Cancer Immunol Immunother (1983) 15:47-53 ancer mmunolggy mmunotherapy

© Springer-Verlag 1983

Inhibition of Pulmonary Metastases of B16 Melanoma with Irradiated Tumor Cells and BCG

Joseph Tai 1, Alev Guclu 1, Tarun Ghose 1, and Stevens Norvell 2

1 Departments of Pathology and Microbiology and 2 Department of Surgery, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4H7

Summary. When the tumor-bearing leg of C57BL/6J mice was amputated 16 days after SC inoculation of 106B16 melanoma cells, all the amputated mice died of pulmonary metastases. Trausfer of lungs from the amputated to normal syngeneic mice revealed tumor cells in the lungs just after amputation. Repeated weekly injections of BCG and irradiated tumor cells, beginning 24 h after amputation of the tumor-bearing limb, prolonged the survival only of mice presensitized to BCG. Injections of BCG or irradiated melanoma cells alone, of neuraminidase- and mitomycin C-treated tumor cells or of Levamisole had no effect, but injections of ConA-coated tumor cells slightly prolonged the survival of the amputated mice. Both BCG and B16 cells induced humoral and cell-mediated immunity but there was no cross-reactivity between BCG and B16 cells.

Introduction

The progressive growth and lethality of micrometastases limit the usefulness of eradicating primary tumors and their macrometastases by surgery or radiation [7]. Immunotherapy, at best, can eradicate only a small number of tumor cells [9, 10, 18] and is therefore more suited to the eradication of micrometastases than to the treatment of lesions with large tumor loads. We report here the inhibition of pulmonary micrometastases of B16 melanoma in BCG-sensitized syn- geneic C57BL/6J mice after immunotherapy with SC injections of irradiated tumor cells and BCG.

To properly evaluate the effectiveness of immunotherapy on tumor micrometastases, we first investigated the lethality and the pattern of spread of the B16 melanoma after IV, IP, and SC inoculation of various numbers of viable tumor cells. We then defined a model in syngeneic C57BL/6J mice, wherein 100% of the ,amputated mice die of pulmonary metastases after amputation of a SC tumor-bearing limb. The therapeutic potential of several protocols were initially assessed on the basis of induction of immunoprophylaxis against a challenge dose of 10 s B16 melanoma cells per adult mouse. We also present the results of several other methods of immunotherapy in this metastatic tumor model: injections of irradiated, neuroaminidase-treated or ConA-coated tumor cells, BCG alone, and the immunomodulating drug, Levamisole.

Reprint requests should be addressed to T. Ghose Abbreviations used: ConA, concanavalin A; SC, subcutaneous; IP, intraperitoneal; IV, intravenous; ID, intradermal; IT, intratumoral; PBS, phosphate-buffered saline (0.01 M sodium phosphate, pH 7.1); VCN, Vibrio cholerae neuraminidase; HBSS, Hank's balanced salt solution; RPMIM, Roswell Park Memorial Institute medium

Materials and Methods

Mice and Tumors. Female C57BL/6J mice (Jackson Lab, Bar Harbor, Maine) aged 8 - 1 4 weeks were used. B16 melanoma, a tumor of spontaneous origin, was obtained from the Jackson Laboratory, Bar Harbor, Maine, and the chemically induced EIA lymphoma from the Chester Beatty Research Institute, London. The melanoma and the lymphoma were maintained by SC and IP passage, respectively, in the syngeneic C57BL/6J mice. Groups of 15-20 mice received injections of a total of 107 BCG/mouse SC into both axillae and groins to sensitize them to BCG. Three weeks later, four random mice from each group were assessed for BCG sensitivity by a footpad test followed by histological examination. When all four mice showed positive evidence of BCG sensitization, the rest of the animals in the group were used for tumor inoculation. Mice were examined and weighed regularly and subjected to necropsy at death. Organs were examined for tumor metastasis and the number of visible metastatic foci were counted. Representative blocks of tissues from all internal organs and tumors were histologically examined.

