Pigment Cell Research 3:l-7 (1990)
Thermochemotherapy for B16 Melanoma: Combination Therapy of Hyperthermia, Melphalan, and CCNU in Mice V. VICENTE,' M. GOMEZ,' M.M. OCHOTORENA,' A. CREMADES,2 M. CANTERAS3 'Department of Pathology, 'Pharmacology, and 'Biostatistics, Faculty of Medicine, University of Murcia, Spain We studied, by means of quantitative histopathological methods, the changes that took place in B16 melanoma implanted into C57BL/6J mice after combination treatment of (CCNU). The hyperthermia,melphalan, and l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea studies were made on animals sacrificed on days 11,18, and 25 after implantation. 'hmors in treated animals showed a progressive delay in growth, a noticeably reduced number of metastases, and diminution of proliferative capacity. However, this treatment did not affect necrosis, stroma, capsule, or cell infiltration. Ultrastructurally,signs of cell damage were constantly present. Key words:
Thermochemotherapy, Melanoma, Hyperthermia, Melaphan
INTRODUCTION The fact that melanoma tumors are refractary to many chemotherapeutic agents is one of the most serious problems in the chemotherapeutic treatment of melanoma. In recent times, several experiments have been conducted in the search for new antitumoral agents and combinations of agents that will enhance the resulting effect and, by the consequent reduction of dosage permitted, thereby lessen undesirable side effects (Gupta, 1984). There has been a renewal of interest in hyperthermia as a method of treatment for cancer patients, and recent research has concentrated on hyperthermia-plus-chemotherapy combinationtreatment (Yamada et al., 1984). However, these studies have not been aimed at assessing the changes produced in the tumor by the drugs. In the present work, we studied, by means of quantitative histopathological methods, the effect of combination treatment with CCNU, melphalan, and whole-body hyperthermia on B16 melanoma transplanted into C57BL/6J mice.
The treatment of the second group commenced on implantation day with the simultaneous intraperitoneal (i.p.1 administration of melphalan (3 mg/kg/day), CCNU (6 mg/kg/day), and whole-body hyperthermia (40°C/45'/day) to each of the animals in the treated group. The hyperthermia was applied by introducing the animals into a Unitemp ventilated hyperthermia chamber. Rectal temperature was measured with a thermoselective pair connected to an ELLAB thermometer and three RMG thermoselective sondes. For sedation all animals, treated and untreated, were given ketamine a t a rate of 3 mg/kg/day/i.p. Thirty animals from the control group and 30 from the treated group were sacrified on days 11, 18, and 25 post implant (subgroups I, 11, and I11 each contained 30 control animals, and subgroups IV, V, and VI each contained 30 treated animals). Samples of tumor and viscera were fixed in 10%buffered neutral formaldehyde, embedded in paraffin, cut into 5 km sections, and stained with hematoxylin and eosin (H&E), Masson Fontana, and Gordon Swett. Animals that died during the experiment were excluded from the study. The mitotic index calculation was based on 1,000 cells taken at random from each tumor. Tbmoral parenchyma evaluation was carried out by means of cell density per unit area (Na) and of volume (Nv), following the technique of Boatman (1986) on microscope pho-
MATERIALS AND METHODS We used 210 C57BL/6J mice weighing 20 g each. The B16-F1 melanoma was obtained from the Institute of Cancer Research, Royal Cancer Hospital, London. The tumor line was maintained by transplants every 15 days into healthy mice, followingthe technique of Geran et al. (1972). Each animal was injected subcutaneously with lo6 viable tumor cclls in 0.5 ml of saline into the dorsal side of ~ _ _ the ferriwal root. The animals then wercI syiaixl.cil I-~LIIArldress reyriiil rcqueda t o L)r. v. ento to 1 )plPl/~, r)rl)ar1111~11t U! domly into two equal groups. In the group used as a con- Pathology, Faculty of Medicine, University of Murcia, Spain. trol, each animal was injected with 0.5 ml of saline on 5 consecutive days, starting on implant day. Received April 5,1989; accepted July 11, 1989. 0 1990 Munksgaard
2
V. Vicente et al.
tographs ( x 1,000). In addition, we evaluated necrosis, tumoral stroma, and host cell infiltration (Li et al., 1984). For the ultrastructural study, fragments 1 mm2 thick were fixed in glutaraldehyde, postfixed in osmium tetroxide, and stained with 2% uranyl acetate. After dehydration and clearing with propylene oxide, they were embedded in Epon. The ultrathin sections then were contrasted with uranyl acetate and lead citrate. For the ultrastructural photographic study, we used a Zeiss EM 1OC electron microscope. In the statistical study, interrelations between the variables were calculated by means of linear regression analysis. Comparison between the groups was done by analysis of double variance and completed with mean contrastsusing a &Studentstatistical table.
