Murine Transitional Cell Carcinoma Research Articles

Preclinical evaluation of a gene therapy treatment for transitional cell carcinoma

Three drugs were compared for their efficacy in treating murine transitional cell carcinoma (TCC) of the bladder. Intravesical gene therapy treatments utilizing expression-targeted plasmids, where the murine cyclooxygenase-2 (Cox-2) promoter was used to drive the expression of exogenously inducible forms of caspases 3 and 9, were compared with treatment modalities employing Bacille Calmette-Guérin (BCG) and celecoxib. When administered via lavage, only the gene therapy regimen was found to be effective at restricting tumor progression following a 7-day incubation of tumor tissues. Celecoxib was also administered via the diet to allow for systemic delivery of the drug. The most efficacious celecoxib use tested yielded tumors with masses of (18.3±8.4 mg) versus the gene delivery method, which yielded tumors with masses of (3.6±7.7 mg). The difference was significant (t-test, n≥4, P<0.025). The results showed that the Cox-2 expression-targeted gene therapy system could efficiently bypass the bladder permeability barrier and more effectively inhibit tumor growth and development than either BCG or celecoxib treatments. Long-term data further demonstrated that the gene therapy system could effectively inhibit tumor growth and elongate life expectancy.

Immunotherapeutic Effect of Concholepas Hemocyanin in the Murine Bladder Cancer Model: Evidence for Conserved Antitumor Properties Among Hemocyanins

We determined the antitumor properties of a newly available hemocyanin obtained from the Chilean gastropod Concholepas concholepas (Biosonda Corp., Santiago, Chile) in a syngeneic heterotopic mouse bladder carcinoma model. Since keyhole limpet hemocyanin (Pierce, Rockford, Illinois) is used increasingly in biomedicine as a carrier for vaccines and an immunotherapeutic agent for bladder transitional cell carcinoma, there is a growing interest in finding new substances that share its potent immunomodulatory properties. Considering that keyhole limpet hemocyanin and Concholepas concholepas hemocyanin differ significantly, it was not possible to predict a priori the antitumor properties of Concholepas concholepas hemocyanin. C3H/He mice were primed with Concholepas concholepas hemocyanin before subcutaneous implantation of mouse bladder tumor-2 cells. Treatment consisted of a subcutaneous dose of Concholepas concholepas hemocyanin (1 mg or 100 mug) at different intervals after implantation. Keyhole limpet hemocyanin and phosphate buffered saline served as positive and negative controls, respectively. In addition, experiments were designed to determine which elements of the immune response were involved in its adjuvant immunostimulatory effect. Mice treated with Concholepas concholepas hemocyanin showed a significant antitumor effect, as demonstrated by decreased tumor growth and incidence, prolonged survival and lack of toxic effects. These effects were similar to those achieved with keyhole limpet hemocyanin. We found that each hemocyanin increased natural killer cell activity but the effect of Concholepas concholepas hemocyanin was stronger. Analysis of serum from treated mice showed an increased interferon-gamma and low interleukin-4, which correlated with antibody isotypes, confirming that hemocyanins induce a T helper type 1 cytokine profile. To our knowledge our results are the first demonstration of the antitumor effect of a hemocyanin other than keyhole limpet hemocyanin. They suggest that this is an ancient conserved immunogenic mechanism shared by those hemocyanins that is able to enhance T helper type 1 immunity and lead to antitumor activity. Therefore, Concholepas concholepas hemocyanin may be an alternative candidate for providing safe and effective immunotherapy for human superficial bladder cancer.

Anticancer Activity of Intravesical Glyceryl Monooleate (GMO)-Paclitaxel Therapy in Murine Superficial Transitional Cell Carcinoma Model Induced by BBN

