Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. VI. Adjuvant therapy after surgery in the inhibition of liver and spleen metastases

Adult DBA/2 mice were injected s.c. with the highly malignant, interferon-resistant 3C18 line of Friend erythroleukemia cells (FLC). Eight or 9 days after established s.c. tumors had developed, the primary tumor was excised and mice were treated i.p. with either mouse interferon alpha/beta or a control preparation. At the time of surgery, mice already had tumor cells in the liver. All control-treated mice died in the ensuing 2 weeks with extensive tumor metastases in the liver and spleen. Interferon treatment resulted in an inhibition of the development of liver and spleen metastases and a markedly increased survival time. We conclude that interferon alpha/beta is effective as adjuvant therapy after surgery for metastatic disease in mice.     

Effectiveness of mouse interferon alpha/beta compared to single-agent chemotherapy in increasing survival time of mice after intravenous inoculation of Friend erythroleukemia cells

DBA/2 mice received an iv injection of 2 X 10(6) Friend erythroleukemia cells (FLCs; approximately equal to 4 X 10(5) lethal dose50), which multiplied rapidly in the liver and spleen and killed all untreated or control treated mice between 7 and 12 days. Daily interferon (IFN) treatment resulted in a very marked increase in survival time and apparent cure of 4 of 22 tumor-inoculated mice. In contrast, treatment of tumor-injected (iv) mice with cyclophosphamide, 5-fluorouracil, and methotrexate increased survival time by only a few days; and treatment of mice with cisplatin, vincristine, doxorubicin, bleomycin, or etoposide was ineffective. However, when FLCs were injected ip, both cytostatic drugs and IFN exerted an antitumor effect. We conclude that IFN alpha/beta was particularly effective in inhibiting the development of liver and spleen metastases and in increasing mouse survival time after iv inoculation of FLCs.     

Biologic and biochemical differences between in vitro and in vivo passaged Friend erythroleukemia cells. I. Tumorigenicity and capacity to metastasize

Cloned interferon-sensitive (745) and interferon-resistant (3Cl-8) Friend erythroleukemia cells (FLC) passaged in vitro, are not very tumorigenic when first injected intraperitoneally (i.p.) into syngeneic DBA/2 mice although they do form solid tumors when injected subcutaneously (s.c.). By serially passaging FLC (either 745 or 3Cl-8 cells) i.p. in DBA/2 mice, we obtained two different FLC lines capable of growing i.p. and inducing tumor ascites. The s.c. injection of DBA/2 mice with these in vivo passaged FLC resulted in tumor metastases in the liver and spleen, whereas metastases were not observed in mice inoculated s.c. with in vitro passaged FLC. The capacity of in vivo passaged FLC to metastasize was acquired after several i.p. passages. This highly malignant behavior was a stable characteristic of these cells. All the clones derived from in vivo passaged FLC and passaged more than 14 times in vitro induced hemorrhagic ascites when injected i.p., and metastasized to the liver and spleen when injected s.c. The phenotype of sensitivity or resistance to the inhibitory effect of alpha/beta mouse interferon on virus replication and cell multiplication was conserved during serial i.p. passages and maintained in the clones derived from in vivo passaged cells. These FLC showed a decreased capacity to differentiate in vitro upon treatment with dimethylsulfoxide (DMSO) and a reduced production of Friend leukemia virus with respect to the original clones passaged in vitro.     

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)     

Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. V. Comparisons with the action of tumor necrosis factor

A number of similarities and dissimilarities in the anti-tumor effects of TNF and interferon alpha/beta have been observed in DBA/2 mice injected with Friend erythroleukemia cells (FLC). Mouse TNF exerted marked anti-tumor effects in mice injected either s.c. or i.p. with FLC lines 3C18 or 3 gamma R8 resistant in vitro to the cytotoxic effects of TNF. Likewise, mouse interferon alpha/beta had anti-tumor activity in mice injected with these FLC, resistant to the action of interferon alpha/beta or gamma in vitro. The results of histopathologic examination and 31P nuclear magnetic resonance analyses of 3C18 FLC s.c. tumors injected with TNF resembled the results previously obtained for 3C18 FLC tumors injected with interferon alpha/beta, although the effects of TNF occurred more rapidly. Injection of mice with antibody to mouse interferon alpha/beta or gamma did not abrogate the anti-tumor effects of TNF in mice injected i.p. with FLC. Our results suggest that in this experimental system the anti-tumor effects of TNF, like interferon alpha/beta, do not result from a direct effect on the tumor cells themselves but are host-mediated.     

