Cancer Immunol Immunother (1983) 14:151-154 ancer mmunology mmunotherapy

© Springer-Verlag 1983

Eradication of Microscopic Hepatic Metastases by Active Specific Immunization

Saraswati Sukumar, James T. Hunter*, Nobukuni Terata, and Herbert J. Rapp*

Laboratory of Immunobiology, National Cancer Institute, Bldg 37, Rm 2B10, National Institutes of Health, Bethesda, MD 20205, USA

Summary. Strain-2 guinea pigs, each with microscopic deposits of line 10 hepatocarcinoma in the liver, were treated by ID immunization with a mixture of irradiated tumor cells and an oil-in-water emulsion containing cell walls of Mycobacterium bovis strain Bacillus Calmette-Gu~rin (BCG CWE). Injection of line 10 hepatoma cells into the hepatic portal vein led to the development of tumor foci in the liver, metastasis in the hepatic lymph node, malignant ascites, and death. Active immunization using irradiated line 10 cells and BCG CWE was effective therapy when administered 1, 7, and 14 days after intraportal injection of line 10 cells. Effective immunization required both irradiated line 10 tumor cells and the BCG cell wall emulsion. Immunization with BCG CWE admixed with irradiated line 1 tumor cells, a hepatoma antigenically distinct from line 10, did not prevent outgrowth of line 10 deposits in the liver. Animals rendered free of disease could reject a challenge of line 10 tumor cells but not of line 1 tumor cells.

Introduction

In the past 5 years, we have reported on the standardization and use of mycobacterial vaccines in the eradication of microscopic metastases remaining after surgical removal of intradermal (ID) implants of the line 10 guinea pig hepatoma [1, 5, 6, 9]. Following ID injection of tumor cells, the line 10 guinea pig hepatoma metastasizes to the regional lymph nodes. Microscopic metastases are present in the first set of draining lymph nodes (clinical stage I), and later in the second set of lymph nodes (clinical stage II). In animals treated with limited surgery, administration of vaccines containing BCG cell walls in oil-in-water emulsion admixed with irradiated tumor cells was effective therapy. Treatment of clinical stage I disease by vaccines was antigenically specific and non-toxic [1].

In this paper we report studies done to test the efficacy of the irradiated line 10 cells-BCG CWE vaccine in elimination of microscopic deposits of line 10 in the liver. Since treatment of hepatic metastases poses an important challenge to the oncologist, we devised a reproducible method for production of liver metastases and studied the effects of immunotherapy in this disease situation.

Reprint requests should be addressed to S. Sukumar * Present address: The Stehlin Foundation of Cancer Research, 1918

Chenevert, Houston, Texas ~ Deceased 25 Sept. 1981 Abbreviations: ID, intradermal; IP, intraperitoneal; BCG CW, cell walls of Mycobacteriurn bovis strain Bacillus Calmette-Gudrin; BCG CWE, BGW CW in oil-in-water emulsion

Materials and Methods

Animals. Sewall-Wright strain 2 male guinea pigs were obtained from the Laboratory Aids Branch, Division of Research Services, National Institutes of Health and from the Experimental Animal Breeding Facility of the National Cancer Institute, Frederick, Maryland. They were housed in groups of six to a cage, and were given NIH guinea pig chow and water, available at all times. The animals were 3 - 4 months old and weighed about 500 g when entered into the experiment.

Tumors. Line 10 and line 1 hepatocellular carcinomas were derived from diethylnitrosamine-induced hepatomas in male strain 2 guinea pigs. After conversion into ascitic variants, tumors were maintained by serial IP passage in male weanling strain 2 guinea pigs. Line 10 was used in its 12th-18th IP transplant generations and line 1, in its 95th-100th IP transplant generations in these experiments. Individually specific tumor rejection antigens have been shown to be present in line 10 and line 1 tumor cells [8].