Purpose: Paclitaxel, on systemic administration, is widely known to be effective in the treatment of bladder cancer. However, the intravesical use of paclitaxel has not been attempted because of its liposolubility and direct toxicity to the bladder mucosa. The purpose of this study was to evaluate the efficacy and toxicity of paclitaxel-loaded glyceryl monooleate (GMO) in the intravesical treatment of superficial bladder cancer, by enhancing its bioadhesiveness and bioavailability. Materials and Methods: 12 mice were divided into two groups, and bladder carcinomas induced by the addition of 0.05% BBN to their drinking water for 12 weeks. Group 1 received an intravesical instillation of 0.1ml GMO-paclitaxel-free buffer and Group 2 an intravesical instillation of 0.1ml GMO-paclitaxel. On day 21, the tumor incidence, bladder weight and toxicity were evaluated. Results: The incidence of cancer in groups 1 and 2 were 83 and 17%, respectively. The incidence of cancer was significantly reduced in group 2 compared to group 1 (p<0.05). There was a tendency for the average bladder weight in group 1 to be heavier than that in group 2, but there was no significant difference (p=0.375). There were no liver, kidney or bone marrow toxicities in either group. Conclusions: Intravesical GMO-paclitaxel therapy may have an inhibitory effect on the growth of superficial bladder cancer in a BBN-induced bladder cancer model; therefore, it could potentially be used in those patients showing little to no response to intravesical Bacillus CalmetteGuerin (BCG) or other anticancer drug therapies. (Korean J Urol 2006; 47:1155-1160) 󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏

75 Intravesically applied immunostimulatory DNA (CPG-ODN) reduces tumour load in an orthotopic murine transitional cell carcinoma (TCC) model (MB-49/C57-BL6)

75 Intravesically applied immunostimulatory DNA (CPG-ODN) reduces tumour load in an orthotopic murine transitional cell carcinoma (TCC) model (MB-49/C57-BL6)

Interleukin-12 Immunotherapy of Murine Transitional Cell Carcinoma of the Bladder: Dose Dependent Tumor Eradication and Generation of Protective Immunity

The antitumor activity of interleukin (IL)-12 has been demonstrated in a number of tumor models but barely tested in bladder cancer models. We evaluated the antibladder cancer activity of this cytokine in syngeneic mice bearing subcutaneous, metastatic and orthotopic tumors. Mice were implanted subcutaneously, intravenously or orthotopically with syngeneic transitional cell carcinoma (TCC) of the bladder. The tumor bearing mice were then treated with IL-12 locally or systemically and monitored for tumor regression and survival. In the subcutaneous model dose dependent suppression of tumorigenesis was observed when IL-12 was administered subcutaneously at a distal site with the MB49 line being more sensitive than MBT-2. IL-12 (10 days) above 50 ng daily was tumor inhibitory, while doses of 500 or 1000 ng daily prolonged survival and cured 70% and 75% of subjects, respectively. Upon re-challenge with parental tumor cells mice previously cured with IL-12 (1000 vs 500 ng daily) exhibited specific protection (70% vs 35% rejection) that was dependent on the earlier dose of cytokine. IL-12 administered intraperitoneally at a dose of 250 ng daily was more potent than subcutaneous administration and complete regression was observed. Metastatic TCC in the lungs and orthotopic tumors in the bladder also favorably responded to systemic or intravesical IL-12 therapy, respectively. Addition of IL-2 to IL-12 therapy increased tumor regression, long-term survival and rejection of re-challenged parental tumor. IL-12 is exceptionally effective for treating murine bladder TCC in subcutaneous, metastatic and orthotopic models. The antibladder cancer activity of this cytokine should be tested in human bladder cancer therapy.

The Essential Role of Interferon-γ During Interleukin-12 Therapy for Murine Transitional Cell Carcinoma of the Bladder

