Enhanced tumor growth and metastatic spread of an MH134 variant lacking a part of the MM antigen: a possible role of antibody-dependent cellular cytotoxicity in control of tumor growth and metastases

The role of antibody-dependent cellular cytotoxicity (ADCC) in host defense against tumor growth and metastasis was investigated with MH134, an MM antigen-positive murine hepatoma, and MH134-M, a variant with enhanced tumorigenesis and metastasis, in syngeneic C3H/HeN mice. When inoculated subcutaneously into C3H/HeN mice, MH134-M tumors grew more rapidly than did MH134 tumors and consistently metastasized to the draining lymph nodes, whereas MH134 tumors did not. Also, MH134-M exhibited a significantly greater lung colonization potential than did MH134, when inoculated intravenously into C3H/HeN mice. In BALB/c nu/nu mice, however, solid MH134 tumors grew and metastasized to the same extent as MH134-M, indicating that there is no significant intrinsic difference between these two tumor lines in proliferative or metastatic capacity. Enzyme-linked immunosorbent assay (ELISA) and immunoblotting, performed after SDS-PAGE analysis of cellular extracts with a monoclonal antibody (MAb) that recognizes a part of the MM-antigen, revealed that the cells of MH134-M lack at least a part of the MM antigen. Sera of C3H/HeN mice bearing solid MH134 tumors were found to contain anti-MM-antigen antibodies, when tested by immunoblotting of SDS-PAGE-developed materials. Cytotoxicity testing in which thioglycollate-induced peritoneal macrophages were used as effector cells revealed that antibodies present in sera strongly induced ADCC to MH134 but not to MH134-M. On the other hand, sera of MH134-M tumor-bearing C3H/HeN mice neither contained anti-MM-antigen antibodies nor induced ADCC to MH134 or MH134-M tumor cells. Intravenous injection of carrageenan into C3H/HeN mice bearing solid MH134 tumors significantly enhanced tumor growth, whereas the growth of subcutaneously injected MH134-M tumors was not influenced by this treatment. These results suggest that the enhanced tumorigenesis and metastasis of the MH134-M line in C3H/HeN mice are based, at least in part, on significant loss of the MM antigen and the resultant inability to induce ADCC-triggering antibody production during tumor growth.     

Suppressive effect of portal venous inoculation with tumor cells on the anti-tumor immune potential

The effect of portal venous (pv) administration of syngeneic tumor cells on the potential to generate anti-tumor immune resistance was examined. C3H/He mice were inoculated with syngeneic X5563 tumor cells via the pv or intravenous (iv) route. A single pv administration of 10,000 R X-irradiated X5563 cells into C3H/He mice not only failed to induce anti-X5563 immunity but also abolished the capability of the mice to develop anti-X5563 in vitro cytotoxic and in vivo protective immunity as induced by intradermal (id) inoculation of viable X5563 cells followed by the surgical resection of the tumor (immunization procedure). Such immunosuppression was also obtained by pv inoculation of tumor cells after the above immunization procedure. The immunosuppression induced by the pv injection of tumor cells before or after the immunization procedure was tumor-specific, since the pv sensitizing regimen using X5563 tumor cells did not interfere with the generation of immunity against another syngeneic tumor, MH134. More importantly, the pv route-induced suppression contrasted with the lack of inhibiting effect of iv inoculation with tumor cells on the induction of the tumor immunity. These results indicate that the host's tumor-specific immune capability is markedly suppressed in an anti-tumor sensitizing stage-independent manner when tumor cells enter the pv circulation.     

