Suppression of Moloney sarcoma virus immunity following sensitization with attenuated virus.

Murine sarcoma virus (Moloney strain) (MSV-M)-induced tumors are unusual in that they regularly appear less than 2 weeks after virus inoculation, progress for 1 to 2 weeks, and are rejected by normal adult BALB/c mice. Rejectio leaves the animals immune to tumor induction. In the present study, presensitization of normal adult BALB/c mice with attenuated MSV-M resulted in an altered pattern of tumor immunity. Injection of active MSV-M into the presensitized animals resulted in tumor induction and rejection similar to that observed in normal animals, but rejection failed to produce protection against the secondary inoculation with MSV-M. After the second inoculation with active MSV-M, tumors appeared and progressed but ultimately were rejected. Over 80% of the mice died, 25% after the primary challenge and the remainder after the secondary challenge. At death, all mice had histological evidence of leukemia which was the probable cause of death. The animals that died following the secondary challenge also had evidence of disseminated MSV-M. Solid tumor nodules were found in skeletal muscle distant from the original site of inoculation, and active MSV-M was isolated from spleen and lungs. The possibility that the results were produced by specific suppression of MSV-Moloney leukemia virus immunity is discussed.     

Studies on Murine Sarcoma Virus; a morphological comparison of tumorigenesis by the Harvey and Moloney strains in mice, and the establishment of tumor cell lines

Anatomic lesions produced by the Moloney and Harvey strains of Murine Sarcoma Virus (MSV-M and MSV-H) in mice have been compared. Erythroblastic splenomegaly is a distinctive feature of disease produced by MSV-H. Solid tumors induced by both viruses were quite similar in morphology, although some consistent differences were noted. The tumors appear to arise by massive recruitment of primitive mesenchymal cells rather than by clonal proliferation. The morphological features of these lesions would suggest that, although MSV-M and MSV-H are similar entities, they are not quite identical. Four transplant lines have been established from the mouse tumors; three MSV-H and one MSV-M. The MSV-M line (CBA strain) was initiated with difficulty and did not release sarcoma virus. The MSV-H lines (two CBA and one BALB/c) were easier to initiate and virus release was demonstrated both in vivo and in vitro. In addition to tumor growth at the site of implantation, the BALB/c line also produced splenic erythroblastosis. The MSV-M and one of the MSV-H CBA lines underwent a distinctive alteration during the course of in vivo passage in which the original spindle-cell lesion evolved into an undifferentiated small-cell tumor.     

Passive local anaphylaxis: demonstration of antitumor activity and complementation of intratumor BCG.

When an extracellular dye, Lissamine green, or 51Cr-labeled spleen cells were injected iv into C3H mice bearing small, partially necrotic 3-methylcholanthrene-induced transplantable fibrosarcomas (McC3), the tumor content of these circulating elements per unit weight was substantially lower than that of other selected organs. The level of these blood-borne materials was, however, significantly augmented by the intratumor induction of passive local anaphylaxis (PLA). The PLA-induced augmentation was inhibited by administration of the histamine and serotonin antagonist cyproheptadine; comparable increases were also induced by the intratumor injection of a histamine and serotonin mixture or BCG. The weekly intratumor induction of PLA in McC3 tumors resulted in the complete regression of a significant number of the tumors, and this therapeutic effect was eliminated by cyproheptadine treatment. The intratumor injection of BCG induced the regression of approximately 50% of injected tumors, and the combination of this immunostimulant treatment with the generation of PLA was more therapeutically effective than either treatment alone. PLA in the vicinity of solid tumors may, by increasing vascular permeability, potentiate antitumor effector mechanisms, particularly when these are BCG-stimulated. Despite this demonstration of a possible role of anaphylactic reactions in tumor immunity, no definitive evidence was found that active reagin-mediated local anaphylaxis occurred in C3H mice bearing the McC3 tumor, whether or not they were treated with immunostimulants.     

