Mitomycin C-treated Tumor Cells Research Articles

Norepinephrine-Mediated Inhibition of Antitumor Cytotoxic T Lymphocyte Generation Involves a β-Adrenergic Receptor Mechanism and Decreased TNF-α Gene Expression

Abstract We have previously shown that norepinephrine (NE) inhibits the in vitro generation of anti-MOPC-315 CTL activity by spleen cells from BALB/c mice rejecting a large MOPC-315 tumor as a consequence of low-dose melphalan (l-phenylalanine mustard (l-PAM)) treatment (l-PAM TuB spleen cells). Since TNF-α plays a key role in the generation of antitumor CTL activity in this system, we determined whether NE mediates this inhibition through inhibition of TNF-α production. Here, we show that NE inhibits the production of TNF-α protein and mRNA by l-PAM TuB spleen cells stimulated in vitro with mitomycin C-treated tumor cells. Flow cytometric analysis of intracellular expression of TNF-α revealed substantial NE-mediated decreases in the percentages of TNF-α+ cells among CD4+ and CD8+ T cells and F4/80+ activated macrophages. NE inhibition of CTL generation was largely overcome by addition of TNF-α to the stimulation cultures. When the β-adrenergic antagonist propranolol was added to the stimulation cultures of l-PAM TuB spleen cells at a concentration that prevented NE-induced cAMP elevation, the NE-mediated decrease in TNF-α mRNA and NE-mediated inhibition of CTL generation were reversed. Collectively, these results suggest that NE inhibits antitumor CTL generation, at least in part, by inhibiting TNF-α synthesis through a mechanism(s) involving β-adrenergic receptor signaling.

Eradication of tumor growth via biolistic transformation with allogeneic MHC genes.

Particle acceleration-mediated biolistics transformation has been rapidly adopted as a versatile technology for gene delivery since its original application as a physical method for delivery of foreign genes into higher plants. We have designed a versatile hand-held gene gun device for the genetic immunization of animals in vivo. The gene gun is driven by air blast and therefore does not require helium gas or vacuum for its function. In this report, we have employed this gene gun device to introduce allogeneic H-2Kb DNA directly into tumor nodules of K36 tumor-bearing AKR/J mice. Expression of the exogenous H-2Kb gene led to the regression of well-established K36 tumors. This tumor regression was correlated with the generation of potent secondary CTL responses against the K36 tumor cells following expression of the exogenous H-2Kb gene in the K36 tumor cells. Furthermore, it was observed that H-2Kb stimulator cells could stimulate the generation of both allogeneic and tumor-specific secondary CTL responses whereas mitomycin-C treated tumor cells could only stimulate tumor-specific secondary CTL responses. This approach of introducing allogeneic MHC genes directly into cutaneous tumor lesions via biolistics transformation of tumor cells in vivo can potentially be developed into an effective method for cancer gene therapy.

Adjuvant immunotherapy in melanoma: a new approach.

Patients with metastatic cutaneous melanoma to two or more regional lymph nodes have an extremely poor prognosis despite radical lymphadenectomy. In an attempt to improve the survival and to determine the safety of a new method of tumor specific adjuvant immunotherapy in such a high risk group of patients, nine patients were studied. Three to four weeks after regional lymphadenectomy, each of them received a single intradermal injection of Bacillus Calmette-Guérin. Three weeks later, they were immunized by allogenic melanoma cells obtained from live donors with distant metastases. Each patient received three vaccinations, each from a different donor (except in one), to avoid development of HLA response, but maintaining exposure to melanoma antigens. No cultured melanoma cells were used. Each vaccine consisted of mitomycin-C treated tumor cells mixed with purified protein derivative (PPD) of tuberculin given intradermally once per month for 3 months. The patients were then observed with no further treatment. Utilizing the leukocyte migration inhibition test, there was some in vitro evidence of tumor specific cell mediated response which seemed to disappear 1-2 months postimmunization. At 5 years, five of the nine patients (55%) were alive free of disease. No autoimmune diseases were detected in any of the immunized patients. A major hindering factor for such an approach was the limited availability of the allogenic melanoma cells.

Production of colony-stimulating factor by tumor cells and the factor-mediated induction of suppressor cells.