Synergistic antitumor effects of BCG and monoclonal antibodies capable of inducing antibody-dependent cell-mediated cytotoxicity

Three monoclonal antibodies (MAbs) of different immunoglobulin subclasses against MH134 murine ascitic hepatoma cells, detecting the same antigenic determinant of tumor-associated antigen of the tumor cells, were tested for their ability to produce a synergistic antitumor effect with Mycobacterium bovis BCG in C3H/HeN mice. 12A2 (IgG2a) induced both antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the tumor cells. C3H/HeN mice were inoculated ip with MH134 tumor cells (day 0), and received an ip injection of BCG (day 1) and/or 12A2 (day 5). The combined therapy with BCG and 12A2 brought about a significant prolongation of the survival period, whereas either BCG or the MAb alone exhibited poor therapeutic effectiveness. 11G2 (IgG1), inducing ADCC but not CDC against MH134 tumor cells, was shown to exhibit antitumor effects as potent as those of 12A2, when used in combination with BCG. However, 7C2 (IgM), capable of inducing CDC but not ADCC to the tumor cells, produced no apparent synergistic effect with BCG. ADCC of BCG-induced peritoneal cells was mediated by the adherent cell population of the cells and abolished by the addition of carrageenan, suggesting that the effector cells of the cytotoxicity were macrophages. Moreover, carrageenan abolished the combined antitumor effect of BCG and these MAbs in the serological Winn assay. These results suggest that activated macrophages play a major role in the synergistic antitumor effect of BCG and MAbs capable of inducing ADCC against MH134 tumor.     

Effect of hemostatics used during operations for digestive organ on cancer cells present in the peritoneal cavity

We investigated effects of hemostatics used during operations for digestive organ on cancer cells present in the peritoneal cavity using BALB/c mice inoculated with Meth A tumor cells (fibrosarcoma) intraperitoneally (i.p.) and C3H/He mice inoculated with MH134 tumor cell (hepatic cell carcinoma) i.p. Microfibrillar collagen hemostat (Avitene) or fibrinogen preparation (Beriplast P) did not affect survivals of those tumor-bearing mice. Gelatin sponge (Spongel)prolonged survivals of MH134 tumor-bearing mice. Liquid form gelatin used instead of Spongel displayed in vitro antitumor effect on MH134 tumor cells at the concentration of 15 mg/ml. Radioactive sodium chromate-labeled MH134 and Meth A tumor cells were not lysed when they were incubated with 15 mg/ml of liquid form gelatin for 24 hours. On the other hand, the tritium thymidine (3H-TdR) uptake by MH134 or RL male 1 tumor cells was suppressed when they were incubated with 15 mg/ml of liquid form gelatin for 24 hours. Proliferation of Meth A tumor cells were not affected by the treatment. Effect of liquid form gelatin on phytohemagglutinin (PHA)-stimulated spleen cells as a benign counter-part of RL male 1 tumor cells (T cell lymphoma) was examined. Liquid form gelatin (15 mg/ml) did not suppress 3H-TdR uptake by PHA-stimulated spleen cells.     

Complete regression of mouse hepatoma transplanted after partial hepatectomy and the immunological mechanism of such regression

We have investigated the effect of partial hepatectomy (HEP) on tumor growth. MH-134 hepatoma cells, which were inoculated in syngeneic C3H/He mice from 3 to 10 days after HEP, grew with a linear increase in size until 7 days, began to regress, and disappeared 14 days after the inoculation. The survival rate was 100%, and the recurrence of tumor was not observed during the following 4 mo. On the other hand, the growth of another syngeneic tumor, X-5563, and of an allogeneic Ehrlich tumor was not affected by HEP. When MH-134 tumor cells were inoculated 7 days before or 15 days after HEP, tumor regression was not observed. The Winn assay showed the presence of tumor-neutralizing activity in spleen cells of MH-134 tumor-regressed mice. Cytotoxic activity against MH-134 tumor cells was also detected in the spleen cells. Analysis by using monoclonal antibodies showed that the effector cells were Thy-1+ and Lyt-2+ cells. Thus, HEP and the following liver cell regeneration may play a role in augmentation of specific immune response to the transplanted hepatoma cells.     