Augmentation of Anti-tumor Immunity in Low-responder Mice by Various Biological Response Modifiers: Analysis of Effector Mechanism

In order to elucidate the role of biological response modifiers (BRMs) in anti-tumor immunotherapy, we examined their effect on the induction of anti-tumor immunity in low-responder mice which hardly exhibit anti-tumor resistance against syngeneic Rous sarcoma virus (RSV)-induced tumors, such as BIO or B10.BR mice. The anti-tumor immunity induction in the low-responder mice was 0% on immunization with mitomycin C-treated syngeneic tumor cells alone. However, if BRMs were used as an adjuvant, BCG cell wall skeleton, OK-432 or lentinan augmented the induction of anti-tumor immunity to 50%, 33% and 33%, respectively. In the low-responder mice treated with BRMs, the anti-tumor immune cells had antigen-specificity at the induction phase of in vitro restimulation but not at the effector phase of target cell lysis by the stimulated cells. When T cells were depleted from immune spleen cells just before in vitro stimulation, cytotoxicity was not induced. Furthermore, cytotoxicity was not induced if accessory cells were removed from immune spleen cells at the induction phase. However, cytotoxicity at the effector phase was not mediated by T-lymphocytes, but by non-T cells. These results suggested that the induced cytotoxicity in low-responder mice was associated with the delayed-type hypersensitivity-like effector mechanism.     

Augmentation of anti-tumor immunity in low-responder mice by various biological response modifiers: analysis of effector mechanism

In order to elucidate the role of biological response modifiers (BRMs) in anti-tumor immunotherapy, we examined their effect on the induction of anti-tumor immunity in low-responder mice which hardly exhibit anti-tumor resistance against syngeneic Rous sarcoma virus (RSV)-induced tumors, such as B10 or B10.BR mice. The anti-tumor immunity induction in the low-responder mice was 0% on immunization with mitomycin C-treated syngeneic tumor cells alone. However, if BRMs were used as an adjuvant, BCG cell wall skeleton, OK-432 or lentinan augmented the induction of anti-tumor immunity to 50%, 33% and 33%, respectively. In the low-responder mice treated with BRMs, the anti-tumor immune cells had antigen-specificity at the induction phase of in vitro restimulation but not at the effector phase of target cell lysis by the stimulated cells. When T cells were depleted from immune spleen cells just before in vitro stimulation, cytotoxicity was not induced. Furthermore, cytotoxicity was not induced if accessory cells were removed from immune spleen cells at the induction phase. However, cytotoxicity at the effector phase was not mediated by T-lymphocytes, but by non-T cells. These results suggested that the induced cytotoxicity in low-responder mice was associated with the delayed-typed hypersensitivity-like effector mechanism.     

A prospective randomized trial of maintenance versus nonmaintenance intravesical bacillus Calmette-Guérin therapy of superficial bladder cancer

Between August 1981 and July 1984, 93 patients with polychronotopic superficial papillary carcinoma (Ta and/or T1), flat carcinoma in situ (Tis), or concomitant superficial papillary and in situ bladder carcinoma were entered into a prospective randomized trial of maintenance v nonmaintenance intravesical bacillus Calmette-Guérin (BCG) therapy. Forty-six patients who received BCG weekly for 6 weeks were compared with 47 patients receiving the six-weekly doses of BCG plus monthly BCG for 2 years. Both groups were evaluated every 3 months by cytology, cystoscopy, and biopsy. A significant reduction in the number of recurrent tumors per patient-month was demonstrated for both groups (P less than .0001); however, the difference in reduction of tumors between the two groups was not significant. Additionally, patients receiving maintenance and nonmaintenance therapy had similar tumor recurrence and progression rates. These results indicate that monthly maintenance BCG does not prevent, delay, or reduce tumor recurrence or progression observed with the 6-week regimen. Maintenance BCG was associated with increased local toxicity, primarily dysuria, frequency, and urgency. Dosage reduction was required in 22 of 47 patients (46.8%). When the data were subjected to multivariate analysis, the presence or absence of tumor following induction BCG and PPD skin test results were found to be significant variables. Controlling for either the presence or absence of tumor following induction BCG, tumor recurrence and progression rates were not significantly different for the two treatment groups. However, the absence of tumor after induction BCG was associated with a longer disease-free duration (P = .00001) and time to progression (P = .095). Patients with a reactive tuberculin skin test before and after induction BCG had significantly less tumor recurrences than patients with different PPD skin tests results (P = .02). Tumor progression was not related to tuberculin skin testing.     