RESULTS Macroscopically,the tumors presented identical characteristics on day 11post implant. %ors of subgroups I and IV had irregular outlines, although these were well defined by the adipose tissue they were compressing. On days 18 and 25 post implant, the tumors in subgroups I1 and I11 were of nodular morphology, atrophyig the epidermis by compressionand well outlined by conjunctive capsules. The tumors found in treated subgroups V and VI showed whitish areas that penetrated from the capsule, irregularly splitting the parenchyma (Fig. 1). The tumor weights in the control group were on the average significantly higher than those in the treated group: F(1/24) = 3,928 ( P < 0.05), particularly in animals sacrificed on day 25 post implant (subgroups 111)("able 1). Microscopically, the tumor in subgroup I presented projection toward the adipose tissue, and in subgroups I1 and
I11the tumors were delineated by conjunctive capsules that engulfedthe adjacent muscular and adipose tissue and even the epidermis. In the control subgroups (I, 11,and 111)the melanocytes were homogeneous and were found in small nests surrounded by thin reticulated tracts, lymphatic vessels, and narrow blood vessels. The nuclei were round or oval, with precise outlines and prominent nucleoli that were eosinophilous and sometimes multiple. The cytoplasm was abundant and eosinophilous with melanotic pigment in fine granules (Fig. 2A). In the treated subgroups (IV, V, and VI), irregular congestive blood vessels were common, as was the alternation of nests of melanocytes of different morphologies. Those of globulous morphology were frequent. The nuclei were irregularlyoutlined, with the chromatin of the periphery thickening the membrane (Fig. 2B). The mitotic index showed very significant differences between treated and control groups F(1/24) = 99.13 ( P < 0.001) and between subgroups ("able 1).It was progressively higher and significantlydifferent in the three treated subgroups compared with the control group. Cellular density per unit of area (Na) and of volume (Nv) was significantly lower in the treated subgroups than in their correspondingcontrols ("able 1).Thmoral necrosiswas constant in all subgroups ("able l),and in the last to be sacrificed it occupied the greater part of the tumoral volume, extending to the periphery and reaching the capsule. At its heart, nests of undamaged melanocytes were common. The necrosis showed similar mean values, ranging between 25% and 75%. The analysis of variance showed no significant differences in the groups of animals sacrificed on the different days F(2/24) = 21.24 ( P < 0.005) ("able 1).
Fig. 1. A Macroscopic aspects of B16 control tumors on days 11,18,and 25 post implant. Sectioned tumors on day 25: B: Control; C:Treated.
Thermochemotherapy for B16 Melanoma
Fig. 2. A An 11 day control is observed to be composed of densely grouped melanocytes with little pleomorphism and numerous small
3
vessels. H&E, x 312.5; B: An 18 day treated tumor shows numerous hiperemic vessels with irregularlumen. H&E, x 312.5.
Typical in the stroma was the presence of prominent vesStroma varied from sparse to moderate, with no significant differences among groups ('Ihble 1). Host's cell infil- sels of irregular lumen, with alteration of the walls and tration was constant and was represented by macrophages, irregularity in the thickness of the basement membrane, polymorphonuclear leucocytes, and lymphocytes. These separation of the adjacent endothelial cells, with the nuclei surrounded the areas of necrosis and the tumoral periph- jutting toward the lumen, and the massive presence of vesery, present along almost the entire outline. Infiltration icles of pinocytosis and vacuoles of different sizes (Fig.5D). Metastases were very infrequent, being found in seven varied from sparse to moderate, with no significant differences in the groups F(2/24) = 8.67 (P < 0.005). animals only (3.3%)and exclusively in the viscera. In the Ultrastructurally, the melanocytes were compactly control animals, there were six cases (five lung, one kidgrouped. They were round or oval and had clear outlines, ney), and the remaining case was found in a treated anias had the nuclei; the euchromatin was disposed in fine mal (liver). granules and the heterochromatin in thick clots, irregularly distributed. The nucleoli were voluminous, with irregDISCUSSION ular outlines, and were frequently multiple (Fig. 3). A number of previous studies that have combined some In the treated group, there was greater nuclear irregularity, with lobular indentations in the nuclei and the het- of the agents we have applied in the present work seem to erochromatin thickening the nuclear membrane. There demonstrate that hyperthermia, by causing vasodilation, were signs of vacuolization and dilation of the nuclear mem- facilitates the access of the drugs to the tumoral parenbrane. In the cytoplasm, phenomena worthy of note were chyma (Minton, 1984), thus bringing about an increase in the varied electrondensity, swelling of the mitochondrias the plasmatic and intratumoral concentrations of the drugs with disorganization of the crests, and the constant pres- (Honnes et al., 1985) and an enhancement of their action ence of lipid vacuoles. The melanosomes were numerous, on nuclear DNA (Minton, 1984). In the present study, our ellipsoidal, irregularly distributed, and presenting lamel- experimental results showed that administration of mellar internal structure. They were in the intermediate stages phalan, CCNU, and hyperthermia for 5 consecutive days of maturation (I1 and 111). There were frequent melano- postimplant brought about a delay in tumoral growth, which is statistically significant when compared with that in the some complexes (Figs. 4,5A-C).
