M5076 Tumor Research Articles

Immunomodulation in an apparently non-immunogenic murine tumor

Madison lung carcinoma (M109), a murine tumor of spontaneous origin, appears to be non-immunogenic, according to 2 commonly employed tests for tumor immunogenicity. However, C.parvum-induced immunopotentiation during the growth of M109 tumor results in post-excision anti-tumor immunity to M109 tumor implants. The C.parvum-potentiated post-excision immunity to M109 is tumor-specific and T-cell-dependent. T cells from mice whose progressive M109 tumors have been excised are capable, on passive transfer, of inhibiting adoptive immunotherapy of T-cell-deficient recipients by spleen cells from mice immunized with an admixture of M109 cells and C.parvum. The data are interpreted as evidence supporting the hypothesis that the apparent lack of anti-tumor immunity in this tumor model is not due to the absence of tumor-associated antigens. We suggest that, instead, in this model the balance between the effector and suppressor arms of the immune response favors tumor-induced immunosuppression, resulting in a magnitude of anti-tumor immunity insufficient for detection by commonly employed tests for tumor immunogenicity. Our study shows that shifting the balance in favor of the effector arm by means of immunopotentiation results in a measurable immune response to an apparently non-immunogenic tumor.

Liposomal muramyl dipeptide therapy of experimental M5076 liver metastases in mice.

The effectiveness of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) or of liposomes containing a lipophilic MDP derivative, MDP-glyceroyldipalmitate MDP-GDP in inhibiting the growth of M5076 reticulum cell sarcoma liver metastases in C57BL/6 mice has been determined. MDP (100 micrograms) or liposomal MDP-GDP (2.5 mumol containing 1 microgram) were equally effective in inhibiting liver metastatic growth when given as a single treatment 3 days before tumor cell injection. Therapeutic treatment, initiated 3 days after tumor cell injection and continued for a period of 2 weeks, failed to inhibit metastatic growth. Activation of thioglycollate-elicited peritoneal macrophages or Kupffer cells in vitro with MDP or liposomal MDP-GDP resulted in the expression of tumoricidal activity against M5076 tumor cells. Adoptive cellular therapy with four injections of 2 x 10(6) macrophages was ineffective: activation of the macrophages with either MDP or liposomal MDP-GDP prior to injection was effective in inhibiting liver metastatic growth. Incorporation of the macrophage toxin dichlorodimethylene diphosphonate within liposomes containing MDP-GDP abolished the ability of such liposomes to induce macrophage or Kupffer cell tumoricidal activity in vitro as well as the antitumor activity when administered 3 days before tumor cell challenge.

Antitumor and DNA-binding properties of a group of oligomeric complexes of platinum(II) and platinum(IV)

The antitumor and DNA-binding properties of a group of oligomeric platinum(II) and platinum(IV) complexes are described. The compounds, having the stoichiometry [cis-PtII(X)2(mu-OH)]2(NO3)2, where X is NH3, NH2CH2CH3, and NH2CH(CH3)2, were found to be inactive or only weakly active against L-1210 leukemia. In vitro studies involving PM2-DNA show that these compounds bind to and unwind closed circular DNA in a manner similar to cis-PtII-(NH3)2Cl2. The Pt(IV) complexes produced by hydrogen peroxide oxidation of the Pt(II) dimers are inactive as antitumor agents and are incapable of unwinding PM2-DNA. The cyclotrimer [cis-PtII(RR-DACH)(mu-OH)]3(NO3)3, where RR-DACH is (R,R)-1,2 diaminocyclohexane, exhibits potent antitumor activity against L-1210 leukemia and modest activities with B-16 and M5076 tumor lines. This compound platinates DNA, causing DNA unwinding and mobility shifts.

