CLIN. EXPL. METASTASIS, 1990, VOL. 8, NO.
1,
3346
Sensitivity to t h e r m o c h e m o t h e r a p y of AKR l y m p h o m a a n d B16 m e l a n o m a variants of m a l i g n a n c y J. L E I B O V I C I t , G. K L O R I N , M. H U S Z A R , S. H O E N I G , O. K L E I N , M . M I C H O W I T Z a n d A. P I N C H A S S O V Department of Pathology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Tel Aviv, Israel
(Received 5 January 1989; accepted 10 April 1989) Drug resistance, which so often accompanies tumor progression, has been shown to be related to changes in membrane properties which may result in decreased drug accumulation in the tumor cell. A correlation between sensitivity to thermochemotherapy and degree of malignancy was found in the A K R lymphoma system. Hyperthermia increased adriamycin (ADR) uptake and concomitantly its cytotoxicity to A K R lymphoma cells. Moreover, these effects were more pronounced on a variant of high malignancy (HM) than on a low malignancy (LM) one. Fluorescent microscopy, as well as cytofluorometry, indicated that lymphoma cells treated by ADR at 43°C were more permeable to the cytotoxic agent than those exposed to the chemotherapeutic substance at 37°C. Cytofluorometry indicated the presence of a minor cell subpopulation with low A D R uptake in the H M variant, not found in the LM one. Fluorocytometry also showed that the temperature-dependent increased ADR uptake was more marked in the H M than in the L M variant, explaining the differential effect of thermochemotherapy on the two lymphoma variants. However, correlation between degree of malignancy and sensitivity to thermochemotherapy is not a general feature. In contrast to the results obtained in the A K R lymphoma system, in the B16 melanoma the low malignancy variant, F1, was more markedly affected by the combined treatment than the F10 variant. The increased cytotoxic effect of ADR by supranormal temperatures in the F1 variant was shown to be due to an augmented drug uptake. T h e results suggest that drug resistance in late stages of tumor progression can be overcome by an agent acting on the cell membrane. However, the data also indicate the necessity of assaying cancer treatment modalities, including those designed to circumvent drug resistance, on various tumor system models.
Introduction A l t h o u g h very c o n s i d e r a b l e i m p r o v e m e n t in cancer t r e a t m e n t has b e e n achieved d u r i n g the past two decades, the cure of the disease still d e p e n d s o n early diagnosis, since the n o w available a n t i n e o p l a s t i c m e a n s are, in m o s t cases, inefficient a g a i n s t the late stages of the disease. C h e m o t h e r a p y has the i m p o r t a n t faculty of acting n o t o n l y o n local b u t also o n d i s t a n t t u m o r g r o w t h . N e v e r t h e l e s s , this p o w e r f u l a n t i m e t a s t a t i c m e a n s , p h y s i c a l l y able to reach a l m o s t a n y site of the b o d y , has often b e e n f o u n d to be b i o c h e m i c a l l y o v e r c o m e b y the g e n e t i c lability of the n e o p l a s t i c cells. T h e process of t u m o r p r o g r e s s i o n is often a c c o m p a n i e d b y the e m e r g e n c e of d r u g - r e s i s t a n t clones. ~Correspondence should be sent to Professor Judith Leibovici, Department of Pathology, Sackler Faculty of Medicine, Tel-Aviv University, 699778 Tel-Aviv, Israel. 0262-0898/90 $3"00© 1990Taylor & FrancisLtd.
