Inconsistent Response of B16 Melanoma to BeG Immunotherapy 1,2 John W. Kreider,3 Gerald L. Bartlett,3,4 and Dallas M. Purnell 5 ,6

Studies of humans and animals suggested that melanocytes may have peculiar immunogenic properties. In a strain of Sinclair swine specifically bred for a high incidence of cutaneous nevi and melanomas, there was a significant incidence of spontaneous regression of the melanotic tumors (1). Local vitiligo occurred at the site of tumor regression and sometimes involved depigmentation of the entire integument. Human melanomas spontaneously regress and do so with the greatest frequency of any human tumor (2). Spontaneously regressing benign nevi ("halo" nevi) in humans may contain a peripheral zone of depigmentation accompanied by an infiltrate of mononuclear leukocytes (3). The peripheral blood of melanoma patients contained lymphocytes cytotoxic to cultured malignant melanoma cells and serum factors that were often cytotoxic per se but occasionally also blocked the lymphotoxicity. The sera of normal healthy blacks possessed "unblocking" activity in in vitro lymphocytotoxicity assays (4). BCG given to human melanoma patients has occasionally produced objective benefits and prolonged survival rates. BCG injected directly into some cutaneous tumor nodules of a patient with a locally recurrent tumor often produced complete regression of the inoculated tumors, and in about 10% of the treated patients, inoculated satellite tumor nodules also regressed (5). Inoculation of cutaneous sites with BCG as an adjunct to conventional therapy for stage II melanoma improved patient survival rates (6). These observations have been interpreted as a BCG-induced augmentation of tumor immunity. There is a need for an experimental model system to guide immunotherapy of human malignant melanoma. Because of the unusual immunogenic properties of melanocytes, BCG immunotherapy of other histologic types of tumors may not be relevant. We believe that an animal model system of malignant melanoma should possess certain attributes: The host should be a relatively small, inexpensive animal with a well-characterized immune response and lymphoreticular system. For maximum precision and reproducibility of experiments, the tumor should be transplantable and detectably both im-

munogenic and melanotic. Preservation of tumorassociated antigens and melanogenesis should be assured by the use of tumors low in transplantation passage numbers. The tumor should be malignant with a biologic pattern of behavior similar to that of human melanoma, with spread both to regional lymph nodes and viscera. Immunotherapy should be applicable to the residual tumor surviving conventional therapies such as surgery. The experimental model should simulate the clinical situation, in which the immunotherapy is applied after the establishment of the tumor. Models in which animals are treated before tumor implantation are not relevant to human immunotherapy. Murine melanomas fulfill most of the above criteria except that they are rare and those available have been transplanted for many years. Of the several murine melanomas, the Bl6 has been most thoroughly studied by immunologic techniques (7, 8). In addition, there have been some previous BCG immunotherapeutic studies in this system (9). The purpose of this investigation was to assess the responsiveness of Bl6 melanoma ..0 BCG immunotherapy. We studied the effects of various treatment protocols including: a) injection of BCG directly into established tumors; b) concurrent. administration of BCG and tumor at separate sites; c) fate of B16 tumor cel1s admixed with BCG and injected together; and d) use of vaccines composed of BCG admixed with living or irradiated Bl6 cells to treat established or residual tumors. MATERIALS AND METHODS

Animals and tumors.-A line of B16 tumor (The Jackson Laboratory, Bar Harbor. Me.) was sequentially doned three times in cell culture (10) and maintained in continuous passage in syngeneic hosts by sc trocar transplantation. Tumors were passaged at about biweekly intervals. Fragments were periodically cultured in thiogylcollate broth for detection of bacterial contamination. The hosts were 4- to 6-week-old male C57BLJ6J mice purchased from The Jackson Laboratory. Preparation of tumor cell suspensions.-Passage tumors about I cm in diameter and free of cutaneous necrosis and ulceration were excised and trimmed of extraneous tissues. The tumor fragments were minced and placed in 0.04% Versene in calcium- and magnesiumfree, phosphate-buffered saline at 37° C. The suspension was stirred in a trypsinizing flask for about 30 minutes and then washed with serum-free Eagle's miniReceived August 28, 1975; accepted October 24, 1975. Supported by Public Health Service '(PHS) contract N01 CB33891 from the Division of Cancer Biology and Diagnosis, National Cancer Institute, and by the Jake Gittlen Memorial Golf Tournament. 3 Departments of Pathology and Microbiology, College of Medicine, The Pennsylvania State University, Hershey, Pa. 17033. 4 Recipient of PHS Career Development Award K04-CA70948 from the National Cancer Institute. 5 Department of PathOlogy, College of Medicine, The Pennsylvania State University. 6 We acknowledge the technical assistance of Susan Webb and Janene Giomariso. 1