Prevention of murine sarcoma virus oncogenesis in offspring of immunized female mice

BALB/c mice born to and nursed by females immunized against MSV-M showed a reduced tumour incidence and a high tumour regression rate following MSV-M injection at 7-14 days of age. Females immunized long before mating could also confer protection to their offspring whereas females immunized after parturition could not. A reduced number of tumours was observed in 3 out of 14 MSV-M injected litters whose mothers had been previously exposed to the virus while nursing infected offspring. Sera from suckling mice born to and nursed by immunized mothers contained MSV-M neutralizing antibody as shown by an in vitro focus reduction assay. Cell-free extracts from mice which developed leukaemia after MSV-M inoculation were tested for oncogenic activity in 1-week old mice. Out of 6 extracts, 4 induced typical MSV-M tumours and 2 caused leukaemias.     

Studies on murine sarcoma virus: II. Detection of group-specific antigens by immunofluorescence

The group-specific (GS) antigen of murine tumor viruses was demonstrated by immunofluorescence in mouse cells recently infected by mouse sarcoma virus, strain Moloney (MSV-M), with the serum of rats carrying long-transplanted MSV-M tumors. GS antigen was detected 15 h post-infection and was also present in various mouse and rat cell lines chronically infected with murine tumor viruses. The antigen was strictly localized in the cytoplasm of infected cells and was also found in mouse and rat cells chronically infected by members of the two major subgroups of murine tumor viruses. Further, the sera employed were shown to contain exclusively GS antibodies and the tumors used for immunization were found by several techniques to be free of virus envelope (V) antigens after a given number of passages in vivo. V antigens were visible only at the cell membrane and the time course of appearance of both GS and V antigens in recently infected cells was parallel. In contrast, GS antigen was not observed in two hamster tumor lines transformed by MSV-M.     

Experimental study on local immunochemotherapy

The purposes of this work were twofold: firstly to determine whether intratumor chemoimmunotherapy was more effective than either treatment alone or systemic therapy and; secondly to study how the intratumor therapy affected on the development of the tumor-specific immunity. Inbred male C3H/He mice and mouse ascited hepatoma 134 (MH 134) of C3H origin were used as host-tumor system. Mitomycin C was used as the chemotherapeutic agent and BCG as the immunopotentiating agent. Intratumor treatment of MMC + BCG led to complete cure in 85 percent of the mice. The lymph node metastases were markedly inhibited in the group treated with MMC + BCG compared to the groups treated with MMC alone or BCG alone. The growth of rechallenged tumor was investigated; 79% of mice treated with MMC + BCG were immune to rechallenge, whereas 57% of mice treated with BCG alone. The number of PFC and DTH against SRBC of the mice treated with MMC intraperitoneally significantly decreased compared to that treated with MMC intratumorally.     

Ratio of IgG: IgM antibodies to sialyl LewisX and GM3 correlates with tumor growth after immunization with melanoma-cell vaccine with different adjuvants in mice