34
J. Leibovici et al.
Antineoplastic drugs should therefore be tested not on static t u m o r models but on t u m o r progression models. Such models m a y consist of variants of malignancy of the same tumor. Variants differing in metastatic capacity have been isolated by various groups [1,3, 10, 21,27] including our own [13, 14, 18]. Studies regarding the comparison of such variants resulted in a massive a m o u n t of information concerning differences existing between cells of high and low malignancy [26, 29]. A large n u m b e r of these studies point to cell m e m b r a n e alterations occurring during t u m o r progression. With the intention of exploiting these cell m e m b r a n e differences, we undertook to examine the effect of agents acting on this cell organelle on t u m o r cells varying in degree of metastatic capacity. W e hoped that, a m o n g such agents, some might be found which could also (or even m o r e efficiently) act on t u m o r cells of high malignancy. We have examined the effect of hyperthermia, the antitumoral effect of which is, at least partly, due to an effect on the cell m e m b r a n e [19, 20] on variants of malignancy of two t u m o r systems: the A K R l y m p h o m a [15, 16] and the B 16 melanoma (unpublished results). In both systems we found a m o r e pronounced inhibitory effect of h y p e r t h e r m i a on the variants of high malignancy than on those of low malignancy. Few studies have c o m p a r e d the effect of h y p e r t h e r m i a on t u m o r cells of varying malignancy [5, 6, 15, 16, 28, 30] and the results diverged. While some authors did not find a correlation between metastatic capacity and sensitivity to h y p e r t h e r m i a [6, 28], other groups, including our own, found that t u m o r cells of high malignancy are m o r e vulnerable to h y p e r t h e r m i c treatment than cells of low metastatic capacity [5, 15, 16, 30]. M e m b r a n e - a c t i n g agents m a y then differentially affect low and high metastatogenic t u m o r cells. T h e s e agents may, in addition, by increasing cell permeability to chemotherapeutic drugs, augment their cytotoxic effect. Indeed, drug resistance, which so often evolves during t u m o r progression, has been found to be related to structural [4, 23, 25] and functional [9, 11] m e m b r a n e changes. H y p e r t h e r m i a has been shown to increase permeability to various cytotoxic agents [2]. In the present study we c o m p a r e d the effect of t h e r m o c h e m o t h e r a p y on two A K R l y m p h o m a variants of low and high malignancy isolated in our laboratory, and on the F1 and F10 variants of B 16 melanoma, originally isolated by Fidler [3], and which have been the subject of n u m e r o u s comparative investigations. In the present study the effect of A D R at various temperatures on the malignancy variants, in these two different t u m o r systems, was compared. We further tried to verify whether the m e c h a n i s m of increased drug efficiency by h y p e r t h e r m i a is related to increased drug uptake. Adriamycin was chosen because of its fluorescence, which enabled us to follow drug uptake at different temperatures, both by fluorescent microscopy and by flow cytometry.
Materials and methods Mice and tumors
T h e A K R l y m p h o m a variants, T A U - 3 9 and T A U - 3 8 , of low and high malignancy respectively, were maintained by subcutaneous (s.c.) inoculation in the neck of A K R / J mice. T h e F1 and F10 variants of B 16 m e l a n o m a were maintained by the same type of inoculation in C 5 7 B L mice. Male mice, 6-10 weeks old, were obtained from the Animal Breeding Center of the T e l - A v i v University. Single cell suspensions of the t u m o r s were obtained as previously described [17].
Metastatic capacity and thermochemotherapy
35
In vitro thermochemotherapeutic treatment of tumor cells H y p e r t h e r m i c in vitro treatment of t u m o r cells, alone and in combination with adriamycin (Farmitalia, Italy) was done as follows: Cells from the L M and H M variants of A K R l y m p h o m a and B16/F1 or B 1 6 / F 1 0 cells (106/ml in R P M I medium) were incubated in thermostated waterbaths ( + 0.2°C) for 1 h at 37°C and at hyperthermic temperatures (42 or 43°C) in the presence or absence of A D R . After incubation the t u m o r cells of the variants were tested for tumorigenicity following inoculation to mice and for A D R uptake by examination in fluorescent microscopy and in F A C S . °
_
Test of tumorigenicity Tumorigenicity following the different treatments was examined by inoculating s.c. 2 × 105 cell in 0"2 ml volume in the neck of different groups of mice (usually five mice per group). T h e data presented are representative results of three or four experiments. T h e concentration of A D R was 0"5 #g/ml for the A K R l y m p h o m a and 1 #g/ml for the B 16 melanoma variants. T h e hyperthermic temperature was 43°C. These moderate conditions were chosen in order to be able to observe differences in combined versus single treatments. T u m o r i g e n i c capacity was evaluated by measuring the diameter of the s.c. tumors two or three times a week, and by daily recording of mouse mortality.
Examination of adriamycin uptake by fluorescent microscopy Examination of A D R uptake by fluorescent microscopy was performed as follows. In the case of A K R l y m p h o m a cells, 106 cells/ml R P M I m e d i u m were incubated in the presence of 50#g/ml A D R for 1 h at 37 ° or 43°C, and were then centrifuged. Cells from the washed pellet, resuspended in PBS, were spread on slides, fixed with formalin vapors and covered with glycerin-gelatin. Cells were examined under an epifluorescence microscope (Zeiss). In the case of the melanoma cells, 106cells/ml in R P M I medium, were incubated as above in the presence of A D R for 1 h at 37°C or 42°C. Cells (2 x 105/0-2ml) were then seeded in 5 8 m m plates (Nunc, Denmark) containing 4 m l R P M I m e d i u m supplemented with 10 per cent fetal calf serum. T h e plates were incubated at 37°C with 5 per cent CO 2 and 100 per cent humidity. At various time intervals after incubation, sample plates were washed three times with saline preheated at 37°C and then fixed with formalin vapors. After covering with glycerin-gelatin, the plates were examined under an epifluorescence microscope (Zeiss).
Examination of adriamycin uptake by F A C S Cytofluorometry was performed on cells treated by A D R (3 or 10 #g/ml for A K R l y m p h o m a variant cells and ! 0/zg/ml for B16 melanoma variant cells at 37°C or 43°C. Cells were analyzed on a Becton Dickinson F A C S IV with Consort 40 data processing. Adriamycin fluorescence was examined with an L P 570 filter.
