Chemopreventive effect of Lactobacillus rhamnosus on growth of a subcutaneously implanted bladder cancer cell line in the mouse.

Lactic acid bacteria are known to have beneficial effects on the host, such as preventing carcinogenesis. The present study was designed to evaluate the chemopreventive effects of Lactobacillus rhamnosus strain GG (LGG) in suppressing bladder cancer formation in a murine subcutaneous model of bladder cancer involving the inoculation of MB49 cells in C57B / L6 mice. After tumor implantation, one group of mice (n = 8) was fed LGG immediately. The remaining mice that had tumors between 0.03 - 0.1 cm(3) were divided into two groups: those fed LGG after 7 days (n = 7) and those fed saline (n = 7). A second group of mice without any inoculation of MB49 cells was fed either LGG (n = 10) or saline (n = 10) and served as non-tumor-bearing controls. LGG was administered orally at 1.6 x 10(8) colony-forming units daily. Mice fed LGG immediately after tumor cell implantation formed smaller tumors and some did not develop tumors (2 out of 8 mice), when the tumor burden was small. The level of spleen CD3, CD4 and CD8a T lymphocytes, as well as natural killer cells in mice fed immediately with LGG was also higher than that in control tumor-bearing mice. There was an increase in lymphocytes and granulocytes in tumor sections, especially from the immediately fed group as compared to the controls. Our results suggest that oral consumption of LGG may prevent tumor growth via modulation of the immune system. The potential of LGG as an adjunct therapy in the treatment of bladder cancer could be further explored.     

Lactobacillus rhamnosus GG induces tumor regression in mice bearing orthotopic bladder tumors

(Cancer Sci 2010; 101: 751–758) The present gold standard for bladder cancer is Mycobacterium bovis, Bacillus Calmette Guerin (BCG) immunotherapy. But it has a non‐responder rate of 30–50% and side effects are common. Lactobacillus casei strain Shirota has been reported to reduce the incidence of recurrence in bladder cancer patients and to cure tumor‐bearing mice. Our aim was to determine if Lactobacillus rhamnosus GG (LGG) could be as efficacious as BCG in a murine model of bladder cancer. MB49 bladder cancer cells secreting human prostate‐specific antigen were implanted orthotopically in female C57BL/6 mice and urinary prostate‐specific antigen levels were used as a marker of tumor growth. Mice were treated with either live or lyophilized LGG given via intravesical instillation, or both oral and intravesical LGG given once a week for a period of 6 weeks starting at day 4 after tumor implantation. A comparison of LGG and BCG immunotherapy was also carried out. LGG therapy (live or lyophilized) significantly (P = 0.006) increased the number of cured mice. Cytokine arrays and immune cell recruitment analysis revealed differences between untreated, treated, cured, and tumor‐bearing mice. LGG therapy restored XCL1 levels to those in healthy bladders. LGG also recruited large numbers of neutrophils and macrophages to the tumor site. Intravesical LGG and BCG immunotherapy had cure rates of 89 and 77%, respectively, compared with 20% in untreated mice. LGG has the potential to replace BCG immunotherapy for the treatment of bladder cancer.     

SA-IL-2锚定修饰治疗表浅膀胱癌的实验研究

Background and Objective:Intravesical administration of Bacillus Calmette-Guèrin(BCG) after transurethral resection is by far the most effective local therapy for superficial bladder cancer,the fifth most common cancer in the world.However,approximately one-third of patients fail to respond and most patients eventually relapse.In addition,there are pronounced side effects of BCG therapy,such as BCG sepsis and a high frequency of BCG-induced cystitis.This study established a novel immunotherapy through immob...     

Both CD133+ and CD133− subpopulations of A549 and H446 cells contain cancer-initiating cells

Tumors have been known to contain a small population of cancer stem cells that initiate tumor growth and promote tumor spreading. CD133 alone or in combination with other markers is currently being used for identification and isolation of the putative cancer stem cell population from malignant tumors. To determine whether the CD133⁺ cells constitute the stem cell populations of lung cancer cells A549 and H446, CD133⁺ and CD133⁻ subpopulations were sorted from A549 and H446 cells by magnetic cell separation and characterized for their in vitro stem cell-like properties. Interestingly, both the CD133⁺ and CD133⁻ cells displayed similar abilities of colony formation, self-renewal, proliferation, differentiation, and invasion, as well as resistance to chemotherapy drugs. Furthermore, colony formation assays showed that more than 40% of cells in both the CD133⁺ cells and CD133⁻ subpopulations could form large colonies capable of regenerating the unsorted populations and forming tumors in nude mice. These results suggest that CD133 alone cannot be used as a stem cell marker for the lung cancer cells A549 and H446, and both the CD133⁺ and CD133⁻ subpopulations contain similar numbers of cancer stem cells. ( Cancer Sci 2009; 100: 1040–1046)     