Importance of interferon alpha in the resistance of allogeneic C57B1/6 mice to the multiplication of Friend erythroleukemia cells in the liver

Friend erythroleukemia cells (FLC) (H-2d) injected intravenously multiply extensively in the livers of syngeneic DBA/2 mice and not at all in the livers of allogeneic C57B1/6 mice. Our results indicate that interferon alpha (IFN-alpha) is an important factor in the resistance of allogeneic mice to the multiplication of FLC in the liver. (a) After i.v. inoculation of FLC there was an inverse correlation between the presence of IFN-alpha in the serum and the capacity of FLC to multiply in the liver. Thus, all 44 FLC-injected adult C57B1/6 mice had circulating IFN-alpha and FLC did not multiply in the liver of any of the mice. Interferon was not detected in the serum of 83% of 41 FLC-injected DBA/2 mice (and was found only at a low titer in 17% of the mice) and FLC multiplied in the liver of all mice. (b) FLC did multiply in the livers of newborn C57B1/6 mice and in the livers of irradiated adult C57B1/6 mice, and IFN-alpha was not detected in their sera. In contrast, after i.v. inoculation of FLC, IFN-alpha was detected in the sera of 3-week-old and athymic nu/nu adult C57B1/6 mice while FLC failed to multiply in the liver. (c) FLC also induced IFN-alpha in congenic B10.D2 (H-2d) mice and FLC did not multiply in the liver. We suggest that, depending on the site of tumor implantation, different host mechanisms have various degrees of importance in controlling the growth and/or rejection of allogeneic tumor cells, and that IFN-alpha is particularly important when FLC are injected i.v.     

Antitumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. I

Interferon-sensitive (745) and interferon-resistant (3C1-8) Friend leukemia cells (FLC) are highly tumorigenic for DBA/2 mice. The phenotype of interferon sensitivity or resistance does not change with in vivo passage. Daily administration of mouse interferon markedly enhanced the survival time of mice injected with either 745 or 3C1-8 cells. Use of quantitative methods for determining the number of FLC (colony formation in agarose and immunofluorescence) permitted us to show that potent, partially purified or highly purified mouse interferon (s.a. 0.5 to 1 X 10(9) u/mg protein) induced a 100- to 1,000-fold decrease in the number of tumor cells in the peritoneal cavity in the days following inoculation of 745 or 3C1-8 cells. Interferon decreased the number of FLC even when treatment was initiated at a time when tumor cells were multiplying exponentially in the peritoneal cavity. There was no evidence that interferon acted as an inducer of FLC differentiation in vivo. The finding that interferon was equally effective in mice inoculated with interferon-resistant cells as in mice inoculated with interferon-sensitive cells suggests that in this experimental system interferon does not act directly on the tumor cells, but that the interferon-induced antitumor activity is mediated by the host.     

Antitumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. III. Inhibition of growth and necrosis of tumors implanted subcutaneously

Administration of highly purified interferon to DBA/2 mice inhibited the growth of interferon-sensitive or interferon-resistant Friend erythroleukemia cells implanted subcutaneously. Injection of interferon at the site of tumor inoculation was more effective than injection of interferon intraperitoneally. Histologic examination of tumors in interferon-treated mice showed extensive areas of tumor-cell necrosis in the absence of an obvious host-cell infiltrate. Interferon inhibited the growth of established subcutaneous tumors and induced complete tumor regression in some mice. Interferon treatment also resulted in an inhibition of tumor metastases in the liver and spleen.     