Protein kinase C levels and protein phosphorylation associated with inhibition of proliferation in a murine macrophage tumor

Treatment of M5076 tumor cells with the phorbol estes 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13 dibutyrate (PdBu) inhibited cellular proliferation, whereas 1,2-dioctanoyl-glycerol (DiC8) and 1-oleoyl2-acetyl-glycerol (OAG) did not affect cell growth. Inhibition of cellular proliferation in this cell line appears to be a consequence of protein kinase C (PKC) down-regulation since phorbol esters, but not a single application of diacylglycerols (DGs) down-regulated cellular PKC levels. By repeated application of DGs, PKC down-regulation was achieved and correlated with inhibition of proliferation. Phorbol ester-induced PKC down-regulation was reversible, upon removal of the phorbol ester, and the reappearance of PKC was associated with resumption of proliferation. The mitogenic responsiveness of these cells to added serum depended upon cellular PKC levels. Phorbol esters also caused the phosphorylation of two proteins which were not phosphorylated in response to DG treatment. Inhibition of growth of M5076 cells appears to be associated with phosphorylation of two novel proteins and/or PKC down-regulation.

Macrophage antigens associated with adhesion: Identification by a monoclonal antibody specific for lewis lung carcinoma cells

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were strongly labeled by Mab 5B5. No binding was detected when adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11–12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.

Role of concomitant resistance in the development of murine lung metastases.

An attempt was made to explain the distinct lung metastatic patterns of 2 mammary adenocarcinomas with a common BALB/c origin: M3, which does not induce spontaneous metastases, and MM3 with an almost 100% incidence. No difference between the 2 tumors was detected with respect to host mononuclear cell content, degree of immunogenicity or lung-colony-forming ability. Conversely, there was a marked difference in the capacity to induce concomitant resistance: M3-bearing mice induced stronger and earlier resistance against i.v. challenge of both M3 and MM3 tumor cells than MM3-bearing mice; this resistance was expressed as lower number of lung metastases and lower tumor-cell proliferation in metastatic nodules. M3 was also able to control the development of spontaneous metastases: metastases developed in all M3-excised mice, compared with none in M3-bearing mice, while MM3-bearing mice also bearing a secondary M3 tumor developed fewer metastases than mice bearing MM3 only. This anti-metastatic effect does not appear to depend on classical immunological mechanisms since no difference could be detected between the 2 tumors in response to T cells, NK, macrophages or antibodies.

Tumor necrosis can facilitate the appearance of metastases.

The non-metastatic murine mammary adenocarcinoma M3 and its metastatic variant MM3 were used to evaluate the role of intratumoral necrosis in cell detachment and metastasis. Accelular extracts from necrotic areas of both tumors increased in vitro cellular detachment from M3 but not from MM3 fragments. Furthermore, the in vivo inoculation of the necrotic extracts within non-metastatic M3 tumors gave rise to pulmonary metastases. Histological studies revealed in M3 a central necrosis limited by an uninterrupted peripheral ring of well preserved cells, while in MM3 necrotic and non-necrotic areas alternated. It is concluded that the distribution of necrosis within the primary tumor by facilitating cell detachment is, at least in part, responsible for the development of metastases.

The anti‐tumor effect of recombinant interferon alpha or gamma is influenced by tumor location

The in vivo anti-tumor effects of a recombinant human hybrid interferon alpha, rHuIFN-alpha A/D, and recombinant murine interferon gamma (rMuIFN-gamma) were evaluated against experimental hepatic metastases and s.c. tumor growth of the murine reticulum cell sarcoma M5076. The 2 interferons were equally active in preventing experimental hepatic metastases. However, the interferons differed in their relative ability to influence the growth of the same tumor when treatment was initiated following injection of tumor cells. Greater efficacy was obtained in the treatment of metastatic foci in the liver with rHuIFN-alpha A/D, while rMuIFN-gamma was more active in the therapy of an s.c. growing M5076 tumor. These results demonstrate that the same tumor growing at different sites can have different relative sensitivities to IFN-alpha and IFN-gamma.