The Roles of CD8+ and CD4+ Cells in Tumor Rejection

In vivo administrations of anti-Lyt-2.2 (CDS) mAb and anti-L3T4 (CD4) mAb selectively eliminated CD8⁺ cells amd CD4⁺ cells, respectively. The relative potencies of CD8⁺ cells and CD4⁺ cells and their roles in primary tumor rejections were studied by investigating the effects of these mAbs on tumor growth. CD8⁺ cells were themselves fully capable of mediating rejection in 5 different tumor rejection systems: two radiation leukemia virus (RadLV)-induced leukemias, B6RV2 and BALBRVD, a radiation-induced leukemia BALBRL♂1, and a plasmacytoma BALBMOPC-70A in CB6F₁ mice, and a Friend virus-induced leukemia B6FBL-3 in B6 mice. On the other hand, CD4⁺ cells were capable of resisting tumor growth of B6FBL-3, but not of the other four tumors. Furthermore, for efficient rejection of CB6F₁UV+^˚l sarcoma by CB6F₁ mice, synergy of CDS⁺ and CD4⁺ cells was necessary. Blocking of UV+^˚ 1 rejection was abrogated by delayed administration of anti-L3T4 (CD4) mAb but not anti-Lyt-2.2 (CDS) mAb, indicating the involvement of CD4⁺ cells in only the initial phase of rejection.     

Monitoring the response of orthotopic bladder tumors to granulocyte macrophage colony-stimulating factor therapy using the prostate-specific antigen gene as a reporter.

PURPOSE: Although orthotopic animal models of cancer best reflect the disease in humans, a major drawback of these models is the inability to monitor tumor growth accurately. Our aims were to produce a bladder tumor cell line (MB49) that secreted human prostate-specific antigen (PSA), analyze the feasibility and accuracy of PSA as a biomarker for monitoring orthotopic bladder tumor growth, and evaluate the effectiveness of granulocyte macrophage colony-stimulating factor (GM-CSF) gene therapy using this model. EXPERIMENTAL DESIGN: PSA secretion was assessed after both s.c. and orthotopic implantation of MB49-PSA cells in C57BL/6 mice. PSA levels in mouse serum and urine samples were monitored at 2- to 3-day intervals by ELISA. Using the orthotopic model, mice with confirmed tumors were given liposome-mediated GM-CSF gene therapy twice a week for 3 weeks intravesically and PSA levels monitored. RESULTS: The MB49-PSA cells behaved similarly as the parental cell line and produced high levels of PSA both in vitro and in vivo. In the s.c. model, the level of PSA produced correlated with tumor volume (r = 0.96). In the orthotopic model, PSA could be detected in serum and urine on the fourth day after implantation. PSA levels over the treatment period indicated that tumor growth was inhibited by GM-CSF gene therapy. Up to 50% of the treated mice were cured. Cytokine array analysis revealed that GM-CSF gene therapy induced the production of other cytokines and chemokines. CONCLUSIONS: MB49 cells modified to secrete PSA are a reliable method to evaluate therapeutic modalities for bladder cancer.     

膀胱癌来源的外泌体通过促进髓系抑制性细胞的扩增介导毒性T细胞的免疫抑制

目的:探究膀胱癌细胞系MB49来源的外泌体(MB49-Exo)对髓系来源的抑制性细胞(MDSCs)的影响及后者对MB49抗原特异性细胞毒性T淋巴细胞(CTL)免疫功能的调控.方法:试剂盒分离MB49-Exo,并应用透射电子显微镜(TEM)观察外泌体形态,纳米粒径颗粒跟踪分析仪(NTA)和Western blot进行鉴定...     