Human melanoma cells (from biopsies and culture) express sialyl-Lewis^x and sialyl Lewis^a, the ligands for ECAM. These ligands may facilitate tumor progression and metastasis in human cancers. To test whether the antibodies to these ligands inhibit tumor progression, IgG and IgM responses to sLe^x and sLe^a were induced in C57BL/6j mice (n = 76) by immunization with human melanoma cells, with or without adjuvants (BCG, MPL, KLH). Control mice were treated with saline or BCG. Tumor growth and antigen expression were monitored after challenge with B16 mouse melanoma cells expressing sLe^x, sLe^a and the ganglioside GM₃. Tumor growth was reduced in mice immunized with BCG alone or cells with BCG or MPL, while tumors in mice receiving cells without adjuvants grew larger than in the control. Augmentation of IgM titers to sLe^x and GM₃ after immunization with BCG, or with cells with BCG or MPL correlated with retarded tumor growth, while increased IgG titers to sLe^x significantly correlated with aggressive tumor growth in mice immunized with cells without adjuvants. SLe^x, sLe^a and GM₃ were expressed in tumors from mice treated with saline or BCG. SLe^x expression, in particular, was lost in tumors growing in mice immunized with cells with or without adjuvants. Anti-sLe^x antibodies may promote or prevent tumor growth by antigenic modulation or by cytotoxic killing of tumor cells. Since early anti-sLe^x IgM correlated with tumor regression, in contrast to anti-sLe^x IgG, it may serve as a potential early endpoint for the effectiveness of melanoma vaccines expressing the antigens. Int. J. Cancer 75:117–124, 1998.© 1998 Wiley-Liss, Inc.     

Effect of isonicotinic acid hydrazide on the intratumor injection of BCG.

Rats with established subcutaneous tumors were treated by repeated intratumor injections of BCG over a 5-week period. Isonicotinic acid hydrazide (INH) was given to some rats to determine if this drug decreased the effect of BCG on local tumor growth. INH alone had no effect on either survival or tumor volume. Rats treated with either BCG or BCG and INH had prolonged survivals and smaller tumor volumes than did controls rats. In these experiments, INH did not decrease the effect of BCG on local tumor growth. However, the intratumor injection of BCG in rats receiving INH systematically was associated with better survival and smaller tumor volumes compared to the intratumor injection of BCG alone.     

Antitumor activity of BCG--inhibition and enhancement of tumor growth by anti-BCG rabbit serum

Administration of heat-killed BCG into BALB/c mice with colon 26 tumors which have common antigens to those of BCG has been shown to enhance tumor growth. The present experiments were undertaken to clarify this phenomenon. It was found that anti-BCG rabbit serum played a role in augmenting tumor growth when injected intraperitoneally into tumor-bearing mice. Interestingly, bi-phasic activity of anti-BCG rabbit serum (inhibition and enhancement of tumor growth) was observed, which was dependent on the concentration of BCG antibodies present. By contrast, immune-complex prepared with BCG antigen-antibody was not considered to be an agent for directly enhancing tumor growth. It was therefore concluded that anti-BCG rabbit serum was a specific agent for augmenting tumor growth.     

Inhibition and promotion of tumor growth by BCG: evidence for stimulation of humoral enhancing factors by BCG

Effects of pretreatment with BCG, strain Japan, on tumor growth were studied using a transplatable methylcholanthrene (MCA)-induced fibrosarcoma in C3H/He mice. Injection of BCG7 weeks before tumor inoculation at a site distant from the tumor caused a slight inhibition of tumor growth. A low dose of tumor cells did not grow at the BCG-primed site when BCG was injected 7 and 11 weeks before the tumor. When a high dose was inoculated into the BCG-primed site, inhibition of the primary tumor occurred in mice which had received BCG 7 weeks previously, but the number of distant metastases in the popliteal lymph node and the lungs was increased in mice pretreated with BCG at any time. Furthermore, post treatment with BCG at a site distant from the tumor caused promotion of tumor growth. Enhanced antibody formation and suppression of delayed type hypersensitivity (DTH) occurred in tumor-bearing mice. BCG treatment of such mice caused a vigorously enhanced antibody formation and a marked suppression of DTH. The sera from tumor-bearing mice enhanced tumor growth. Tumor growth was suppressed in splenectomized mice. These findings suggested that antibodies against tumor-specific antigens enhanced tumor growth in this system and that BCG treatment of tumor-bearing mice stimulated formation of antibodies probably acting as blocking factors.     