Interaction of IFN α/β with host cells essential to the early inhibition of friend erythroleukemia visceral metastases in mice

We have previously shown that an intact immune system was essential to the increase in survival time of IFN-α/β-treated mice injected i.v. with an IFN-α/β-resistant line of Friend erythroleukemia cells (FLC) highly metastatic to the liver and spleen. Here, we have investigated the early interactions of IFN α/β with host cells prior to the development of the immune response. IFN α/β treatment resulted in 50- to 100-fold inhibition of FLC multiplication in the liver and spleen of normal DBA/2 mice shortly after tumor inoculation, as evaluated by colony formation in agarose. IFN treatment was far less effective in inhibiting the multiplication of FLC in the livers of NK-cell-deficient DBA/2 beige mice, or in immunocompetent DBA/2 mice treated with antibody to asialo GMI, or silica, or in mice subjected to sub-lethal irradiation. Injection of antibody to CD4 or CD5 did not affect the early inhibitory action of IFN α/β on FLC multiplication but did decrease survival time. Light- and electron-microscope examination of the livers of IFN-treated, FLC-injected mice confirmed the early inhibition of FLC multiplication in the liver and spleen. Our results indicate that IFN α/β inhibits the development of FLC visceral metastases by acting first on host cells, such as NK cells and macrophages, and then continues to act in consort with the developing immune response. © 1994 Wiley-Liss, Inc.     

Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant friend leukemia cells. IV. Definition of optimal treatment regimens

Mouse interferon alpha/beta exerted a similar anti-tumor effect in DBA/2 mice injected i.p. with Friend erythroleukemia cells (FLC) either sensitive or resistant to interferon as determined by both in vitro and in vivo assays. Using this tumor system we attempted to define optimal treatment regimens for interferon administration. Interferon was most effective when injected at the site of tumor inoculation rather than at a distant site. Two factors seemed of especial importance: the amount of interferon injected and the frequency of interferon administration. Thus, for daily administration of interferon, the antitumor effect was directly related to the amount of interferon injected. For a given total dose of interferon, repeated administration of small doses of interferon was more effective than administration of a larger dose at more widely spaced intervals. The anti-tumor efficacy of interferon was independent of the number of FLC inoculated when 10(2) to 10(5) FLC were injected, but interferon treatment was less effective when 10(6) or 10(7) FLC were injected. The relevance of these results to the use of interferon in patients with cancer is discussed.     

Antitumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. II. Role of host mechanisms

We have attempted to determine what host mechanisms are responsible for inducing a rapid decrease in the number of Friend leukemia cells (FLC) in the peritoneal cavity of interferon-treated mice. By injecting radiolabelled FLC, we showed that there was a greater loss of radioactivity from individual interferon-treated mice than from control mice. Thus, it was likely that fewer cells were recovered from the peritoneal cavity of interferon-treated mice because of cell destruction. Treatment of mice with interferon limited to the period preceding tumor-cell inoculation conferred some degree of antitumor activity, although this regimen was far less effective than when interferon treatment was initiated and continued daily after tumor-cell inoculation. We have been unable to transfer any antitumor activity with peritoneal washings containing macrophages and lymphocytes from interferon-treated donor mice to tumor-inoculated recipient mice. Inoculation of silica particles i.p., which destroys macrophage function and may affect NK cell activity, did not abrogate interferon's antitumor activity. We suggest that interferon induces a host-mediated antitumor effect by mechanisms which are not mediated by easily recoverable soluble factors or by cytotoxic cells. The nature of this potent interferon-induced host mechanism remains unknown.     