New potential therapy for orthotopic bladder carcinoma by combining HVJ envelope with doxorubicin

Purpose To establish a new therapeutic method to treat bladder carcinoma, we investigated the therapeutic potential of doxorubicin hydrochloride (DXR) combined with hemagglutinating virus of Japan-envelope vector (HVJ-E) in an orthotropic mouse bladder cancer model. Methods DXR and/or HVJ-E were instilled into the bladder after implantation of MB49 cells. Antitumor effects of combination therapy were evaluated by histological analysis of the bladder on day 14 after tumor implantation. The survival rate of MB49-disseminated mice was examined for 60 days after single or double administration of DXR alone or DXR/HVJ-E. The surviving mice were re-challenged with intravesical injection of MB49 cells, and the bladder was observed after 3 weeks. Results Combined intravesical instillation of HVJ-E and DXR resulted in a significantly higher rate of tumor-free mice (11/21) compared with mice treated using DXR alone (3/19, P < 0.05). Median survival was >60 days for intravesical instillation of HVJ-E and DXR, compared with the 29 days for DXR instillation alone (P < 0.05). After combination therapy, surviving mice formed no tumors in the bladder following intravesical re-instillation of MB49. Conclusions HVJ-E increased antitumor effects in combination with chemotherapeutic agent (DXR). Antitumor immunity appeared to be enhanced using HVJ-E.     

Sequential T Cell Response Involved in Tumor Rejection of Sarcoma, Meth A, in Syngeneic Mice

We investigated the type of T cell response involved in Meth A tumor rejection in primary immune and hyperimmune syngeneic mice. It was found that a CD4⁺ T cell-mediated delayed-type hypersensitivity (DTH) response activating non-specific killer cells such as macrophages, NK and LAK cells, without a specific CD8⁺ cytotoxic T lymphocyte (CTL) response, was the major immune response leading to Meth A tumor rejection in primary immune mice. In contrast, the specific CD8⁺ CTL response was the major response leading to the tumor rejection, in addition to CD4⁺ T cell-mediated DTH response, in hyperimmune mice. Analysis of CD4⁺ T cell clones established from primary immune and hyperimmune spleen cells indicated that a CD4⁺ T cell clone (C9) of primary immune mice (although only one clone was established) was of Th1 type, and induced cytotoxicity in accessory cells by classic DTH in vitro. Eight CD4⁺ T cell clones were established from hyperimmune spleen cells. Six out of the eight clones were of the Th2 type and two were Th0-like. However, no Th1-type CD4⁺ T cell clone was established from hyperimmune spleen cells. All of these CD4⁺ T cell clones, even the Th2-type clones, were capable of inducing cytotoxicity in vitro in T cell-depleted accessory cells, as in an in vitro DTH response. We postulate on the basis of these results that the T cell response leading to Meth A tumor rejection in vivo sequentially changed from a CD4⁺ T cell-mediated classic DTH response to a CD8⁺ CTL response, in addition to a cellular response mediated probably by Th2-type cells, during the process of repeated immunization.     

Antitumor immune responses derived from transgenic expression of CD40 ligand in myeloma cells

Tumor cells engineered to express immunogenes have been used for cancer vaccines to induce the antitumor immunity and study the antitumor immune mechanisms derived from the immunogene expression. In the present study, we engineered a mouse myeloma cell line J558 with a cloned CD40 ligand (CD40L) gene. We demonstrated that (i) the engineered J558/CD40L tumor cells expressing the CD40 ligand molecule lost their tumorigenicity in syngeneic mice, and (ii) the inoculation of J558/CD40L tumor cells further lead to the protective immunity against wild-type J558 tumors. In animal studies using T-cell subset depleted mice, we further showed that the primary rejection of J558/CD40L tumors did not require T cells, but was mainly mediated by NK cells, whereas the effector phase of the protective immunity is mediated by CD8+ T cells. In addition, our data, for the first time, showed that the inoculation of engineered J558/CD40L tumor cells is able to stimulate stronger activation of dendritic cells with enhanced expression of B7-1 and ICAM-1 molecules than the wild-type J558 tumor cells Taken together, we demonstrated the antitumor effect of engineered J558/CD40L tumor cells that is mediated by the activation of the host dendritic cells in vivo. Our data indicate that the introduction of co-stimulatory CD40 ligand molecule will be useful as a new strategy of immunogene therapy against tumors.     

