The effect of Taurolidine on adherent and floating subpopulations of melanoma cells.

The annual incidence of malignant melanoma is estimated at 10-12 per 100000 inhabitants in countries of Central Europe and the US, with more recent estimates showing a dramatic upward trend. Taurolidine (Carter/Wallace, Cranberry, NJ) is a novel, potentially effective, antitumor chemotherapeutic agent. We hypothesized that Taurolidine could inhibit the growth, induce apoptosis, affect the cell cycle and change morphology of melanoma cells. We expected this process to be different in adherent and floating subpopulations that may be reflective of solid tumors and their metastases. Analysis of MNT-1 human and B16F10 murine melanoma cells showed that at 72 h the IC(50) of Taurolidine was 25.4+/-3.3 microM for MNT-1 human melanoma cells and 30.9+/-3.6 microM for B16F10 murine melanoma cells. Taurolidine induced DNA fragmentation of melanoma cells in a dose-dependent manner. Taurolidine (75 and 100 microM) induced 52-97% Annexin-V binding (apoptosis), respectively. Evaluation of cell cycle after 72 h exposure to Taurolidine (0-100 microM) revealed that the percentage of melanoma cells in S phase increased from 27 to 40% in the adherent subpopulation and from 33 to 49% in the floating subpopulation. Phase contrast microscopy revealed a marked swelling of melanoma cells and decreasing cell numbers in adherent subpopulation starting at 24 h with 25 microM Taurolidine. Shrinkage of cells dominated at 75-100 microM Taurolidine. Using Cytospin assay in the floating population, we observed swelling of melanoma cells induced by 25-100 micro Taurolidine and appearance of giant (multinuclear) forms resulting from exposure to 75-100 micro Taurolidine. Some floating cells with normal morphology were observed with low concentrations of Taurolidine (0-25 microM). These data show that effects of Taurolidine may be different in adherent and floating subpopulations of melanoma cells. More importantly, floating subpopulations that may contain some viable melanoma cells, may be reflective of potential metastasis after treatment of solid tumors in vivo.

Immunotherapy of mice with an irradiated melanoma vaccine coupled with interleukin-12.

Interleukin (IL)-12 can activate cytotoxic lymphocytes, stimulate natural killer cell activity, induce the production of INF-gamma and inhibit the development of various experimental tumors. We previously demonstrated that immunotherapy of melanoma bearing mice with an irradiated melanoma vaccine (IMV) coupled with IL-2 or GM-CSF had beneficial effects against primary melanoma growth and against subsequent spontaneous metastasis. We also had found that treatment of melanoma bearing mice with IL-12 (300 ng/day) for 4 weeks inhibited the development of primary melanoma tumors in 40% of mice. The purpose of this study was to investigate the efficacy of combined therapy of experimental melanoma with an IMV prepared from B16F10 melanoma cells coupled with IL-12 treatment. C57BL/6 mice were challenged subcutaneously in the tail with B16F10 melanoma cells and by the 45th day, more than 50% of the mice had developed visible primary melanoma tumors at the injection site. Subsequent immunotherapy of mice with IMV, when coupled with IL-12, provided partial inhibition of primary melanoma tumor growth. Optimal results against primary tumor growth were observed when IMV therapy was coupled with IL-12 at a dose of 50 ng/day. Combination of IMV with IL-12 at a dose of 100 ng/day significantly reduced melanoma metastasis to the lungs compared with control mice, and an improvement in mean survival time was observed in mice treated with a combination of IMV with IL-12 (300 ng/day).

A new mouse model of experimental melanoma for vaccine and lymphokine therapy.

The annual incidence of malignant melanoma is estimated at 10-12 per 100,000 inhabitants in countries of central Europe and the United States, and alarmingly there has been a dramatic upward trend in that estimate. The B16 murine melanoma is a rapidly growing metastatic tumor of spontaneous origin, as are human malignant melanomas. Melanoma cells produce specific antigens which are uniquely different from normal cellular antigens, and the expression of such antigens is the cornerstone for preparation of anti-melanoma vaccines. One major problem in evaluating the effectiveness of vaccination and other biologic therapies is the variability of experimental tumor models. A new metastatic model of experimental melanoma which was developed in our laboratory imitates the major clinical stages of malignant metastatic melanoma: stage I, primary (local) tumor growth and bone marrow invasion; stage II, regional lymph node involvement; and stage III, metastasis to distant organs, such as the lungs. This model has been used successfully for screening vaccines constructed in our laboratory. Immunization with formalinized vaccines (of extracellular antigens, intact melanoma cells, or B700 antigen) or irradiated vaccines (of intact melanoma cells) partially inhibit primary melanoma tumor growth, reduce metastasis to regional lymph nodes and lungs, and significantly increase mean survival time. These anti-tumor effects were improved when polyvalent and monovalent vaccines were combined with IL-2 therapy. We also compared the immunogenic activity of vaccines made from B16 melanoma cells transfected with genes encoding murine IL-2 or GM-CSF, and effects on tumor bearing mice were compared with or without therapy using the corresponding lymphokines. In sum, comparison of antibody production, growth of primary melanoma tumors, number of surviving mice, mean survival time, and percent of mice with lung metastases, showed that the best course of immunotherapy involves vaccination of mice with irradiated B16 melanoma cells transfected to secrete GM-CSF, coupled with GM-CSF therapy.