1H-NMR relaxation times and water compartmentalization in experimental tumor models

The present studies were conduced with RIF-1, M5076 and Panc02 subcutaneous tumor models to assess the relationship between tissue-free water compartmentalization and observed tissue T1 and T2 changes at 10 MHz. Observed T1 was shown to correlate directly with total extracellular water and interstitial water volumes. T1 and T2 were also inversely related to intracellular water volumes. T1 and T2 decreases after dexamethasone treatment were, however, most closely correlated with changes in tumor extracellular water and not changes in cell or total water volumes. Studies to assess Gd-DTPA-dimeg dose dependent T1 and T2 modification in model serum protein solutions indicated that although the Gd concentration that reduced T2 by 50% was about 2.5 fold greater than that required to reduce T1 equally, the of the concentration dependent T1 and T2 modifications were similar. In studies with tumor models, the injected dose of Gd-DTPA-dimeg that reduced T1 by 50% was inversely correlated with tumor extracellular water volumes. The slopes for dose dependent T1 modification in all tumors were similar and similar to that observed for model protein solutions. Gd-DTPA-dimeg had a different effect on observed T2 values for the 3 tumor models. Exponential slopes were about twice that observed for T2 modification of serum protein solutions, and Gd-DTPA-dimeg doses that reduced observed tumor T2 ranged from 9 to 50 times that necessary to similarly reduce T1. The results from these studies indicate that the observed T1, for these tumors, was dominated by relaxation of water protons in interstitial water but that the observed T2 was most strongly influenced by proton relaxation in water compartments that were unavailable to the Gd labeled probe.

The measurement of extracellular water volumes in tissues by Gadolinium modification of 1H-NMR spin lattice (T1) relaxation

The present studies were conducted in RIF-1, M5076 and Panc02 murine tumor models to compare extracellular water measurements made by 51Cr-EDTA dilution techniques and a new method which exploits the concentration dependent modification of 1H-NMR proton relaxation by Gadolinium-DTPA-dimethyl glucamine (Gd-DTPA-dimeg) in plasma and tissues. The time dependent changes in T1 modification in tissue and plasma were determined at various intervals after i.v. injection of 0.1 ml of 100 mM Gd-DTPA-dimeg and Gd concentrations determined from standard curves of Gd concentration dependent T1 modification of mouse plasma. Comparison of tissue and plasma Gd concentrations permitted the calculation of Gd-DTPA-dimeg distribution volumes. In unperturbed RIF-1, M5076 and Panc02 tumors, Gd-DTPA-dimeg distribution volumes determined by the T1 modification technique were similar to extracellular water spaces determined by 51Cr-EDTA dilution assays. In mouse liver, the 51Cr-EDTA assay resulted in artifactually high extracellular water space estimates due to internalization of the probe; Gd-DTPA-dimeg distribution volumes determined in liver with both the 153Gd-DTPA-dimeg and by the T1 modification method were approximately 150 ul/gram. The Gd-DTPA-dimeg T1 modification method also provided good approximations of changes in extracellular water volumes in RIF-1 tumors after dexamethasone and cyclophosphamide treatments. These results indicate that Gd-DTPA-dimeg modification of proton relaxation may be extremely useful for monitoring changes in tumor water dynamics during cancer therapy.

Levels of plasmatic fibronectin in mice bearing adenocarcinomas of different metastasizing ability

The levels of fibronectin (FN) were assayed in plasma of BALB c mice subcutaneously inoculated with a mammary adenocarcinoma of moderate metastatic ability (M3) and a related variant tumor with higher metastasizing potential (MM3). The mean plasmatic FN concentration increased in parallel with increased M3 and MM3 size and weight. Highest and earliest FN increases were observed in mice inoculated with the rapidly growing M3 tumor. A strong correlation between the level of plasma fibronectin and the number of lung metastases was only found in MM3 inoculated mice. Plasma fibronectin level is a good biological marker of tumoral growth rate in these adenocarcinoma tumors, but its role in the metastatic process warrants investigation.

Effect of hydrocortisone on the macrophage content, growth and metastasis of transplanted murine tumors.