Statistical evaluation Statistical evaluation was done by Student's t-test for data of t u m o r size and for data of cumulative incidence of tumors and mouse mortality in cases in which all the mice in the group developed tumors. For t u m o r incidence and mouse mortality in cases in which some of the mice did not develop tumors, and therefore survived, the logrank method [24] was used.
J. Leibovici et al.
36
Results T h e effect of combined A D R - h y p e r t h e r m i c treatment on the tumorigenic capacity of cells derived from the two A K R lymphoma variants is presented in figure 1. While A D R alone, at the concentration used (0"5/t/gml), did not inhibit any of the variants and hyperthermic treatment (43°C) resulted in a delay of mouse mortality only in those inoculated with the highly malignant variant, combined A D R - h y p e r t h e r m i c treatment resulted in a very strong inhibition of cell tumorigenicity of the two variants. However, the variant of high malignancy was more markedly affected, since all the mice of this group survived. Figure 2 shows a comparison of fluorescence intensity of cells treated with A D R at 37°C and 43°C of the H M variant. Cells treated at 43°C show a higher intensity of fluorescence. Some of the cells treated by A D R at this temperature have a ghost-like appearance, indicating cell destruction. Similar results were seen with the L M cells (not shown).
TAU - 39 I00-
O•37*43°
_J •
:>
~ 0 R , 43" //--0
50-
O0
,~f, I0
2b
3b DAY
,0
AFTER
TUMOR
5;
T A U -- 38 I00-
-//
50.
~
80
ADR, 4 3 *
#--O
INOCULATION
43*
>
== (n ~'
37*
ADRo3
~o
20 DAY
AFTER
,~ TUMOR
5b
n
so
#-'1
80
INOCULATION
Figure 1. Effect of combined adriamycin-hyperthermic treatment on tumorigenicity of ceils derived from a low-metastasizing (TAU-39) and a high-metastasizing (TAU-38) variant of AKR lymphoma. The LM and HM variant cells, treated or non-treated with ADR (0-5/tg/106 cells/ml), were incubated at 37°C or 43°C for 1 h and then inoculated s.c. (2 × 105 cells) to different groups of mice.
Metastatic capacity and thermochemotherapy
37
Figure 2. Effect of hyperthermia on adriamycin uptake of AKR lymphoma cells-examination by fluorescent microscope. Tumor cells were treated with ADR 50 #g/106 cell/ml) at 37°C or 43°C for 1 h. After washing, the cells were spread on slides and fixed by formalin vapors. Magnification: left side x 52; right side x 300.
F i g u r e 3 shows the cytofluorimetric profile of the A D R content of cells treated at 37°C and 43°C, at two A D R concentrations, 3 and 10 #g/ml, of the l o w - m a l i g n a n c y variant ( u p p e r part), and the h i g h - m a l i g n a n c y one (lower part). It is evident (as also shown by fluorescent m i c r o s c o p y ) that b o t h variants are m o r e p e r m e a b l e to the cytotoxic agent at 43°C than at 37°C. However, examination by F A C S p e r m i t t e d the detection of differences between the two variants. (1) W h i l e the L M variant consists of a single cell p o p u l a t i o n in relation to A D R uptake, the H M variant presents two s u b p o p u l a t i o n s , a major one with an A D R p e r m e a b i l i t y similar to L M (see table 1) and a m i n o r one with a lower p e r m e a b i l i t y which peaks at channels 88 and 123 with 3 and 10 #g A D R , respectively. (2) T h e difference in A D R uptake between 37°C and 43°C is m o r e m a r k e d in the variant of high than in the one of low m a l i g n a n c y
J. Leibovici et al.
38
LM
255I AOR LII 3
AflR
le 37--):7--)~i--43(--43
.-I -J 0
~J yz
255
FIg RDR
HM
3
32->
--
. . . .
6~
120
68 128 RELATIVE FLUORESCENCE 180
248
188
218
Figure 3. Comparison of hyperthermic effect on adriamycin uptake of cells derived from a low- (LM) and a high-metastasizing (HM) variant of AKR lymphoma--examination by FACS. ADR was used at 3 #g/ml (left side curves) and 10#g/ml (right side curves). LM, Low-malignancy variant; HM, high-malignancy variant.
Table 1.
Comparison of h y p e r t h e r m i c i n c r e a s e d a d r i a m y c i n uptake of a low- and a high m a l i g n a n c y variant of AKR l y m p h o m a : c o m p a r i s o n of m a i n peak in FACS.