Tumor Cell Preparations. Single cell suspensions from SC transplants of B16 melanoma were prepared by digesting finely minced tumor with 0.25% trypsin (Gibco, Grand Island, NY, USA) in PBS for 10min. Cell clumps and undigested fragments were removed by filtration through glass wool. Cellular viability was assessed by dye exclusion (0.1% trypan blue in PBS), which regularly showed -> 90% impermeable cells. The cells were washed in PBS and then adjusted to appropriate concentrations.

Irradiation. Tumor cells (107 cells/ml HBSS) were exposed to 25,000 fads of gamma radiation from a Gamma Cell 220 6°Co irradiation unit (Atomic Energy of Canada Ltd. Ottawa, Ontario, Canada).

Treatment of B16 Melanoma Cells with VCN or ConA. Melanoma cells (106 cells/ml HBSS) were incubated for i h at 37 ° C, pH 7.0, with 25 U of VCN from Vibrio cholerae strain 74 (Gibco) or with 50 ~tg ConA (grade III, Sigma Chemical Co., St Louis, MO, USA). The thiobarbituric acid assay method [22] revealed that 1.25 ~tmol of sialic acid was released from 109 B16 cells by VCN digestion. More than 90% tumor cells remained viable after digestion as assessed by trypan blue exclusion. Binding of ConA on the tumor cell surface was confirmed by immunofluorescence, using fluoresceinated goat anti-ConA globulin (Miles Lab Inc., Kanakakee, IL). All

48

viable B16 cells were treated with mitomycinC (Sigma Chemical Co., St Louis, MO, USA) and washed in PBS before being injected into C57BL/6J mice (25 ~tg mitomycin C/106 cells, 37 ° C, I h).

Amputation of Tumor-Bearing Limbs. Under pentobarbital anesthesia (50 mg/kg IP), an incision was made proximal to the tumor, near the groin. After ligation of vessels, the femoral head was disarticulated and removed along with the inguinal lymph node. Tumors were weighed after removal.

BCG Vaccine. Freeze-dried vaccine (Connaught Medical Research Lab, Toronto) was reconstituted in cold PBS before use. Each 40-mg vial contained 6,000 million BCG organ- isms.

Assessment of Cell-Mediated Immune Response to B16 Mela- noma Cells and BCG. Delayed hypersensitivity to BCG and tumor cells was assayed as described before [9, 10]. In brief, 0.03 ml HBSS containing either irradiated tumor cells (108 cells/ml suspension) or 1.5 mg BCG (heat-inactivated at 56 ° C for 30 min) was injected ID into the left footpad of immune or previously untreated control mice. The appropriate diluent was injected into the fight footpad for comparison. The thickness of the footpads was measured immediately before injection and at 24, 48, and 72 h after challenge. Footpads excised from normal and immunized mice at 24, 48, and 72 h after challenge were fixed in 10% formalin. Sections from paraffin blocks were stained with hematoxylin and eosin or carbol fuchsin to detect acid-fast bacilli [9].

The migration inhibition test according to Falk and Zabriskie [6] was used to assess cell-mediated immunity in vitro. Suspensions of spleen cells obtained from normal and immunized mice were treated with 0.84% NH4C1 (4 ° C, 8 min) to lyse red blood cells. Heat-inactivated BCG was mixed in varying ratios with the nucleated spleen cells. Migration areas were traced on papers after a 24-h incubation at 37 ° C in 5% CO2 in chambers containing RPMIM 1640 supplemented with 10% fetal calf serum (Gibco, Grand Island, NY, USA). A minimum of triplicates was set up for each test.

Cell-mediated tumor immunity was also evaluated by a Winn-type assay [9, 10]. Tumor cells were mixed with nucleated spleen cells from the immunized mice at specified ratios. After 30 min incubation at 37 ° C with gentle shaking, these mixtures were inoculated SC into previously untreated syngeneic mice. To assess tumor suppression or enhancement by nucleated spleen cells, the mean survival of the mice was compared with that of control groups. Control groups had been inoculated with aliquots containing the same number of tumor cells incubated with either nucleated spleen cells from previously untreated mice or HBSS alone.