Wound-induced tumor progression: a probable role in recurrence after tumor resection.

OBJECTIVE: To determine the effect of several wound factors on melanoma growth in a mouse model. DESIGN: Cohort analytic study. SETTING: Animal research facility of Roger Williams Medical Center, Providence, RI. STUDY GROUP: Seventeen groups of 5 C57BL/6 mice each. INTERVENTIONS: A surgical wound was created in 1 hind limb, after which different concentrations of B16F10 melanoma cells were injected in adjacent subcutaneous tissue. The nonwounded hind limb in the same mouse served as a control. In this fashion, a critical tumor cell dose was determined that showed tumor growth in the wounded but not the control hind limb. Tumor growth in control hind limbs then was compared with that in the "artificially wounded" hind limbs, which were co-injected with mouse wound fluid or growth factors. Early (day 1) and late (day 10) wound fluids and tumor growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF), both combined, and interleukin 6 (IL-6) were used. MAIN OUTCOME MEASURE: Wound factors increase tumor growth, indicating potentiation of tumor recurrence at a surgical wound. RESULTS: The critical tumor cell dose was 10(3) cells. All growth factors and both wound fluids showed increased tumor growth over time except IL-6. Hind limbs injected with early wound fluid showed increased tumor growth over time when compared with those injected with late wound fluid (P<.001), TGF-beta (P<.001), bFGF (P<.001), and IL-6 (P<.001). Combined TGF-beta and bFGF co-injection resulted in increased tumor growth compared with TGF-beta (P<.001) and bFGF (P<.001), but did not differ significantly from early wound fluid (P<.07). CONCLUSIONS: The healing wound and its mediators in wound fluid or purified growth factors significantly enhanced tumor growth. Combining TGF-beta and bFGF increased tumor growth to a level closer to wound fluid. The inflammatory response provoked by wound healing mediators may be an important mechanism in tumor growth after ablative surgery.

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A.

Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naive mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vbeta and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vbeta T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B 16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls. These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.

Immunization of mice with irradiated melanoma tumor cells transfected to secrete lymphokines and coupled with IL-2 or GM-CSF therapy.

We compared the immunogenic activity of irradiated vaccines prepared from B16 F10 melanoma cells with one made from B16 F10 melanoma cells transfected with genes encoding murine IL-2 or GM-CSF. Vaccines were studied in the conditions of treatment of C57BL/6 mice with or without the corresponding lymphokines. Control and prevaccinated mice were challenged with parental B16 F10 murine melanoma cells (5 x 10(5)) subcutaneously in the midtail to examine growth of the primary (local) tumor in the middle of the tall and metastases to the lungs. This experimental model is very close to the clinical stages of metastatic melanoma. The effectiveness of preimmunization of mice was determined by the levels of antibody production to a melanoma-associated antigen termed B700. The comparison of antibody production, growth of primary melanoma tumors, number of mice surviving at the end of the observation period, mean survival time and per cent mice with metastases in the lungs showed that the best course of immunotherapy was prevaccination of mice with a vaccine of irradiated B16 F10 melanoma cells transfected to secrete GM-CSF, coupled with GM-CSF therapy.

Intrasplenic vaccination against experimental melanoma.