Mechanisms for recognition of tumor antigens and mediation of anti-tumor effect by noncytolytic Lyt-2+ T cell subset

The mode of anti-tumor function in vivo of noncytolytic Lyt-2+ T cells from C3H/He mice hyperimmune to syngeneic MH134 hepatoma was investigated in a double diffusion chamber system which was recently established in our laboratory. C3H/He mice were implanted intraperitoneally with the double diffusion chamber unit in which each chamber contained either L3T4+ T cell-depleted MH134-hyperimmune spleen cells plus mitomycin C-treated MH134 tumor cells or other syngeneic X5563 viable tumor cells plus normal spleen cells as a source of macrophages. Inclusion of anti-MH134 Lyt-2+ T cells together with MH134 tumor cells in one chamber resulted in comparable growth inhibition of viable X5563 tumor cells in the other chamber to that obtained by unfractionated MH134-hyperimmune spleen cells. The induction in the Lyt-2+ T cell-containing chamber of anti-tumor effect to be delivered into the other chamber was dependent on the co-existence of Ia-positive adherent cells along with Lyt-2+ T cells. Although adherent cell-depleted Lyt-2+ T cells regained the inducibility of anti-tumor immunity when supplemented with splenic adherent cells, the addition of adherent cells pretreated with chloroquine failed to restore the ability of Lyt-2+ T cells to induce their anti-tumor effect. In addition, paraformaldehyde-treated MH134 tumor cells instead of untreated tumor cells were not capable of activating Lyt-2+ T cells. These results indicate that a portion of Lyt-2+ T cells exerts their anti-tumor effect by a mechanism distinct from direct tumor cell lysis and that their activation for mediation of this type of tumor immunity requires the recognition of tumor antigens processed and presented by Ia-positive adherent cells.     

Increased protective activity against acid precipitation of poly(U) in the serum of tumor-bearing mice

Transplantation of leukemia L1210 cells into DBA/2 mice and of Ehrlich ascites tumor cells into BALB/C mice resulted in a significant increase of protective activity against acid precipitation of poly (U) in the serum. The increase was observed as early as one day after the tumor transplantation and seems to be connected with cancer growth, since inoculation of L1210 cells into BALB/C mice did not affect the protective activity, evidently as a result of their well established inability to cause cancer in this strain. Furthermore, no increase of activity was observed when bacteria were inoculated into mice, or when the latter were partially hepatectomized. The results suggest that the protective activity against acid precipitation of poly (U) could prove to be a tumor marker for the early detection of cancer growth.     

Immunomodulatory and Antitumor Activity of Biophytum sensitivum Extract

An alcoholic extract of Biophytum sensitivum was studied for its immunomodulatory and antitumor activity. Theextract was 100% toxic at a concentration of 0.5 mg/ml to Dalton’s lymphoma ascites (DLA) and Ehrlich ascitescarcinoma (EAC) cells. B. sensitivum extract was also found to be cytotoxic towards L929 cells in culture at aconcentration of 0.1 mg/ml. Administration of B. sensitivum extract (500μg/dose/animal) could inhibit the solid tumordevelopment in mice induced with DLA cells and increase the lifespan of mice bearing Ehrlich ascites carcinomatumors by 93.3%. B. sensitivum treatment significantly (p<0.001) reduced the tumor cell glutathione (GSH) levels aswell as serum gamma glutamyl transpeptidase (GGT) and nitric oxide (NO) levels in ascites tumor bearing animals.The total WBC count was also increased to 14,087 cells/mm3 on the 12th day in BALB/c mice. The number of plaqueforming cells also enhanced significantly (p<0.001), and bone marrow cellularity and β-esterase positive cells werealso increased by the administration of B. sensitivum extract.     

Selective suppression of the generation of anti-tumor L3T4+ but not of Lyt-2+ T cell-mediated immunity in the tumor-bearing 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-bearing state.     