4
V. Vicente et al.
Fig. 3. Electron micrograph oE A An 11day control tumor shows melanoma cells with regular outlines, as the nuclei had. x 3,500; B:
An 18 day control tumor shows melanocytes compactly grouped with regular outlines and multiple voluminousnucleoli. x 3,500.
Fig. 4. Electron micrograph of an 18day treated tumor. A Melanocytes with varied cytoplasmicelectrondensity. x 3,500;B: Melano-
cytes with irregular nuclear outlines and presence of lipid vacuoles (arrows). X 3,500.
Thermochemotherapyfor B16 Melanoma
Fig. 5. Electron micrograph of 25 day treated tumor. A: Nuclear irregularity and melanosomes 11-IIIin detail. x 7,000,B: Idem. Dilation of the nuclear membrane and endoplasmic reticulum. x 7,000;C:
5
Swollen organelles. x 7,000; D: Prominent irregular vessels in t,he stroma. x 4,000.
V. Vicente et al.
6
TABLE 1. Modifications of Different Parameters in B16 Melanoma After the Tkeatmentt nmor group Control1 Control11 Control111 TreatedIV TreatedV TreatedVI
'hmor weight
Mitotic index
% Necrosis
% Stroma
% Host cell
infiltration
Na
Nv
Capsule
0 . 1 8 f 3.24 2 . 0 4 f 0.10 3.09 f 0.32 0.68 f 0.12 1.69 2 0 . 1 1 2.01 f 0.27*
10.2 f 0.37 12 f 0.70 10.4 f 2.29 1 5 . 8 f 1.62 26.2 *3.05* 39.8 f 2.55*
25 0.24 6 5 f 0.24 80 f0.24 4 0 f 0.24 65 f 0.24 75 f 0.37
53.3f 0.24 53.3 k 0.24 80 f 0.24 60 t 0.2 60 f 0.37 53.3 t 0.24
53.3 2 0.24 66.6-c 0 80 0.24 46.6 t 0 . 2 4 66.6 f 0 66.6 f 0
151 f 9.14 152 f 11.18 141.4k 5.42 11.4 f 8.02* 117.82 7.49* 113.6 f 5.93*
29.61 f 1.97 2 8 . 5 2 f 2.79 24.39-c 1.28 21.07 f 1.67* 22.08 f 1.92* 20.04 f 1.36
2.2 f 0.2 2.2 f O . 3 7 2.2 f 0.2 2 f0 2 . 6 f 0.24 2.8 f 0.2
*
tValues are mean f SEM. *Significant differences between treated and control groups.