2

JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 56, NO.4, APRIL 1976

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SUMMARY-We evaluated the potential of the B16 melanoma of mice as a model system for BCG immunotherapy of malignant melanoma. We studied a variety of treatment protocols: a) BCG given simultaneously but separately with a small number of B16 cells significantly inhibited tumor growth in only three of eight experiments. b) BeG injected directly into the tumor stimulated tumor growth in three of three experiments; the stimulation was at least partially attr,lbutable to the nutrient medium in which the BCG was suspended. c) The B16 tumor was weakly immunogenic and the addition of BCG to a tumor cell vaccine offered little improvement in subsequent resistance to tumor cell challenge. d) In a model of postsurgical residual tumor, metastatic to regional lymph nodes, BeG and tumor cell vaccination did not alter the development of nodal metastases. The B16 melanoma was not a useful model system for BeG immunotherapy, because the tumor inhibition was feeble, inconsistent, and not associated with augmented tumor immunity.-J Natl Cancer Inst 56: 803-810, 1976.

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KREIDER, BARTLETT, AND PURNELL

each animal's uninoculated foot was subtracted from the weight of the tumor-bearing foot. Alternatively, the thickness of tumor-bearing feet was directly measured with a carpenter's calipers. Most experiments were terminated before cutaneous ulceration of the tumor developed. Ulceration and associated wound infections and cannibalism made it almost impossible to accurately assess the volume or mass of large Bl6 tumors. Statistical comparisons employed Student's t-test. Production of residual tumors.-A suspension of 10 5 ?r. lOB trypan blue:excluding BI6 cells in 25 .p.l was mJected mto the hmd footpads of mice. At intervals after the injection, l\lice were anesthetized with Nembutal and the tumor-bearing hind foot was disarticulated through the joint capsule, with the aid of an electrocautery. No deaths or wound infections resulted f~om the amputation. Mice were observed and the poplIteal nodes palpated weekly for 2-3 months. All animals that died during the observation period and those killed at the end of the experiment were autopsied, and visceral metastases were identified. RESULTS Concurrent Administration of BCG and Tumor at Separate Sites

We assessed the therapeutic effect of BCG on a minimal initial tumor load representing the small mass of residual tumor surviving a primary cytoreductive treat~ent such as surgery. Bl6 cells (10 5 or 106 ) were injected mto the hind footpad and BCG was given sc into the back. We studied the consequences of varying the doses of tumor cells and BCG and the total number of BCG treatments. In the first series of experiments, the tumor ceIlinoculated mice were divided into groups of 10 and treated with different BCG doses. After 2-7 weeks (depending on the size of the tumor), the mice were anesthetized and both hind feet were amputated and weighed. The mice were observed for subsequent popliteal lymph node metastases and death. At autopsy, the extent of disseminated tumor was noted. Four identical experiments were performed (table I): In the first, BCG doses of 105 , 1()6, and 107 CFU significantly inhibited the growth of 105 tumor cells; however, only the highest BCG dose (10 7 CFU) inhibited the growth of 106 tumor cells. In the second, significant acceleration of the growth of 10 5 tumor cells was obtained with all BCG doses. The third experiment produced no significant results, and in the fourth, only 107 CFU significantly suppressed the growth of 105 tumor cells. In all four experiments, the incidence of tumor dissemination to popliteal lymph nodes and lungs closely reflected the fate of the primary tumor. In those animals with primary tumor growth retarded by BCG, nodal metastases developed with reduced frequency. Conversely, metastases were frequent and large in animals with primary tumors not inhibited by BCG. Some mice in experiments 1 and 4 failed to develop residual tumor after amputation. These animals belonged to both BCGtreated and untreated groups and were challenged with an sc injection of 1(}6 B16 tumor cells. There was no difference in the growth rate of challenge tumor in mice previously exposed to Bl6 tumor and/or BCG, We also studied the effect of multiple sc injections at weekly intervals on the growth of BI6 footpad tumors. '1

N -2-hydroxyethylpi perazine-N'·2·ethanesulfonic acid.