Hydrocortisone (HC) reduced the macrophage content of four murine tumors to less than half of control values. Since HC causes monocytopenia and inhibits the recruitment of mononuclear phagocytes at sites of inflammation but does not affect the viability and proliferative capacity of macrophages, this finding suggests that the maintenance of macrophage levels in growing tumors is in part dependent upon the entry into the neoplasm of circulating monocytes. HC inhibited the growth of these tumors. The effect of HC was most marked on the mFS6 and MN/MCA1 sarcomas and 3LL carcinoma, with only marginal inhibition in the M109 carcinoma. HC augmented spontaneous and/or artificial (i.v.) metastasis in these tumor models. Cells from metastatic foci of the mFS6 sarcoma and M109 carcinoma inoculated i.m. were as susceptible to HC as cells from primary tumors. The effects of HC on macrophage content, growth and metastasis of the M109 carcinoma and mFS6 sarcoma were similar in mice with defective T-cell function (nude or thymectomized) or defective NK activity (beige or antiasialo GM1-treated) and in controls. The in vitro growth of the mFS6 and MN/MCA1 sarcomas was not modified by HC. Some inhibition of tumor-cell proliferative capacity was observed with the 3LL and M109 tumors. Tumor cell had high-affinity binding sites for glucocorticoid hormones, but these were not correlated to susceptibility in vivo. Thus it is unlikely that a direct interaction of HC with tumor cells accounts for the inhibition of tumor growth observed in vivo. To evaluate whether HC affected tumor growth by reducing the macrophage content of tumors, mFS6 sarcoma cells were transplanted, mixed with peritoneal macrophages into normal or HC-treated mice. Macrophages did not affect the growth of tumor cells in normal mice, whereas in HC-treated animals lesions from mixtures of macrophages and sarcoma cells appeared earlier and weighed more than those from tumor cells alone or tumor cells and thymocytes. However, macrophages did not reconstitute growth of the other sarcoma (MN/MCA1) in HC-treated mice. These results are consistent with the hypothesis that HC inhibited growth of some poorly immunogenic transplanted murine tumors, at least in part, by interfering with the macrophage levels in neoplastic tissues, tumor-associated macrophages providing the conditions for optimal tumor-cell proliferation at least in some neoplasms.

Morphologic studies on a murine reticulum cell sarcoma (histiocytic sarcoma) of histiocytic origin and its metastases.

The M5076 tumor, a reticulum cell sarcoma of histiocytic origin that arose spontaneously in the ovary of a C57BL/6 mouse, is highly invasive and metastatic. Regardless of the site of the primary tumor, this neoplasm rapidly and preferentially metastasizes to the liver and spleen, killing the host. Numerous other organs also are involved, including the lungs and bone marrow. This tendency to metastasize to osseous tissues appears to be a characteristic of the M5076 tumor that rarely is found with other rodent neoplasms. However, these extrahepatic foci are evident only microscopically and are seen relatively late in the progression of tumor growth. We describe the sequential gross and microscopic lesions that develop in syngeneic mice during the metastatic spread of the M5076 tumor. Since viable tumor cells form small tumor colonies in most organs, we suggest that the gross pattern of metastasis, with the apparent predilection for hepatic tissue, is caused by variations in tumor cell proliferation rather than by distinctive patterns of neoplastic cell spread and entrapment.

Potentiation of nonspecific immunotherapy of experimental lung metastases by indomethacin.

Intraperitoneal treatment with the interferon inducer, maleic anhydride-divinyl ether copolymer (MVE), has previously been demonstrated to effectively reduce metastatic growth in the lungs and prolong survival times of BALB/c mice bearing the syngeneic Madison lung (M109) carcinoma. Resistance to lung metastasis formation induced by MVE appears to result from an activation of alveolar macrophage function. Since E-type prostaglandins (PGE) suppress the cytotoxic activity of activated macrophages, we sought to determine the effect of indomethacin, a prostaglandin synthetase inhibitor, on the antimetastatic activity of MVE. An artificial metastasis model was developed in which single-cell suspensions of the M109 tumor were injected i.v. into BALB/c mice. A 52,600 molecular weight fraction of MVE (MVE-5) was administered i.p. at 20 mg/kg two days prior to tumor inoculation. MVE-5 treatment produced greater than 80 percent reduction in macroscopic lung lesion formation at Day 15 and Day 19 after tumor inoculation and a resultant 45 percent increase in lifespan. Chronic administration of indomethacin in the drinking water at 10 micrograms/ml potentiated the MVE-5 antitumor induced macrophage activation in vivo. In the absence of any evidence for an interaction between indomethacin and circulating M109 cells, it was felt that the potentiating effect could be best explained in terms of interference with PGE-mediated feedback inhibition of macrophage functional activity.