control animals, and also resulted in an inhibiting effect on the spontaneous formation of metastases. The synergic effect of hyperthermia in combinationwith chemotherapeutic agents was described by Luck (1956), who used melphalan on melanoma in mice; this effect was later confirmed in human and animal melanoma cultures by Parsons (1984). It has also been pointed out by use of a combination of hyperthermia with nitrosoureas, applied initially by Hahn et al. (1975) in the form of hyperthermal perfusion and more recently by Yamada et al. (1984)in melanoma in humans and B16 transplanted into mice. Most of the studies have been directed toward discoveringthe sensitivity of the tumor cells to the agents, whereas studies aimed at discovering the changes that take place in the tumor and in the host have been few. The studies by Yamada et al. (1984, 1985) indicate the synergic effect of nitrosoureas plus hyperthermia on the inhibition of tumoral growth and describe their effects onthe tumor: tumoral parenchyma with involutive phenomena in the melanocytes and partial substitution by fibrous tissue. However, the work by Li et al. (1984) is the only one we have been able to find that makes an attempt to quantify the alterations brough about by thermochemotherapy. Li et al. (1984) quantified replacement of tumoral necrosis by viable tumoral tissue in either treated or control animals; also, in our study, tumoral parenchymal necrosis, conjunctive capsules, and host cell infiltration evolved with the passage of time and showed no significant differences between treated and control animals, indicating that these variables were not affected by the treatment. We confirm the study by Li et al. (1984) in our observation of a diminution of cellular density of the viable tumoral parenchyma, which would point to excessive drug-caused cell death. We also observed, as did Li et al., an increase in the proliferative capacity of the tumoral parenchyma that did remain viable; this increase is reflected in the fact that the mitotic index was significantly higher in these tumors than in the those found in the control group. Morphologically, the treated subgroups presented melanocytes with irregularly shaped nuclei with margination of the chromatin, which was seen thickening the nuclear menbrane. Cytoplasm was markedly eosinophilous. These characteristics, described in previous works as typical of
the alterations caused by various alkylant agents and also by radiotherapy, are considered to be the consequences of cell damage following the action of agents of different natures (Li et al., 1984). Ultrastructurally, the alterations corresponded to the margination, with vacuolization, of the heterochromatin, and dilation of the nuclear membrane, swelling of organelles, frequent lipid vacuoles, and peripheral localization of melanosomes. We found no appreciable differences between treated and control groups in the alterations in melanosomes with regard to morphology, internal structure, or degree of melanization. We encountered constant vascular alterations in the treated tumors, which were similar to those observed by Emami et al. (1981) at temperatures above 42°C. These had not been observed previously at a temperature of 40°C. We believe that our findings are due to our having applied 40°C for 5 consecutive days. Metastasizingcapacity seems very variable, ranging from nil for Demopoulos et al. (1965) to constant for Wosko et al. (19841, who observed it exclusively in viscera. In our study it was rarely encountered (in 3.3% of animals) and then always in viscera as Aubert et al. (1989) recently observed. This low incidence, compared with the work of Wosko et al. (1984), might be due to the different transplant technique; however, it also differs from the high percentages obtained by Kancklertz and Chapman (1986), who used the same implant technique, the same number of cells, and a tumoral line obtained from the same Centre as ours. The treatment used seems to diminish metastasizing capacity. Further studies with highly metastatic cell lines, such as B16-Fl0 line (developed by Dr. Liotta, N.I.H.), should be done to support what w;ts observed in the works of Fidler (1973) and Hanna and Fidler (1981), who each used a different therapeutic procedure, each again different from that used in the present work.
ACKNOWLEDGMENTS The authors thank the skillful technical assistance of Miss A. Pina, E. Sanchez, and F.C. Smilg. This work has been partially supported by a grant from the DGICYT PM88-0093, Ministerio de Educacion y Ciencia.
Thermochemotherapyfor B16 Melanoma REFERENCES Aubert, G., C. Voulot, F. Rouge, V. F'irisi, and Galindo, J.R. (1989) New variants of B16 mouse melanoma: Differentiation and metastatic properties. Pigment Cell Res. 2:17-25. Boatman, E.S. (1986)Digitizingand quantitation. In: Ultrastructure Techniques for Microorganism. H.C. Aldrich and W.J. ?bod, eds. Plenum Press, New York, pp 365-398. Demopoulos, H.B., T. Kasuga, A.A. Channing, and H. Bagdoyan (1965) Comparison of ultrastructure of B16 and S 9 1 mouse melanoma, and correlation with growth patterns. Lab. Invest., 14: 109-121. Emami, B., G.H. Nussbaum, N. Hahn, A.J. Piro, A. Dritschilo, and F. Quimby (1981) Histopathological study on the effects of hyperthermia on microvasculature. Int. J Radiat. Oncol. Biol. Phys., 7:w-348. Fidler, I.J. (1973) Selection of successive tumor lines for metastasis. Nat. New. Biol., 242A48-149. Geran, R.I., N.H. Greenberg, M.M. McDonald, A.M. Schumacher, and B.J. Abbott (1972) Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother. Rep., 3-3:7-63. Gupta, V. (1984) Experimental chemotherapy of melanoma. In: Clinical Management of Malignant Melanoma. Constanzi, ed. Boston, pp. 150-165. Hahn, G.M., J. Braun, and I. Har-Keeder (1975) Thermochemotherapy: Synergism between hyperthermia (42O-43")and Adriamycin (or bleobycin) in mammalian cell inactivation. Proc. Natl. A&. Sci. U.S. A., 72937-940. Hanna, N., and I.J. Fidler (1981) Relationship between metastatic potential and resistance to natural killer cell-mediated cytotoxicityinmurinetumorsystem. U.S. Natl. Cancer Inst., 66:1183-1190.