Detection of Tumor-Specific Antibodies. Cytoplasmic and membrane immunofluorescence assays of mouse tumor cells and other control preparations were performed using fluo- resceinated goat antiserum against mouse globulin (Hyland Laboratories, Los Angeles, CA, USA) [9, 10]. Comple- ment-dependent cytotoxicity was assayed by trypan blue permeability of tumor cells incubated with test sera and rabbit complement [9, 10].

Statistical Evaluation. Student's t-test was used to compare survival time between groups in which all the mice had died, while a modified Wilcoxon's test [1] was used for groups containing living mice at the time of calculation.

TaMe 1. Survival of C57BL/6J mice with various numbers of B16 melanoma cells in different sites a

Site/method of inoculation

Mean survival (days + SD) in mice inoculated with b, c

104 cells 10 s cells 106 cells

Left axilla SC 43.0 _ 6.3 28.1 _ 4.3 27.6 _+ 4.1 Right flank SC 49.5 _ 8.9 29.8 _+ 5.7 22.8 + 4.3 IP 25.5 _+ 3.2 22.8 _+ 2.8 24.4 _+ 2.1 IV 29.3_+ 2.1 22.3 + 1.6 19.3 + 1.0 Left footpad SC 44.9 _+ 15.9 30.6 + 8.2 Not done

a Each group contained at least 10 adult female mice b The cells were inoculated in 0.1 ml PBS c The mice given 107 B16 cells SC in the left axilla or right flank died

after 21.1 __+ 5.2 and 20.7 _+ 4.5 days, respectively

Results

Survival of C57BL/6J Mice Inoculated with Various Numbers of B16 Melanoma Cells by Different Routes

Table I shows that the length of survival of mice given tumor IP or SC varied inversely with the size of tumor inocula. In the groups inoculated SC, the mice receiving 104 tumor cells survived longer than those given 105 tumor cells (P < 0.001). Mice inoculated with 105 cells SC in the right flank survived longer than those receiving 106 cells in the same site (P < 0.001). No such difference could be seen between groups inoculated SC in the axilla with 105 and 106 cells. Mice inoculated SC in the flank or axilla with 105 cells survived longer than those inoculated with 107 cells (P < 0.001). After IP inoculation, 18 of 18 mice given 103 B16 cells died after 29.2 + 6 days but only eight of 14 mice given 102 cells died (mean survival of dead mice 48.2 + 5.8 days). Two of 10 mice given a single B16 cell attached to a fragment of cover slip died of histologically confirmed malignant melanoma after 45 and 63 days during an observation period of 18 months.

In groups receiving the same number of tumor cells, survival varied with the route of tumor inoculation. With 104 and 105 tumor cells, animals inoculated IP or IV died earlier than those given tumor SC (P < 0.001).

Spontaneous regression of an established B16 melanoma or failure of tumor take after inoculation of -> 104 viable B16 cells per syngeneic mouse has never been observed in our laboratory.

Spread of B16 Melanoma in C57BL/6J Mice. Autopsy of mice dying of SC-transplanted B16 melanoma (104 to 107 tumor cells/mouse) usually revealed metastasis in the draining lymph nodes and a maximum of 20 grossly visible pigmented metastatic nodules in the lungs. Gross and histological examinations did not reveal tumor deposits in any other internal organs.

Growth of B16 Melanoma in BCG-Sensitized Mice. There was no difference between BCG-sensitized and previously untreated mice when the weight of tumor-bearing legs amputated on the 16th day after SC inoculation of 106 B16 cells/mouse was compared. Weight of tumor-bearing legs was 3.6 + 0.6 g in BCG-sensitized mice (n = 116) and 3.5 _+ 0.8 g in unsensitized mice (n = 122). Amputated limbs were pooled from mice used for evaluating various immunotherapeutic protocols for inhibition of pulmonary metastases.