The immunodominant component of a formalinized extracellular antigen (fECA) vaccine prepared from B16 F10 melanoma cells is the melanoma-associated antigen B700. We now demonstrate that a single prophylactic intrasplenic inoculation of B700 antigen (1-10 mu g) stimulates the production of antibodies which have antiproliferative effects on B16 F10 melanoma cells in vitro. In addition, potential cytotoxic effects of splenocytes from B700 antigen inoculated mice were evaluated for two cellular immune effector functions, natural killer (NK) cell activity and lymphokine activated killer (LAK) cell activity; both activities were increased following B700 antigen inoculation. Intrasplenic injection of B700 antigen elicited an increase in the expression of the CD25 surface antigen (IL-2 R alpha) by T lymphocytes and up-regulated the expression of IL-2 R alpha mRNA. Thus both humoral and cellular cytotoxic immune responses might play roles in the decreased growth of primary tumors in B700 antigen inoculated mice and in the higher survival rate in this group of animals.

Further characterization of a clinically relevant model of melanoma metastasis and an effective vaccine.

A major problem in evaluating the effectiveness of tumor cell vaccination and other biological therapies is the variability of experimental models. In this study we have further developed and characterized a model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination: stage I--growth of the primary (local) tumor, stage II--dissemination to regional lymph nodes, and stage III--metastasis to distant organs (lungs). C57BL/6 mice were challenged subcutaneously with B16 F10 murine melanoma cells in the midtail, and within 3 weeks 100% of the mice had local tumors growing in their tails. By 5-7 weeks after challenge, most of the mice had developed metastases to the inguinal lymph nodes and subsequently had metastatic colonies in the lungs and in the bone marrow. Preimmunization of mice with a formalinized extracellular antigen vaccine, derived from B16 F10 melanoma cells, provided partial inhibition of the growth of the primary melanoma tumors, as well as reducing the number of metastases to the regional (inguinal) lymph nodes and lungs along with concomitantly increasing survival time. This model for melanoma metastasis provides a reasonable and reproducible test system for the study of anti-melanoma immunity and the different cellular and humoral mechanisms involved.

Novel experimental approaches to melanoma diagnosis and therapy.

We have investigated the potential use of immune therapies on the growth of melanoma metastases in a new animal model that more closely approximates the clinical situation. We have found that significant benefits towards decreased metastatic growth and subsequent animal survival can be achieved by treatment of tumor-bearing mice with melanoma-specific monoclonal antibodies or alternatively, with various types of monovalent or polyvalent vaccines. The beneficial effects of those vaccines can be significantly enhanced by concomitant interleukin-2 therapy.

Nude mouse model to study passive humoral immunotherapy directed against B16 F10 murine melanoma.

A model to study passive humoral immunotherapy of experimental melanoma was generated by subcutaneous injection of B16 F10 murine melanoma cells in the midtail of BALB/C nude (nu/nu) mice. Mice were challenged with melanoma cells pretreated: (1) with complete culture medium, (2) with 10% adjuvant control serum, (3) with 10% anti-fECA (formalinized extracellular antigens) immune serum, or (4) with a monoclonal antibody (mAB H2-3-3) specific for the B700 melanoma-associated antigen. All control mice challenged with melanoma cells pretreated either with culture medium or with medium containing adjuvant control serum (Groups I and II) died during the observation period of 84 days. At day 84, 60% of the mice challenged with melanoma cells pretreated with anti-fECA immune serum (Group III) survived, as did 100% of the mice challenged with cells pretreated with mAb H2-3-3 (Group IV). Injection of melanoma cells pretreated with mAb H2-3-3 was associated with the greatest reduction of subsequent local tumor growth and the lowest number of metastatic lung tumors. The inhibitory effects of immune sera in vivo also correlated with in vitro effects of anti-fECA immune serum and mAb H2-3-3, determined on B16 F10 melanoma target cells using assays for DNA synthesis and antibody dependent cellular cytotoxicity (ADCC). In sum, this nude mouse model for the study of passive humoral immunotherapy of experimental melanoma was utilized to demonstrate significant protective effects against B16 F10 melanoma cell challenge by treatment with anti-fECA immune sera or a melanoma-specific monoclonal antibody.

Antitumor effects of polyvalent and monovalent vaccines coupled with interleukin-2 in a metastatic melanoma model.

We have previously reported that preimmunization of mice with formalinized extracellular antigens (fECA) derived from melanoma cells, in combination with interleukin 2 (IL-2) treatment and surgical resection, decreased subsequent tumor growth and increased survival of mice in a new model for spontaneous metastasis of melanoma. In this study, we have modified the sequence of tumor growth and therapy to more closely mimic the clinical situation. Mice were challenged subcutaneously in the tail with 5 x 10(5) B16 F10 melanoma cells and, by day 21, all of them had developed localized melanoma tumors. The primary tumor-bearing tails of control and experimental animals were then resected distal to the base of the tail, and therapy of the mice was initiated the following day. Groups of mice received different polyvalent and monovalent murine melanoma vaccines (including native or formalin treated extracellular antigens, intact melanoma cells, or purified B700 antigen), with or without concomitant low doses of IL-2. The results demonstrate that the vaccine therapies elicited significant increases in survival of the mice, accompanied by reductions in the size of lymph nodes and in the number of pulmonary metastases. These effects, particularly with the intact melanoma cell vaccine, could be improved even further with concomitant IL-2 treatment.

