B16 Melanoma Tumor Cells Research Articles

Disruption of STAT3 Signaling in Antigen Presenting Cells Represents a Novel Strategy To Overcome Tumor-Induced Antigen-Specific CD4+ T-Cell Tolerance.

Tumor antigen-specific T-cell tolerance imposes a significant barrier to the development of effective therapeutic cancer vaccines. Bone marrow-derived antigen presenting cells (APCs) are critical in the induction of this unresponsive state. Recently, we have identified STAT3 signaling in APCs as an important regulatory pathway that determines the functional outcome of antigen-specific CD4+ T-cells in response to cognate antigen. Indeed, while disruption of this signaling pathway in APCs led to effective T cell priming, enhanced STAT3 activity resulted in the induction of T cell unresponsiveness1.Given the above results, we explored in this study whether disruption of STAT3 signaling in APCs may preserve the responsiveness of antigen-specific CD4+ T-cells during the growth of a tumor that induces antigen specific T-cell tolerance. First, mice with a genetic disruption of Stat3 in macrophages, neutrophils and a sub-population of myeloid DCs (LysMcre/Stat3flox/− mice) or control C57BL/6 mice were given subcutaneously 1x106 B16 melanoma tumor cells engineered to express Ovalbumin as a model tumor antigen (B16-OVA). Four days later, naive CD4+ T-cells (1x106) specific for a MHC class II-restricted epitope of Ovalbumin (OT-II cells) were adoptively transferred intravenously into tumor bearing mice as well as into tumor-free controls. Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses to in vitro re-stimulation with OVA-peptide were evaluated. As expected, antigen-specific T cells re-isolated from tumor-bearing C57BL/6 mice were fully tolerant (lack of HA-specific proliferation and cytokine production). In sharp contrast, anti-OVA CD4+ T-cells isolated from tumor bearing LysMcre/Stat3flox/− mice remained fully functional as determined by their capacity to proliferate and produce IL-2 and IFN-gamma in response to cognate OVA-peptide. The demonstration that tumor-induced antigen-specific CD4+ T-cell tolerance occurs in mice with an intact STAT3 signaling in APCs, but not in mice with genetic disruption of this signaling pathway, led us next to evaluate the efficacy of pharmacologic inhibitors of STAT3 in preventing and or overcoming tumor-induced T-cell tolerance. In vivo treatment of tumor bearing mice with Tyrphostin AG490 (0.5 mg/ i.p. /twice a day x 5 days), a well-known inhibitor of STAT3 signaling, also resulted in preservation of the responsiveness of tumor-antigen specific CD4+ T-cells. Furthermore, in vitro treatment of APCs with this compound led to effective priming of naive antigen-specific T cells and breaking of antigen-specific T-cell anergy. More recently, we have evaluated the efficacy of a novel STAT3 inhibitor, compound 295558, which efficiently inhibits the DNA-binding activity of STAT3. Treatment of DCs or macrophages with this specific inhibitor led to the generation of inflammatory APCs capable of restoring the responsiveness of tolerized CD4+ T-cells isolated from tumor bearing mice.Taken together, our findings establish a critical role for STAT3 signaling in the induction of tolerance to tumor antigens in vivo. Inhibition of this signaling pathway in APCs provides a novel molecular target to overcome the remarkable barrier that tolerance to tumor antigens imposes to cancer vaccination strategies.

Sequential plasmid vaccinations targeting normal melanocytes protects against challenge with melanoma in a syngenic murine model

2570 Background: Malignant melanoma is one of a handful of tumors responsive to immunotherapy. Tumor regression correlates with an expanded epitope response and induction of vitiligo. In fact, expansion of the immune response to include multiple melanoma differentiation antigen (MDA) targets correlates with clinical response in newer vaccine approaches. We hypothesized that a vaccine designed to induce epitope spreading against melanocytic antigens may be useful in melanoma. By directly targeting normal melanocytes in situ, we hoped to induce both autoimmunity and a broad anti-melanocytic response. To accomplish this, we utilized sequential plasmid vaccinations in an immune competent murine model for melanoma. Methods: C57/b6 mice were vaccinated with plasmid DNA encoding either the gene for chicken ovalbumin (cOVA) or empty vector intramuscularly (IM) weekly for two weeks. Mice (n=9) were then vaccinated intradermally (ID) with either cOVA or a control gene under the control of the tyrosinase promoter weekly for three weeks. Mice were challenged with subcutaneous injections of 105 B16 melanoma tumor cells or B16 containing the transgene cOVA (B16cOVA). Tumor size and survival were followed. Results: Vaccination with the cOVA gene IM resulted in 90% to 100% protection against B16cOVA tumor cells compared to 0% to 10% of control plasmid injected mice. Challenge with B16 resulted in equally poor survival between the two groups with 100% of mice being sacrificed by day 26. Surprisingly, if the mice were vaccinated with the cOVA gene ID under the control of the tyrosinase promoter after the initial IM vaccination with cOVA and subsequent tumor challenge, B16 growth was delayed compared to controls. Delay was not observed without the initial IM cOVA vaccination. Conclusions: Ectopic expression of antigens for which a strong pre-existing cellular immune response exists may allow targeting of an immune response to normally tolerant antigens. This system provides a relatively simple approach in understanding the requirements of both autoimmunity and cancer immunotherapy. No significant financial relationships to disclose.