Recombinant interleukin (IL)-12 and adenoviral IL-12 gene therapy have been shown to be potent therapeutic interventions for murine transitional cell carcinoma (TCC) of the bladder in vivo. We investigated the mechanisms through which IL-12 induces antibladder cancer immunity. The ability of IL-12 to enhance interferon-gamma (IFN-gamma) expression, a major T-helper type 1 cytokine, was analyzed in murine serum, urine and splenocyte cultures. MB49, a murine TCC line, was treated with IFN-gamma and evaluated for its proliferation, surface molecule expression and sensitivity to splenocyte mediated cytotoxicity. Neutralizing antiIFN-gamma antibody was applied to test the role of IFN-gamma in the IL-12 therapy of MB49 tumor. IL-12 was observed to significantly increase IFN-gamma concentrations in serum and urine as well as in splenocyte cultures. While IL-12 had no direct activity against TCC in vitro, IFN-gamma showed potent dose dependent antiproliferative and pro-apoptotic activity, which was further enhanced by supplementation of tumor necrosis factor-alpha. In addition, IFN-gamma substantially up-regulated the expression of surface immune molecules on TCC cells, including MHC-I, MHC-II, ICAM-I, B7.1, B7.2 and Fas. Maximum splenocyte mediated cytotoxicity against TCC was enhanced by pretreatment of target bladder cancer cells with IFN-gamma plus tumor necrosis factor-alpha. Furthermore, IL-2 in combination with IL-12 further enhanced splenocyte mediated cytotoxicity. The in vivo antibladder cancer activity of IL-12 was abolished by concurrent treatment with antibodies to IFN-gamma. This study strongly suggests that IFN-gamma has an essential role in IL-12 induced antibladder tumor immunity. Activation of host effector immune cells by IL-12 is also required for induction of optimal tumor destruction in IL-12 therapy.

MURINE IL-2 SECRETING RECOMBINANT BACILLUS CALMETTE-GUÉRIN AUGMENTS MACROPHAGE-MEDIATED CYTOTOXICITY AGAINST MURINE BLADDER CANCER MBT-2

This study was to establish a more effective anti-cancer immunomodulating agent by constructing recombinant (r) Bacillus Calmette-Guérin (BCG) secreting alpha-antigen (alpha-Ag) fused murine (m) interleukin (IL)-2, and to study its biological activity on cell-mediated cytotoxicity against murine bladder cancer cell, MBT-2, in vitro. pSO246 plasmid vector ligated with mIL-2 gene was introduced into BCG by electroporation. Thioglycollate-elicited murine peritoneal exudate cells (PEC) were stimulated in vitro with parental BCG or rBCG and their cytotoxic activity and the cytokine production was studied. Cytokines were assayed by an enzyme-linked immunosorbent assay (ELISA) and L929 bioassay. Cytotoxicity was measured by 51Cr releasing assay. rBCG (alpha-Ag-IL-2) secreted functional IL-2 and augmented more efficient cytotoxicity to MBT-2 and cytokines such as IL-12, tumor necrosis factor and interferon (IFN)-gamma in PEC than parental BCG did. rBCG (alpha-Ag) had the same activity as BCG. Anti-IL-2 antibody reduced rBCG (alpha-Ag-IL-2)-mediated cytotoxicity and IFN-gamma production. Exogenous IL-2 also enhanced BCG-mediated cytotoxicity, but 100 times more IL-2 was required to express the same activity as rBCG (alpha-Ag-IL-2). Anti-IL-12 neutralizing antibody and the depletion of T cells and NK cells reduced IFN-gamma production by PEC stimulated with rBCG (alpha-Ag-IL-2), suggesting that T cells, NK cells and IL-12 participate in the enhancement of IFN-gamma production. rBCG secreting IL-2 showed significant antitumor activity and cytokine production and this will be a promising agent for bladder cancer patient to reduce both clinical dose and side effects of BCG for immunotherapy.

Intravesical Therapy With Vinorelbine Tartrate: Antitumor Activity in Orthotopic Murine Cell Carcinoma of the Bladder

Objectives To ascertain intravesical vinorelbine tartrate (VNR) antitumor activity against MB-49, a murine transitional cell carcinoma of the bladder (TCC), in an in vivo setting. Materials and Methods C57Bl/6J female mice were intravesically implanted with 5 × 10 [4] MB-49 cells and treated locally with VNR. Tumor incidence and volume analyses, as well as survival studies were carried out. Results Tumor incidence was significantly lower in VNR-treated mice (48%, n = 23) than in controls (84%, n = 19), as evaluated sixteen days after MB-49 orthotopic inoculation. Intravesical tumor volume was also significantly smaller in treated mice respect to controls (median [range]: 0.5 [0.4 to 61.8] mm. [3] versus 47.7 [4.2 to 179.7] mm. [3] respectively, p <0.001 Kruskal-Wallis test). Median survival duration of the animals treated with VNR was 68 [21 to 68] days, and was significantly greater (p = 0.01, Kruskal-Wallis test) than that of untreated controls (18 [16 to 20] days). Conclusion Intravesical VNR treatment demonstrated an evident antitumor effect against the TCC model assayed. The results obtained suggest a potential use of VNR as intravesical treatment for superficial TCC following transurethral bladder tumor resection to prevent recurrence or retard tumor growth.