Selective Suppression of the Generation of Anti-tumor L3T4+ but Not of Lyt-2+ T Cell-mediated Immunity in the Tumor-hearing State

C3H/He mice hyperimmune against syngeneic MH134 hepatoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated id challenges with viable tumor cells. Winn assays performed utilizing spleen cells from these mice have revealed that both Lyt-2⁺ and L3T4⁺ T cell subsets from MH134-hyperimmune mice produced complete tumor protection. The in vivo tumor-neutralizing activity was also found in spleen cells from tumor-bearing mice at various times after id implantation of MH134 tumor cells. However, in contrast to comparable tumor-neutralization by Lyt-2⁺ and L3T4⁺ T subsets from hyperimmune mice, only the Lyt-2⁺ T cell subset from tumor-bearing mice was capable of mediating the in vivo protective immunity. L3T4⁺ T cell-mediated immunity was not detectable in the tumor-bearing state irrespective of the length of the sensitization period with a primary growing tumor, but emerged in the mice which resisted the first tumor challenge after the resection of the primary tumor. These results indicate that the emergence of L3T4⁺ T cell-mediated anti-tumor immunity is stage-dependent and the Lyt-2⁺ T cells represent the main functional subset in the tumor-bearing state, although both subsets of T cells are potentially capable of effecting anti-tumor in vivo immunity. The results are discussed in relation to the selective suppression of the L3T4⁺ but not of Lyt-2⁺ T cell function in the tumor-hearing state.     

Combinational immunotherapy for established tumors with engineered tumor vaccines and adenovirus-mediated gene transfer

There are currently extensive studies relating to cancer vaccines using tumor cells engineered to express immunogenes and cancer gene therapy using adenovirus (AdV)-mediated gene transfer. In this study, a mouse tumor cell line, VKCK, was cotransfected with genes coding for tumor necrosis factor-alpha (TNF-alpha) and costimulatory B7-1 molecule to enhance immunogenicity. The transfectant cell line VKCK-TNF-alpha/B7-1 showed reduced tumorigenicity and tumor regression. Its inoculation further induced protective immunity; both CD4+ and CD8+ T cells were involved in the induction phase, whereas only CD8+ T cells mediated the effector phase. Susceptible mice bearing VKCK tumors developed a T helper type 2-dominant response, whereas resistant mice with VKCK-TNF-alpha/B7-1 tumor regression developed a T helper type 1-dominant response to VKCK, indicating that the tumor regression was related to a shift in the cytokine profile of the host from type 2 to type 1. Vaccination of VKCK-TNF-alpha/B7-1 cells inhibited tumor formation derived from a single dose of 3 x 10(6) VKCK cells and eradicated 3-day tumors but not 10-day tumors. AdV-mediated TNF-alpha gene transfer by intratumoral injection of AdV-TNF-alpha significantly inhibited tumor growth but failed to eradicate any well-established tumors. However, combinational immunotherapy with vaccination of VKCK-TNF-alpha/B7-1 cells and AdV-mediated TNF-alpha gene transfer not only significantly inhibited tumor growth but also eradicated 10-day VKCK tumors in three of eight mice. Therefore, the present study may be useful not only in understanding the mechanisms responsible for an efficient antitumoral immunity, but also in establishing a more effective immunotherapeutic approach for cancer patients.     

Sequential Involvement of Two Distinct CD4+ Regulatory T Cells during the Course of Transplantable Tumor Growth and Protection from 3–Methylcholanthrene-induced Tumorigenesis by CD25–depletion

The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4⁺CD25⁺ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4⁺CD25⁻ T cells downregulated the tumor rejection response in the late stage. Both CD4⁺CD25⁺ and putative CD4⁺CD25-T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3–methylcholanthrene (3–MC) inoculation retarded tumor occurrence and prolonged survival.     

Negative p53/Positive p21 Immunostaining Is a Predictor of Favorable Response to Chemotherapy in Patients with Locally Advanced Bladder Cancer

The relationship between clinical response to DNA-damaging drugs and p53 and p21 status in patients with locally advanced transitional cell carcinoma (TCC) of the bladder was assessed. The response to intraarterial chemotherapy (IAC) comprising 100 mg/m² of cisplatin (CDDP) and 40 mg/m² of pirarubicin (THP) and the prognosis were assessed in 23 patients (the mean follow-up period was 19 months). The p 53 gene status of tumors was analyzed at exons 5–8 using polymerase chain reaction-single strand conformation polymorphism analysis in 19 patients, and paraffinembedded tumor sections were immunostained for p⁵³ and p²¹ in 23 patients. The overall objective response rate (incidence of good responders) was 70%. The negative p53 group ( n =17) showed a significantly higher objective response rate than the positive p53 group ( n =6) (82% vs. 33%; P =0.045). The p 53 gene status or p21 staining status was not significantly associated with responsiveness. When the p53 and p21 immunostaining results were combined, good responders were more accurately predicted than by p53 staining status alone; the negative p53/positive p21 group ( n =12) showed an objective response rate of 92%, which was significantly higher than that of the positive p53 and/or negative p21 group (45%, n =11) ( P =0.027). Cause-specific survival of the negative p53 group was significantly superior to that of the positive p53 group ( P =0.015). Negative p53/positive p21 immunostaining is a possible predictor of favorable chemotherapeutic response in patients with TCC of the bladder.     

