as compared to those of Kinsey et al. [7]. However, PRT induces an increase in temperature at the borderline level between its having no effect and its producing a hyperthermic therapeutic one. Laser irradiation applied on its own seems to induce a dose-dependent toxicity ht vitro ( 2 = 4 8 8 - 5 1 4 nm) and an increase in the growth rate in vivo (2 = 630 nm): uncontrolled photodynamic reactions could be responsible for this phenomenon. Histopathological examination indicates that there seems to be no difference in the toxic effects produced by either kind of irradiation. This is especially important for endoscopic applications where it is sometimes easier to use external rather than interstitial irradiation.
Acknowledgement This work was supported by Grant No. 83 N O 657 from the M.I.R.
Discussion References The results obtained here in the complete absence o f hyperthermia fully confirm the study o f Andreoni et al. [2], which is unique in that it compared cells of the same origin. However, it is important to know whether their 'normal' thyroid cells were in fact normal since these were established as a continuous cell line. In our study, selectivity was observed in only one experimental model, and it is difficult to extrapolate the results to other tumor types, especially solid tumors. It has recently been shown [5] that even a short period Of hyperthermia can induce a tumor response. The use of collagen gel in our in vitro experiments as the culture
1 Gomer C.J. & Dougherty T.J. (1979) Cancer Res. 39, 146-151 2 Andreoni A. & Cubeddu R. (1983) Cancer Res. 43, 2076-2080 3 Lanotte M. (1984) Biol. Cell. 50, 107-120 4 Patrice T. & Le Bodic M.F. (1983) CancerRes. 43, 2876-2880 5 Urano M. & Kahn J. (1985) Cancer Res. 45, 2527-2532 6 Gomer C.J. & Rucker N. (1985) Cancer Res. 45, 1973-1977 7 Kinsey J.H. & Cortese D.A. (1983) CancerRes. 43, 1562-1567
923
Experimental aspects of in vitro and in vivo photochemotherapy Thierry PATRICE I, Vincent PRALORAN 2, Marie-Fran¢oise LE BODIC 3 and Louis LE BODIC I I D~partement Laser, H6pital G. et R. La6nnec, B.P. 1005, Nantes, and 2Laboratoire d'Hdmatologie and 3Laboratoire d'Anatomie Pathologique A et B, CHR de Nantes, Nantes, France (Received 6-2-1986, accepted 26-3-1986)
S u m m a r y - The selectivity of bt vitro photodynamic reactions and the in vivo effects induced by PRT, whether the irradiation is applied interstitially or externally, still remains unclear, hi vitro studies were performed using leukemic cell lines and syngeneic normal hemopoietic progenitors. For these, cells incubated with hematoporphyrin derivative (HPD) and non-incubated cells were irradiated with an argon laser. Data were obtained as the count of cell colonies found after a 7-day incubation period on semi-solid collagen gel medium. In vivo studies employed the HT 29 tumor model grafted into nude mice. Both animals injected with HPD and non-infected controls were irradiated with a dye laser pumped by an argon laser (Coherent) using a 400 tzm optic fiber located either at a distance of 65 mm from the skin or inserted into the tumor. The temperature increase occurring during PRT was measured using non-absorbing thermocoupies. ht vitro, after HPD treatment and argon irradiation leukemic cells showed a greater phototoxicity ( > 2 logl0) than did the normal cells (0.25 logl0), hz vivo, when the heat rise is very similar ( < 4°C) in both the tissues irradiated externally and those irradiated interstitially after HPD injection, histological examination of these did not reveal any quantitative differences (90°7o of tumor mass). These results are discussed.
hematoporphyrin derivative I hi vitro photocylotoxicity / L 1210, P 388, HT 29 / argon laser / dye laser
R~sum6 - Aspects exp~rimentaux de la photochimioth~rapie in vitro et in vivo. La s61ectivit~ des rdactions photochimiqttes in vitro, les efrets anatomo-pathologiques indttits in vivo par la r6action photochimique, lors d'une irradiation soit externe soit interstitielle demettrent real connus. Les ~tudes in vitro ont ~td tnendes sttr des modules celhtlaires lettcdntiques et sttr des prog~nitettrs hdntopo't~tiqttes syngdniqttes. Dans ce grottpe d"expdriences, l'irradiation des celhdes sottmises ott 11011~t tree incubation de deux hettres avec H P D est r6alis6e en utilisant le laser gt l'argon ionis& Les rdsuitats sont obtenus par comptage de colonies celhtlaires aprbs sept jours d'incubation sur ttn miliett demi-solide de gel de collagkne. Les 6tudes in vivo ont reposd sttr le tnodble tttntoral H T 29, addnome colique humain greff~ sur la sottris nttde. L "irradiation pratiqu6e gt 632 nm (Dyelaser pomp6 par laser argon ionis6 Coherent©) a 6t~ r~alis6e soit ?t l'aide d'une fibre 400 tzm maintentte gt 65 m m de la peatt (irradiation externe) soit par bnplantation de la fibre optiqtte. Des ntesures d'dldvation thermiqlte par therntocouple non absorbant flnplantd ont ~t6 r6alis6es. In vitro, notts avons ptt ntettre en 6vidence ttne phototoxicit6 de phts de 2 log~o sur les celhdes leuc6tniqttes trait~es par H P D et laser, mais seulement de 0,25 Ioglo sttr des celhdes prog6nitrices normales. In vivo, alors que les ~16vations thermiqttes sont comparables avec les dettx m~thodes d'irradiation, hlfdriettres ?t 4°C, l'~tttde anatomo-pathologique ne met pas en dvidence de diffdrences quantitatives quant ?t la ndcrose tissulaire htduite par laser + HPD. Ces r~sttltats sont discutds. h6matoporphyrine ddriv~e I photoc.vtotoxicit~in vitro ! L 1210, P 388, H T 291 laser argon I laser h colorants