Preimmunization of mice with formalinized extracellular antigens of melanoma in combination with IL-2 and surgical resection increased survival and tumor control in metastatic melanoma model.

Recently we found that immunization with formalized extracellular antigens (FECAs) could induce the production of specific antimelanoma antibodies and increase the defense mechanisms of antimelanoma cellular and humoral immunity. In experiments we used pathogen-free female mice C57BL/6 18-20 g. We injected FECA (0.02 mg of protein/per S.C.--subcutaneous injection) for 1 month, once per week. Concurrently we injected S.C. human recombinant IL-2: 100 U/g of weight (2,000 U/per mouse). Interleukin-2 (IL-2) was injected for 1 month, 5 days/week. On days 7, 14, 21, and 28 we took retroorbital blood from mice for the study of anti-FECA and anti-IL-2 antibody production with ELISA. Control and experimental mice were then given a subcutaneous injection with 0.5 x 10(6) cells B16-F10 melanoma in 25 microliters into the middle of the tail. By 18 days 100% developed local melanoma tumors. We resected tails of all control and experimental animals 5 mm distal the base of the tail under metaphan anesthesia. The production of antibodies to FECA and IL-2 started after the 21st day and was higher in the group of mice immunized with FECA and with IL-2 than in control animals. Combining preimmunization with FECA and IL-2 and resection of local melanoma tumors decreased the mortality and the number of mice with local recurrence and metastatic melanoma tumors to the lungs.

Generation of cytotoxic antibodies to the B16 murine melanoma using a formalinized vaccine.

The goal of our experiments was to determine the extent to which the humoral response to a melanoma vaccine elicits the production of cytotoxic antibodies in tumor-challenged mice. Mice were immunized with a vaccine produced from formalinized extracellular antigens (FECA) derived from B16 F10 melanomas. The production of antibodies that recognized the vaccine preparation was determined by ELISA, as was their cross-reactivity with the B700 melanoma antigen. The antibodies were shown to be anti-proliferative by inhibition of tritiated thymidine incorporation into the DNA of cultured target cells and cytotoxic by assays for complement-mediated and antibody-dependent cellular cytotoxicity. Flow cytometric analyses indicated that approximately 60% of the target cells specifically bound antibody from the immune sera. These results confirm that B700 is a significant antigenic component of the FECA vaccine, and provide encouragement for this approach to developing useful melanoma vaccines.

B700 antigen as a component of an antimelanoma vaccine: formalinized extracellular antigens.

Formalin fixation has enjoyed widespread use in the preparation of antibacterial and other vaccines, but rather less use in antitumor vaccines. Previous studies from our laboratories have demonstrated the efficacy of antimelanoma vaccines in mice, produced from formalinized antigens shed by cultured melanoma cells. In this study, we provide evidence that the immunodominant component of that vaccine is the well-characterized B700 melanoma antigen.

Induction of interleukin-2, natural killer cell activity and anti-melanoma antibodies resulting from immunization of mice with formalinized extracellular antigens (FECA) of murine melanoma.

Extracellular products from melanoma cells may play an important role in the pathogenesis of metastatic melanoma. Studies were designed to evaluate the effect of vaccination with formalinized extracellular antigens (FECA) of murine melanoma cells (MMM B16-F10) on survival and immune response of C57BL/6 mice. The cellular immune response was evaluated by assessing interleukin-2 (IL-2) production and natural killer cell activity, whereas the humoral immune response was examined by measuring the production of specific antibodies to extracellular antigens (ECA). IL-2 production by the splenocytes from immunized animals was significantly higher (4.7 U/ml and 3.7 U/ml) than that of controls (1.38 U/ml). The splenocytes from immunized mice revealed significantly higher natural killer cell activity. Similarly, immunized animals responded by producing specific antibodies against the extracellular melanoma antigens as detected by ELISA. The peak production of antibodies against ECA was observed on the 21st day post-immunization. These results suggest that FECA are immunogenic and may enhance active cellular and humoral anti-melanoma immunity.