MART-1 adenovirus-transduced dendritic cell immunization in a murine model of metastatic central nervous system tumor.

Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially. Genetic modifications of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector. Sixty-two C57BL/6 mice were immunized subcutaneously with AdVMART-1-transduced DCs (n = 23), untransduced DCs (n = 17), or sterile saline (n = 22). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes from representative animals in each group were harvested for standard cytotoxicity (CTL) and enzyme-linked immunospot (ELISPOT) assays. The remaining mice were followed for survival. Immunization of C57BL/6 mice with DCs transduced with an adenoviral vector encoding the MART-1 antigen elicited the development of antigen-specific CTL responses. As evidenced by a prolonged survival curve when compared to control-immunized mice with intracranial B16 tumors, AdMART-1-DC vaccination was able to elicit partial protection against central nervous system tumor challenge in vivo.

Taurolidine improves survival by abrogating the accelerated development and proliferation of solid tumors and development of organ metastases from circulating tumor cells released following surgery.

Surgical trauma is partly responsible for enhancing tumor growth through a variety of mechanisms that are still not fully elucidated. The use of perioperative immunostimulants may modulate these effects. This study examined the effect of administration of taurolidine after laparotomy or laparoscopy on the growth of solid tumor, the establishment of hepatic and lung metastases, and effects on natural killer (NK) and lymphokine-activated killer (LAK) cell function. B16 melanoma right flank tumors were established in mice (n = 180). These animals underwent anesthesia only (control) or laparotomy or laparoscopy (n = 60 per group) and were randomized to receive either saline or taurolidine (n = 30 per group) at specific time points. Survival was determined in each group, and in a further 90 mice tumor growth was followed over 10 days postoperatively. The experiment was repeated in 540 mice, which underwent one of the three procedures and were treated with either saline or taurolidine. NK and LAK cell cytotoxicity (NKCC, LAKCC) was determined at several time points postoperatively. In a further experiment, B16 melanoma tumor cells were delivered via tail vein injection (n = 180) and intrasplenic injection (n = 180). The effect of saline or taurolidine administration on survival after the establishment of metastases was determined, and again in a further 180 mice the establishment of metastatic deposits in the liver or lungs was determined after 8 days. Survival appeared to be significantly decreased in both the solid-tumor model and the metastatic models undergoing laparotomy compared to laparoscopy and controls (P < 0.0001) and to a lesser extent in the laparoscopy group compared to controls (P < 0.001). Flank tumor growth and metastatic tumor formation were more significant in laparotomy groups compared to laparoscopy groups and controls, but also to a lesser extent in laparoscopy groups compared to controls (P </= 0.05). NKCC and LAKCC were significantly decreased in the same patterns (P < or = 0.03). However, treatment with taurolidine abolished these effects, restoring NKCC and LAKCC (P < or = 0.04) and laparoscopy groups (P < or = 0.001). It appears that changes that occur after the trauma of laparotomy, and to a lesser extent, after laparoscopy, significantly enhance the growth of primary tumors as well as the development of metastases from circulating tumor cells and are associated with a suppression of host antitumor surveillance mechanisms. This not only affected tumor progression in the immediate postoperative period, but also ultimately affected survival. Taurolidine, a known immunostimulant, appears to abrogate the effects of surgical trauma on primary and metastatic tumor growth and also enhances survival. This may have significant value in the management of tumor-bearing patients undergoing resection.

Recombinant adenovirus-transduced dendritic cell immunization in a murine model of central nervous system tumor

Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially. Genetic modification of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector (AdVMART1). Sixty-two C57BL/6 mice were immunized by subcutaneous injection of AdVMART-1-transduced DCs (23 mice), untransduced DCs (17 mice), or sterile saline (22 mice). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes obtained from representative animals in each group were harvested for standard cytotoxicity and enzyme-linked immunospot assays. The remaining mice were followed for survival. Immunization of C57BL/6 mice with DCs transduced with AdVMART1-DC elicited the development of antigenspecific CTL responses. As evidenced by a prolonged survival curve when compared with control-immunized mice harboring intracranial B16 tumors, AdMART1-DC vaccination was able to elicit partial protection against central nervous system (CNS) tumor challenge in vivo. However, this CNS antitumor immunity was weaker than that previously demonstrated against subcutaneous B16 tumors in which the same vaccination strategy was used. These data suggest that immune responses generated against CNS tumors by DC-based vaccines may be different from those obtained against subcutaneous tumors.