Exposure to Vinorelbine Inhibits in Vitro Proliferation and Invasiveness of Transitional Cell Bladder Carcinoma

To study the effect of vinorelbine (VNR) on in vitro cell proliferation, invasiveness, cell adhesion to substrate, cell motility and metalloproteinase secretion of MB-49, a murine transitional cell carcinoma of the bladder (TCC). The colorimetric MTS assay, which depends upon viable versus nonviable mitochondria, was used to evaluate the effect of graded concentrations of VNR on in vitro MB-49 cell growth. Chemoinvasion and cell motility were studied in TCC cells exposed for 24 hours to a noncytotoxic dose of VNR, through their ability to migrate across Matrigel-coated or Type IV collagen-coated 8-microns. pore filters. Zymographic studies in gelatin-embedded polyacrylamide gels were done to investigate gelatinolytic activity in conditioned media from treated and untreated MB-49 cells. Vinorelbine inhibited MB-49 cell growth in a dose-dependent manner (IC(50)40 ng./ml.). In vitro cell invasive capacity of MB-49 cells pretreated for 24 hours with VNR at noncytotoxic doses (1 and 10 ng./ml.) was significantly lower than that of untreated cells. The decreased invasion of VNR-treated cells was not accompanied by a diminished adhesion to Matrigel or type IV collagen nor by a significant reduced secretion of gelatinolytic metalloproteinases. Instead, motility of MB-49 cells exposed to noncytotoxic concentrations of VNR was inhibited in a dose-response fashion similar to that of invasion. Vinorelbine proved to be an effective drug to inhibit tumor cell growth and invasion in a transitional cell bladder carcinoma model. The results obtained would justify preclinical studies to evaluate the effectiveness of VNR as a potential treatment of TCC.

Exposure to Vinorelbine Inhibits in Vitro Proliferation and Invasiveness of Transitional Cell Bladder Carcinoma

Purpose To study the effect of vinorelbine (VNR) on in vitro cell proliferation, invasiveness, cell adhesion to substrate, cell motility and metalloproteinase secretion of MB-49, a murine transitional cell carcinoma of the bladder (TCC). Materials and Methods The colorimetric MTS assay, which depends upon viable versus nonviable mitochondria, was used to evaluate the effect of graded concentrations of VNR on in vitro MB-49 cell growth. Chemoinvasion and cell motility were studied in TCC cells exposed for 24 hours to a noncytotoxic dose of VNR, through their ability to migrate across Matrigel -coated or Type IV collagen-coated 8-micrometer. pore filters. Zymographic studies in gelatin-embedded polyacrylamide gels were done to investigate gelatinolytic activity in conditioned media from treated and untreated MB-49 cells. Results Vinorelbine inhibited MB-49 cell growth in a dose-dependent manner (IC 50 40 ng./ml.). In vitro cell invasive capacity of MB-49 cells pretreated for 24 hours with VNR at noncytotoxic doses (1 and 10 ng./ml.) was significantly lower than that of untreated cells. The decreased invasion of VNR-treated cells was not accompanied by a diminished adhesion to Matrigel (R) or type IV collagen nor by a significant reduced secretion of gelatinolytic metalloproteinases. Instead, motility of MB-49 cells exposed to noncytotoxic concentrations of VNR was inhibited in a dose-response fashion similar to that of invasion. Conclusion Vinorelbine proved to be an effective drug to inhibit tumor cell growth and invasion in a transitional cell bladder carcinoma model. The results obtained would justify preclinical studies to evaluate the effectiveness of VNR as a potential treatment of TCC.