Variant Low malignancy Adriamycin concentration (#g/ml)
Temperature (°C)
Relative fluorescence (mean channel) _+SD
3 3 10 10
37 43 37 43
116_+12 136_+ 11 163_+ 7 187_+10
Differential fluorescence between 37°C and 43°C 20 24
High malignancy Relative fluorescence (mean channel) _+SD 127_+9 165+9 159_+6 194_+7
Differential fluorescence between 37°C and 43°C 38 35
(table 1); (3) the minor subpopulation of low A D R uptake seen at 37 ° in the H M variant disappears completely at 43°C, indicating that, at this supranormal temperature, the cells of this resistant subpopulation probably overcome their A D R resistance, due to an increased drug uptake. T h e effect of combined A D R - h y p e r t h e r m i a on the tumorigenic capacity of the F1 and F10/B 16 melanoma variants was compared. We also tried to examine
Metastatic capacity and thermochemotherapy
39
whether the increased A D R effect at supranormal temperatures is due to an increase in drug uptake. Figures 4 and 5 show the effect of A D R and hyperthermia, in single and combined treatments, on the tumorigenicity of B 16/F1 and B 16/F10, by the criteria of tumor size and mice mortality, respectively. Tables 2 and 3 show the statistical evaluation of the experimental data presented in the figures, and of another experiment.
30 -
F__!
20-
~T
/ / /..E E
IO-
w
0 I,,-
~
oF I0
w
NT 20-
/ .J
I0-
O-
!
o
~o
20 oA~
AFTER
30 TUMOR
~o
,NOCULA.,O.
Figure 4. Effect of adriamycin and h y p e r t h e r m i a on the F1 and F 1 0 variants of B 1 6 melanoma: size of tumors. T h e F1 and F 1 0 m e l a n o m a cells, treated or non-treated by A D R (1/zg/106 cells/ml), were incubated at 37°C for I h and then inoculated s.c. (2 x 10 s cells) to different groups o f C 5 7 B L mice.
J. Leibovici et al.
40
NT.
5o
o-
~ f
,
~
,
O
,
,
~
70
so
9o
•"
v
.J
0
p----/
0
'/'--"'-1
20
30
,to
~o
6o
12o
DAY AFTER TUMOR INOCULATION F i g u r e 5.
E f f e c t o f a d r i a m y c i n a n d h y p e r t h e r m i a o n t h e F1 a n d F 1 0 v a r i a n t s o f B 1 6 m e l a n o m a : m o r t a l i t y o f m i c e . E x p e r i m e n t a l p r o c e d u r e as in f i g u r e 4.
T h e F1 variant appears to be slightly more sensitive to A D R than the F10. By contrast, the variant of higher malignancy was more sensitive to the hyperthermic treatment. Combined A D R - h y p e r t h e r m i c treatment resulted in a more efficient reduction in tumorigenicity of the F1 variant as compared to the F10. In fact, in the combined treatment, A D R only slightly improved the antineoplastic activity of hyperthermia against B 16/F10 while it strongly augmented the efficiency of the hyperthermic effect on B 16/F1. According to most data, including those of t u m o r cumulative incidence (not shown), the inhibition by the combined treatment was statistically more significant in the case of F1 than with the F10. Figure 6 presents the fluorescent microscopy picture of F1 and F10 culture plates seeded with cells pretreated in vitro during 1 h with A D R at 37°C or 42°C. F r o m figure 6 it is evident that A D R uptake of F1 cells at 37°C is more efficient than drug uptake of the F10 cells. T r e a t m e n t by A D R at 42°C inhibited the growth of both F1 and F10, as compared to A D R pretreatment at 37°C, as evidenced by the paucity of cells seen. However, the fluorescence of the remaining cells at 42°C was more intense as compared to those at 37°C. Figure 7 presents the cytofluorimetric profile of F1 and F10 cells treated by A D R at 37°C. T h e data show that at 37°C the F10 cells consist of two subpopulations with regard to A D R uptake; one with a low permeability, peaking at channel 155 and a second, with a higher uptake potential, with a peak at channel 192. T h e A D R uptake
Metastatic capacity and thermochemotherapy Table 2.
41
Effect of a d r i a m y c i n a n d h y p e r t h e r m i c t r e a t m e n t , a l o n e a n d in c o m b i n a t i o n , on c e l l s o f the F1 a n d F10/B16 m e l a n o m a variants: s i z e of t u m o r s .