Surgical Procedure for Intraportal Injection of Line 10. Guinea pigs were anesthetized by inhalation of a mixture of methoxy- flurane (Metafane, Pitman-Moore Inc., Washington Crossing, N.J.) and oxygen, using a veterinary anesthesia machine (Heidbrick, Model 970). A mid-ventral incision was made starting below the xiphoid process, and the intestines were rotated laterally towards the left side of the animal. The portal vein was readily seen anterior to the right kidney. The exposed organs were kept moist with physiological saline and 0.2 ml of a line 10 cells suspension was injected into the portal vein with a 30G needle using a 1-ml syringe. The point of insertion of the needle was through the adjoining fatty tissue; the needle was held in place for about 15 s after injection of line 10 for tumor cells to be flushed away from the needle. Very little, if any, bleeding was seen at the point of injection. About 10 ml physiological saline was poured into the abdominal cavity to prevent adhesions. The peritoneum and fascia were sutured with continuous Black 00 silk sutures and the skin closed with metal wound clips. Extreme care was taken throughout the procedure to prevent leakage of line 10 cells into the peritoneal cavity, since a single living line 10 cell injected IP will multiply and result in death of the recipient within about 40 days (J. T. Hunter and H. J. Rapp, unpublished observations). Each animal was given an IM injection of 0.1 ml solution containing 20 mg cephaloridine (Eli Lilly & Co., Indianapolis, Ind., USA) the day after tumor implantation and again l week later. Surgical mortality was about 5%.

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Preparation of Oil-in-Water Emulsions Containing BCG CW. Cell wails of Bacillus Calmette-Gudrin (BCG CW, Lot 286) were obtained from Dr Edgar Ribi, Rocky Mountain Labo- ratories, Hamilton, Montana. Oil-in-water emulsions were prepared by grinding BCG CW to a smooth paste with mineral oil (Drakeol 6 VR) and Tween-80 (0.2%) in normal saline (0.85%) in a tissue grinder as described elsewhere [9]. The final composition of the ground preparation per milliliter was: 1.87 mg CW, 30 ~tl oil, and 2 ~1 Tween.

Irradiation of Tumor Cells. Ascites containing line 10 and line 1 cells was harvested, and the cells washed twice with Medium 199; tumor cells were exposed to 10,000 rad of gamma-irra- diation. Then 40 ml cell suspension in a 50-ml polypropylene centrifuge tube (No. 25335, Corning Glass Works, Coming, N.Y.) was placed in an upright position on the rotating platform inside the irradiation chamber of a J.L. Sheperd Mark I irradiator (Model 68, 137Cs source; dose rate 2,383 Roentgen/min). The cells were washed after irradiation. Suspensions containing more than 90% trypan blue excluding cells were used in vaccine preparations.

Composition of Mixtures used for Treatment. Mixtures of irradiated tumor cells and BCG CW emulsions were prepared as described elsewhere [2]. Oil emulsion was added in a dropwise fashion to the pelleted cells, kept dispersed by continuous vortexing at low speed. The mixture was injected ID within 2 - 3 h of preparation, during which time the tumor cells were 90%-100% viable. Each guinea pig received a mixture of 0.4ml CW emulsion plus an 0 .4 -0 .6ml of irradiated tumor cell suspension containing 100 x 106 tumor cells.

Immunotherapy of Animals with Line 10 in the Liver. At 1, 7, or 14 days after receiving line 10 by intraportal injection, guinea pigs were treated by ID administration of vaccines. Each guinea pig received the vaccine equally divided among four sites, in a vertical line along the left thoracic flank. One week later the same vaccine was injected on the opposite flank. Untreated animals died with hepatic tumor, tumor in the hepatic lymph node, and malignant ascites.

Measurements. Animals that died within 2 weeks of surgery did not show any gross liver pathology and death was usually attributable to obstruction of the gastrointestinal tract due to herniation through loose abdominal sutures. Guinea pigs were examined at weekly intervals after surgery to check for weight loss and other signs of debilitation. At death, guinea pigs were necropsied; pieces of liver, hepatic node, and lung were sent for histology. Animals were observed for a minimum of 120 days. The significance of differences between groups was determined by Fisher's exact test and Chi-square analysis (two-tailed).