Requirements of adherent cells for activating Lyt-1+2- T cells as well as for functioning as antitumor effectors activated by factor(s) from Lyt-1+2- T cells

The mechanism by which tumor-specific Lyt-1+2- T cells exhibit their antitumor effect in collaboration with an adherent cell population was investigated with the use of a double diffusion chamber. The double diffusion chamber was prepared by separating the two chambers from each other by a Millipore membrane and implanted in the peritoneal cavity of C3H/He mice. When one chamber contained normal C3H/He spleen cells plus syngeneic viable MH134 hepatoma cells and the other contained normal C3H/He spleen cells plus syngeneic viable X5563 plasmacytoma cells, tumor cells in both chambers continued to proliferate. In contrast, the injection of spleen cells immunized to the MH134 tumor into one (the first) chamber containing MH134 tumor cells not only resulted in the growth inhibition of MH134 tumor cells, but also exhibited an appreciable inhibitory effect on the growth of X5563 tumor cells admixed with normal spleen cells in the other (second) chamber. The growth inhibition of X5563 tumor cells in the second chamber was mediated by Lyt-1+2- T cells specific for MH134 tumor cells admixed in the first chamber. Such tumor-specific Lyt-1+2- T cell function was dependent on the existence of adherent cells in the first chamber, and adherent cells in the second chamber were also required for the X5563 growth inhibition. In addition, when the second chamber containing adherent cells, instead of the connection to the first chamber, was provided with macrophage-activating factor (MAF), X5563 growth inhibition was also observed. These results indicate that adherent cells are required for activating tumor-specific Lyt-1+2- T cells and for functioning as nonspecific antitumor effector cells activated by factor(s) such as MAF from Lyt-1+2- T cells.     

Murine tumor cell lysis by antibody-dependent macrophage-mediated cytotoxicity using syngeneic monoclonal antibodies

Four monoclonal antibodies to MH134 murine syngeneic hepatoma cells, 3H1, 7C2, 11G2, and 12A2, were produced by hybridomas constructed by fusing P3-X63-Ag8-U1 murine myeloma cells with spleen cells of a C3H/HeN mouse immunized with the syngeneic tumor cells. Immunodiffusion analysis with rabbit anti-mouse immunoglobulin antisera showed that 3H1, 7C2, 11G2, and 12A2 are IgG2a, IgM, IgG1, and IgG2a, respectively. Enzyme-linked immunosorbent assay using cells of five syngeneic tumor lines, MH134, MM102, MM46, MM48, and X5563, and lymph node cells of C3H/HeN and C57BL/6 mice showed that 3H1 specifically bound to MH134 tumor cells, whereas 7C2, 11G2, and 12A2 reacted not only with MH134 but also with MM102 and MM46 tumor cells. None of these monoclonal antibodies bound either to cells of MM48 or X5563 tumor lines or to normal lymph node cells. These results strongly suggest that MH134 tumor cells display at least two kinds of tumor-associated antigens on their cell surfaces: one is expressed uniquely by MH134 tumor cells, which are recognized by 3H1; the other is commonly shared by MH134, MM102, and MM46 tumor cells, which are determined by the other three antibodies. 3H1, 11G2, and 12A2 but not 7C2 were found to be able to induce antibody-dependent cellular cytotoxicity (ADCC) against MH134 tumor cells. Target specificity of ADCC induced by these monoclonal antibodies was identical with that seen in enzyme-linked immunosorbent assay. 3H1, 7C2, and 12A2 but not 11G2 exhibited complement-dependent cytotoxicity, showing the same specificity in target cell lysis as that seen in enzyme-linked immunosorbent assay or ADCC. Pretreatment of MH134 tumor cells with 7C2 inhibited ADCC of both 11G2 and 12A2. Pretreatment of the tumor cells with 11G2 inhibited complement-dependent cytotoxicity of both 7C2 and 12A2. Neither ADCC nor complement-dependent cytotoxicity of 3H1 was inhibited by the pretreatment of the cells with 7C2 or 11G2. These results strongly suggest that tumor-associated antigens recognized by 3H1 are located apart from that recognized by 7C2, 11G2, and 12A2 and that the binding sites of the latter three antibodies are closely associated with, or identical with, each other in the tumor-associated antigen. The ability of 12A2 to induce ADCC against MH134 tumor cells was significantly stronger than that of 3H1 or 11G2.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Mechanisms for Recognition of Tumor Antigens and Mediation of Anti-tumor Effect by Noncytolytic Lyt-2+ T Cell Subset*1