Spleen mononuclear cells of C3H/HeN mice were cultivated with mitomycin C-treated tumor cells, X5563, MH134, MM48, MM46, and FM3A/R, all of which were of syngeneic origin, in a medium containing normal syngeneic mouse serum but not FCS. There was a proliferative response to X5563, MH134, and MM48, but not to the two other tumor cells, MM46 and FM3A/R. The responder spleen cells were found to be nonadherent cells with a phenotype of Thy-1-L3T4-Lyt2-Ig-Macl-, which were neither mature T and B cells nor mature macrophage/granulocytes. It was also found that the proliferation of these nonadherent no-marker cells was mediated by tumor cell-derived soluble factors but not by direct stimulation with tumor cells. The responsible factor was a molecule(s) with a Mr of 23 to 25 kDa, which had a CSF activity inducing granulocyte (G)-, macrophage (M)- and G + M-colonies in the bone marrow cells. Neutralization tests of this factor-induced proliferation of spleen cells revealed that a major part of the factor may be GM-CSF or a molecule closely related to it. Incubation of spleen mononuclear cells with these GM-CSF-like tumor cell factors resulted in induction of myeloblastic/promyelocytic cells with a phenotype of Mac-1+2+Ia+ Thy-1-L3T4-Lyt2-Ig- in the spleen cell cultures, which could suppress mitogenic responses of the spleen cells to T and B cell mitogens. GM-CSF-like activity could also be detected in the serum of mice bearing X5563, MH134, and MM48, but not in those bearing MM46 and FM3A/R. Subcutaneous inoculation of C3H/HeN mice with these X5563, MH134, and MM48 tumor cells generated massive metastasis in the lung and lymph nodes, whereas MM46 and FM3A/R produced no macroscopic tumor cell metastasis. These results strongly suggest the possibility that in some tumor cell-host systems, a GM-CSF-like factor(s) produced constitutively by the tumor cells may play an important role in the development of tumor metastasis, mediating through suppression of lymphoid tissues of the host.

Host Response to Myeloma: I. Induction of Cytotoxic and Suppressor T Cells by In Vivo Immunization with MOPC 104E Plasmacytoma

Spleen cells from BALB/c mice immunized with mitomycin C-treated MOPC 104E plasmacytoma cells demonstrated negligible cytotoxic activity (less than 10% specific cytotoxic activity) in the 51Cr release assay. These cells exhibited increased cytotoxic activity when they were secondarily sensitized in vitro with mitomycin C-treated MOPC 104E cells. Spleen cells from normal mice showed tumor-specific cytotoxic activity when cocultured with mitomycin C-treated tumor cells at the optimal responder to stimulator ratio of 25:1. The level of cytotoxic activity obtained by in vivo primed and secondarily in vitro sensitized spleen cells did not exceed the level of activity obtained by in vitro primary sensitized cells. Significant suppression of the cytotoxic activity of in vitro primary sensitized cells was observed when cocultured with in vivo primed spleen cells during primary sensitization in vitro at a responder to suppressor cell ratio of 1:1. Suppressor cells of in vivo primed mice were removed by treatment with anti-Thy 1.2 and complement. These results suggest that spleen cells from in vivo primed mice consisted of at least two subpopulations of cells, a cytotoxic (prekiller) and suppressor T cells. Attempts to induce cytotoxic cells in vivo might have failed because of the appearance of suppressor T cells.

Cytotoxic Activity of Peripheral Blood Lymphocytes Grown with Interleukin 2 against Autologous Cultured Tumor Cells in Patients with Renal Cell Carcinoma: Preliminary Report

To examine the use of interleukin 2 as a therapeutic agent for human renal cell carcinoma the peripheral blood lymphocytes derived from patients with renal cell carcinoma were grown in interleukin 2 and tested in a 4-hour 51chromium release cytotoxicity assay against autologous cultured tumor cells. Peripheral blood lymphocytes, which were cultured separately in medium alone, mitomycin C-treated tumor cells, 20 per cent interleukin 2 and tumor cells plus interleukin 2, were used as effector cells. In all patients neither peripheral blood lymphocytes in medium alone nor in mitomycin C-treated tumor cells could lyse their own tumor cells. However, in 5 of 7 patients peripheral blood lymphocytes grown in interleukin 2 regardless of the presence of mitomycin C-treated tumor cells were capable of causing 5.7, 9.4, 12.7, 36.1 and 52.5 per cent lysis at effector/target cell ratios of 25, 50, 50, 50 and 25, respectively. However, more significant cytotoxicity was not obtained by peripheral blood lymphocytes cultured with tumor cells and interleukin 2. These results suggest that interleukin 2 might become a useful agent for patients with renal cell carcinoma.

Inhibition of pulmonary metastases of B16 melanoma with irradiated tumor cells and BCG.

When the tumor-bearing leg of C57BL/6J mice was amputated 16 days after SC inoculation of 10(6)B16 melanoma cells, all the amputated mice died of pulmonary metastases. Transfer of lungs from the amputated to normal syngeneic mice revealed tumor cells in the lungs just after amputation. Repeated weekly injections of BCG and irradiated tumor cells, beginning 24 h after amputation of the tumor-bearing limb, prolonged the survival only of mice presensitized to BCG. Injections of BCG or irradiated melanoma cells alone, or neuraminidase- and mitomycin C-treated tumor cells or of Levamisole had no effect, but injections of ConA-coated tumor cells slightly prolonged the survival of the amputated mice. Both BCG and B16 cells induced humoral and cell-mediated immunity but there was no cross-reactivity between BCG and B16 cells.