Single versus double preoperative intralesional BCG in 13762 mammary adenocarcinoma

Ninety Fischer 344 female rats were injected subcutaneously with a syngeneic 13762 mammary adenocarcinoma. In order to study if repeated doses of preoperative intralesional (IL) BCG were more effective than single doses of IL BCG, and whether the amount of tumor burden influenced the effectiveness of single and/or double doses of BCG, 40 rats had single tumor implants and 50 had double tumor implants. If tumor was allowed to grow for 22 days before resection, all animals eventually died from spontaneous disseminated metastasis (survival for rats with single tumor = 72.8 +/- 12.1 days; for those with double tumors = 63.3 +/- 14.0 days). For rats with single tumors, one dose of IL BCG given 11 days before resection of tumor results in 25% longterm cures. But repeated doses of IL BCG gave much better survival results (43% cures). For rats with two subcutaneous tumors, single doses of preoperative BCG was not effective. Double injections of BCG significantly improved survival results and achieved occasional long-term cures (25%) if the combined tumor volumes were relatively small.     

Suppressor T cells in BCG-treated mice interfere with an in vivo specific antitumoral immune response

The interference by BCG in the induction and expression of a specific antitumoral immune reaction was studied in B6 mice, using the in vivo Winn assay and also active immunization. T cells immunized against MCA-induced fibrosarcoma (MC B6-1) transferred together with the tumour cells protected the syngeneic host against tumour take. Pretreatment of normal B6 mice with moderate or high doses of BCG prevented the development of a protective immune response after immunization. Moreover, a single dose of 1 mg, or 2 doses of 0.01 mg BCG, completely eliminated an established antitumour immunity. Suppressor cells are involved in the BCG-induced inhibitory effect; they interfered (1) with the expression of the antitumour response, since their addition to immune T cells in the Winn test resulted in decreased protection and (2) with the induction of the antitumour response, since injection of spleen cells from BCG-treated mice (BCG SpC) into normal mice before immunization inhibited the development of immunity. Treatment of BCG SpC with anti Thy 1.2 and anti Lyt 1.2 antibodies plus complement before injection into normal mice significantly decreased the suppressive activity, showing that the suppressor cells induced by BCG are T cells expressing the Lyt 1+ phenotype. The partial increase in protection obtained after IL-2 administration to BCG-treated mice suggests that the suppressive action of BCG SpC on the IL-2 producing capacity of helper T cells is only one of a number of possible mechanisms of T-cell-mediated suppression.     

Histological and combined chemoimmunostimulation therapy studies against a murine leukemia.

A Graffi murine leukemia was utilized as a model system to investigate the effect of chemoimmunostimulation therapy. Subcutaneous inoculation of approximately 1.0 times 10(6) tumor cells resulted in a rapidly growing tumor at the site of inoculation and subsequent development of splenomegaly and lymphoadenopathy. All animals succumbed to the leukemia within 24 to 30 days. Treatment of diseased animals with two courses of cytoxan over a 2-week period resulted in a remission period of approximately 16 to 18 days before relapse and eventual death of approximately 70% of the drug-treated animals. A significant number of long-term survivors (50 to 83%) was obtained in groups of animals that received combined drug plus BCG or C. parvum therapy. In contrast, the administration of MER (a methanol-extracted residue of BCG) to animals in a drug-induced remission period was no more effective than drug alone. The protective effect afforded by BCG and C. parvum was dependent on the time interval between drug therapy and the administration of the immunostimulators. Treatment of leukemic animals with BCG, C. parvum, or MER alone proved ineffective as all mice died at approximately the same time as untreated control animals. No leukemic cells were observed in any of the histologically examined tissues taken from long-term survivors. The implication of these results for cancer therapy is discussed.     

Effect of pretreatment with immune serum on murine sarcoma virus (Moloney) tumour induction and growth.