The mode of anti-tumor function in vivo of noncytolytic Lyt-2⁺ T cells from C3H/He mice hyperimmune to syngeneic MH134 hepatoma was investigated in a double diffusion chamber system which was recently established in our laboratory. C3H/He mice were implanted intraperitoneally with the double diffusion chamber unit in which each chamber contained either L3T4⁺ T cell-depleted MH134-hyperimmune spleen cells plus mitomycin C-treated MH134 tumor cells or other syngeneic X5563 viable tumor cells plus normal spleen cells as a source of macrophages. Inclusion of anti-MH134 Lyt-2⁺ T cells together with MH134 tumor cells in one chamber resulted in comparable growth inhibition of viable X5563 tumor cells in the other chamber to that obtained by unfractionated MH134-hyperimmune spleen cells. The induction in the Lyt-2⁺ T cell-containing chamber of anti-tumor effect to be delivered into the other chamber was dependent on the co-existence of la-positive adherent cells along with Lyt-2⁺ T cells. Although adherent cell-depleted Lyt-2⁺ T cells regained the inducibility of anti-tumor immunity when supplemented with splenic adherent cells, the addition of adherent cells pretreated with chloroquine failed to restore the ability of Lyt-2⁺ T cells to induce their anti-tumor effect. In addition, paraformaldehyde-treated MH134 tumor cells instead of untreated tumor cells were not capable of activating Lyt-2⁺ T cells. These results indicate that a portion of Lyt-2⁺ T cells exerts their anti-tumor effect by a mechanism distinct from direct tumor cell lysis and that their activation for mediation of this type of tumor immunity requires the recognition of tumor antigens processed and presented by la-positive adherent cells.     

Immunological studies on mouse mammary tumors. II. Characterization of tumor-specific antibodies against mouse mammary tumors

After injection of isografted mammary tumor MM2 sensitized with rabbit antiserum, resistance was induced in C3H/He mice after repeated challenges with MM2. The serum taken from these mice was found to be cytotoxic against MM2 cells. The serum also inhibited the outgrowth of transplant of primary tissue culture cells of spontaneous mammary tumor of C3H/He mice. A series of transplanted mammary tumors recently converted into ascitic form in our laboratory (MM3, MM4-1, MM4-2, MM4-3, MM6, MM8, MM9, MM11, MM12 and MM13), and Ehrlich ascites tumor were found to be susceptible to this serum, as tested by trypan blue uptake in vitro. Outgrowth of these tumors was also inhibited when tumor premixed with this serum was injected. No cytotoxic effect was observed against normal mouse mammary gland cells of a C3H/He mouse. Sera obtained from hyperimmunized syngeneic C3H/He mice were able to fix complement with MM2 tumors. They were partially inactivated by heating at 56° C and by treatment with 2-mercaptoethanol. After gel filtration through Sephadex G-200, complement fixing and cytotoxic activity were found in both 19S and 7S fractions. The 7S fractions, after DEAE cellulose column chromatography, gave a precipititin line at the IgG position in immunoelectrophoresis. From the above evidence, it is concluded that the target cells of the cytotoxic factors of this serum are primary cultured cells or isografted cells of mammary tumors of C3H/He mice. The cytotoxic factors present in the serum are considered to be antibodies against isografts of mammary tumors in C3H/He mice.     