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mum essential medium (MEM) with HEPES 7 buffer. The concentration of trypan blue-excluding cells was determined in a hemacytometer and the cell suspension appropriately adjusted with the medium. X-irradiation of tumor cells.-Cell suspensions in a 60-mm plastic petri dish were irradiated with a 250 kV peak source at a 13-cm distance in an 8-cm field wi th a 2-mm aluminum filter for 10 minutes (total dose: 10,000 rads). Animal inoculations.-BI6 tumor cells in 25-p.1 were injected with the use of a microsyringe. Cell suspensions were placed either sc or intradermally (id) on the back or beneath the skin of the hind foot. In early studies, the tumor cells were placed in the (ventral) footpad. This practice was soon replaced by use of the dorsal foot site because of an unpredictable tendency of the tumor cells to grow proximally along the Achilles tendon from ventral inocula. In foot inoculation experiments, the popliteal nodes were routinely palpated for metastases. Unless otherwise specified, each experimental group contained 10 mice. BCG.-The Phipps strain of BCG (TMC # 1029) was obtained from the Trudeau Institute, Saranac Lake, New York. The reported concentration of this material was 2.1 X 108 colony-forming units (CFU)/ml. Phipps BCG was used as the "BCG" in all experiments except where otherwise noted. Dilutions where appropriate were made in 7H9 medium (Baltimore Biological Laboratories, Cockeysville, Md.) Tice strain of BCG (lyophilized) at a reported concentration of 4 X 108 CFU /ml was obtained from the University of Chicago, Chicago, Illinois. BCG cell walls were provided by Dr. Edgar Ribi (Rocky Mountain Laboratory, Hamilton, Mont.) and were used to prepare a vaccine of cell walls in oil droplets (CWOD), according to the methods previously described (11). Measurement of plating efficiency of 7H9-treated B16 cells.-A continuous culture line of Bl6C3 cells (10) was resuspended with 0.04% Versene, washed in MEM, and divided into two 5-ml aliquots. One aliquot was then washed once in 7H9 medium and the other in MEM with 10 fetal calf serum (FCS). Both aliquots were then resuspended in 1.0 ml of the appropriate fresh medium. The cell suspensions were incubated at room temperature in a tightly capped tube. At 0, I, and 4 hours, the tubes were agitated, a small volume was removed from the incubation mixture, and the trypan blue-excluding cells were counted in a hemacytometer. The cell suspensions were then adjusted to 102 dye-excluding cells/ml and three 60-mm petri dishes (# 3002; Falcon Plastics, Oxnard, Calif.) were seeded wi th 1 or 2 ml from each suspension. (The petri dishes were previously filled with 5 ml MEM with 10% FCS and equilibrated in a humidified 95% air-5,% CO 2 incubator.) After 6 days, the medium was discarded and the cells were washed, fixed with methanol, and stained with Giemsa. The colonies per plate were counted with a dissecting microscope. The plating efficiency was calculated as the percent colonies yielded from the original cell numbers of the inocula. Measurements of tumor size.-Subcutaneous tumors on the backs of mice were excised, trimmed of extraneous tissue, and weighed on an analytic balance. The weights of tumors in the hind footpad were established by an indirect method. We amputated both hind feet of dead, tumor-bearing mice by cutting through the joint capsule of the tibiotalar articulation. The weight of

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B16 MELANOMA AND BeG TABLE

I.-Response of B16 footpad tumors to BCG treatment" Tumor weight (mg)

Tumor cell inoculum

Tumor growth (wk)

Expt No.

b

BCG dose

Control 105

106

2 2

10106

142±19 139±14

3±2 c 131±14

3±3 c 97±22

5±4 c 4±2 c

2

4

10-

375±214

1,280±349 c

800±334 c

983±304 c

3

7 4

5

10 106

387±51 262±56

737±83 291±110

213±43 371±98

463±70 171±63

4

5 2

105 10 6

47±21 25±9

55±27 15±4

28±20 20±7

1O±6 c 20±6

• Injections given same day, B16 in footpad and BCG sc in dorsum . • Mean±sE. • P

Inconsistent response of B16 melanoma to BCG immunotherapy.

We evaluated the potential of the B16 melanoma of mice as a model system for BCG immunotherapy of malignant melanoma. We studied a variety of treatmen...
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