49

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80- Fig. 1. Survival of groups of C57BL/6J mice ,~ amputated 6, 9, 12, and 16 days after SC > inoculation of 106 B16 cells into the leg. Mice that did not undergo amputation Of the " 60 tumor-bearing limb survived for 23.2 _+ 2.5 days. m Inset shows photos of a C57BL/6J mouse before and after amputation of a limb bearing a z 16-day-old tumor transplant. The number of ~ 40 mice varied from a minimum of six to a ,,,, maximum of 24 per group. (111) Amputation 6 days after tumor inoculation; (©) Amputation 9 days after tumor inoculation; (A) Amputation 20 12 days after tumor inoculation; (x) Amputation 16 days after tumor inoculation; (&) Amputation 16 days after tumor inoculation-BCG + ve mice O"

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Fig. 2A-D. Lungs from C57BL/6J mice. (A) Lungs without any visible tumor nodule, obtained from a mouse 16 days after SC inoculation of 106 B16 melanoma cells into the left leg; (B) Lungs showing pigmented metastatic nodules (arrows) 5 days after amputation of the tumor-bearing leg; (C) Lungs showing larger metastatic nodules (arrows) 12 days after amputation; (D) Lungs showing almost complete replacement by tumor tissue 24 days after amputation

Establishing a Metastatic B16 Melanoma Model. Figure 1 shows the survival of C57BL/6J mice inoculated with 106 B16 cells/mouse SC in the left leg followed by its amputation on days 6, 9, 12, 14, and 16 post-tumor inoculation. Though all the mice in the groups in which legs were amputated on the 14th and 16th days died of pulmonary metastases, the survival of the mice whose limbs were amputated on the 14th day (i.e., from 39-73 days) varied more widely than that of mice in which amputation was performed on the 16th day (i.e., 46.3 + 8.4

days). There was no grossly detectable tumor metastasis in the lungs of mice on the 16th day after tumor inoculation. However, in all mice amputated on the 16th day, metastases could be seen in the lungs on the 5th day after amputation. On the 12th day after amputation, large tumor nodules were seen projecting from the surface of the lungs, and on the 24th day after amputation both the lungs appeared to be completely replaced by tumor tissue (Fig. 2). Two groups of mice, one with intact 16-day-old tumor transplants in the left legs and the

50

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80

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Fig. 3. Survival of groups of C57BL/6J mice (not sensitized to BCG) that were given various modalities of immunotherapy after amputation of the tumor-bearing limb. Limbs were amputated 16 days after SC inoculation of 106 melanoma cells. Treatment began 1 day after surgery and was given once a week for 8 weeks. Each group contained at least 15 mice. BCG negative groups: (e) No AMP; (x) AMP only; (&) AMP + 106 BCG/4 sites SC/wk; (D) AMP + 106 BCG/4 sites SC 10 days 107 y-IRRAD B16 cells/ 4 sites SC; (O) AMP + mixture of 106 BCG + 107 y-IRRAD B16 cells/4 sites SC/wk; ( I ) AMP + 106 BCG IV/wk; (&) AMP + 10 7 v-IRRAD B16 cells/ 4 sites SC/wk

Table 2. Survival of C57BL/6J mice a treated by IT injection of BCG b 10 days after inoculation of 10 6 B16 cells/mouse SC in the right flank

Treatment Mean survival (days + SD)

BCG + ve BCG - ve

BCG once 27.1 + 4.0 26.4 + 5.7

BCG once every week 29.3 + 4.3 24.4 + 2.8

PBS once every week 28.9 + 3.1 26.9 + 5.5

Each group contained a minimum of nine mice 106 BCG in 0.1 ml PBS were injected directly inside the tumor nodule

other with the legs amputated on the 16th day after tumor inoculation (106 cells/mouse), were exsanguinated to death by cardiac puncture. When fragments of lungs from these two groups were inoculated IP into previously untreated mice (both the lungs from a donor to one recipient), the recipients died of histologically proven IP melanoma after 32.6 + 10.3 days and 23.3 + 15.1 days, respectively. However, no tumor could be detected within an observation period of at least 200 days in two other groups of mice that received cardiac blood from the two groups of mice exsanguinated on the 16th day after SC tumor inoculation.

Immunotherapy of B16 Melanoma

Intratumoral BCG Therapy of SC B16 Melanoma Transplants. When BCG-sensitized or previously untreated mice were given 106 tumor cells SC and then received injections with 106 BCG IT either on a single occasion (on the 10th day after tumor inoculation) or repeatedly (once a week, beginning on the 10th day after tumor inoculation, till death), survival was not prolonged beyond that in tumor-inoculated control groups given HBSS IT (Table 2).