Regressor serum from MSV-M-infected mice markedly reduced MSV-M oncogenesis when administered i.p. (0-1 ml/mouse) as much as 30 days before i.m. MSV-M infection in adult BALB/c mice. The regressor serum activity appeared to be directly dependent on the amount of IgG, as shown by: (1) inactivity of sera which have low virus-neutralizing antibody content; (2) high effectiveness only of the IgG serum fraction; (3) inactivity of regressor serum incubated with anti-mouse gamma-globulin serum. The regressor serum activity was specific and could not be ascribed to interferon or interferon-inducing factors, antigen-antibody complexes or free antigen. The activity was not suppressed by sublethal irradiation (380 rad) of recipient mice. These results suggest that the activity of regressor serum administered before MSV-M infection is mediated through sensitization of host cells which are not radiosensitive.     

Treatment of bladder carcinomas using recombinant BCG DNA vaccines and electroporative gene immunotherapy

Intravesical immunotherapy with live Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the treatment of choice for superficial bladder cancers. Nevertheless, a significant proportion of patients do not respond to this therapy, and adverse effects are common. Here, we report the cloning of recombinant mycobacterial DNA vaccines and demonstrate the ability of multicomponent and multisubunit DNA vaccines to enhance Th1-polarized cytokine-mediated responses as well as effector cell responses. Splenocytes from immunized groups of mice were restimulated in vitro and examined for cytotoxicity against murine bladder tumur (MBT-2) cells. We used four combined recombinant BCG DNA vaccines (poly-rBCG) for electroporative gene immunotherapy (EPGIT) in vivo, and found that tumor growth was significantly inhibited and mouse survival was prolonged. Increased immune cell infiltration and induction of apoptosis were noted after treatment with poly-rBCG alone, with the murine interleukin-12 (mIL-12) vaccine alone, and-most significantly-with the poly-rBCG+mIL-12 vaccine combination. Electroporation of poly-rBCG+mIL-12 resulted in complete tumor eradication in seven of eight mice (P<.01) within 28 days. Thus, EPGIT using multicomponent multisubunit BCG is highly effective in the treatment of bladder cancer. This approach presents new possibilities for the treatment of bladder cancer using recombinant BCG DNA vaccines.     

Enhanced host resistance to transplantable murine lymphosarcoma in Swiss mice by combined immunostimulation with BCG and polyinosinic-polycytidylic acid

Combined immunostimulation with BCG and double-stranded polyinosinic-polycytidylic acid (poly I . poly C) was more effective than single-modality immunostimulation in suppressing tumor growth in inbred Swiss mice. BCG sensitization followed by administration of poly I . poly C on the day of tumor cell injection significantly prolonged the survival period against parental lymphosarcoma (LS) and its ascites variant (LS-A). BCG and poly I . poly C given together on the day of tumor cell injection suppressed only LS-A and not LS. BCG or poly I . poly C given alone did not result in tumor cures. Silica injection given 2 days before poly I . poly C injection completely abrogated the antitumor effect of sequential treatment with BCG and poly I . poly C. Silica treatment given on and beyond the day of poly I . poly C injection did not abrogate the antitumor effect. This observation indicated that intact macrophage effector function was essential at the time of tumor cell inoculation to obtain an effective antitumor action.     

Induction of tumor resistance with BCG-associated tumor antigen.

Soluble material enriched in tumor-associated antigen was prepared by affinity chromatography from a KCl extract of the chemically-induced D-23 rat hepatoma. Microgram quantities of the above material bound spontaneously to living BCG when the two were incubated briefly in vitro. When injected into normal syngeneic rats, the BCG-associated tumor antigen induced a measure of resistance against challenge with D-23 tumor cells. Peritoneal exudate cells (PEC) obtained from such actively immunized subjects were able to suppress the growth of D-23 tumor cells at a test site in muscle. In contrast, immunization with either BCG alone, tumor protein alone, or tumor protein admixed with BCG in circumstances designed to impede association of the protein, failed to provoke the formation of tumor suppressor PEC. The results encourage of the belief that binding of tumor antigen to BCG favors the induction of a cell-mediated tumor suppressive response.