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Honnes, D.J., J. Donaldson, P. Workman, and N.M. Bleehen (1985) The effect of systemic hyperthermia on melphalan pharmacokinetics in mice. Br. J. Cancer, 51:77-84. KancMertz, A., and J.D. Chapman (1986) The treatment of animal tumors and their metastases with 4-hydroxyanisole. Br. J. Cancer, 54:693-698. Li, X., G. Paulus, G. Atassi, and N. Buyssens (1984) Growth response of B16 melanoma to in vivo treatment with 1-(2-~hloroethyl)-3cyclohexyl-1-nitrosourea(CCNU) at the initial stage after tumor transplantation. Am. J. Pathol., 115:403-411. Luck,J.M. (1956)Action of fi(2-chloroethyl)-amine-Lphenyl-alanine on Harding-Passey mouse melanoma. Science, 123:984-993. Minton, J.P. (1984) The staging and surgical management of primary malignant melanoma. In: Clinical Management of Malignant Melanoma. Costanzi, ed. Boston, pp. 1-59. Parsons,P.G. (1984)Dependence on treatment time of melphalan resistance and DNA cross-linking in human melanoma cell lines. Cancer Res., 44:2773-2778. Wosko, T.J., D.T. Ferrada, and L.S. Sartori (1984)Histological comparison on the B16 melanoma and its F1 variant. Cancer Lett. 24:57-63. Yamada, K., T. Someya, S. Shimada, K. Ohara, and A. Kukita (1984)Thermochemotherapy for malignant melanoma: combination therapy of ACNU and hyperthermia in mice. J. Invest. Dermatol. 82:180-184. Yamada, K., T. Someya, S. Shimada, H. Nakagawa, A. Kukita, H. Tokita, and N. Tanaka (1985) Thermochemotherapy for malignant melanoma: Overcomigheterogeneity in drug sensitivity. J. Invest. Dermatol., 85:43-46.
Thermochemotherapy for B16 Melanoma: Combination Therapy of Hyperthermia, Melphalan, and CCNU in Mice V. VICENTE,' M. GOMEZ,' M.M. OCHOTORENA,' A. CREMADES,2 M. CANTERAS3 'Department of Pathology, 'Pharmacology, and 'Biostatistics, Faculty of Medicine, University of Murcia, Spain We studied, by means of quantitative histopathological methods, the changes that took place in B16 melanoma implanted into C57BL/6J mice after combination treatment of (CCNU). The hyperthermia,melphalan, and l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea studies were made on animals sacrificed on days 11,18, and 25 after implantation. 'hmors in treated animals showed a progressive delay in growth, a noticeably reduced number of metastases, and diminution of proliferative capacity. However, this treatment did not affect necrosis, stroma, capsule, or cell infiltration. Ultrastructurally,signs of cell damage were constantly present. Key words:
Thermochemotherapy, Melanoma, Hyperthermia, Melaphan
INTRODUCTION The fact that melanoma tumors are refractary to many chemotherapeutic agents is one of the most serious problems in the chemotherapeutic treatment of melanoma. In recent times, several experiments have been conducted in the search for new antitumoral agents and combinations of agents that will enhance the resulting effect and, by the consequent reduction of dosage permitted, thereby lessen undesirable side effects (Gupta, 1984). There has been a renewal of interest in hyperthermia as a method of treatment for cancer patients, and recent research has concentrated on hyperthermia-plus-chemotherapy combinationtreatment (Yamada et al., 1984). However, these studies have not been aimed at assessing the changes produced in the tumor by the drugs. In the present work, we studied, by means of quantitative histopathological methods, the effect of combination treatment with CCNU, melphalan, and whole-body hyperthermia on B16 melanoma transplanted into C57BL/6J mice.
