Stimulation of tumor growth in mice by high doses of BCG.

The growth of Krebs-2 carcinoma in BCG-prevaccinated virgin female C57BL/6, CC57BR/M, and C3Hf mice was studied in relation to the number of living mycobacteria in the organism. When the number of mycobacteria was high, tumor growth was stimulated. After the bacteria were eliminated, tumor growth was inhibited. The effect of BCG was based, on the one hand, on the diversion of effector cells, presumably macrophages, responsible for tumor defense and, on the other hand, on the activation of the pool of these cells. The conclusions were reached that high doses of BCG may be dangerous in human cancer immunotherapy and that patients predisposed to neoplastic disease should be revaccinated with BCG.     

High-dose inhibition and low-dose enhancement of murine sarcoma growth exhibited by BCG vaccine

An in vivo assay for BCG anti-cancer efficacy was developed, utilizing subcutaneous injection into CFW Swiss-Webster mice of cultured S180 sarcoma cells and mixed with freeze-dried TiceTM BCG vaccine. A BCG dose of 0.156-1.56 mg dry weight significantly inhibited tumor formation, but a BCG dose of 0.156-1.56 micrograms significantly enhanced tumor growth, evidenced by increased tumor incidence, volume and initial growth rate. These antagonistic activities may contribute to the high variability of BCG anti-cancer efficacy seen in animals and humans and indicate the need to exercise caution when employing even low doses of BCG for cancer immunotherapy.     

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 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) BCG injected directly into the tumor stimulated tumor growth in three of three experiments; the stimulation was at least partially attributable 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, BCG and tumor cell vaccination did not alter the development of nodal metastases. The B16 melanoma was not a useful model system for BCG immunotherapy, because the tumor inhibition was feeble, inconsistent, and not associated with augmented tumor immunity.     

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.     

Inhibition of murine sarcoma virus oncogenesis with living BCG

Intramuscular inoculation of murine sarcoma virus of Moloney (MSV-M) leads to the development of tumors at the site of injection. The effect of BCG treatment on tumor induction by MSV-M in BALB/c mice was studied. Mice injected with BCG 28 days before induction of tumors with MSV-M alone had shorter latent periods for tumor development and longer survivals than mice not previously exposed to BCG. When BCG was mixed with the MSV-M used for tumor induction there was a significant decrease in mortality, tumor incidence, and tumor size compared to those seen in mice inoculated with MSV-M alone. Previous exposure of the mice to BCG before attempted induction of tumors with the mixture of BCG and MSV-M completely inhibited tumor development. In addition, these mice were effectively immunized against tumor development when subsequently challenged with a lethal dose of MSV-M. The mechanisms of BCG's impressive activity are discussed.     

Effect of lymphokine-containing sera on adenocarcinoma BW10232 and melanoma B16 in C57BL/6 mice

Lymphokine-containing sera (LKS) inhibited the growth of adenocarcinoma BW10232 and melanoma B16 in C57BL/L mice. Resistance to tumor growth was conferred by daily injections into the challenge site. The injection of LKS did not provide long-term resistance, inasmuch as termination of treatment permitted resumption of tumor growth at the primary tumor site. Whether LKS exerted a direct or indirect effect on the tumor cells to retard their was not certain.     

不同微生物佐剂S-180瘤苗的免疫效应和抑瘤作用

 目的　比较肿瘤主动特异性免疫治疗中具有佐剂作用的白色念珠菌、李斯特菌及卡介苗的免疫调节效应和抑瘤作用。方法　选择白色念珠菌、李斯特菌、卡介苗各自联合小鼠S-180肿瘤疫苗,皮下免疫接种荷瘤小鼠,检测小鼠脾细胞IL-2、IFN-产生水平,腹腔MTNF-α产生水平,观察荷瘤小鼠瘤体重量和存活期。结果　三种微生物佐剂均能协同瘤苗增强免疫小鼠IL-2、IFN-和TNF-α的产生能力(P<0.01,0.05),有效抑制足垫部移植瘤的生长,抑制率在62.8%～75.8%之间,显著高于单独注射瘤苗组(P<0.01),延长腹腔植瘤小鼠平均存活期7～14天,白色念珠菌、李斯特菌协同提高腹腔植瘤小鼠存活率20%。结论　白色念珠菌和李斯特菌与卡介苗具有同样的佐剂效应,能够协同瘤苗有效抑制肿瘤生长。     

Effectiveness of cancer vaccine therapy using cells transduced with the interleukin-12 gene combined with systemic interleukin-18 administration