924
T. Patrice et al.
Introduction Photodynamic reactions mediated by hematoporphyrin derivative have been applied for many years in experimental cancer therapy, but many problems still remain, even regarding such fundamental points as selectivity. The preferential uptake o f the sensitizer by neoplastic tissues was first demonstrated for solid tumors using 3H- or 14Clabelled H P D [1]. More recently, Andreoni et al. [2] demonstrated that P R T produced greater cytotoxicity in virus transformed thyroid cells than in normal cells. In this preliminary in vitro study, a new colony-forming assay [3] was used in which no temperature rise was involved at any moment. It was thus possible to assess the efficacy o f P R T on LI210 cells, as well as the selectivity of this treatment on normal syngeneic cells. The effect of PRT treatment #z vivo is also unclear as regards the role o f laser-induced hyperthermia or the efficiency o f interstitial or external laser delivery. With our H T 29 tumor model, we attempted to clarify these two points.
Materials and melhods In vitro studies An argon ion (multiwavelength) laser was used which is strongly absorbed by HPD. The laser beam was transmitted by an index step silica-silicon optic fiber of core diameter 400/.zm, cooled by a stream of CO2. The tip of the fiber was maintained at 65 mm from the target, and an energy of 50-150 J/cm 2was delivered, the quantity depending on the exposure time. 25-cm 2 flasks each containing 5 ml of 5× 105 cells/ml were irradiated directly. Two kinds of cells were used: LI210 cells (lymphocytic leukemia induced by methylcholanthrene in DBA2 mice) and normal progenitors obtained by flushing out marrow cells from the femurs of syngeneic Fj mice. In both cases the cells were seeded on collagen gel medium after irradiation. The plating efficiency was established after a 7-dayincubation period. HPD was prepared according to the method of Gregorie and Lipson. In vivo studies A human colonic carcinoma type tumor HT 29 was grafted into albino nude mice by bilateral injection of crushed tumors. 5 mg/kg of HPD was injected intravenously 24 h before irradiation. The laser source was a CR 99 dye laser pumped by an Innova 90 (Coherent R) argon laser emmitting 200 mW at the fiber tip at 632 nm. For external irradiation (14 tumors) the fiber tip was maintained at a distance of 65 mm from the mouse's skin. For interstitial irradiation (12 tumors) the tip was embedded in the tumor at a depth of 5 mm. Temperature measurements were made with non-absorbing thermocouples
from Lelt (Lorient, France). Histopathological examinations were performed on mice killed 48 h after laser irradiation. Control groups each made up of I0 animals with tumors were used for each experiment.
Results In vitro studies The results are summarized in Table I. There was an absence o f toxicity of H P D in the concentration range used for the incubations. Laser irradiation on its own produced a dose-dependent alteration o f plating efficiency. A highly cytotoxic effect of combined H P D and laser treatment was evident with leukemic clonogenic cells, which was dependent on both H P D concentration and laser dose. H P D at a concentration o f equal to or greater than 20 t~g/ml PlUs laser irradiation of equal or greater than 50 J / c m 2 always reduced plating efficiency to 1.8 lOgl0 or 3 lOgl0 under optimal conditions. Normal progenitor cells under the same conditions showed no decrease o f plating efficiency either with H P D alone (max. H P D concentration = 30 ~g/ml) or with laser alone (50 or 100 J/cm2). However, combined H P D plus argon laser treatment (50 J / c m 2) decreased the plating efficiency to 0.25 logl0.