The treatment of the second group commenced on implantation day with the simultaneous intraperitoneal (i.p.1 administration of melphalan (3 mg/kg/day), CCNU (6 mg/kg/day), and whole-body hyperthermia (40°C/45'/day) to each of the animals in the treated group. The hyperthermia was applied by introducing the animals into a Unitemp ventilated hyperthermia chamber. Rectal temperature was measured with a thermoselective pair connected to an ELLAB thermometer and three RMG thermoselective sondes. For sedation all animals, treated and untreated, were given ketamine a t a rate of 3 mg/kg/day/i.p. Thirty animals from the control group and 30 from the treated group were sacrified on days 11, 18, and 25 post implant (subgroups I, 11, and I11 each contained 30 control animals, and subgroups IV, V, and VI each contained 30 treated animals). Samples of tumor and viscera were fixed in 10%buffered neutral formaldehyde, embedded in paraffin, cut into 5 km sections, and stained with hematoxylin and eosin (H&E), Masson Fontana, and Gordon Swett. Animals that died during the experiment were excluded from the study. The mitotic index calculation was based on 1,000 cells taken at random from each tumor. Tbmoral parenchyma evaluation was carried out by means of cell density per unit area (Na) and of volume (Nv), following the technique of Boatman (1986) on microscope pho-
MATERIALS AND METHODS We used 210 C57BL/6J mice weighing 20 g each. The B16-F1 melanoma was obtained from the Institute of Cancer Research, Royal Cancer Hospital, London. The tumor line was maintained by transplants every 15 days into healthy mice, followingthe technique of Geran et al. (1972). Each animal was injected subcutaneously with lo6 viable tumor cclls in 0.5 ml of saline into the dorsal side of ~ _ _ the ferriwal root. The animals then wercI syiaixl.cil I-~LIIArldress reyriiil rcqueda t o L)r. v. ento to 1 )plPl/~, r)rl)ar1111~11t U! domly into two equal groups. In the group used as a con- Pathology, Faculty of Medicine, University of Murcia, Spain. trol, each animal was injected with 0.5 ml of saline on 5 consecutive days, starting on implant day. Received April 5,1989; accepted July 11, 1989. 0 1990 Munksgaard
2
V. Vicente et al.
tographs ( x 1,000). In addition, we evaluated necrosis, tumoral stroma, and host cell infiltration (Li et al., 1984). For the ultrastructural study, fragments 1 mm2 thick were fixed in glutaraldehyde, postfixed in osmium tetroxide, and stained with 2% uranyl acetate. After dehydration and clearing with propylene oxide, they were embedded in Epon. The ultrathin sections then were contrasted with uranyl acetate and lead citrate. For the ultrastructural photographic study, we used a Zeiss EM 1OC electron microscope. In the statistical study, interrelations between the variables were calculated by means of linear regression analysis. Comparison between the groups was done by analysis of double variance and completed with mean contrastsusing a &Studentstatistical table.
RESULTS Macroscopically,the tumors presented identical characteristics on day 11post implant. %ors of subgroups I and IV had irregular outlines, although these were well defined by the adipose tissue they were compressing. On days 18 and 25 post implant, the tumors in subgroups I1 and I11 were of nodular morphology, atrophyig the epidermis by compressionand well outlined by conjunctive capsules. The tumors found in treated subgroups V and VI showed whitish areas that penetrated from the capsule, irregularly splitting the parenchyma (Fig. 1). The tumor weights in the control group were on the average significantly higher than those in the treated group: F(1/24) = 3,928 ( P < 0.05), particularly in animals sacrificed on day 25 post implant (subgroups 111)("able 1). Microscopically, the tumor in subgroup I presented projection toward the adipose tissue, and in subgroups I1 and
I11the tumors were delineated by conjunctive capsules that engulfedthe adjacent muscular and adipose tissue and even the epidermis. In the control subgroups (I, 11,and 111)the melanocytes were homogeneous and were found in small nests surrounded by thin reticulated tracts, lymphatic vessels, and narrow blood vessels. The nuclei were round or oval, with precise outlines and prominent nucleoli that were eosinophilous and sometimes multiple. The cytoplasm was abundant and eosinophilous with melanotic pigment in fine granules (Fig. 2A). In the treated subgroups (IV, V, and VI), irregular congestive blood vessels were common, as was the alternation of nests of melanocytes of different morphologies. Those of globulous morphology were frequent. The nuclei were irregularlyoutlined, with the chromatin of the periphery thickening the membrane (Fig. 2B). The mitotic index showed very significant differences between treated and control groups F(1/24) = 99.13 ( P < 0.001) and between subgroups ("able 1).It was progressively higher and significantlydifferent in the three treated subgroups compared with the control group. Cellular density per unit of area (Na) and of volume (Nv) was significantly lower in the treated subgroups than in their correspondingcontrols ("able 1).Thmoral necrosiswas constant in all subgroups ("able l),and in the last to be sacrificed it occupied the greater part of the tumoral volume, extending to the periphery and reaching the capsule. At its heart, nests of undamaged melanocytes were common. The necrosis showed similar mean values, ranging between 25% and 75%. The analysis of variance showed no significant differences in the groups of animals sacrificed on the different days F(2/24) = 21.24 ( P < 0.005) ("able 1).
Fig. 1. A Macroscopic aspects of B16 control tumors on days 11,18,and 25 post implant. Sectioned tumors on day 25: B: Control; C:Treated.