We introduced the interleukin-12 (IL-12) gene into mouse renal cell carcinoma (RenCa) cells to develop a tumor vaccine and to examine mechanisms of tumor rejection. IL-12-secreting RenCa (RenCa/IL-12) cells were completely rejected when implanted into syngeneic BALB/c but not athymic nude mice, suggesting that T cells were involved in this antitumor effect. Depletion of natural killer (NK) cells in nude mice did not affect the tumor growth of RenCa/IL-12. The simultaneous injection of mitomycin C-treated RenCa/IL-12 inhibited the tumor growth of parental RenCa injected at a distant site, whereas injection of mitomycin C-treated parental RenCa did not. The antitumor effect of RenCa/IL-12 as a cancer vaccine was induced by CD8+ T cells and NK cells and was inhibited by CD4+ T cells. Although the systemic administration of recombinant IL-18 (rIL-18) alone did not inhibit the tumor growth, it did enhance the cancer vaccine effect of RenCa/IL-12. The combination therapy of RenCa/IL-12 and the systemic administration of rIL-18 retarded even the growth of established tumors. The effector cells of this combination therapy consist not only of CD8+ T cells and NK cells but also of CD4+ T cells. This synergistic cancer vaccine effect of in situ secretion of IL-12 and the systemic administration of rIL-18 may be attributed to a functional change of CD4+ T cells.     

In vivo antitumor activity of various forms of delayed-type hypersensitivity in mice.

Relationships among various forms of delayed-type hypersensitivity (DTH) and nonspecific resistance to Lewis lung tumor were studied in syngeneic and semisyngeneic mice. Only the tuberculin type of DTH obviated a virulent inoculum of 10(6) tumor cells. The Jones-Mote type of DTH, even modified by cyclophosphamide pretreatment, produced a significant local inflammatory reaction which was unable to destroy tumor cells. The antitumor effect of the tuberculin type was observed in BCG-or in Corynbacterium parvum-immune mice and also in sheep red blood cell-immunized mice, but only after the modulating effect of BCG. The antitumor activity of the DTH reaction was anatomically restricted and time related, and it required local persistence of specific antigen. A minimal number of bacteria, 1 times 10(6) living or heat-killed BCG organisms, were equally able to eradicate 10(5) tumor cells in BCG-immune mice. Biphasic effects on tumor growth were observed when systemic specific inflammatory reactions were elicited in BCG-immune mice. However, tumor-specific immunity was never observed, inasmuch as BCG-immune mice surviving injection of a mixture of BCG and tumor cells did not resist a second tumor cell challenge.     

S180瘤苗和BCG抗癌作用及诱发肿瘤细胞凋亡

 用S180瘤苗(可溶性抗原)及BCG皮内注射治疗荷瘤小鼠, 显示肿瘤生长受抑, 脾脏CTL活性增强, 与对照组或单一治疗组(瘤苗组、BCG组)相比P<0.01, 而荷瘤前6天治疗更显著。 治疗组的瘤组织作超微形态观察, 有核质凝聚胞浆固缩等细胞凋亡的改变，作流式细胞术DNA直方图显示在G1期前具凋亡特征的AP峰较对照明显升高P<0.01。 实验显示, 瘤苗BCG可能通过CTL启动细胞凋亡途径抑制肿瘤生长。     

Antitumor effect of intralesional injection with formalin-fixed Toxoplasma gondii organisms on Lewis lung carcinoma in Toxoplasma-infected mice

The antitumor effect of formalin-fixed Toxoplasma organisms (f-Tp) as an immunostimulant was examined in Toxoplasma-infected female C57BL/6 mice using a syngeneic Lewis lung carcinoma (3LL). Toxoplasma-infected mice, intradermally inoculated with the tumor cells mixed with 10(5), 10(6) or 10(7) f-Tp, developed a marked antitumor effect, inhibition of tumor growth and prolongation of life-span, in direct relation to the strength of the delayed-type hypersensitivity (DTH) reaction induced by f-Tp. The antitumor effect could also be observed even if an intralesional injection with f-Tp was performed 1, 3 or 5 days after the tumor inoculation. In a control, the injection with 2.5 X 10(6) live Mycobacterium bovis (BCG) in BCG-sensitized mice induced a significant antitumor effect, only when the BCG was injected in the mixture of tumor cells. These results demonstrate that the injection with f-Tp can induce a potent antitumor activity in mice with Toxoplasma infection.     

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.     

Relative tumor inhibitory and stimulatory activities of BCG vaccine preparations, lots and substrains in a quantitative mouse sarcoma bioassay.