No. of experiment
Variant
Treatment
Average diameter of t u m o r ( r a m ) +_ S D
P compared to 37°C
P compared to A D R
P compared to 43°C
F1 F1 F1 F1
37°C 37°C-ADR 43°C 43°C-ADR
26"0___ 1"41 2 1 " 0 _ 4"74 9-8 _ 8-52 0 - 0 _ 0-00
t> 0-025 < 0"0025 < 0"0005
< 0"0005
< 0-025
F10 F10 F10 F10
37°C 37°-CADR 43°C 43°C-ADR
28-6___ 1-02 25-6 ___0-80 3"4___5-42 1"4___1-74
< 0"0005 < 0-0005
1,
3346
Sensitivity to t h e r m o c h e m o t h e r a p y of AKR l y m p h o m a a n d B16 m e l a n o m a variants of m a l i g n a n c y J. L E I B O V I C I t , G. K L O R I N , M. H U S Z A R , S. H O E N I G , O. K L E I N , M . M I C H O W I T Z a n d A. P I N C H A S S O V Department of Pathology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Tel Aviv, Israel
(Received 5 January 1989; accepted 10 April 1989) Drug resistance, which so often accompanies tumor progression, has been shown to be related to changes in membrane properties which may result in decreased drug accumulation in the tumor cell. A correlation between sensitivity to thermochemotherapy and degree of malignancy was found in the A K R lymphoma system. Hyperthermia increased adriamycin (ADR) uptake and concomitantly its cytotoxicity to A K R lymphoma cells. Moreover, these effects were more pronounced on a variant of high malignancy (HM) than on a low malignancy (LM) one. Fluorescent microscopy, as well as cytofluorometry, indicated that lymphoma cells treated by ADR at 43°C were more permeable to the cytotoxic agent than those exposed to the chemotherapeutic substance at 37°C. Cytofluorometry indicated the presence of a minor cell subpopulation with low A D R uptake in the H M variant, not found in the LM one. Fluorocytometry also showed that the temperature-dependent increased ADR uptake was more marked in the H M than in the L M variant, explaining the differential effect of thermochemotherapy on the two lymphoma variants. However, correlation between degree of malignancy and sensitivity to thermochemotherapy is not a general feature. In contrast to the results obtained in the A K R lymphoma system, in the B16 melanoma the low malignancy variant, F1, was more markedly affected by the combined treatment than the F10 variant. The increased cytotoxic effect of ADR by supranormal temperatures in the F1 variant was shown to be due to an augmented drug uptake. T h e results suggest that drug resistance in late stages of tumor progression can be overcome by an agent acting on the cell membrane. However, the data also indicate the necessity of assaying cancer treatment modalities, including those designed to circumvent drug resistance, on various tumor system models.
Introduction A l t h o u g h very c o n s i d e r a b l e i m p r o v e m e n t in cancer t r e a t m e n t has b e e n achieved d u r i n g the past two decades, the cure of the disease still d e p e n d s o n early diagnosis, since the n o w available a n t i n e o p l a s t i c m e a n s are, in m o s t cases, inefficient a g a i n s t the late stages of the disease. C h e m o t h e r a p y has the i m p o r t a n t faculty of acting n o t o n l y o n local b u t also o n d i s t a n t t u m o r g r o w t h . N e v e r t h e l e s s , this p o w e r f u l a n t i m e t a s t a t i c m e a n s , p h y s i c a l l y able to reach a l m o s t a n y site of the b o d y , has often b e e n f o u n d to be b i o c h e m i c a l l y o v e r c o m e b y the g e n e t i c lability of the n e o p l a s t i c cells. T h e process of t u m o r p r o g r e s s i o n is often a c c o m p a n i e d b y the e m e r g e n c e of d r u g - r e s i s t a n t clones. ~Correspondence should be sent to Professor Judith Leibovici, Department of Pathology, Sackler Faculty of Medicine, Tel-Aviv University, 699778 Tel-Aviv, Israel. 0262-0898/90 $3"00© 1990Taylor & FrancisLtd.