Results

Immunotherapy of Guinea Pigs with Microscopic Deposits of Line 10 in the Liver

Microscopic deposits of tumor in the liver of guinea pigs were established by intraportal injection of different doses of line 10 cells. Animals were immunized 1 day after intraportal injection, followed by a booster 1 week later. The results of

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Fig. 1 A-C . Immunotherapy of guinea pigs with line 10 deposits in the liver by immunization with vaccines containing irradiated line 10 cells and BCG CWE. Each guinea pig received 103 (A), 104 (B), or 10 s (C) line 10 cells intraportally on day 0. Solid lines, no treatment after injection of line 10 cells; broken lines, immunized with vaccine containing 108 irradiated line 10 cells and 750 ~tg BCG CWE on day 1 (left flank) and day 8 (right flank)

Table 1. Survival of guinea pigs given intraportal injection of 105 line 10 hepatocarcinoma cells: effect of immunization 1, 7, and 14 days after line 10 tumor injection

Group Immunization a Survivors/ total no. of animals in group

1 None 0/12 2 Day 1 and day 8 11/12 b 3 Day 7 and day 14 9/12 b 4 Day 14 and day 21 5/12 °

a Guinea pigs were given intraportal injection of 105 syngeneic line 10 hepatoearcinoma cells on day 0; on various days after line 10 injection, animals were immunized with vaccine containing 750 Ixg BCG CWE and 108 irradiated line 10 cells. Experiment was evaluated at 150 days after intraportal injection of line 10 cells

b Significantly different from group 1 (P < 0.005 by Fisher's exact test)

c Significantly different from group 1 (P < 0.05 by Fisher's exact test)

this experiment (Fig. 1) showed that intraportal injection of 10 g or 105 line 10 cells resulted in death of all the animals in the groups. Of the animals immunized with irradiated line 10 and BCG CWE 80%-100% were alive and tumor-free at the end of the observation period. In a second experiment, treatment was initiated 1, 7, or 14 days after intraportal injection of 105 line 10 cells. The vaccine afforded decreasing, but significantly effective protection when treatment was initiated 7 or 14 days after intraportal injection of 105 line 10 (Table 1).

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Fig. 2 A and B. Survival of guinea pigs with line 10 deposits in the liver treated by active immunization. Experimental animals received an intraportal injection of 10 4 (A) or 10 5 (B) line 10 cells on day 0, followed by no treatment (solid lines) or by vaccines (broken lines). Vaccines contained BCG CWE alone (O), irradiated line 10 cells alone (O), BCG CWE plus irradiated line 10 cells (&), or BCG CWE plus irradiated line 1 cells (V). Vaccines were administered ID at four sites along the left flank on day 1 and along the right flank on day 8

Specificity of Immunization and the Need for a Complete Vaccine for Successful Immunotherapy

This experiment was designed to test the need for both mycobacterial and specific tumor cell components in the vaccine used for immunization of animals with microscopic deposits of line 10 in the liver. Groups of guinea pigs, each with an implant of 105 line 10 cells in the liver, were immunized with vaccines containing (1) BCG CWE alone, (2) irradiated line 10 tumor cells alone, (3) irradiated line 1 tumor cells admixed with BCG CWE, or (4) irradiated line 10 tumor cells admixed with BCG CWE. Survival of guinea pigs treated by active immunization using complete vaccine following intraportal injection of 104 line 10 cells is shown in Fig. 2A. The results presented in Fig. 2B show that immunization with vaccines containing irradiated line 10 cells alone or the adjuvant BCG CWE alone 1 day following intraportal injection of 105 line 10 cells was ineffective in preventing outgrowth of liver tumor. Use of line 1 tumor cells (an antigenically distinct tumor) admixed with BCG CWE was also ineffective treatment; all the animals died and no prolongation of survival was seen. Immunization using a complete vaccine containing both irradiated line 10 cells and BCG CWE resulted in the protection of 12 of 14 animals. In contrast, animals that received no treatment died with liver tumor.

Specific Immunity to Line 10 Rechallenge in Cured Animals

Twenty-four animals that had been rendered tumor-free by immunization were challenged 150 days after intraportal injection with ID injections of 106 line 10 cells on one flank and 3 x 106 line 1 on the contralateral flank. At the end of 10 days,

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24 of 24 guinea pigs showed no growth at the line 10 challenge site, whereas the line 1 challenge grew during this period, thus demonstrating the presence of specific antitumor immunity as a result of treatment.

D i s c u s s i o n

This study demonstrates that active specific immunotherapy with irradiated syngeneic tumor cells admixed with BCG CWE is effective therapy for microscopic deposits of line 10 hepatoma in the liver of strain 2 guinea pigs. This model for liver disease is an artificial one derived by injection of line 10 tumor cells via the portal vein and has limitations comparable to those of lung metastases growing as a result of IV injection of tumor cells [2-4 , 7].