Systemic Immunotherapy with Irradiated Tumor Cells and~or BCG in the Amputated Mice. A comparison of several protocols for the induction of immunoprophylaxis against this melanoma supported reports from other laboratories [2, 8, 13]

that repeated injections of irradiated B 16 cells alone or tumor cells and BCG were essential for inducing systemic immunity against this tumor. The protocols that were effective for immunoprophylaxis were adapted for immunotherapy in the amputated mice. Starting 24 h after excision of the tumor-bear- ing limb, the mice received either 106 BCG SC or IV, 107 irradiated B 16 cells, or a mixture of 106 BCG and 107 irradiated B16 cells. One group of mice was given BCG SC 24 h after amputation and irradiated tumor cells 10 days later (i.e., a split-adjuvant protocol) [11]. In the remaining groups, injec- tions were repeated once a week till death or, if the mice survived, for a total of eight injections. All SC injections were given in four equally divided doses into both axillae and groins. In mice not sensitized to BCG, none of the immunotherapeutic procedures added to the prolongation of survival that resulted from amputation alone (Fig. 3). On the other hand, in the BCG-sensitized mice, immunotherapy with weekly injections of BCG and irradiated tumor cells led to longer survival than in groups subjected to amputation alone or amputation followed by other methods of immunotherapy (P < 0.001) (Fig. 4). Of 38 mice in this group, five survived tumor-free, and two of these five survivors also survived a challenge with 105 B16 cells SC in the right flank 9 months after the initial tumor challenge. The remaining three mice, which died of tumor after this second challenge, survived longer (i.e., 48, 59, 123 days) than did the identically challenged but previously untreated mice (survival 29.8 + 5.7 days).

Immunotherapy with Levamisole, or with Neuramini- dase-Treated or ConA-Coated B16 Cells, 16 Days after Amputation of the Tumor-Bearing Leg. Levamisole, at 12 mg or 24 mg/kg IP three times a week, did not affect the survival of the amputated mice (Fig. 5). Figure 5 shows that active immunotherapy with injections of 107 neuraminidase- and mitomycin C-treated B16 cells on alternate days for a total of six injections also had no effect on the survival of the amputated mice. However, injection of ConA- and mitomycin C-treated B16 cells following the same schedule prolonged survival of the treated mice (P < 0.02).

Immune Response of C57BL/6J Mice to B16 Melanoma and BCG: Cell-Mediated Immune Response. The footpads of mice that were sensitized to BCG alone (106 BCG S C , 17 days

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Fig. 4. Survival of groups of C57BL/6J mice that were sensitized to BCG (i.e., a single dose of 107 viable BCG inoculated SC into both axillae and groins) before tumor inoculation and were then subjected to amputation and immunotherapy as in Fig. 3. Each group contained at least 15 mice. BCG positive groups: (O) No AMP; (x) AMP only; (A) AMP + 106 BCG/4 sites SC/wk; ([3) AMP + 106 BCG/4 sites SC 10 days 107 y-IRRAD B16 cells/4 sites SC; (O) AMP + mixture of 106 BCG + 107 7-IRRAD B16 cells/4 sites SC/wk; (in) AMP + 106 BCG IV/wk; (A) AMP + 10 v v-IRRAD B16 cells/4 sites SC/wk

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SURVIVAL ( days )

Fig. 5. Survival of C57BL/6J mice that, 1 day after amputation of the tumor-bearing limb, were given levamisole (either 24 mg/kg or 12 mg/kg) IP three times a week till death, 107 neuraminidase- and mitomycin C-treated B16 ceils, or 107 ConA-coated B16 cells on alternate days for a total of six injections. The number of mice varied from a minimum of six to a maximum of 11 per group. (O) No AMP; (x) AMP only; (/x) AMP + high dose Levamisole; (A) AMP + low dose levamisole; ([]) AMP + neuraminidase treated B16 cells; (11) AMP + ConA-coated B16 cells

before challenge) or to both BCG and B16 cells (106 BCG and 107 irradiated B16 cells) showed more swelling when chal- lenged with the sensitizing antigen than did the contralateral footpads injected with PBS or appropriate antigen-injected footpads of previously untreated mice. Footpads of BCG-sen- sitized mice did not show any such increased swelling when challenged with irradiated B 16 cells. Histological examination of footpads challenged with BCG in BCG-sensitized mice showed predominant perivascular mononuclear cellular infil-