Immunogene therapy for murine melanoma using recombinant adenoviral vectors expressing melanoma-associated antigens.

Adenoviral vectors expressing tumor-associated antigens can be used to evoke a specific immune response and inhibit tumor growth. In this study, we tested the efficacy of adenoviral vectors encoding human gp100 (Ad2/hugp100), murine gp100 (Ad2/mugp100), or murine TRP-2 (Ad2/muTRP-2) for their ability to elicit a specific cellular immune response and inhibit the growth of B16 melanoma tumor cells in the mouse. C57BL/6 mice were immunized with Ad2/hugp100, Ad2/mugp100, or Ad2/muTRP-2 either 2 weeks prior to B16-F10 tumor challenge (prophylactic treatment) or 3 days after tumor challenge (active treatment). Ad2/hugp100 and Ad2/muTRP-2 administered to two or more intradermal (i.d.) sites inhibited subsequent subcutaneous tumor growth in > or = 80% of the mice and elicited an antigen-specific cytotoxic T lymphocyte response, whereas other administration routes were not as effective. Ad2/mugp100 administered to two i.d. sites did not inhibit tumor growth or provoke cellular immunity. Immunization was less effective with active treatment where tumor growth was not significantly inhibited by a single dose of either Ad2/muTRP-2 or Ad2/hugp100. However, increasing the number of intradermal immunization sites and the number of doses resulted in progressive improvements in protection from tumor growth in the active treatment model. In conclusion, breaking host tolerance to elicit protective immunity by using adenoviral vectors expressing melanoma-associated antigens is dependent upon the choice of antigen, the site of administration, and the number of doses. These observations provide insights into the clinical applicability of adenoviral vaccines for immunotherapy of malignant diseases.

Induction of Antitumor Immunity with Dendritic Cells Transduced with Adenovirus Vector-Encoding Endogenous Tumor-Associated Antigens

Dendritic cells (DCs) are professional Ag-presenting cells that are being considered as potential immunotherapeutic agents to promote host immune responses against tumor Ags. In this study, recombinant adenovirus (Ad) vectors encoding melanoma-associated Ags were used to transduce murine DCs, which were then tested for their ability to activate CTL and induce protective immunity against B16 melanoma tumor cells. Immunization of C57BL/6 mice with DCs transduced with Ad vector encoding the hugp100 melanoma Ag (Ad2/hugp100) elicited the development of gp100-specific CTLs capable of lysing syngeneic fibroblasts transduced with Ad2/hugp100, as well as B16 cells expressing endogenous murine gp100. The induction of gp100-specific CTLs was associated with long term protection against lethal s.c. challenge with B16 cells. It was also possible to induce effective immunity against a murine melanoma self Ag, tyrosinase-related protein-2, using DCs transduced with Ad vector encoding the Ag. The level of antitumor protection achieved was dependent on the dose of DCs and required CD4+ T cell activity. Importantly, immunization with Ad vector-transduced DCs was not impaired in mice that had been preimmunized against Ad to mimic the immune status of the general human population. Finally, DC-based immunization also afforded partial protection against established B16 tumor cells, and the inhibition of tumor growth was improved by simultaneous immunization against two melanoma-associated Ags as opposed to either one alone. Taken together, these results support the concept of cancer immunotherapy using DCs transduced with Ad vectors encoding tumor-associated Ags.

An in vitro model fails to demonstrate aerosolization of tumor cells.

We investigated the ability of pressurized CO2 gas to aerosolize B16 melanoma (B16) tumor cells in an in vitro model. The experimental apparatus consisted of an 18.9-L plastic cylindrical vessel and a compliant latex pouch was attached to the top. Two 5-mm ports penetrated the vessel; insufflation and desufflation were carried out through them. A culture dish containing 20 million B16 cells in liquid culture media was placed at the base within the container. In the first experiment, the vessel was insufflated with CO2 gas to a static pressure of 15 or 30 mm Hg with the outflow port closed. After 10 min, the outflow port was opened and the gas was desufflated through a collecting device containing sterile culture medium. In a second experiment, a continuous flow of CO2 through the vessel was maintained after a pressure of 15 or 30 mm Hg was established. A total of 10 L CO2 was cycled through the vessel. In both experiments, 24 determinations were carried out at each pressure. Each experimental culture dish was microscopically scanned for 2 weeks for the presence of tumor cells. The third and fourth experiments tested for the presence of aerosolized nonviable tumor cells in the expelled gas. Using the model described above, after 10 mins of 30 mm Hg static pressure, the CO2 gas was expelled directly onto a glass slide and cytofixed. Alternately, after 10 mins at 30 mm Hg static pressure, the gas was expelled through a saline-filled Soluset (Abbott Laboratories), centrifuged, and the residue cytofixed onto a glass slide. Each of the five slides per experiment were examined microscopically for the presence of cells. In the first and second experiments, no cells or growth were observed in any of the 96 experimental dishes. In experiments three and four, no cells were detected on any of the slides. It was not possible with this model to aerosolize tumor cells in a pressurized CO2 environment. Our results suggest that aerosolization of tumor cells is not the mechanism of port site recurrences after laparoscopic surgery for malignant disease.