34
J. Leibovici et al.
Antineoplastic drugs should therefore be tested not on static t u m o r models but on t u m o r progression models. Such models m a y consist of variants of malignancy of the same tumor. Variants differing in metastatic capacity have been isolated by various groups [1,3, 10, 21,27] including our own [13, 14, 18]. Studies regarding the comparison of such variants resulted in a massive a m o u n t of information concerning differences existing between cells of high and low malignancy [26, 29]. A large n u m b e r of these studies point to cell m e m b r a n e alterations occurring during t u m o r progression. With the intention of exploiting these cell m e m b r a n e differences, we undertook to examine the effect of agents acting on this cell organelle on t u m o r cells varying in degree of metastatic capacity. W e hoped that, a m o n g such agents, some might be found which could also (or even m o r e efficiently) act on t u m o r cells of high malignancy. We have examined the effect of hyperthermia, the antitumoral effect of which is, at least partly, due to an effect on the cell m e m b r a n e [19, 20] on variants of malignancy of two t u m o r systems: the A K R l y m p h o m a [15, 16] and the B 16 melanoma (unpublished results). In both systems we found a m o r e pronounced inhibitory effect of h y p e r t h e r m i a on the variants of high malignancy than on those of low malignancy. Few studies have c o m p a r e d the effect of h y p e r t h e r m i a on t u m o r cells of varying malignancy [5, 6, 15, 16, 28, 30] and the results diverged. While some authors did not find a correlation between metastatic capacity and sensitivity to h y p e r t h e r m i a [6, 28], other groups, including our own, found that t u m o r cells of high malignancy are m o r e vulnerable to h y p e r t h e r m i c treatment than cells of low metastatic capacity [5, 15, 16, 30]. M e m b r a n e - a c t i n g agents m a y then differentially affect low and high metastatogenic t u m o r cells. T h e s e agents may, in addition, by increasing cell permeability to chemotherapeutic drugs, augment their cytotoxic effect. Indeed, drug resistance, which so often evolves during t u m o r progression, has been found to be related to structural [4, 23, 25] and functional [9, 11] m e m b r a n e changes. H y p e r t h e r m i a has been shown to increase permeability to various cytotoxic agents [2]. In the present study we c o m p a r e d the effect of t h e r m o c h e m o t h e r a p y on two A K R l y m p h o m a variants of low and high malignancy isolated in our laboratory, and on the F1 and F10 variants of B 16 melanoma, originally isolated by Fidler [3], and which have been the subject of n u m e r o u s comparative investigations. In the present study the effect of A D R at various temperatures on the malignancy variants, in these two different t u m o r systems, was compared. We further tried to verify whether the m e c h a n i s m of increased drug efficiency by h y p e r t h e r m i a is related to increased drug uptake. Adriamycin was chosen because of its fluorescence, which enabled us to follow drug uptake at different temperatures, both by fluorescent microscopy and by flow cytometry.
Materials and methods Mice and tumors
T h e A K R l y m p h o m a variants, T A U - 3 9 and T A U - 3 8 , of low and high malignancy respectively, were maintained by subcutaneous (s.c.) inoculation in the neck of A K R / J mice. T h e F1 and F10 variants of B 16 m e l a n o m a were maintained by the same type of inoculation in C 5 7 B L mice. Male mice, 6-10 weeks old, were obtained from the Animal Breeding Center of the T e l - A v i v University. Single cell suspensions of the t u m o r s were obtained as previously described [17].
Metastatic capacity and thermochemotherapy
35
In vitro thermochemotherapeutic treatment of tumor cells H y p e r t h e r m i c in vitro treatment of t u m o r cells, alone and in combination with adriamycin (Farmitalia, Italy) was done as follows: Cells from the L M and H M variants of A K R l y m p h o m a and B16/F1 or B 1 6 / F 1 0 cells (106/ml in R P M I medium) were incubated in thermostated waterbaths ( + 0.2°C) for 1 h at 37°C and at hyperthermic temperatures (42 or 43°C) in the presence or absence of A D R . After incubation the t u m o r cells of the variants were tested for tumorigenicity following inoculation to mice and for A D R uptake by examination in fluorescent microscopy and in F A C S . °
_
Test of tumorigenicity Tumorigenicity following the different treatments was examined by inoculating s.c. 2 × 105 cell in 0"2 ml volume in the neck of different groups of mice (usually five mice per group). T h e data presented are representative results of three or four experiments. T h e concentration of A D R was 0"5 #g/ml for the A K R l y m p h o m a and 1 #g/ml for the B 16 melanoma variants. T h e hyperthermic temperature was 43°C. These moderate conditions were chosen in order to be able to observe differences in combined versus single treatments. T u m o r i g e n i c capacity was evaluated by measuring the diameter of the s.c. tumors two or three times a week, and by daily recording of mouse mortality.
Examination of adriamycin uptake by fluorescent microscopy Examination of A D R uptake by fluorescent microscopy was performed as follows. In the case of A K R l y m p h o m a cells, 106 cells/ml R P M I m e d i u m were incubated in the presence of 50#g/ml A D R for 1 h at 37 ° or 43°C, and were then centrifuged. Cells from the washed pellet, resuspended in PBS, were spread on slides, fixed with formalin vapors and covered with glycerin-gelatin. Cells were examined under an epifluorescence microscope (Zeiss). In the case of the melanoma cells, 106cells/ml in R P M I medium, were incubated as above in the presence of A D R for 1 h at 37°C or 42°C. Cells (2 x 105/0-2ml) were then seeded in 5 8 m m plates (Nunc, Denmark) containing 4 m l R P M I m e d i u m supplemented with 10 per cent fetal calf serum. T h e plates were incubated at 37°C with 5 per cent CO 2 and 100 per cent humidity. At various time intervals after incubation, sample plates were washed three times with saline preheated at 37°C and then fixed with formalin vapors. After covering with glycerin-gelatin, the plates were examined under an epifluorescence microscope (Zeiss).
Examination of adriamycin uptake by F A C S Cytofluorometry was performed on cells treated by A D R (3 or 10 #g/ml for A K R l y m p h o m a variant cells and ! 0/zg/ml for B16 melanoma variant cells at 37°C or 43°C. Cells were analyzed on a Becton Dickinson F A C S IV with Consort 40 data processing. Adriamycin fluorescence was examined with an L P 570 filter.