tration at 24 and 48 h, and typical early granuloma formation at 72 h. Footpads of B16 melanoma-sensitized mice, examined 24-72 h after challenge with irradiated tumor cells, showed predominant mononuclear cellular infiltration. No granulomas were seen in the footpads of these mice. Migration inhibition was assayed using, per chamber, 108 splenocytes from immunized mice (106 BCG and 107 irradiated B16 cells SC) and 0.1 mg heat-inactivated BCG or 0.2 mg of 3 M KC1 extract of B16 melanoma cells. Inhibition index (i.e., area of migration in the presence of antigen/area of migration in medium not containing antigen x 100) was 57.9 with 0.1 mg inactivated BCG and 70.2 with 0.2 mg 3 M KC1 extract of B16 cells. This compared, respectively, with indices of 99.7 and 94.2 when the assays were performed with splenocytes from nonimmune mice (P < 0.001 with both the antigens).

Winn Assay., When previously untreated mice were given SC into the right flank 105 B16 cells incubated in vitro with 107 nucleated spleen cells from mice killed i week after completion of immunization with 107 irradiated B16 cells IP once a week for 4 weeks, their survival (27.3 + 3.4 days) was no longer than in mice that received 105 B16 cells (24.1 + 3.3 days) or 105 B16 cells incubated with 107 nucleated spleen ceils from previously untreated mice (23.3 + 3.9 days).

Humoral Response. On immunofluorescence assay, pooled sera from normal C57BL/6J mice did not react with either B16 cells or BCG organisms. Pooled sera from SC B16 melano- ma-bearing mice and the immune mice used in the Winn assay showed low titers (i.e., -< 4) of reactivity with B16 melanoma cells but not with BCG organisms. Serum obtained from mice immunized with 106 BCG IP once a week for 4 weeks reacted only with BCG (titer -< 16) and not with B16 melanoma cells. C57BL/6J mice immunized with both BCG and B16 cells produced low titers of antibody (-< 16) against both BCG and B16 cells. In the complement-mediated cytotoxicity assay, none of these sera were cytotoxic in vitro to B16 cells.

52

Discussion

Our results confirm that even though B16 melanoma is capable of provoking both humoral and cell-mediated immune re- sponses in the syngeneic C57BL/6J host [3, 8], the tumor grows progressively and invades both locally and systemically. The primary tumor or its metastases ultimately kill the host unless treatment is instituted shortly after tumor inoculation.

The inverse correlation between the number of cells inoculated SC or IV and survival makes posttumor inoculation survival a useful parameter of tumor load [9, 10]. The development of tumor in mice given, IP, the fragments of lungs from mice whose limbs are amputated 16 days after SC inoculation of 106 cells confirms the presence of tumor cells in the lungs just after amputation of the tumor-bearing limb and establishes the validity of this micrometastasis model. Like Chassoux and Salomon [4], we also failed to observe any therapeutic effect of intralesional BCG in established SC transplants of B 16 melanoma. Further, systemic immunother- apy was effective in inhibiting pulmonary micrometastases only when (a) mice were given both irradiated tumor cells and BCG and (b) injections were administered repeatedly. Single injections of BCG and irradiated tumor cells (i.e., the split-adjuvant protocol) were ineffective. Preexisting sensitiv- ity to BCG was also essential for the success of immunother- apy. The results of footpad and migration-inhibition assays demonstrated the induction of cell-mediated immunity to both BCG and B16 melanoma cells after immunization with BCG and B16 cells. However, injection of BCG alone induced delayed-type hypersensitivity to BCG only and not to B16 melanoma cells. The lack of cross-reactivity between our line of B16 melanoma and the Connaught strain of BCG was also seen at the humoral level. It is possible that during immu- notherapy with both BCG and irradiated tumor cells, preex- isting sensitivity to BCG induced an effective tumor-suppres- sive antitumor immunity earlier than in BCG-unsensitized mice. In mice not sensitized to BCG, the metastatic tumor cells in the lungs probably proliferated to a size beyond the control of immunotherapy during the longer lag period [9, 18, 251.