In vitro sensitization of human lymphocytes to a myeloma cell-related antigen

Peripheral blood lymphocytes from normal human donors were cocultivated with cells from two established human multiple myeloma cell lines, RPMI 8226 and K-737, and with lymphoblastoid cells from a third B cell line, RAMM. After a comparison of three methods of lymphocyte sensitization, a 6-day incubation protocol with equal numbers of normal lymphocytes and mitomycin C-treated tumor cells was selected. Cells from the RPMI 8226 myeloma line stimulated the differentiation of lymphocytes into cytotoxic effector cells as measured by 51Cr release from labeled target cells. The RPMI 8226-sensitized lymphocytes were cytotoxic for myeloma cells (RPMI 8226 and K-737) and for lymphoblastoid cells (RAMM) but not for cells from human lung tumor lines (A549, A427, MB9812), a breast carcinoma line (ALAB), a normal diploid fibroblast line (HSBP), or normal lymphocytes.

Role of delayed hypersensitivity and cytostatic macrophages in the rejection of an allo-transplantable murine tumor.

Growth of an allo-transplantable tumor, sarcoma-180, was suppressed in ICR mice immunized with mitomycin C-treated tumor cells in Hanks' solution or heat-killed tumor cells mixed with complete Freund's adjuvant 7 days before subcutaneous inoculation of tumor cells. In those mice, cytotoxic lymphocytes were not detected in spleen, lymph node or peritoneal exudate cells before or after tumor inoculation. The activities of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity were not found in sera. Cytostatic activity was detected in peritoneal glass-adherent cells, but not in glass-nonadherent cells. Such an activity was also conferred on normal peritoneal glass-adherent cells by incubation with the culture supernatant of lymph node cells from preimmune mice and tumor cells. Cytostatic macrophages mediated by lymphokine-producing lymphocytes may be at least one of the main effector mechanisms in resistance against such an allo-transplantable tumor line.

Augmentation of delayed-type hypersensitivity and resistance against allogeneic or syngeneic methylcholanthrene-induced tumors in mice preimmunized with the tumor extracts.

Delayed-type hypersensitivity (DTH) responses against methylcholanthrene-induced fibrosarcomas in C3H/He and BDF1 mice were developed in BDF1 mice by sc injection of the respective mitomycin C-treated tumor cells. The DTH responses to the allogeneic and the syngeneic tumor cells were accelerated and enhanced tumor-specifically by priming 7 days previously with KCl extracts of the respective tumors. The ability in the mice primed with the tumor extracts enhancing the DTH response against the tumor cells could be transferred to recipient mice by the spleen cells, but not by the T-cell-depleted spleen cells. Rejection of allogeneic tumor was accelerated under the development of accelerated and enhanced DTH response against the allogeneic tumor antigens. Moreover, resistance to syngeneic tumor growth increased significantly with the development of accelerated and enhanced DTH response against the syngeneic tumor antigens. Thus, the augmentation of DTH response by preimmunization with tumor extracts was accompanied by the increased resistance to tumor growth, suggesting that T cells involved in the augmentation of tumor-specific DTH response play some role in increasing the resistance to tumor growth.

In vitro lymphocyte immunization to cultured human tumor cells: parameters for generation of cytotoxic lymphocytes.

Conditions for optimal immunization of allogeneic lymphocytes against human tumor cells were studied. Maximal cell-mediated cytotoxic responses were generated usually with 2-4 X 10(4) tumor cells and 1 X 10(6) lymphocytes after a 5-day incubation. A further increase in the number of stimulating cells usually suppressed the immunization of lymphocytes. Viable untreated tumor cells were generally more immunogenic than were the mitomycin C-treated tumor cells. Lymphocytes (1 X )0(6)) sensitized in small volumes (0.5-1 ml) of medium were invariably transformed into cytotoxic effector cells, whereas the same number of lymphocytes sensitized in relatively larger volumes (4 ml) of medium had little or no cytotoxic activity. Occasional shaking of mixed culture during the sensitization provided better immunization than that in similar stationary culture. The replacement of culture fluids with fresh medium during incubation decreased the cytotoxicity level of sensitized cells.

In Vitro Induction of Cytotoxicity against Syngeneic Mastocytoma and Its Suppression by Spleen and Thymus Cells from Tumor-Bearing Mice

P815 times 2 mastocytoma cells, when injected subcutaneously into syngeneic DBA/2 mice, induced T lymphocytemediated cytotoxicity in the mice. During the course of tumor growth this cytotoxic activity decreased and ultimately the tumor killed the mice. When spleen cells from mice with tumors in the early stages of growth were incubated in vitro with mitomycin C-treated tumor cells, specific cytotoxicity mediated by T lymphocytes was developed. In contrast, spleen cells, taken from mice with tumors at a later stage in their growth, did not develop cytotoxic activity. The unresponsiveness of spleen cells from mice with tumors in the latter stages of growth seemed to be due to the presence of suppressor cells since in vitro development of cytotoxicity by spleen cells from early tumor-bearing mice were inhibited by the addition of spleen cells or thymocytes from mice with progressively growing tumors. Normal spleen cells or thymocytes did not affect the response.