原位膀胱癌动物模型的建立及应用

背景与目的:表浅性膀胱癌术后膀胱灌注丝裂霉素等药物进行化疗,肿瘤仍有较高的复发率.有研究报道往膀胱内灌注小型干扰RNA(siRNA)可抑制裸鼠膀胱肿瘤生长.本研究目的是建立荷人膀胱癌的原位动物模型,通过磁共振成像(magnetic resonance imaging,MRI)监测肿瘤生长过程,并利用此模型评价靶向Survivin的干扰质粒对丝裂霉素的增效作用.方法:直视下经尿道机械损伤BALB/c裸鼠膀胱粘膜,将人膀胱癌细胞T24经尿道种植于25只裸鼠膀胱,建立荷人膀胱癌原位动物模型.以钆-二乙三胺五乙酸作为膀胱造影剂,用MRI监测肿瘤的生长,同时取裸鼠膀胱组织标本行HE染色进行病理学检查.同法建立膀胱癌裸鼠动物模型18只,分为对照组、丝裂霉素组和联合组3组,每周两次膀胱灌注,联合组为靶向Survivin的干扰质粒和丝裂霉素交替用药;膀胱灌注6次后荷瘤膀胱称重.结果:25只裸鼠在种植T24细胞后均形成膀胱肿瘤.种植后7天裸鼠膀胱MRI检查无明显变化,14、21、28天MRI检查均可发现膀胱不同程度的充盈缺损,MRI图像与肿瘤实际大小吻合.病理检查显示:种植后7天,肿瘤生长于裸鼠膀胱粘膜或浅层肌肉;14～28天局限于肌层;35天时侵及浆膜层.丝裂霉素组和联合组的抑瘤率分别为33.45%、56.34%,联合组优于丝裂霉素组(P＜0.05).结论:成功建立了裸鼠原位膀胱癌动物模型,肿瘤生长基本模拟了人膀胱癌的发生、发展过程;MRI检查可作为对裸鼠膀胱原位肿瘤动态观察的可靠方法.靶向Survivin的干扰质粒增加了丝裂霉素的抗肿瘤作用.     

Tumor-promoting macrophages induce the expression of the macrophage-specific receptor CD163 in malignant cells

Tumor-associated macrophages (TAMs) represent a distinct malignancy-promoting phenotype suggested to play a key role in tumor formation and metastasis. We aimed to investigate the expression of the monocyte/macrophage-restricted receptor CD163 in bladder tumor biopsies and assess the potential mechanism inducing the CD163 expression in tumor cells. A high CD163 mRNA expression (n = 87) was significantly associated with a poor 13-year overall survival (log-rank test, χ(2) = 8.931; p = 0.0028). Moreover, CD163 mRNA expression was significantly increased in muscle invasive (T2-T4), p = 0.017, and aggressive (grade III/IV) cancers (p = 0.015). The expression strongly correlated with local expression of IL-6 (r = 0.72; p <0.0001) and IL-10 (r = 0.75; p <0.0001), mediators known to induce CD163 expression in vitro. CD163 immunostaining (n = 46) confirmed the association between dense TAM infiltration and histologically advanced disease. In 39% of the biopsies, CD163 immunoreactivity was also observed in tumor cells, and CD163-expressing metastatic cells were identified in lymph node biopsies (n = 8). Bladder cancer cell lines did not express CD163; however, when cocultured with macrophages the bladder cancer cell expression of CD163 was significantly induced in an IL-6/IL-10 independent manner. In conclusion, we show a strong association between CD163 mRNA expression in bladder cancer biopsies and poor patient outcome. CD163 expression was not confined to the infiltrating TAMs, but was also expressed by a significant portion of the malignant cells in both tumors and lymph nodes. CD163 expressing tumor cells may constitute a subpopulation of tumor cells with a phenotypic shift associated with epithelial-to-mesenchymal transition (EMT) and increased metastatic activity induced by TAMs.     