The mice that survived after immunotherapy retained their antitumor immunity for at least 9 months. On rechallenge with B16 melanoma cells, they either survived longer than untreated tumor-challenged mice or were tumor-free. The Winn assay failed to reveal any tumor-inhibitory effect of splenic cells from the immune mice. The 1 0 0 : a ratio of nucleated spleen cells to tumor cells has been reported to be optimal for inhibition of B16 cells by sensitized lymphoid cells [5]. It is therefore possible that other cell population(s) had the predominant effecfor role in antitumor immunity.

In a B16 melanoma pulmonary metastatic model com- parable to ours (but not using mice presensitized to BCG), Proctor et al, [19] reported that a single injection of 107 irradiated B16 cells or 104 irradiated tumor cells together with 25 ~tg or 2.5 ~tg BCG increased the number of pulmonary metastases. On the other hand, single injections of 106 irradiated B16 cells alone or together with 250 btg BCG decreased the number of pulmonary metastases. However, in keeping with our observations, immunotherapy with BCG alone had no effect on the incidence of pulmonary metastases. The size of the tumor inoculum (103 cells/mouse) and the size of the tumor at the time of amputation (mean weight 1.19 g) in the model used by Proctor et al. were smaller than in our study (i.e., tumorl inoculum: 106 cells/mouse; mean tumor weight at

the time of amputation: 2.42 g). Thus, our failure to observe tumor inhibition after immunotherapy in BCG-unsensitized mice might be due to the larger number of tumor cells in the lungs from larger and more rapidly growing tumor transplants [15, 24] and anergy associated with larger tumor mass [23]. Apar t from Proctor et al., several other groups have observed tumor enhancement after immunoprophylaxis and immuno- therapy with various combinations of BCG and irradiated and VCN-treated B16 cells [5, 8, 13]. Our use of 106 BCG/mouse/injection was based on our previous finding that, at this dose, the Connaught strain of BCG was effective for both immunoprophylaxis and immunotherapy of EL4 lym- phoma in C57BL/6J mice. At this dose of BCG, we have never observed enhancement in vivo of either the B16 melanoma or of several other tumors we investigated [9, 10].

Like ourselves, Froese et al. [8] also failed to confirm any tumor-inhibitory effect of VCN-treated B16 cells, which was reported by Rios and Simmons [20]. We also failed to confirm inhibition of pulmonary metastases by Levamisole, which was reported by Proctor et al. [19]. However, our parameter of tumor inhibition was the interval between tumor inoculation and death (associated with replacement of functional lung tissue by metastatic tumor cells); Proctor et al. counted the number of metastatic foci in the lungs 31 days after tumor inoculation. The short but significant increase in the survival of amputated mice after treatment with ConA-coated B16 melanoma cells is interesting because, in spite of preexisting sensitization to ConA, immunoprophylaxis with ConA-coated tumor cells had no effect on several ascites tumor models [9, 10]. Since Mathr ' s demonstration of the therapeutic effective- ness of BCG [17], these organisms or their methanol extract residues have been subjected to extensive clinical trials. Results of such trials have either been inconclusive [16] or shown limited success [12, 14, 21]. Our results support the conclusion of the Southwest Oncology Group (based on a recent clinical study on small-cell carcinoma of the lung) that, though BCG does not have any effect on large tumor burdens, it may be worthwhile to further explore its role in controlling minimal residual tumors after achieving complete clinical remission by other therapeutic modalities [14].

Acknowledgments. We are grateful to Dr A. C. Irwin, Department of Preventive Medicine for help in the statistical evaluation of the data, and to Mrs M. Mammen and Mr D. Sadi for their technical help.

This study was supported by grants from the M.R.C. and the Kidney Foundation of Canada.