Thermochemotherapy for B16 Melanoma
Fig. 2. A An 11 day control is observed to be composed of densely grouped melanocytes with little pleomorphism and numerous small
3
vessels. H&E, x 312.5; B: An 18 day treated tumor shows numerous hiperemic vessels with irregularlumen. H&E, x 312.5.
Typical in the stroma was the presence of prominent vesStroma varied from sparse to moderate, with no significant differences among groups ('Ihble 1). Host's cell infil- sels of irregular lumen, with alteration of the walls and tration was constant and was represented by macrophages, irregularity in the thickness of the basement membrane, polymorphonuclear leucocytes, and lymphocytes. These separation of the adjacent endothelial cells, with the nuclei surrounded the areas of necrosis and the tumoral periph- jutting toward the lumen, and the massive presence of vesery, present along almost the entire outline. Infiltration icles of pinocytosis and vacuoles of different sizes (Fig.5D). Metastases were very infrequent, being found in seven varied from sparse to moderate, with no significant differences in the groups F(2/24) = 8.67 (P < 0.005). animals only (3.3%)and exclusively in the viscera. In the Ultrastructurally, the melanocytes were compactly control animals, there were six cases (five lung, one kidgrouped. They were round or oval and had clear outlines, ney), and the remaining case was found in a treated anias had the nuclei; the euchromatin was disposed in fine mal (liver). granules and the heterochromatin in thick clots, irregularly distributed. The nucleoli were voluminous, with irregDISCUSSION ular outlines, and were frequently multiple (Fig. 3). A number of previous studies that have combined some In the treated group, there was greater nuclear irregularity, with lobular indentations in the nuclei and the het- of the agents we have applied in the present work seem to erochromatin thickening the nuclear membrane. There demonstrate that hyperthermia, by causing vasodilation, were signs of vacuolization and dilation of the nuclear mem- facilitates the access of the drugs to the tumoral parenbrane. In the cytoplasm, phenomena worthy of note were chyma (Minton, 1984), thus bringing about an increase in the varied electrondensity, swelling of the mitochondrias the plasmatic and intratumoral concentrations of the drugs with disorganization of the crests, and the constant pres- (Honnes et al., 1985) and an enhancement of their action ence of lipid vacuoles. The melanosomes were numerous, on nuclear DNA (Minton, 1984). In the present study, our ellipsoidal, irregularly distributed, and presenting lamel- experimental results showed that administration of mellar internal structure. They were in the intermediate stages phalan, CCNU, and hyperthermia for 5 consecutive days of maturation (I1 and 111). There were frequent melano- postimplant brought about a delay in tumoral growth, which is statistically significant when compared with that in the some complexes (Figs. 4,5A-C).
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V. Vicente et al.
Fig. 3. Electron micrograph oE A An 11day control tumor shows melanoma cells with regular outlines, as the nuclei had. x 3,500; B:
An 18 day control tumor shows melanocytes compactly grouped with regular outlines and multiple voluminousnucleoli. x 3,500.
Fig. 4. Electron micrograph of an 18day treated tumor. A Melanocytes with varied cytoplasmicelectrondensity. x 3,500;B: Melano-
cytes with irregular nuclear outlines and presence of lipid vacuoles (arrows). X 3,500.
Thermochemotherapyfor B16 Melanoma
Fig. 5. Electron micrograph of 25 day treated tumor. A: Nuclear irregularity and melanosomes 11-IIIin detail. x 7,000,B: Idem. Dilation of the nuclear membrane and endoplasmic reticulum. x 7,000;C:
5
Swollen organelles. x 7,000; D: Prominent irregular vessels in t,he stroma. x 4,000.
V. Vicente et al.
6
TABLE 1. Modifications of Different Parameters in B16 Melanoma After the Tkeatmentt nmor group Control1 Control11 Control111 TreatedIV TreatedV TreatedVI
'hmor weight
Mitotic index
% Necrosis
% Stroma
% Host cell
infiltration
Na
Nv
Capsule
0 . 1 8 f 3.24 2 . 0 4 f 0.10 3.09 f 0.32 0.68 f 0.12 1.69 2 0 . 1 1 2.01 f 0.27*
10.2 f 0.37 12 f 0.70 10.4 f 2.29 1 5 . 8 f 1.62 26.2 *3.05* 39.8 f 2.55*
25 0.24 6 5 f 0.24 80 f0.24 4 0 f 0.24 65 f 0.24 75 f 0.37
53.3f 0.24 53.3 k 0.24 80 f 0.24 60 t 0.2 60 f 0.37 53.3 t 0.24
53.3 2 0.24 66.6-c 0 80 0.24 46.6 t 0 . 2 4 66.6 f 0 66.6 f 0
151 f 9.14 152 f 11.18 141.4k 5.42 11.4 f 8.02* 117.82 7.49* 113.6 f 5.93*
29.61 f 1.97 2 8 . 5 2 f 2.79 24.39-c 1.28 21.07 f 1.67* 22.08 f 1.92* 20.04 f 1.36
2.2 f 0.2 2.2 f O . 3 7 2.2 f 0.2 2 f0 2 . 6 f 0.24 2.8 f 0.2
*
tValues are mean f SEM. *Significant differences between treated and control groups.
