Effects of differential hypothermia and 5-fluorouracil on C3H mouse mammary tumors: Histologic study

13, 347-353

CRYOBIOLOGY

(1976)

Effects of Differential Hypothermia on C,H Mouse Mammary Tumors: PAVA Dep,artments

POPOVIC,

of Physiology

V. POPOVIC,

and Pathology,

Many tumors appear to be more sensitive to heat exposure than normal tissues (1-5, 8, 10, 11, 23). This fact is used in the treatment of patfents with tumors. Beneficial effects of hyperthermic treatment have been described int the literature (1, 6, 7, 12, 24, 25). A different approach, generalized cooling of the whole body with localized tumor warming and systemic chemotherapy, was used in the present study. This approach was undertaken because of the well-known protective effects of generalized hypothermia. The reason for combining generalized body coo1in.g with local tumor warming (differential hypothermia) was to decrease systemic toxicity of the administered anticancer agent and to concentrate the full effect of the anticancer agent on warm, metabolically active tumors. After such treatment that lasted only 1 hr, transplanted tumors in hamsters (9, 1820), virus-induced brain tumors in hamsters (12), chemically induced tumors in rats ( 21), and spontaneous mammary tumors in C&H mice (14, 15) regressed or even disappeared. This ‘approach was applied to human patients with recurrent gliomas of the brain with good success (12). The present publication is a continuation of the previous experimental work on C,H mice, with e:mphasis on histologic examination of spontaneous mammary tumors before and after prolonged differential hypoReceived

February

Emory

University,

Georgia

AND

30322

hypothermia

METHODS

Animals. Thirty-five C3H mice with spontaneous mammary tumors, raised in our own colony, were used in experiments. The raising stock came from Jackson Laboratory (Bar Harbor, Me. ). Each experimental animal was kept in a separate cage and had food and water ad libitum. Tumor size of these animals was measured once per week (three dimensions) by calipers. During the measurement each animal was lightly anesthetized by halothane.l There was no mortality from anesthesia, and the animals did not lose any body weight. Tumor measurements were always done by the same person. Since measurement by calipers is not a precise method, our criterion that a tumor regressed was b’ased on a decrease of 40% or more in the tumor size. Each animal in the differential hypothermia group combined with chemotherapy had a PE 10 polyethylene cannula implanted in the jugular vein 1 week before the experiment (13). This chronic cannula allowed administration of the drug into the circulation of the hypothermic animals where drug absorption is much decreased. The implantation was done under Nembutal anesthesia. 1 Fluothane, tories.

Inc. reserved.

Atlanta,

MATERIALS

5, 1975.

0 1976 by Academic Press, of reproduction in any form

D. WEATHERS

thermia and I-hr differential with chemotherapy.

347 Copyright All rights

AND

and 5-Fluorouracil Histologic Study

kindly

supplied

by Ayerst

Labora-

348

POPOVIC,

POPOVIC

Induction of differential hypothermia. The animals were cooled to a body temperature of 15°C by the hypercapnic-hypoxic technique ( 16, 17). The animals were then placed on a tray and covered with crushed ice in order to keep their body temperature between 12 and 15°C. The tumor was not covered with ice and was warmed by ultrasound equipment 2 to a temperature of 39°C. During the whole experiment, the temperature of the tumor was stable ‘and was recorded by two needle thermocouples (Ga 25) #and a Honeywell recorder. One thermocouple monitored the temperature of the upper part of the tumor and the second thermocouple the temperature of the base of the tumor. The third and the fourth thermocouples recorded the deep colonic temperature and the chest muscle temperature of the animal, respectively. At the end of the experiment each animal was rewarmed to a normal body temperature. In the ,diff erential hypothermia-chemotherapy group, when the deep tumor temperature reached 39”C, 5-fluorouracil (5 FU), 100 mg/kg, was injected through the chronically implanted cannula. The cannula was then rinsed with saline and flame sealed. Histology. The tissue for histologic examination (biopsy or the whole excised tumor) was immediately placed in 10% neutral buffered formalin and kept at least 24 hr. The tissue was paraffin embedded, cut to 6-pm length, and stained with hematoxylin and eosin. Interpretation of the slides was done without knowledge of the previous experimental procedure. All animals were randomly separated into four groups, each group having five animals. An additional 15 animals were used only for histologic studies. Group 1. Normothermic animals with tu17~)~sbut without chemotherapy. The animals in this group were not cooled, and 2 Kindly Pasadena,

supplied Calif.