References

1. Burdette WJ, Gehan EA (1970) In: Planning and analysis of clinical studies. Charles C. Thomas, Springfield, p 72

2. Burger DR, Hu F, Pasztor LM, Malley A (1973) A model for immunity to melanomas in mice. Proc Soc Exp Biol Med 144 : 426

3. Bystryn JC, Bart RS, Livingston P, Kopf AW (1974) Growth and immunogenicity of routine B-16 melanoma. J Invest Dermatol 63 : 369

4. Chassoux D, Salomon JC (1975) Therapeutic effect of intratu- moral injection of BCG and other substances in rats and mice. Int J Cancer 16:515

5. Chee DO, Bodurtha AJ (1974) Facilitation and inhibition of B16 melanoma by BCG in vivo and by lymphoid cells from BCG-treated mice in vitro. Int J Cancer 14:137

6. Falk RE, Zabriskie JB (1971) The capillary technique for measurement of inhibition of leucocyte migration: an assessment of cell-mediated immunity. In: Bloom BR, Glade PR (eds) In

53

vitro methods in cell-mediated immunity. Academic Press, New York, p 301

7. Fidler IJ (1978) Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res 38:2651

8. Froese G, Berczi I, Sehon AH (1974) Neuraminidase-induced enhancement of tumor growth in mice. J Natl Cancer Inst 52: 1905

9. Ghose T, Gucln A, Tai J, Norvell ST, MacDonald AS (1976) Active immunoprophylaxis and immunotlierapy in two mouse lymphoma models. J Natl Cancer Inst 57:303

10. Ghose T, Guclu A, Tai J, Mammen M, Norvell ST (1977) Immunoprophylaxis and immunotherapy of EL4 lymphoma. Eur J Cancer 13 : 925

11. Hawrylko E, Mackaness GB (1973) Immunopotentiation with BCG. III. Modulation of the response to a tumor-specific antigen. J Natl Cancer Inst 51:1677

12. Hersh EM, Gutterman JU, Mavligit GM (1977) BCG as adjuvant immunotherapy for neoplasia. Annu Rev Med 28:489

13. Kreider JW, Bartlett GL, Purnell DM (1976) Inconsistent response of B16 melanoma to BCG immunotherapy. J Natl Cancer Inst 56:803

14. McCracken JD, Chen T, White J, Samson M, Stephens R, Coltman CA Jr, Saiki J, Lane M, Bonnet J, McGavran M (1982) Combination chemotherapy radiotherapy, and BCG immunother- apy in limited small-cell carcinoma of the lung: a Southwest Oncology Group study. Cancer 49:2252

15. Martinez C, Miroff G, Bittner J (1956) Effect of size and/or rate of growth of a transplantable mouse adenocarcinoma on lung metastasis production. Cancer Res 16:313

16. Mastrangelo MJ, Berd D, Bellet RE (1976) Critical review of previously reported clinical trials of cancer immunotherapy with nonspecific immunostimulants. Ann NY Acad Sci 277:94

17. Math6 G (1969) Approaches to the immunological treatment of cancer in man. Br Med J 4 :7

18. Math6 G (1976) Surviving in company of BCG. Cancer Immunol Immunother 1 : 3

19. Proctor JW, Auclair BG, Stokowski L, Mansell PWA, Shibata H (1977) Comparison of effect of BCG, glucan and levamisole on B16 melanoma metastases. Eur J Cancer 13:115

20. Rios A, Simmons RL (1974) Active specific immunotherapy of minimal residual tumor: excision plus neuraminidase-treated tumor cells. Int J Cancer 13:71

21. Varella AD, Bandiera DC, DeAmorim AR, Calvis LA, Santos IO, Escaleira N, Gentil F (1981) Treatment of disseminated malignant melanoma with high-dose oral BCG. Cancer 48 : 1353

22. Warren L (1959) The thiobarbituric acid assay of sialic acids. J Biol Chem 234:1971

23. Whitney RB, Levy JG, Smith AG (1974) Influence of tumor size and surgical resection on cell-mediated immunity in mice. J Natl Cancer Inst 53 : 111

24. Wood JS Jr/Holyoke ED, Clason WPC, Sommers SC, Warren S (1954) An experimental study of the relationship between tumor size and number of lung metastases. Cancer 7:437

25. Zbar B (1972) Tumor regression mediated by Mycobacteriurn bovis (strain BCG). Natl Cancer Inst Monogr 35:341

Received July 26, 1982/Accepted September 12, 1982