Effect of withaferin A on ehrlich ascites tumor cells. II. Target tumor cell destruction in vivo by immune activation

Intraperitoneal administration of Withaferin A 24 h after inoculation of Ehrlich ascites carcinoma resulted in an immediate inhibition of tumor growth, followed by complete disappearance of the malignant cells after 3-4 days. During this period a striking proliferation of macrophages in the peritoneal cavity and clustering of viable acid-phosphatase-rich macrophages around Ehrlich ascites tumor cells was observed. In addition, giant cell formation in the peritoneal cavity of the tumor-rejecting mice was commonly seen. No significant difference was found in the number of lymphocytes and polymorphonuclears of the peritoneal exudate of the tumor-rejecting mice as compared to untreated tumor-bearing mice. A similar sequence of changes with an even more striking proliferation of macrophages terminating again in complete tumor regression was observed after reinoculation of Ehrlich ascites cells to Withaferin A cured mice. Growth of Ehrlich ascites tumor cells in normal mice was prevented by passive transfer of serum or peritoneal cells, but not by spleen cells taken from immune mice. Humoral antibodies in the serum of immune mice were demonstrated by the complement fixation, the passive cutaneous anaphylaxis, and the cytotoxic tests. One-day-old suckling litters born to immune mothers were also refractory to Ehrlich ascites implantation. However, Ehrlich ascites cell growth was not affected if the litters were injected previously with a cell-free tumor homogenate.     

Anti-tumor activity of ceramides and glycosphingolipids in a murine tumor system

The anti-tumor activity of 7 sphingolipids, 2 ceramides and 5 glycosphingolipids against the syngeneic murine ascitic tumors MH 134 and MM 102 in C3H mice was examined. Five of these compounds showed anti-tumor activity against the tumors, ceramide type-IV (Cer-lv) having the highest activity without cytotoxic or cytostatic activity. These results indicate that the fatty acid in ceramide and sugar chains binding to it affect the anti-tumor activity in vivo. The anti-tumor activity of Cer-IV depended on the time of treatment. Mice treated with Cer-IV one day after tumor implantation showed the highest rate of survival. The cured mice were resistant to rechallenge with the same tumor (MH 134 → MH134, MM102 → MM102) but not with a heterologous tumor (MH 134 → X5563, MM102 → X5563), indicating that the effect of Cer-IV may be due to in vivo induction of specific immunity. Studies with various antibodies demonstrated that the anti-tumor effect of Cer-IV was inhibited by all the antibodies tested (L3T4, Lyt-2, and Thy-1.2 T cells, macrophages, and TNFα) in the induction phase (before Cer-IV administration) and by the antibodies of L3T4 and TNFα in the effector phase (after Cer-IV administration). Therefore, the anti-tumor effect of Cer-IV in this system depended on the host immune response rather than on its direct cytotoxic and/or cytostatic action.     

Tumor growth and food intake in interleukin-6 gene knock-out mice.

Interleukin (IL)-6 has potential antitumor activity and at the same time is responsible for tumor-derived cachexia. Using IL-6 gene knock-out (GKO) mice, we investigated the effect of IL-6 deficiency on the survival time, tumor growth and daily food intake of mice inoculated with Ehrlich ascites carcinoma. IL-6 GKO mice gained weight due to tumor growth more rapidly than the wild-type mice. The daily food intake of wild-type mice declined on day 2 after tumor inoculation and was only 37% on day 8. In contrast, the daily food intake of IL-6 GKO mice was constant for the first 7 days after tumor inoculation. Although wild-type mice suffered from cachexia, their survival time was significantly longer than that of IL-6 GKO mice. We propose that both IL-6 secretion and cancer cachexia syndrome may be involved in the defense mechanism against tumor progression.     