by Mettler

Electronics

Corp.,

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WEATHERS

the chemotherapy was not administered. Growth of the tumors and life span of the animals were followed for 5 weeks. Group 2. Normothermic animals with tumors and with chemotherapy. These animals received a single lOO-mg/kg ip injection of 5-FU but were not cooled. The tumor growth was measured and life span recorded as for Group 1. Group 3. Eight-hour differential hypothermia without chemotherapy. The animals in this group were cooled to a body temperature of 1%15°C while their tumors were kept at a temperature of 3739°C. Thus induced differential hypothermia was kept for 8 hr. The anticancer agent was not administered. After 8 hr, the animals were rewarmed and the tumors excised for histologic study. Group 4. One-hour diferential hypothermia with chemotherapy. The animals were cooled to a colonic temperature of 15°C by the hypoxic-hypercapnic technique, and their tumors were kept warm for 1 hr. These animals received a single NO-mg/kg iv injection of 5-FU. Two weeks later the tumors were excised for histologic study. RESULTS

Group 1. Normothermic Animals with Tumors but without Chemotherapy In this group of five animals, tumor growth was followed during the period of 5 weeks. The tumors of all normothermic animals grew. All tumors were malignant but without any histologic changes. The average life span of tumor-bearing animals was 90 days. One animal had a slow-growing tumor. It was sacrificed on the 188th day and the tumor prepared for histologic examination. This tumor was also malignant. The tumor growth of these animals and their life spans are given in Table 1. Histologic studies of mammary tumors were done on an additional group of five animals randomly selected (biopsy examinations). Tumor growth was not followed

HYPOTHERMIA

AND

5-FLUOROURACIL TABLE

Tumor

Growth

of Normothermic

ON CgH MOUSE

TUMORS

349

1 C&H Mice Mammary

Tumors

Number

0 Day

1 Week

2 Weeks

3 Weeks

4 Weeks

5 Weeks

Life span (days)

B C D G P

136” 273 357 273 575

272 380 369 323 439

202 396 544 740 480

249 443 465 640 315

450 554 746 1020 448

987 678 1015 1081 480

89 163 54 124 188*

0 Values are given in cubic millimeters. * Sacrificed for pathologic examination. because of biopsy. All tumors were found to be malignant. Five non-tumor-bearing animals also had mammary gland tissue examined. Mammary glands of 10 animals with untreated tumors showed a poorly circumscribed epithelial tumor composed of rather uniform round cells with a moderate amount of cytoplasm and a vesicular nucleus. Nuclear hyperchromatism was present to a moderate degree and mitoses were fre-

quently seen. The cells were arranged in a duct-like configuration (Fig. 1). In some areas compression resulted in the formation of a solid syncytium. Occasional fields showed an adenoid cystic pattern. The supporting fibrous connective tissue stroma was scant, but the tumor was highly vascular with formation of large “blood lakes.” The histologic diagnosis was adenocarcinoma of mouse breast.

FIG. 1. A representative field of the untreated mouse mammary tumor. The tumor is composed of epithelial cells forming duct-like structures. There is some pleomorphism and hyperchromatism. Invasion into striated muscle can be seen at left. Magnification 200X.

POPOVIC,

350

POPOVIC TABLE

Tumor Number

83 84 1 O-6 O-189

Growth 0 Day

399a 557 349 168 957

of Normothermic

AND

WEATHERS

2

and 5-FU ip Injected

CaH Mice

Mammary

Tumors

1 Week

2 Weeks

3 Weeks

4 Weeks

5 Weeks

736 548 717 387 997

Died 903 792 745 2041

3156 613 3700 5730

Died 1019 7856 9721

2518 Died 15,725

Life span (days)

24 71 61 69

a Values are given in cubic millimeters.

Mammary glands of five animals without tumors did not show any histologic evidence of malignancy. Mammary glands of animals without clinical evidence of tumors showed a sparse, well-organized ductal arrangement with scant fibrous connective tissue stroma. Few, if any, acini were noted. The fibroglandular units were surrounded by both normal ilipocytes and “brown fat.” The histologic diagnosis was normal mouse breast.