A quantitative in vivo assay for BCG anticancer efficacy was developed to maximize detection of tumor-antagonistic mechanisms. Cultured S180 sarcoma cells admixed with various quantities of Mycobacterium bovis-BCG organisms were injected subcutaneously into CFW Swiss-Webster mice and response was measured as tumor incidence 14 days after injection. Assay of various BCG substrains, lots and killed preparations revealed characteristic patterns of BCG dose-dependent tumor inhibition and enhancement that suggest the existence in the vaccine of multiple active components, the relative concentrations of which vary among cultures. Inhibition of tumor growth by high doses of BCG (greater than 10 micrograms dry weight) was found to be a function of total cell mass and not of the bacterial viability, suggesting that this activity is dependent upon one or more heat-stable components.     

Responses of tumors induced in inbred guinea pig strain JY=1 and strain Hartley/F to BCG.

A transplantable fibrosarcoma induced in inbred JY-1 guinea pig strain by 3-methylcholanthrene (MCA) and designated J4, an allotransplantable subline of J4 (JH4) which was obtained by the transplantation of J4 into the inbred Hartley/F guinea pig strain and maintained by passages in this strain, and a syngeneic liposarcoma H10 induced in a Hartley/F guinea pig by MCA were tested for their immunotherapeutic response with BCG. The growth of J4 and H10 tumors was suppressed in most of the animals when tumor cells were mixed with BCG before being injected sc into BCG-immune or BCG-nonimmune recipients. The growth of the JH4 tumor was suppressed at the sites of injection with a mixture of tumor cells and BCG in BCG-immune recipients but not in nonimmune animals. All guinea pigs surviving the injection of a tumor cell-BCG mixture resisted a second tumor cell challenge. When subcutaneous sarcomas grew to about 8-15 mm in diameter, BCG was injected into the tumors. The growth of JH4 tumor was not influenced by the injection in either BCG-immune or BCG-nonimmune animals, while the regression of the established J4 transplants was produced in 2 of 3 nonimmune recipients. The growth of the H10 tumor was not inhibited with an intratumor injection into nonimmune guinea pigs, while the H10 tumor regressed in BCG-immune animals for 4-5 weeks after intratumor injection and thereafter grew progressively. Skin reactions in animals that received repeated intradermal injections of the tumor cells and BCG were tested with 10(6) viable tumor cells as eliciting antigens. Typical delayed-type hypersensitivity reactions that were specific to the homologous antigens were observed. The possible reasons for the different responses to BCG among the guinea pig tumors, including line-10 hepatocarcinoma in strain-2 guinea pigs, were discussed.     

Effect of Corynebacterium parvum on tumor growth in normal and athymic (nude) mice.

The effect of systemic or local injection of Corynebacterium parvum at the tumor site on the growth of various murine tumors was studied in intact and congenitally athymic BALB/c mice. Systemic injection of C. parvum usually had a marked antitumor effect in both types of mouse. Two lymphomas, which regressed spontaneously in untreated intact mice but not in athymic mice, grew progressively in intact mice given systemic C. parvum, though their growth was inhibited in similarly treated athymic mice. Local injection into the site of the tumor markedly inhibited tumor growth in intact mice but was without effect in athymic mice. C. parvum was believed to exert its antitumor effects by two different mechanisms, only one of which was T-cell dependent. The mechanism not dependent on T-cells was particularly activated by systemic C. parvum injection.     

Failure of immunotherapy with neuraminidase-treated tumor cell vaccine in mice bearing established 3-methylcholanthrene-induced sarcomas

C3H/HeJ mice bearing MC-80 fibrosarcomas were given immunotherapy consisting of multiple injections of a Vibrio cholerae neuraminidase (VCN)-treated tumor cell vaccine at a site remote from the established tumor. In five separate experiments we were unable to show either partial or complete tumor regression or prolongation of survival for vaccine-treated mice compared to appropriate controls. Further, the use of BCG in addition to VCN-treated tumor cells failed to show any therapeutic efficacy. We could not confirm the successful immunotherapy results reported by others despite multiple efforts of reproduce the immunotherapy model as carefully and precisely as possible.     