Cloning, sequence analysis and expression of the cDNAs encoding the canine and equine homologues of the mouse double minute 2 (mdm2) proto-oncogene

The immunomodulatory and antimetastatic/antitumor activity of thymosin alpha(1) (Talpha(1)) was evaluated in BALB/c-mice. Daily subcutaneous application (7 consecutive days, 0.01-10 microg of Talpha(1)/injection per mouse) upregulated the number of thymocytes and peripheral blood cells in tumor bearing mice. To check the influence of Talpha(1) treatment on growth of experimental metastases, RAW H10 lymphosarcoma cells or L-1 sarcoma cells were intravenously injected into BALB/c-mice to establish liver or lung metastases. Local tumor growth was induced by subcutaneous injection of L-1 sarcoma cells. Talpha(1) was subcutaneously administered daily for 7 consecutive days starting 24 h after tumor cell challenge. Organ colonization, as well as local tumor growth, were investigated on day 14 after tumor cell inoculation, and demonstrated a statistically significant (P<0.05) reduction of experimental liver and lung metastases and local tumor growth for Talpha(1) treated mice.     

31P-nuclear magnetic resonance analysis of interferon-induced alterations of phospholipid metabolites in interferon-sensitive and interferon-resistant Friend leukemia cell tumors in mice

Adult DBA/2 mice were given injections s.c. with either interferon-sensitive (745) or -resistant (3Cl-8) Friend erythroleukemia cells (FLC). After tumor nodules had developed, mouse interferon-alpha/beta was injected daily into the tumor. 31P-Nuclear magnetic resonance (NMR) spectroscopy examinations were undertaken on freshly dissected tumors at different days of treatment with either interferon or control preparations. Analysis of 745 FLC tumors in untreated mice at different days of tumor growth (day 8 to 13 after tumor implantation) showed marked increases in the levels of phosphorylcholine (PCho), glycerophosphorylethanolamine (GroPEtn) and glycerophosphorylcholine (GroPCho). In contrast high levels of PCho, GroPEtn and GroPCho were already detectable in the 3Cl-8 FLC tumors on day 8, and no significant changes were observed during subsequent tumor growth. The intracellular pH value remained practically constant in both FLC tumors. Daily intratumoral administration of either partially purified (10(7) IU/mg of protein) or highly purified (10(9) IU/mg of protein) mouse interferon-alpha/beta to both cell tumors resulted in decreases in the levels of PCho, GroPEtn and GroPCho and in increases in the intracellular pH with respect to tumors treated with control preparations or left untreated. Two days of daily treatment of mice with interferon sufficed to induce these metabolic changes which preceded the appearance of necrosis in the tumors. Treatment of FLC tumors with X-rays on day 12 of tumor growth did not result in any comparable metabolic changes 2 days after irradiation. Changes in the levels of phospholipid metabolites were not observed when 745 or 3Cl-8 cells were cultivated in the presence of interferon. As interferon induced these changes in both interferon-sensitive and -resistant tumors we conclude that interferon treatment results in host-mediated effects on the biosynthesis and/or catabolism of tumor cell phospholipids.     

Microvascular injury in pathogenesis of interferon-induced necrosis of subcutaneous tumors in mice

DBA/2 mice were injected sc with cells from the highly malignant Friend erythroleukemia cell (FLC) 3Cl8 subline, which is resistant to mouse interferon alpha/beta, or with the ESb lymphoma. When interferon alpha/beta was injected intratumorally or peritumorally, tumor growth was markedly suppressed, and established vascularized tumor nodules became progressively necrotic. Tumor necrosis was of the coagulation type that usually results from deprivation of blood flow. Morphologic examination of approximately 1,000 blood vessel profiles and approximately 2,000 endothelial cells in 1-micron Epon sections of sc 3C18 FLC tumors showed that interferon treatment resulted in rapid and pronounced vascular endothelial cell damage that preceded tumor necrosis. No inflammatory cell infiltrate was observed. Our results suggest that interferon alpha/beta exerted an antitumor effect in these tumor models by damaging tumor blood vessels, causing disruption of tumor blood flow, which led to ischemic tumor necrosis.     