Antitumor effect of 6-phenyl-7(6H)-isoselenazolo[4,3-d]pyrimidone on the growth of Ehrlich ascites tumor

Two selenium compounds have a strong antitumor effect on Ehrlich ascites tumor; 6-phenyl-7(6H)-isoselenazolo[4,3-d]pyrimidone (ISP) especially, markedly inhibited the growth of tumor inoculated i.p. in mice, inducing almost complete regression of tumors at doses of 100 micrograms/mouse per day X 10 with no sign of toxicity. The antitumor activity of 4,5-dihydro-4-methyl-6-oxo-5-phenyl-6H-pyrazolo [4,5-c]isoselenazole(PIS) was weaker than that of ISP. The total lipid and phospholipid contents in the tumor cells treated with ISP were significantly decreased. In addition, the fatty acid pattern of cholesterol esters, phosphatidyl choline and phosphatidyl ethanolamine from the ISP-treated Ehrlich ascites tumor cells differed markedly from that of the corresponding lipids from the control tumor cells.     

Antitumor activity of murine tumor necrosis factor (TNF) against transplanted murine tumors and heterotransplanted human tumors in nude mice

The antitumor activity of highly purified tumor necrosis factor (TNF) was tested against eight kinds of murine tumor and five kinds of human tumor heterotransplanted into nude mice. Mice were treated by intravenous or intratumoral injection of TNF, commencing when the tumors were well established. TNF showed an excellent curative effect against all kinds of murine and human tumors tested. Meth A sarcoma, Colon 26, Ehrlich, sarcoma 180, MM 46, MH 134, B16 melanoma, and Lewis lung tumors transplanted into mice underwent tumor necrosis and regression following a single injection of TNF. Sometimes a complete cure was observed in Meth A sarcoma, sarcoma 180, Ehrlich, and MM 46 tumors. Human cancers, SEKI, HMV-I, KATO-III, MKN 45, or KB, heterotransplanted into nude mice, also exhibited tumor necrosis and regression in size following several intratumoral injections of TNF. A great difference in curative effects of TNF was observed in Meth A sarcomas between those transplanted into BALB/c nu/+ and into BALB/c nu/nu mice: following a single intravenous administration the effect was stronger in BALB/c nu/+ than in nu/nu mice. In contrast, tumor necrosis was almost the same in nu/+ and nu/nu mice following intratumoral administration. The present results thus indicate that TNF from mice had an antitumor activity against not only murine tumors but also human tumors. In addition to direct cytotoxicity against tumor cells, TNF induced a host-mediated factor which contributed to the antitumor effects.     

Therapy of murine liver metastases by administration of MDP encapsulated in liposomes

In a reproducible murine model of liver metastases, it was demonstrated that liposomal muramyl dipeptide (MDP) as an adjuvant therapy reduces and prevents the development of metastases. C26 colon adenocarcinoma cells were injected into the spleen (5 x 10(4) cells per mouse) of syngeneic BALB/c mice. On day 3, the spleen was removed to prevent a large tumor burden in the spleen. On day 17, 100% of the mice had developed tumor foci in the liver. Liposomal MDP treatment consisted of the i.v. or i.p. administration of 1 mumol of liposomal lipid containing 5 micrograms of MDP per mouse for ten consecutive days. When therapy was initiated two days after tumor cell inoculation, the number of metastases that had developed on day 17 was strongly reduced compared to control mice. Approximately 20% of the mice were free of liver metastases. Initiation of therapy two days prior to tumor cell inoculation enhanced the effect significantly: about 45% of the mice were free of metastases on day 17. The treatment protocol for survival studies was slightly different; liposomal MDP was administered on the first six consecutive days followed by administration twice weekly, through day 24. Control mice died between day 21 and 33 after tumor cell inoculation, whereas liposomal MDP treated mice died between day 26 and 46 with 1 out of 25 mice surviving for more than 120 days. The mortality of the liposomal MDP treated mice that were free of liver metastases was caused by a local tumor at the site of operation.(ABSTRACT TRUNCATED AT 250 WORDS)