Statistical evaluation Statistical evaluation was done by Student's t-test for data of t u m o r size and for data of cumulative incidence of tumors and mouse mortality in cases in which all the mice in the group developed tumors. For t u m o r incidence and mouse mortality in cases in which some of the mice did not develop tumors, and therefore survived, the logrank method [24] was used.
J. Leibovici et al.
36
Results T h e effect of combined A D R - h y p e r t h e r m i c treatment on the tumorigenic capacity of cells derived from the two A K R lymphoma variants is presented in figure 1. While A D R alone, at the concentration used (0"5/t/gml), did not inhibit any of the variants and hyperthermic treatment (43°C) resulted in a delay of mouse mortality only in those inoculated with the highly malignant variant, combined A D R - h y p e r t h e r m i c treatment resulted in a very strong inhibition of cell tumorigenicity of the two variants. However, the variant of high malignancy was more markedly affected, since all the mice of this group survived. Figure 2 shows a comparison of fluorescence intensity of cells treated with A D R at 37°C and 43°C of the H M variant. Cells treated at 43°C show a higher intensity of fluorescence. Some of the cells treated by A D R at this temperature have a ghost-like appearance, indicating cell destruction. Similar results were seen with the L M cells (not shown).
TAU - 39 I00-
O•37*43°
_J •
:>
~ 0 R , 43" //--0
50-
O0
,~f, I0
2b
3b DAY
,0
AFTER
TUMOR
5;
T A U -- 38 I00-
-//
50.
~
80
ADR, 4 3 *
#--O
INOCULATION
43*
>
== (n ~'
37*
ADRo3
~o
20 DAY
AFTER
,~ TUMOR
5b
n
so
#-'1
80
INOCULATION
Figure 1. Effect of combined adriamycin-hyperthermic treatment on tumorigenicity of ceils derived from a low-metastasizing (TAU-39) and a high-metastasizing (TAU-38) variant of AKR lymphoma. The LM and HM variant cells, treated or non-treated with ADR (0-5/tg/106 cells/ml), were incubated at 37°C or 43°C for 1 h and then inoculated s.c. (2 × 105 cells) to different groups of mice.
Metastatic capacity and thermochemotherapy
37
Figure 2. Effect of hyperthermia on adriamycin uptake of AKR lymphoma cells-examination by fluorescent microscope. Tumor cells were treated with ADR 50 #g/106 cell/ml) at 37°C or 43°C for 1 h. After washing, the cells were spread on slides and fixed by formalin vapors. Magnification: left side x 52; right side x 300.
F i g u r e 3 shows the cytofluorimetric profile of the A D R content of cells treated at 37°C and 43°C, at two A D R concentrations, 3 and 10 #g/ml, of the l o w - m a l i g n a n c y variant ( u p p e r part), and the h i g h - m a l i g n a n c y one (lower part). It is evident (as also shown by fluorescent m i c r o s c o p y ) that b o t h variants are m o r e p e r m e a b l e to the cytotoxic agent at 43°C than at 37°C. However, examination by F A C S p e r m i t t e d the detection of differences between the two variants. (1) W h i l e the L M variant consists of a single cell p o p u l a t i o n in relation to A D R uptake, the H M variant presents two s u b p o p u l a t i o n s , a major one with an A D R p e r m e a b i l i t y similar to L M (see table 1) and a m i n o r one with a lower p e r m e a b i l i t y which peaks at channels 88 and 123 with 3 and 10 #g A D R , respectively. (2) T h e difference in A D R uptake between 37°C and 43°C is m o r e m a r k e d in the variant of high than in the one of low m a l i g n a n c y
J. Leibovici et al.
38
LM
255I AOR LII 3
AflR
le 37--):7--)~i--43(--43
.-I -J 0
~J yz
255
FIg RDR
HM
3
32->
--
. . . .
6~
120
68 128 RELATIVE FLUORESCENCE 180
248
188
218
Figure 3. Comparison of hyperthermic effect on adriamycin uptake of cells derived from a low- (LM) and a high-metastasizing (HM) variant of AKR lymphoma--examination by FACS. ADR was used at 3 #g/ml (left side curves) and 10#g/ml (right side curves). LM, Low-malignancy variant; HM, high-malignancy variant.
Table 1.
Comparison of h y p e r t h e r m i c i n c r e a s e d a d r i a m y c i n uptake of a low- and a high m a l i g n a n c y variant of AKR l y m p h o m a : c o m p a r i s o n of m a i n peak in FACS.