Sequential Immunocytological Evaluation of Murine Transitional Cell Carcinoma During Intralesional Bacillus Calmette-Guerin and Interleukin-2 Immunotherapy

The antitumor effect of intralesionally administered recombinant interleukin-2 was highly effective (90% complete response) in murine bladder cancer. We postulated that interleukin-2 may be integral to the Bacillus Calmette-Guerin-induced antitumor response in human bladder cancer. Flow cytometric evaluation of the tumor infiltrates was compared before and after intralesional treatment of an established, untreated murine bladder tumor model with recombinant interleukin-2, Bacillus Calmette-Guerin or saline. Large increases in the number of tumor infiltrating immune cells occurred between the day of randomization and the second day (one day after the first treatment) in all three groups. However, since tumor volume was reduced by treatment, the ratios of the immune cells to tumor volume was increased. The ratios of Thelper, Tcytotoxic/suppressor cells, macrophages, and natural killer cells to tumor volume were 1.5 to 3.4 times higher in the interleukin-2 and Bacillus Calmette-Guerin groups in comparison to the saline group. The ratio of Thelper/Tcytotoxic/suppressor cells however, remained approximately the same despite treatment. Over the next 22 days all subpopulations of tumor infiltrating immune cells decreased in number and frequency to less than measurable levels. The similar modulation of infiltrating immune cell subpopulations by Bacillus Calmette-Guerin and interleukin-2 may indicate that the production of interleukin-2 is part of the tumor modulating mechanism of Bacillus Calmette-Guerin.

Effects of Photodynamic Therapy in Combination with Intravesical Drugs in a Murine Bladder Tumor Model

This study was designed to evaluate the interaction of photodynamic therapy (PDT) and intravesical drugs (thiotepa, adriamycin, mitomycin C and BCG) in a murine transitional cell carcinoma (MBT-2) model. C3H/He mice with implanted MBT-2 flank tumors were treated with either thiotepa (TT), adriamycin (ADM), mitomycin C, Bacillus Calmette-Guerin (BCG), photodynamic therapy (PDT) or a combination of the drug and PDT. The MBT-2 tumor showed sensitivity to adriamycin, MMC or PDT compared to control. PDT combined with either adriamycin, MMC or BCG, produced a greater retardation in the growth of the MBT-2 tumor than monotherapy with adriamycin, MMC, BCG or PDT.PDT combined with the anticancer agents currently used in intravesical therapy for bladder cancer is well tolerated. The combination of PDT and intravesical drugs may enhance the tumoricidal effect of either treatment used alone.

Immunotherapy of Murine Transitional Cell Carcinoma of the Bladder Using Alpha and Gamma Interferon in Combination with Other Forms of Immunotherapy

BCG immunotherapy is very effective in the treatment of superficial transitional cell carcinoma of the bladder, but its significant toxicity may limit its use in some patients. In an effort to find less toxic and potentially more effective treatments we investigated the possible immunotherapeutic potential of combinations of Alpha Interferon (1000 IU) and Gamma Interferon (500 IU) with bacillus Calmette Guerin (BCG) (10(7) cfu), Interleukin-2 (4000 IU), and Keyhole Limpet Hemocyanin (50 micrograms.) in the MBT2 murine bladder cancer model. Significant reductions (p less than 0.05) in tumor incidence relative to the saline control, 83%, Day 35) was observed in groups receiving alpha interferon (42%), Keyhole limpet hemocyanin (42%), interleukin-2 (25%), alpha interferon + Keyhole limpet hemocyanin (17%), alpha interferon + interleukin-2 (33%), gamma interferon + BCG (42%), and gamma interferon + interleukin-2 (17%). All treatment groups with the exception of the group receiving gamma interferon had significantly reduced tumor volume (p less than 0.05) relative to the saline control. Combination treatment groups were significantly more effective than single agent treatments (p = 0.0057). The exhibited anti-tumor effect of these immunotherapeutic agents alone and in combination suggest that they may prove to be effective forms of immunotherapy for transitional cell carcinoma of the bladder.