Responsiveness of the Madison 109 lung carcinoma to maleic vinyl ether copolymer.

This study establishes the responsiveness of mice bearing the Madison 109 lung carcinoma (M109), a tumor relatively resistant to chemotherapy, to the immunomodulator maleic vinyl ether (MVE-2; molecular weight 15,500). BALB/c mice inoculated with 5 X 10(5) M109 cells into the hind footpad developed a primary tumor which metastasized to the lung within 7 days and resulted in death of the host between 36 and 42 days. Early in the disease, weekly intratumor (i.t.) or intravenous administration of MVE-2 (25 mg/kg) inhibited the growth of the primary tumor and significantly prolonged the life span of the host. During the pulmonary metastatic process, systemic administration of MVE-2 alone or MVE-2 coupled with surgical excision or radiotherapy of the primary tumor became decreasingly efficacious. Late in the disease only MVE-2 introduced directly into the metastatic tumor bed by intrapleural injection proved to be effective in prolonging life span. These studies indicate that both the primary and metastatic M109 tumors are sensitive to MVE-2 and suggest that the efficacy of MVE-2 treatment is largely dependent upon its distribution in the tumor-bearing mice.

Monoclonal antibody and an antibody-toxin conjugate to a cell surface proteoglycan of melanoma cells suppress in vivo tumor growth.

A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

Laminin inhibits the adhesion of a murine tumor of macrophage origin

The adhesive behavior of the M5076 reticulum cell sarcoma, a highly metastatic murine tumor of macrophage origin, was investigated in vitro both in the presence and in the absence of purified exogeneous laminin. Although laminin enhanced the adhesion of other murine cell lines to collagen-coated and plastic surfaces, it reduced the attachment to both substrates of M5076 cells, peritoneal macrophages and the macrophage cell line WEHI-3. Thus, the inhibition appeared to be related to the macrophage nature of the M5076 tumor. The effects of laminin were reversible and did not cause loss of viability or functional capacity of the M5076 cells. Laminin appeared to exert this inhibitory effect by binding to the substrates rather than binding to the cells. These studies indicate that laminin, a glycoprotein from the basement membrane, may either stimulate or inhibit cell attachment, depending on the type of cell.

Evaluation of radiosensitizers in combination with chemotherapeutic agents in solid tumors

The electron-affinic compounds misonidazole (MISO), metronidazole (MET), desmethylmisonidazole (DMM), and CB1954 were shown to enhance the activity of alkylating agents in M5076 ovarian tumor. MISO, MET, and DMM enhanced the antitumor activity of cyclophosphamide by factors of 1.7, 1.5, and 1.7, respectively. MIS, MET, and CB1954 enhanced the antitumor activity of melphalan by factors of 2.2, 2.4, and 1.8, respectively. MISO also enhanced the antiitumor activity of mitomycin C by a factor of 2.0. MISO did not enhance the activity of chlorozotocin given as a single dose and only slightly enhanced the antitumor activity of this agent when the combination was given on a q4D × 3 schedule. MISO administered 1 hr prior to the antimetabolite 5-FU on a q7D × 3 schedule was no more effective than 5-FU alone against colon carcinoma 38. MISO given simultaneously with or 1 hr after 5-FU strongly inhibited the antitumor activity of 5-FU in this tumor. Data obtained in this study indicate that several radiation sensitizers including three nitroimidazoles and a dinitrobenzamide can enhance the antineoplastic activity of alkylating agents. The degree of enhancement in the M5076 tumor appears to be independent of the electron affinity of the sensitizer. Enhancement was observed with relatively ineffective as well as highly active alkylating agents.