Table 1. Selective photodynamic cytotoxicity of leukemic cells (LI210) compared to normal hemopoietic progenitors (CFU-GM). Normal cells Leukemiccells (CFU-GM, day 7) (LI210),x 103/ml × I05/ml Controls
113.0 +__15 (100%)
Treated cells 20tzg/ml, 50 J/cruZ73.5 _+0.7 64.5 +0.7 20t~g/ml, 100 J/cm 2 31 _+8 27.5+7.7
64 + 5 (100%)
1.2+0.3 (1.8%) 0.7+0.1 (---1% o)
I n vivo s t u d i e s Temperature measurements (Figs. I, 2) using an intrarectal reference thermocouple taken either during external or interstitial irradiation indicated a heat increase of less than 4°C during the first
Experimental aspects of in vitro and in vivo pl~otochemotherapy
AT"
EXTERNAL
d 65 me P 26 O mw
4
P 2 mm .
I
1
925
J
l
I
.
.
.
.
i
3
.
.
.
!
4
t~~.~
2.8 "C .
.
.
I
5
I
6
I
7
8
1
time iaia
Fig. 1. Heat increase during external PRT irradiation (optic fiber-skin surface distance 65 mm). Cu~'ves from top to bottom: intratumoral thermocouple depth 2 ram; intratumoral thermocouple depth 4 mm (after skin incision); intratumoral thermocouple depth 4 mm (without skin incision). The increase in skin temperature was 2.8°C.
I
Z~T
d O,F,/thermo couple (mm) I
1
2
5
8
~T
4,5
4
3
1
maximale (*C)
1
10 0,5
EMBEDDED FIBER
]~
P200 mw
•
I I
•
I 2
time
rain
Fig. 2. Heat increase during interstitial PRT irradiation. Curve a (top): optic fiber-thermocouple distance 2.5 mm. Curve b (bottom): optic fiber-thermocouple distance 5 mm.
926
T. Patrice et al.
10 min of exposure to laser irradiation. In the case of interstitial irradiation the initial temperature increase gradient was very steep, while during the external irradiation the increase in skin temperature was 2.8°C. Dye laser irradiation 24 h after H P D injection induced a decrease in growth rate as compared to controls ( + 1.4 v s + 3.7 relative units, respectively), whereas tumors irradiated by laser alone showed an increase in growth rate as compared to controls ( + 5.7 vs + 3.7 relative units). While no macroscopic change was evident immediately after external irradiation following H P D injection, histological examination revealed that definite skin necrosis had occurred by the time o f sacrifice. Mice that had only received H P D injections showed rare areas o f scanty punctiform necrosis with persistence of more than 90°7o o f the tumor mass as described earlier [4]. Laser-irradiated mice showed no differences as compared with control mice. Tumors treated with laser plus H P D showed necrosis, affecting more than 90070 of the tumor mass. No quantitative differences were detectable between externally irradiated or interstially irradiated mice.
medium, apart from being more physiological than agar, avoids any hyperthermia. In our #1 vivo experiments the heat increase was never above 4°C. The results are in agreement with those o f Gomer et aL [6] but are underestimated as compared to those of Kinsey et al. [7]. However, PRT induces an increase in temperature at the borderline level between its having no effect and its producing a hyperthermic therapeutic one. Laser irradiation applied on its own seems to induce a dose-dependent toxicity ht vitro ( 2 = 4 8 8 - 5 1 4 nm) and an increase in the growth rate in vivo (2 = 630 nm): uncontrolled photodynamic reactions could be responsible for this phenomenon. Histopathological examination indicates that there seems to be no difference in the toxic effects produced by either kind of irradiation. This is especially important for endoscopic applications where it is sometimes easier to use external rather than interstitial irradiation.
Acknowledgement This work was supported by Grant No. 83 N O 657 from the M.I.R.
Discussion References The results obtained here in the complete absence o f hyperthermia fully confirm the study o f Andreoni et al. [2], which is unique in that it compared cells of the same origin. However, it is important to know whether their 'normal' thyroid cells were in fact normal since these were established as a continuous cell line. In our study, selectivity was observed in only one experimental model, and it is difficult to extrapolate the results to other tumor types, especially solid tumors. It has recently been shown [5] that even a short period Of hyperthermia can induce a tumor response. The use of collagen gel in our in vitro experiments as the culture
1 Gomer C.J. & Dougherty T.J. (1979) Cancer Res. 39, 146-151 2 Andreoni A. & Cubeddu R. (1983) Cancer Res. 43, 2076-2080 3 Lanotte M. (1984) Biol. Cell. 50, 107-120 4 Patrice T. & Le Bodic M.F. (1983) CancerRes. 43, 2876-2880 5 Urano M. & Kahn J. (1985) Cancer Res. 45, 2527-2532 6 Gomer C.J. & Rucker N. (1985) Cancer Res. 45, 1973-1977 7 Kinsey J.H. & Cortese D.A. (1983) CancerRes. 43, 1562-1567