Treatment of Murine Transitional Cell Carcinoma with Intralesional Interleukin 2 and Murine Interferon Gamma

The antitumor effect of intralesionally administered recombinant interleukin-2, alone or in combination with recombinant interferon gamma was studied in murine transitional cell carcinoma, MBT2. In the initial prophylactic model treatment was started at day one at the site of tumor inoculation. Maximal and significant reduction in tumor volume occurred in groups receiving 4,000 units of recombinant interleukin 2 and 107 colony forming units Bacillus Calmette Guerin (p <0.00001 vs saline control). In the same experiment, a reduction in tumor incidence and increase in survival occurred in groups receiving 4,000 units of recombinant interleukin 2, 1,000 units of recombinant interleukin 2 plus 2,000 units of recombinant interferon gamma, as well as 107 colony forming units Bacillus Calmette Guerin relative to saline control (p <0.005).The dose-response effect of recombinant interleukin 2 alone was also tested in a model of an established transitional cell carcinoma. Intralesional injection treatments were initiated after tumors were palpable. Reduction in tumor volume was observed in the group receiving 8,000 units of recombinant interleukin 2 (p = 0.01 vs saline control), but no significant advantage in survival was noted.

Modulation of hematoporphyrin derivative-sensitized phototherapy with corynebacterium parvum in murine transitional cell carcinoma

The interaction of photodynamic therapy (PDT) with hematoporphyrin derivative (Hpd) and immunotherapy with Corynebacterium parvum (CP) was studied in a murine transitional cell carcinoma (MBT-2) model. C3H/He mice were transplanted subcutaneously in the hind limb with 2.5 × 10 5 tumor cells. One day after transplantation, mice were randomized into groups to receive saline (control), PDT, CP 25 μg, CP 250 μg, CP 25 μg + PDT, and CP 250 μg + PDT. PDT was administered by intraperitoneal (IP) injection of Hpd (12.5 μg/g body weight), followed twenty-four hours later by photoirradiation. CP was given intralesionally at the same time as IP injection of Hpd (24 hours before photoirradiation). A low dose of CP (25 μg) was shown to enhance the effect of PDT while PDT reduced the benefit obtained with high dose of CP (250 μg). In a second series of experiments, CP (250 μg) treatment after photoirradiation was shown to give significantly greater benefit than CP treatment before photoirradiation. The study thus indicates that the effectiveness of combined immunophototherapy is dependent on the sequence of the combination and its intricate relationship with the dosage of CP. The enhancement of PDT by low dose of CP in this model suggests the usefulness of this combined immunophototherapy in enhancing tumor control and in lessening deleterious side effects.

Ethylchlorformate polymerized tumor protein immunotherapy of the murine bladder tumor.

Using murine transplantable transitional cell carcinoma (MBT2), the effect of ethylchlorformate (ECF) polymerized tumor protein was compared with that of bacillus Calmette-Guerin (BCG). Seventy-five C3H/He mice were challenged with an intradermal inoculation of 5 X 10(5) viable MBT2 tumor cells and divided into five groups. Each group was intradermally administered with 0.01 mg of ECF (low ECF), 0.25 mg of ECF (high ECF), 0.1 mg of ECF and 10(6) CFU BCG (ECF/BCG), 10(6) CFU of BCG alone or normal saline (control) weekly for 10 weeks. The mean survival rate for the treatment groups was 64 to 73 days and significantly longer than that for the control group (P less than 0.001, Savage). The incidence of biologically active tumor progression was significantly less for the treatment groups (low ECF, 53%; high ECF, 33%; ECF/BCG, 7%; BCG, 27%) compared with the control group (87%; P less than 0.5, chi-square. The mean rate of tumor growth was significantly lower for all treatment groups than for the control group (P less than 0.001, ANOVA and SNK), and the ECF/BCG group had the lowest growth rate despite a higher incidence of local granulomatous reaction. In this study, immunotherapy significantly prolonged the survival rate, decreased the incidence of biologically active tumor progression, and slowed the rate of tumor growth. The combination of ECF polymerized tumor protein and BCG had the greatest effect, suggesting that the effect of the vaccine was increased with BCG.