Anti-tumor effects of interleukin-2 and interleukin-1 in mice transplanted with different syngeneic tumors

We have studied the anti-tumor effects of human recombinant IL-2, alone or in association with LAK cells, in mice transplanted subcutaneously (s.c.) with the following syngeneic tumors: highly metastatic Friend leukemia cells (FLC), nonmetastatic FLC, lymphoma RBL-5 cells and HeJ16 fibrosarcoma cells. In these tumor models, peri-tumoral injections of IL-2 were more effective in inhibiting tumor growth than a systemic treatment. Although s.c. IL-2 treatment resulted in marked inhibition of tumor growth in mice injected s.c. with highly metastatic FLC, it was not effective in inhibiting growth of FLC in the liver and spleen. IL-2 therapy was more effective at increasing survival time in mice transplanted with non-metastatic FLC or with RBL-5 cells. In mice transplanted with HeJ16 fibrosarcomas, s.c. IL-2 treatment resulted in highly significant anti-tumor effect and survival of 70% of tumor-injected mice. No general correlation was found between in vitro sensitivity or resistance to the cytolytic activity of LAK cells and the anti-tumor effects observed in vivo. Subcutaneous injection of IL-1 beta in mice transplanted with highly metastatic FLC resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. Combined treatment of IL-1 beta and IL-2 produced a synergistic anti-tumor effect: 60% of mice injected with highly metastatic FLC survived. Combined IL-1/IL-2 treatments exerted no anti-tumor activity either in DBA/2 mice injected with antibody to Thy 1.2 antigen or in nude mice, indicating that T cells play important roles during IL-1/IL-2 therapy. In vitro treatment of FLC with IL-1 beta resulted in a slight inhibition of cell multiplication, whereas even high doses of IL-2 did not affect FLC multiplication. Our results indicate that local combined treatments with IL-1 and IL-2 can induce potent, host-dependent (T cell-mediated) anti-tumor effects against highly malignant tumors.     

Inhibition of mouse alpha/beta-interferon of the multiplication of alpha/beta-interferon-resistant Friend erythroleukemia cells cocultured with mouse hepatocytes

Administration of alpha/beta-interferon (IFN) exerts a marked antitumor effect in DBA/2 mice given injections i.v. of large numbers of IFN-alpha/beta-resistant erythroleukemia cells (FLC). To investigate the possible mechanisms of FLC tumor inhibition in the liver of interferon-treated mice, we developed an in vitro model consisting of a coculture of IFN-alpha/beta-resistant 3Cl8 FLC and syngeneic mouse hepatocytes. Whereas IFN-alpha/beta did not inhibit the multiplication of 3Cl8 FLC cultivated alone, it effectively inhibited the multiplication of 3Cl8 FLC in coculture with hepatocytes. The inhibitory effect was directly proportional to the amount of IFN-alpha/beta added to the cocultures, and more than 90% inhibition of FLC multiplication was noted with 1.6 x 10(5) IU/ml of IFN-alpha/beta on Day 3 of coculture. When FLC were separated from the monolayer of hepatocytes by a pored membrane (0.4 microns), the inhibitory effect on FLC proliferation was unchanged, indicating that a soluble factor(s) released from IFN-treated hepatocytes was most important in the inhibition of FLC multiplication. An inhibitory activity of FLC multiplication was detected only in the conditioned medium of IFN-treated hepatocytes but not in the conditioned medium of control hepatocytes nor in extracts of IFN-treated or control hepatocytes. The inhibitory factor(s) in the conditioned medium of IFN-treated hepatocytes was retained by an ultrafiltration membrane (Mr cut off 10,000), and its activity was completely abrogated by trypsin digestion. Its stability to treatment with 1 M acetic acid as well as lack of correlation between the antiproliferative effect and the amount of L-arginine in the medium distinguished this factor(s) from liver arginase which was also found to be a potent inhibitor of FLC multiplication in vitro. The inhibitory factor(s) was also distinguishable in its biological activity from IFN gamma, interleukin 1 alpha and beta, and transforming growth factor beta 1 and beta 2. These results suggest the possibility that the inhibitory effect of IFN-alpha/beta on the development of 3Cl8 FLC in the livers of IFN-treated mice may be mediated by an IFN-induced inhibitor of FLC multiplication.     