Group 2. Normothermic Animals with Tumors and with Chemotherapy The tumors were measured 1 week prior to injection (5-FU, 100 mg/kg ip); subsequent tumor measurements were once per week. In this group of five animals, one animal died from toxicity 1 week after administration of the drug. The life span of these animals was shorter than for Group 1 (Table 2).

FIG. 2. Tumor exposed to 8-hr differential hypothermia architecture and a focus of necrosis. Magnification, 200x.

shows

disruption

of the

tumor

HYPOTHERMIA

AND

5-FLUOROURACIL

ON C:,H MOUSE

TUMORS

FIG. 3. Tumor exposed to I-hr differential hypothermia and 5-FU infusion. architecture is seen here with a large focus of necrosis. Magnification 200X.

Histologic study (biopsy of tumors) was done on an additional group of five animals 2 weeks post-administration of 5-FU. There was no tumor regression. Tumor growth was not followed due to biopsy. Mammary tumors of all 10 animals were typical, as described in Group 1. In some cases, broken cells were -noticed. Group 3. Eight-Hour-Long Differential Hypothermia without Chemotherapy Since, from our previous work, it was shown that l-hr differential hypothermia does not induce regression of mammary tumors in C&I mice, we wanted to see if prolonged differential hypothermia has any effect on tumors. Five animals in this group were cooled to a body temperature of 12 15°C while their tumors were kept at a temperature of 37-39°C. Thus, induced differential hypothermia was kept for 8 hr. The anticancer agent was not administered. These animals were sacrified as soon as the experiment was over (immediately after

Typical

tumor

rewarming), and their tumors were histologically examined. This was done because these animals cannot be permanently recovered after ,a profound hypothermia of such a long duration ( 16, 17). Histologic findings: Tumors exposed to S-hr-long differential hypothermia without chemotherapy showed that there was intercellular and intracellular edema with distortion and disruption of the overall architecture. Thrombosis of the blood lakes was seen, and a few small localized areas of necrosis were seen in three animals (Fig. 2). Group 4. One-hour Differential Hypothermia with Chemotherapy In this group of experiments, five animals were ,injected with 5FU through the venous cannula as soon as differential hypothermia was established. One hour later the animals were rewarmed. Two weeks after the experiment, tumor size was measured and tumors then excised for pathologic examination. All animals were sacrificed, and

x7.2

POPOVIC,

POPOVIC

AND

WEATHERS

FIG. 4. Tumor exposed to I-hr differential hypothermia and 5-FU infusion shows marked distortion of the architecture. Bizarre cells are also noted which probably represent cytotoxic effects of the treatment. Magnification, 200x.

the proper position of the cannula in the jugular vein was established and checked. This way we were sure that the drug was injected systemically without any leakage. From five animals in this group, one died from toxicity after 1 week. Two animals had tumors that regressed more than 50% in size. Two animals had tumors that remained unchanged in size. Histologic findings: In tumors treated with 1-hr differential hypothermia, ultrasound warming, and 5-FU therapy, there were obvious edema and degenerative cellular changes with marked distortion of the architecture. Large areas of coagulative necrosis were seen in two tumors and small areas in another. The amount of degeneration and necrosis appeared to be quantitatively greater in this group than in the others (Figs. 3 and 4). DISCUSSION

Protective effects of hypothermia have been known for a long time. Using this

protective effect in differentially cooled animals, it has been possible to protect the healthy tissues from the toxic and leukopenic effects of tumor chemotherapy while at the same time expose the tumors to full or even an enhanced effect of the drugs (9, 12, 14, 15, 18-22). Histologic studies in this paper illustrate some of the morphologic changes of tumors in C&H mice that are induced after prolonged differential hypothermia and after 1-hr differential hypothermia combined with chemotherapy. SUMMARY

No regression of mammary tumors of CSH mice was observed in normothermic animals with or without 5-fluorouracil chemotherapy. After prolonged differential hypothermia, histologic changes of necrosis and of cell degeneration were prominent. However, when differential hypothermia of 1 hr and chemotherapy were combined, tumors regressed in 50% of the cases. Histologic changes were more striking and cor-

HYPOTHERMIA

responded tumors.

AND

5-FLUOROURACIL

to the decrease in size of the 13. REFERENCES

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