Influence of Transplantation Site on Metastatic Ability of Mouse Bladder Carcinoma Sublines

The influence of the primary implantation site on the metastatic behavior of a murine transitional cell carcinoma line (MBT-2) and three metastatic sublines (L3F1, L3F2, and L3F3) was studied. The parent MBT-2 cell line produced a low incidence of lung metastasis after intravenous injection and no metastases from the primary tumor when injected either subcutaneously in the right hind flank or in the footpad. Intramuscular implantation of the MBT-2 cells in the right hind flank resulted in a significant increase over the subcutaneous, footpad, and intravenous sites in the incidence and number of lung metastases. Three in vivo/in vitro selected metastatic sublines (L3F1 L3F2, and L3F3) were highly metastatic when injected subcutaneously, intramuscularly, and intravenously. A low number of pulmonary metastases was observed after footpad implantation of the three sublines. This study demonstrated a definite implantation site-influence on the metastatic ability of the parent MBT-2 line and the three selected sublines. Intramuscular implantation was the most permissive implantation site for the development of spontaneous metastasis for the MBT-2 line and the L3F1, L3F2, and L3F3 sublines.

Phagocytic and Natural Killer Cytotoxic Responses of Murine Transitional Cell Carcinoma to Postsurgical Immunochemotherapy

Postsurgical immunochemotherapy with Corynebacterium parvum (CP) and cis-diamminedichloroplatinum (II) (CDDP) was evaluated in mice with transitional cell carcinoma (MBT-2). C3H/He mice were transplanted subcutaneously in the hind limb with 5 × 105 tumor cells. Ten to 14 days later when the tumor reached a diameter of five to seven mm., it was surgically removed. Mice were then randomized into four groups to receive a total of three treatments on days 1, 3 and 5 after surgery: 1) saline (control group); 2) CP, 250 pg. into the surgical site; 3) CDDP, 5μg./gm. body weight intraperitoneally; and 4) combined CP and CDDP. Recurrence of tumor occurred in 70%, 52%, 55% and 28% of mice receiving surgery only, CP, CDDP, and combined CP and CDDP respectively. In the second part of the experiment, phagocytic activity using chemiluminescence assay and natural killer (NK) activity using chromium-51 release assay were determined with cells from the peritoneum, spleen and inguinal lymph nodes. CP or CDDP alone enhanced the phagocytic and NK activity. The most significant enhancement was obtained with cells from the inguinal lymph nodes of mice receiving combined CP and CDDP, the group with the lowest tumor recurrence. These results suggest that combination of CP and CDDP may be useful in control of postsurgical recurrence of bladder cancer. (J. Urol., 140: 660–663, 1988)

Role of the implantation site on metastatic ability of the murine MBT-2 transitional cell carcinoma.

The influence of implantation site on the metastatic behavior of a murine transitional cell carcinoma line (MBT-2) was examined. MBT-2 cells were injected into one of four anatomic sites; subcutaneously, intramuscularly, intravenously or into the footpad, to evaluate the influence of implantation site on the formation and number of metastases. The MBT-2 cell line produced a low incidence of lung metastases after intravenous injection with a mean of 1.1 lung tumors per mouse. Injection of MBT-2 cells into the footpad or subcutaneously did not produce metastases from the primary tumor. Intramuscular implantation, however, resulted in a sixty percent incidence of metastasis with a mean of 8.2 lung nodules per mouse. This study demonstrated a definite implantation site influence on the metastatic ability of the MBT-2 line.

The Efficacy of Chemotherapeutic Agents Against Murine Bladder Metastasis

Three chemotherapeutic agents, methotrexate, cyclophosphamide and cis-diamminedichloroplatinum (cis-platinum), were examined for their effectiveness against metastases in a murine transitional cell carcinoma model. Systemic treatment of the drugs was applied against a MBT-2 derived subline which generates 100% incidence of lung metastases in C3H mice by five weeks. The drugs were examined for their effect against the number of metastases, incidence of metastasis and size of the subcutaneously implanted primary tumor. All three compounds significantly reduced both the number of lung metastases and the incidence when compared to untreated animals. None of the agents proved 100% effective against metastatic tumors. These results suggest the existence of a chemotherapeutic resistant population of metastatic cells. Administration of methotrexate and cis-platinum effectively reduced the size of the primary tumor as compared to untreated animals. Cyclophosphamide did not significantly affect primary tumor size. The response of the antineoplastic agents against the metastatic tumor cells indicates that the L3F2 metastatic cell line is an effective model to study agents against metastatic bladder cancer.