control animals, and also resulted in an inhibiting effect on the spontaneous formation of metastases. The synergic effect of hyperthermia in combinationwith chemotherapeutic agents was described by Luck (1956), who used melphalan on melanoma in mice; this effect was later confirmed in human and animal melanoma cultures by Parsons (1984). It has also been pointed out by use of a combination of hyperthermia with nitrosoureas, applied initially by Hahn et al. (1975) in the form of hyperthermal perfusion and more recently by Yamada et al. (1984)in melanoma in humans and B16 transplanted into mice. Most of the studies have been directed toward discoveringthe sensitivity of the tumor cells to the agents, whereas studies aimed at discovering the changes that take place in the tumor and in the host have been few. The studies by Yamada et al. (1984, 1985) indicate the synergic effect of nitrosoureas plus hyperthermia on the inhibition of tumoral growth and describe their effects onthe tumor: tumoral parenchyma with involutive phenomena in the melanocytes and partial substitution by fibrous tissue. However, the work by Li et al. (1984) is the only one we have been able to find that makes an attempt to quantify the alterations brough about by thermochemotherapy. Li et al. (1984) quantified replacement of tumoral necrosis by viable tumoral tissue in either treated or control animals; also, in our study, tumoral parenchymal necrosis, conjunctive capsules, and host cell infiltration evolved with the passage of time and showed no significant differences between treated and control animals, indicating that these variables were not affected by the treatment. We confirm the study by Li et al. (1984) in our observation of a diminution of cellular density of the viable tumoral parenchyma, which would point to excessive drug-caused cell death. We also observed, as did Li et al., an increase in the proliferative capacity of the tumoral parenchyma that did remain viable; this increase is reflected in the fact that the mitotic index was significantly higher in these tumors than in the those found in the control group. Morphologically, the treated subgroups presented melanocytes with irregularly shaped nuclei with margination of the chromatin, which was seen thickening the nuclear menbrane. Cytoplasm was markedly eosinophilous. These characteristics, described in previous works as typical of
the alterations caused by various alkylant agents and also by radiotherapy, are considered to be the consequences of cell damage following the action of agents of different natures (Li et al., 1984). Ultrastructurally, the alterations corresponded to the margination, with vacuolization, of the heterochromatin, and dilation of the nuclear membrane, swelling of organelles, frequent lipid vacuoles, and peripheral localization of melanosomes. We found no appreciable differences between treated and control groups in the alterations in melanosomes with regard to morphology, internal structure, or degree of melanization. We encountered constant vascular alterations in the treated tumors, which were similar to those observed by Emami et al. (1981) at temperatures above 42°C. These had not been observed previously at a temperature of 40°C. We believe that our findings are due to our having applied 40°C for 5 consecutive days. Metastasizingcapacity seems very variable, ranging from nil for Demopoulos et al. (1965) to constant for Wosko et al. (19841, who observed it exclusively in viscera. In our study it was rarely encountered (in 3.3% of animals) and then always in viscera as Aubert et al. (1989) recently observed. This low incidence, compared with the work of Wosko et al. (1984), might be due to the different transplant technique; however, it also differs from the high percentages obtained by Kancklertz and Chapman (1986), who used the same implant technique, the same number of cells, and a tumoral line obtained from the same Centre as ours. The treatment used seems to diminish metastasizing capacity. Further studies with highly metastatic cell lines, such as B16-Fl0 line (developed by Dr. Liotta, N.I.H.), should be done to support what w;ts observed in the works of Fidler (1973) and Hanna and Fidler (1981), who each used a different therapeutic procedure, each again different from that used in the present work.
ACKNOWLEDGMENTS The authors thank the skillful technical assistance of Miss A. Pina, E. Sanchez, and F.C. Smilg. This work has been partially supported by a grant from the DGICYT PM88-0093, Ministerio de Educacion y Ciencia.
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