Variant Low malignancy Adriamycin concentration (#g/ml)
Temperature (°C)
Relative fluorescence (mean channel) _+SD
3 3 10 10
37 43 37 43
116_+12 136_+ 11 163_+ 7 187_+10
Differential fluorescence between 37°C and 43°C 20 24
High malignancy Relative fluorescence (mean channel) _+SD 127_+9 165+9 159_+6 194_+7
Differential fluorescence between 37°C and 43°C 38 35
(table 1); (3) the minor subpopulation of low A D R uptake seen at 37 ° in the H M variant disappears completely at 43°C, indicating that, at this supranormal temperature, the cells of this resistant subpopulation probably overcome their A D R resistance, due to an increased drug uptake. T h e effect of combined A D R - h y p e r t h e r m i a on the tumorigenic capacity of the F1 and F10/B 16 melanoma variants was compared. We also tried to examine
Metastatic capacity and thermochemotherapy
39
whether the increased A D R effect at supranormal temperatures is due to an increase in drug uptake. Figures 4 and 5 show the effect of A D R and hyperthermia, in single and combined treatments, on the tumorigenicity of B 16/F1 and B 16/F10, by the criteria of tumor size and mice mortality, respectively. Tables 2 and 3 show the statistical evaluation of the experimental data presented in the figures, and of another experiment.
30 -
F__!
20-
~T
/ / /..E E
IO-
w
0 I,,-
~
oF I0
w
NT 20-
/ .J
I0-
O-
!
o
~o
20 oA~
AFTER
30 TUMOR
~o
,NOCULA.,O.
Figure 4. Effect of adriamycin and h y p e r t h e r m i a on the F1 and F 1 0 variants of B 1 6 melanoma: size of tumors. T h e F1 and F 1 0 m e l a n o m a cells, treated or non-treated by A D R (1/zg/106 cells/ml), were incubated at 37°C for I h and then inoculated s.c. (2 x 10 s cells) to different groups o f C 5 7 B L mice.
J. Leibovici et al.
40
NT.
5o
o-
~ f
,
~
,
O
,
,
~
70
so
9o
•"
v
.J
0
p----/
0
'/'--"'-1
20
30
,to
~o
6o
12o
DAY AFTER TUMOR INOCULATION F i g u r e 5.
E f f e c t o f a d r i a m y c i n a n d h y p e r t h e r m i a o n t h e F1 a n d F 1 0 v a r i a n t s o f B 1 6 m e l a n o m a : m o r t a l i t y o f m i c e . E x p e r i m e n t a l p r o c e d u r e as in f i g u r e 4.
T h e F1 variant appears to be slightly more sensitive to A D R than the F10. By contrast, the variant of higher malignancy was more sensitive to the hyperthermic treatment. Combined A D R - h y p e r t h e r m i c treatment resulted in a more efficient reduction in tumorigenicity of the F1 variant as compared to the F10. In fact, in the combined treatment, A D R only slightly improved the antineoplastic activity of hyperthermia against B 16/F10 while it strongly augmented the efficiency of the hyperthermic effect on B 16/F1. According to most data, including those of t u m o r cumulative incidence (not shown), the inhibition by the combined treatment was statistically more significant in the case of F1 than with the F10. Figure 6 presents the fluorescent microscopy picture of F1 and F10 culture plates seeded with cells pretreated in vitro during 1 h with A D R at 37°C or 42°C. F r o m figure 6 it is evident that A D R uptake of F1 cells at 37°C is more efficient than drug uptake of the F10 cells. T r e a t m e n t by A D R at 42°C inhibited the growth of both F1 and F10, as compared to A D R pretreatment at 37°C, as evidenced by the paucity of cells seen. However, the fluorescence of the remaining cells at 42°C was more intense as compared to those at 37°C. Figure 7 presents the cytofluorimetric profile of F1 and F10 cells treated by A D R at 37°C. T h e data show that at 37°C the F10 cells consist of two subpopulations with regard to A D R uptake; one with a low permeability, peaking at channel 155 and a second, with a higher uptake potential, with a peak at channel 192. T h e A D R uptake
Metastatic capacity and thermochemotherapy Table 2.
41
Effect of a d r i a m y c i n a n d h y p e r t h e r m i c t r e a t m e n t , a l o n e a n d in c o m b i n a t i o n , on c e l l s o f the F1 a n d F10/B16 m e l a n o m a variants: s i z e of t u m o r s .
No. of experiment
Variant
Treatment
Average diameter of t u m o r ( r a m ) +_ S D
P compared to 37°C
P compared to A D R
P compared to 43°C
F1 F1 F1 F1
37°C 37°C-ADR 43°C 43°C-ADR
26"0___ 1"41 2 1 " 0 _ 4"74 9-8 _ 8-52 0 - 0 _ 0-00
t> 0-025 < 0"0025 < 0"0005
< 0"0005
< 0-025
F10 F10 F10 F10
37°C 37°-CADR 43°C 43°C-ADR
28-6___ 1-02 25-6 ___0-80 3"4___5-42 1"4___1-74
< 0"0005 < 0-0005