In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity

In vivo delivery of immunomodulatory genes is a promising strategy for solid tumor vaccination. A drawback is that it necessitates induction of a large effect from transgene expression in a small percentage of tumor cells. Although the B7 family is known to be the most potent of the costimulatory molecules, gene transduction of B7 alone has not been effective in inducing antitumor immunity in nonimmunogenic tumors by ex vivo methods, much less in vivo. We have developed a novel approach where a gene encoding soluble B7-1, a fusion protein of the extracellular domain of murine B7-1 and the Fc portion of human IgG1, is delivered to tumor cells in vivo in the context of an oncolytic replication-competent herpes simplex virus, and the gene product is secreted by tumor cells rather than expressed on the cell surface. Defective herpes simplex virus vectors containing the B7-1-immunoglobulin (B7-1-Ig) fusion transgene (dvB7Ig) were generated using G207 as a helper virus and tested in the poorly immunogenic murine neuroblastoma, Neuro2a, in syngeneic A/J mice. Intraneoplastic inoculation of dvB7Ig/G207 at a low titer successfully inhibited the growth of established s.c. tumors, despite the expression of B7-1-Ig being detected in only 1% or fewer of tumor cells at the inoculation site, and prolonged the survival of mice bearing intracerebral tumors. Immunohistochemistry of dvB7Ig/G207-inoculated tumors revealed a significant increase in CD4+ and CD8+ T-cell infiltration compared with control tumors inoculated with defective vector expressing alkaline phosphatase (dvAP/G207). The antitumor effect of dvB7Ig/G207 was not manifested in athymic mice. In vivo depletion of immune cell subsets in A/J mice further revealed that CD8+ T cells, but not CD4+ T cells, were required. Animals cured of their tumors by dvB7Ig/G207 treatment were protected against rechallenge with a lethal dose of Neuro2a cells but not SaI/N cells. The results demonstrate that the use of soluble B7-1 for immune gene therapy is a potent and clinically applicable means of in situ cancer vaccination.     

An improved intravesical model using human bladder cancer cell lines to optimize gene and other therapies

Orthotopic implantation of human bladder cancer cells into immunodeficient mice is an important tool for studying the biology and effects of therapy. Nevertheless, the incidence of tumor implantation and growth by transurethral instillation of the human bladder cancer cells into murine bladders has been low or not reproducible. However, using a modified intravesical technique and the human bladder cancer cell lines, KU-7 and UM-UC-2, we have been able to obtain a high and reproducible incidence of superficial bladder tumors. Furthermore, intravesical administration of the LacZ adenovirus vector resulted in significant beta-galactosidase expression in these bladder tumors as well as the normal urothelium, which was associated with the removal of the glycosoaminoglycan layer. Because this modified technique produces a high incidence of superficial human tumor growth and allows the efficacy of gene transfer to be evaluated, it should be a useful model for the study of intravesical gene therapy for human bladder cancer.     

Endogenous Tumor Necrosis Factor Induction with Bordetella pertussis Vaccine as a Triggering Agent and Its Therapeutic Effect on MM46 Carcinoma-bearing Mice

Induction of endogenous tumor necrosis factor (TNF) by administration of Bordetella pertussis vaccine (BPV) as a triggering agent and its therapeutic effect against MM46 carcinoma were investigated in C3H/He mice. Test triggering agents were injected intravenously into mice after intravenous injection of 4-fold dilution of macrophage activating factor (MAF) or 10⁴ units of murine interferon-γ (Mu-IFN-γ). Then sera were obtained from the mice, and their TNF activities were assayed on L-929 cells by the method of Ruff and Gifford. The triggering activity of BPV was the highest among those of conventional triggers, such as lipopolysaccharide (LPS) of Escherichia coli , and OK-432. The levels of serum TNF activity triggered by BPV (4 × 10⁹ cells), LPS of E. coli (3 μg) and OK-432 (3 KE) were 5350, 85 and 102 units/ml, respectively. Growth of MM46, a spontaneous mammary carcinoma cell line of C3H/He was observed for 35 days after tumor inoculation and was suppressed significantly by intravenous injection of MAF and BPV (4 × 10⁹ cells). On local injection of BPV (2 × 10⁹ cells) into murine tumors, complete regression was observed in 67% of the mice tested with or without MAF priming on day 25 after tumor inoculation, and intratumoral TNF activity was observed even in the case of the single injection of BPV.