Limited studies on histopathology of liver sections at 1, 3, 7, 10, 14, and 21 days after injection of 10 s cells show that clusters of two to three tumor cells were visible in the vicinity of venules as early as 3 days after injection of tumor cells. By the 21st day, one or two l-ram foci were grossly visible on the surface of the liver.

In earlier studies, vaccines consisting of irradiated line 10 cells and BCG CWE were shown to be effective in the treatment of microscopic metastases in the regional lymph nodes of animals surgically cured of dermal tumor implants and palpable lymph node metastases and against IV-implanted microscopic tumor deposits in the lung [1, 5, 9]. In this study we have shown that intraportal injection of 104 or 105 line 10 cells resulted in the death of all animals in the group. Active specific immunization using syngeneic tumor cells and BCG CWE was effective in curing a significant number of animals with deposits of line 10 cells in the liver up to 14 days after injection of 10 s tumor cells. Specificity of tumor cells in the vaccine and the need for the presence of BCG CWE for effective therapy were demonstrated. Long-term survivors had demonstrable tumor-specific immunity in that they were able to reject a challenge of line 10 but not line 1 tumor cells. No complications of active immunization with irradiated tumor cell-BCG CWE vaccine were seen other than the formation of ulcers at the sites of vaccination, which healed within 2 months of vaccination.

Line 10 tumor has been shown to be weakly antigenic by its inability to induce a tumor rejection response following immunization with attenuated line 10 cells alone. However, immunotherapeutic efficacy of vaccines containing line 10 cells admixed with adjuvants has been demonstrated in a variety of experimental situations [1-9] . The results of this study indicate that under the conditions described, active immuno- therapy was effective in the eradication of microscopic metastases in the liver. Tumor cells in the liver were accessible to immune manipulations provided there was evidence of tumor immunogenicity. With the caveat that these studies were carried out in an artificial system and with a limited tumor load, the results suggest that in treatment of hepatic cancer or of microscopic metastases in the liver, active immunotherapy with surgery or chemotherapy may be an adjunct worthy of consideration. However, further experimentation is needed to determine the extent of tumor load that can be effectively eradicated and any improvement that might be possible in the vaccine and its mode of administration.

Acknowledgements. The authors thank Dr Chao Kuang Hsu, Com- parative Medicine Program, University of Maryland Medical School, for histology consultations and Dr Berton Zbar for reviewing the manuscript.

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References

1. Ashley MP, Zbar B, Hunter JT, Rapp HJ, Sugimoto T (1980) Adjuvant-antigen requirements for active specific immunotherapy of microscopic metastases remaining after surgery. Cancer Res 40:4197

2. Bartlett GL, Kreider JW, Purnell DM, Hockley AJ (1978) Treatment of visceral tumor with BCG-tumor cell vaccine. Cancer Immunol Immunother 4:15

3. Hanna MG Jr, Peters LC (1978) Specific immunotherapy of established micrometastases with Bacillus Calmette-Gudrin tumor cell vaccine. Cancer Res 38:204

4. Hanna MG Jr, Peters LC (1981) Morphological and functional aspects of active specific immunotherapy of established pulmonary metastases in guinea pigs. Cancer Res 41:4001

5. Hunter JT, Ashley MP, Sukumar S, Sugimoto T, Zbar B, Rapp HJ, Yarkoni E (1981) Treatment by limited surgery and specific immunization of guinea pigs with stage II experimental cancer. J Exp Med 154:253

6. Yarkoni E, Ashley MP, Zbar B, Sugimoto T, Rapp HJ (1982a) Eradication by active specific immunotherapy of established tumor transplants and microscopic lymph node metastases. Cancer Res 42 : 2544

7. Yarkoni E, Hunter JT, Sukumar S, Rapp HJ (1982b) Active specific immunotherapy of guinea pigs with visceral tumor implants. Cancer Immunol Immunother 12:273

8. Zbar B, Bernstein ID, Tanaka T, Rapp HJ (1970) Tumor immunity produced by intradermal inoculation of living tumor cells and living Mycobacterium boris (strain BCG). Science 170:1217

9. Zbar B, Canti G, Ashley MP, Rapp HJ, Hunter JT, Ribi E (1979) Eradication by immunization with mycobacterial vaccines and tumor cells of microscopic metastases remaining after surgery. Cancer Res 39:1597

Received October 13, 1982/Accepted November 26, 1982