Sensitized T lymphocytes render DBA/2 beige mice responsive to IFN α/β therapy of friend erythroleukemia visceral metastases

Interferon α/β (IFN α/β) is highly effective in inhibiting the development of Friend erythroleukemia cell (FLC) visceral metastases in DBA/2 mice injected intravenously (i.v.) with FLC, but does not protect FLC-injected DBA/2 beige (bg/bg) mice. Use of IFN α/β-resistant FLC indicated that IFN was acting through host mechanisms in DBA/2 mice and thus pointed to a defect in some host mechanism in bg/bg mice essential for IFN's anti-metastatic action. We undertook experiments to restore in bg/bg mice the marked anti-FLC meta-static effect of IFN a/p observed in DBA/2 and +/bg mice. Adoptive transfer of spleen cells from normal syngeneic mice to IFN-treated bg/bg mice was ineffective, but the transfer of splenic T lymphocytes from FLC-immunized DBA/2 or +/bg mice markedly increased the survival time of FLC-injected bg/bg mice provided that these mice were also treated with IFN a/p. Neither treatment alone resulted in an increase in survival time. As few as 1 × 10⁷ immune spleen cells were effective in IFN-treated FLC-injected bg/bg mice. The T-cell immune response to FLC of bg/bg mice was diminished compared with that of +/bg mice. Likewise, only combination therapy of immune spleen cells and IFN α/β resulted in an increased survival time of ESb-lymphoma-injected bg/bg mice. Our results indicate the essential participation both of T-cell-mediated immune mechanisms and of IFN α/βin the inhibition of FLC visceral metastases.     

Synergistic anti-tumor effects of combined IL-1/IFN-alpha/beta therapy in mice injected with metastatic Friend erythroleukemia cells

Peritumoral injection of relatively low doses of either mouse interferon (IFN)-alpha/beta (10,000-20,000 units/injection) or of recombinant human interleukin-1 (IL-1) beta (125-250 ng/injection) in mice transplanted s.c. with Friend erythroleukemia cells (FLC) resulted in some inhibition of primary tumor growth, inhibition of liver and splenic metastases and increased survival time. A synergistic anti-tumor effect was observed in mice injected with both IL-1 and IFN-alpha/beta. Highly purified mouse IFN-beta also exerted a synergistic anti-tumor effect when combined with IL-1-beta in mice injected with FLC. The anti-tumor action of IL-1/IFN was markedly reduced in mice treated with antibodies to CD4 antigens. Antibodies to asialo-GM1 also diminished the anti-tumor effect by the combined cytokine treatment. The combined IL-1/IFN therapy was effective in NK-deficient bg/bg mice, although the extent of the anti-tumor response in these mice was less than that observed in bg/+mice.     

Antimetastatic effect of biological response modifiers in the "double grafted tumor system"

The antimetastatic effect of biological response modifiers (BRM) in a new experimental mouse model was studied. Intratumoral administration of BRMs (PSK, OK-432, interferon alpha A/D) strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumors. Subsequently, the antimetastatic effect of BRMs was examined in the "double grafted tumor system," in which mice first received simultaneous intradermal inoculations of Meth-A in the right (10(6) cells) and left (2 X 10(5) cells) flanks and were then injected with BRMs in the right tumor on day 3. PSK and interferon (IFN) significantly inhibited the growth of the left (non-treated) tumor. This finding suggests that intratumoral BRM immunotherapy in one region has an effect on tumor growth in another region. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of BRMs and had rejected reinoculated tumors. One hour after intravenous injection of cyclophosphamide (2 mg/mouse), immunized spleen cells (2 X 10(7) cells/mouse) were injected into the Meth-A tumor on day 3. Adoptive transfers of PSK and IFN immunized spleen cells caused the complete regression of Meth-A tumors. These results suggest that the intratumoral administration of BRMs might induce cytotoxic cells in the left non-treated tumor of the "double grafted tumor system" and bring about the regression of metastatic tumors.