Mouse Lewis lung carcinoma and hepatoma ascites treatment by combination of liposome chemotherapy and non-specific immunotherapy

Liposomes have been used as biological carriers of anti-tumor drugs, and their potential use has been tested in mouse Lewis lung carcinoma and hepatoma ascites tumor models. Ara-C3 given by the intraperitoneal i.p. route either at 35 mg/kg in a single dose or at 2.5 mg/kg/dose with 5 doses/day for 3 days had no effect on the average survival time of i.v. implanted LLC. However, the same single dose of Ara-C encapsulated in positively charged MLV significantly improved the average survival time of LLC-bearing mice. MTX was chosen as a test drug for the treatment of hepatoma ascites. Non-encapsulated MTX given at either 3 or 30 mg/kg by the i.p. route had little effect on the average survival of i.p.-implanted hepatoma ascites. However, MTX encapsulated in SUV at a 3 mg/kg dose by the i.p. route significantly improved the average survival time of tumor-bearing mice. A combination of chemotherapy and non-specific immunotherapy has also been tested with these 2 tumor models. Two non-specific microbial immune stimulators, Bacillus Calmette Guérin (BCG) and Corynebacterium parvum (CP) were tested by both the i.v. and i.p. routes. A combination of BCG therapy with non-encapsulated anti-tumor drugs was not effective for either of the tumor models. A combination of BCG therapy with liposome therapy appeared to improve the average survival time of LLC-bearing mice. In particular, BCG treatment by the i.p. route in combination with liposome therapy resulted in a 20% long-term survival rate among treated mice. However, BCG therapy by either route in combination with SUV encapsulated MTX therapy had no effect on the average survival time of hepatoma ascites-bearing mice. Immunostimulation with CP at a given dose appears to be superior to BCG therapy for both tumor models. In the treatment of LLC, injection of CP by either the i.v. or i.p. route appears to be equally effective in combination with liposome therapy. However, for the treatment of hepatoma ascites, CP was only effective by the i.p. route, in combination with liposome therapy.     

Ubiquitinated proteins enriched from tumor cells by a ubiquitin binding protein Vx3(A7) as a potent cancer vaccine

Background

Our previous studies have demonstrated that autophagosome-enriched vaccine (named DRibbles: DRiPs-containing blebs) induce a potent anti-tumor efficacy in different murine tumor models, in which DRibble-containing ubiquitinated proteins are efficient tumor-specific antigen source for the cross-presentation after being loaded onto dendritic cells. In this study, we sought to detect whether ubiquitinated proteins enriched from tumor cells could be used directly as a novel cancer vaccine.

Methods

The ubiquitin binding protein Vx3(A7) was used to isolate ubiquitinated proteins from EL4 and B16-F10 tumor cells after blocking their proteasomal degradation pathway. C57BL/6 mice were vaccinated with different doses of Ub-enriched proteins via inguinal lymph nodes or subcutaneous injection and with DRibbles, Ub-depleted proteins and whole cell lysate as comparison groups, respectively. The lymphocytes from the vaccinated mice were re-stimulated with inactivated tumor cells and the levels of IFN-γ in the supernatant were detected by ELISA. Anti-tumor efficacy of Ub-enriched proteins vaccine was evaluated by monitoring tumor growth in established tumor mice models. Graphpad Prism 5.0 was used for all statistical analysis.

Results

We found that after stimulation with inactivated tumor cells, the lymphocytes from the Ub-enriched proteins-vaccinated mice secreted high level of IFN-γ in dose dependent manner, in which the priming vaccination via inguinal lymph nodes injection induced higher IFN-γ level than that via subcutaneous injection. Moreover, the level of secreted IFN-γ in the Ub-enriched proteins group was markedly higher than that in the whole cell lysate and Ub-depleted proteins. Interestingly, the lymphocytes from mice vaccinated with Ub-enriched proteins, but not Ub-depleted proteins and whole cell lysates, isolated from EL4 or B16-F10 tumor cells also produced an obvious level of IFN-γ when stimulated alternately with inactivated B16-F10 or EL4 tumor cells. Furthermore, Ub-enriched proteins vaccine showed a significant inhibitory effect on in vivo growth of homologous tumor, as well as allogeneic tumor, compared with Ub-depleted proteins and tumor cell lysate. Tumor growth was regressed after three times of vaccination with Ub-enriched proteins in contrast to other groups.

Conclusion

These results indicated that Ub-enriched proteins isolated from tumor cells may have a potential as a potent vaccine for immunotherapy against cancer.     

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.     

不同时期应用细胞瘤苗对红白血病荷瘤小鼠抗瘤作用的研究

目的：探讨不同时间应用细菌瘤苗对红细胞病荷瘤小鼠的抗瘤作用。方法：在不同时间内应用瘤苗免疫治疗红白血病荷瘤小鼠，观察小鼠生存期，皮下肿瘤大小及肿瘤，注射部位病理变化。结果：3天，7天瘤苗治疗的小鼠生存期延长，皮下肿瘤生长缓慢，病理可见瘤组织坏死及大量以单个核细胞为主的炎细胞浸润，与PBS组及10天瘤苗组相比，有显著性差异。结论：3天，7天瘤苗比10天瘤苗组抗肿瘤作用强。