In vivo anti-tumor effects of local adoptive transfer of mouse and human cultured lymphoid cells.

Mouse and human lymphoid cells were cultivated in the presence of T-cell growth factor (TCGF) and evaluated for their in vivo anti-tumor effect in mice. Cultured spleen cells of normal BALB/c mice or of mice bearing the M109 tumor had a high level of cytotoxic activity in vitro against a variety of tumor target cells and had characteristics of natural killer cells. These cultured cells were evaluated for their in vivo cytotoxic activity by a mixture with [125I]dUrd-labelled M109 tumor cells (2 x 10(5)) at a 30:1 ratio and inoculation of the mixture into the footpads of BALB/c mice. The level of radioactivity remaining in the footpad was determined at various periods following inoculation of radiolabelled tumor cells. The presence of cultured cells in the inocula caused a marked decrease in the footpad radioactivity by 24 h. However, the slope of clearance of the radiolabel then became similar to that in the control mice. The cultured cells delayed, but did not prevent, tumor appearance, and did not influence the subsequent rate of growth of the tumors. The transient effects of the cultured cells contrasted with the more prolonged in vivo effects of alloimmune lymphocytes, and this may be due to their short survival period. Ninety-nine percent of [125I]dUrd-labelled cultured mouse lymphoid cells were eliminated within 48 h of i.f.p. inoculation. Cultured human lymphoid cells, initiated from the blood of normal donors, also had a high level of cytotoxic activity in vitro and were evaluated for their in vivo effects by intra-footpad inoculation into nude mice, together with radiolabelled human tumor cell lines, G-11 or HT-29. In vivo cytotoxic activity of the human cultured lymphoid cells correlated with their cytotoxic effect in vitro. These results indicate that cultured mouse an human effector cells have appreciable in vivo cytotoxic effects against tumor cell lines. However, the transient duration of these effects may limit their immunotherapeutic potential.

Radioisotope assay for evaluation of in vivo natural cell-mediated resistance of mice to local transplantation of tumor cells.

AbstractA radioisotopic assay based on the rate of elimination of [125I]dUrd‐labelled tumor cells from the local site of inoculation was used to study anti‐tumor natural cellmediated immunity in vivo. [125I]dUrd‐labelled YAC‐I, RL♂ I, and M109 tumor cells were inoculated into the footpads of mice and the radioactivity remaining at the site was measured at various times. This method allowed quantitation, in individual mice, of the actual number of tumor cells inoculated and their local clearance. It was found that 90–99% of inoculated YAC‐I or RL♂ I cells were eliminated in the first 24–48 h following transplantation. Participation of NK cells in the local elimination of the transplanted tumor cells was supported by the following findings: (1) The rate of elimination of NK‐sensitive tumor cells was higher than that of the NK‐resistant M 109 tumor cells. (2) There was a positive correlation between the level of in vitro spleen‐cell NK activity of the different strains of mice and the rate of in vivo elimination of YAC‐I. (3) Inoculation of poly I:C or cyclophosphamide increased or decreased, respectively, the clearance of tumor cells in vivo. (4) Local adoptive transfer of normal spleen cells accelerated the rate of tumor cell elimination in vivo. (5) This adoptive transfer of the cytotoxicity was mediated by non‐adherent, non‐T cells that expressed asialo GMI. Despite these correlations between NK activity and the in vivo assay, two major discrepancies were found: YAC‐I cells were eliminated at the same rate in young and old mice, and beige mice were as effective for in vivo clearance of tumor cells as were normal C57BL/6 mice. These data suggested that other natural effector cells may participate in the in vivo destruction of the transplanted tumor cells or that different regulatory processes may operate in vivo. Our findings provide further evidence that natural cell‐mediated immunity can play an important role in the in vivo elimination of the transplanted tumor cells and indicate that this can occur at the local site of tumor inoculation as well as in the lungs or other organs.