Nonspecific cell-mediated cytotoxicity in the seawater teleosts (Sparus aurata andDicentrarchus labrax): Ultrastructural study of target cell death mechanisms

In higher vertebrates, cytolytic lymphocytes lyse their targets by the mechanisms of cell death called necrosis and apoptosis. However, the mechanisms of target cell death mediated by fish cytotoxic cells have not been established. We report the ultrastructure of target cell death mediated by the seawater teleosts gilthead seabream (Sparus aurata) and sea bass (Dicentrarchus labrax). Head-kidney, blood, and peritoneal exudate leukocytes were incubated with HeLa or B16 melanoma tumor cells, as targets, and processed for transmission electron microscopic studies. After incubation of tumor cells with leukocytes, substantial ultrastructural changes typically associated with both necrosis and apoptosis were observed in the plasmalemma, nucleus, and cytoplasm of the tumor cells. These morphological changes included loss of microvilli, the formation of long cytoplasmic processes and blebs, condensation and margination of the chromatin, swelling of the organelles, and vacuolation and condensation of the cytoplasm. Our results indicate that leukocytes from gilthead seabream and sea bass are able to kill their targets by the mechanisms of necrosis and apoptosis, in a similar way to mammalian cytotoxic cells.

5-Fluorouracil's cytotoxicity is enhanced both in vitro and in vivo by concomitant treatment with hyperthermia and dipyridamole.

We obtained evidence that the cytotoxic effect of 5-fluorouracil (5-FU) is augmented when the drug is given in combination with hyperthermia (HYP) and dipyridamole (DP). Nontoxic levels of DP enhanced the combined cytotoxicity of 5-FU and HYP against B16 melanoma and human tumor cells in vitro as measured by the succinate dehydrogenase inhibition (SDI) test. Growth of B16 melanoma that had been subcutaneously implanted into the feet of C57 BL mice was inhibited by treatment with the combinations of 5-FU and HYP, of 5-FU and DP, and of 5-FU, HYP and DP as compared with the administration of 5-FU alone. Treatment with HYP plus DP did not alter the body weight of mice that received 5-FU. The administration of DP plus HYP seemed to render the tumor cells more sensitive to 5-FU. The combination of 5-FU, HYP and DP shows promise for the treatment of patients suffering from malignant disease.

Successful immunotherapy of murine melanoma metastases with 7-thia-8-oxoguanosine

We have recently reported that a synthetic nucleoside, 7-thia-8-oxoguanosine (7T8OG) is a potent activator of a number of effectors which are involved in anti-tumor immune responses. 7T8OG was found to induce interferon (IFN) production, to activate asialo-GM1 positive (AGM+1) killer cells, and to enhance specific antibody responses. In the present study, we investigated the effect of 7T8OG on growth of the murine pulmonary B16 melanoma and on formation of metastases. C57BL/6 mice were injected i.p. with 50-150 mg/kg 7T8OG before or after i.v. inoculation of B16 melanoma tumor cells, and 17-19 days after tumor inoculation, the number of metastases in the lungs were counted. 7T8OG given systemically in a single or a divided dose 24 h prior to the challenge of tumor cells reduced the number of lung tumor metastases by 89-99% which is highly significant as compared to untreated control (P less than 0.001). Occasional extra pulmonary tumor growth in the thoracic cavity and neck lymph node was also completely inhibited. The reduction in the number of tumor nodules was dose dependent. A single dose of 150 mg/kg of 7T8OG was also effective in inhibiting the growth of 3-5 day old metastatic tumors. The cytotoxic activity of killer cells induced in vivo by 7T8OG was completely abolished by in vitro treatment of cells with anti-AGM1 antibody plus complement. Administration of anti-AGM1 antibody following the 7T8OG treatment completely abrogated the anti-tumor effect of 7T8OG, resulting in a massive increase in the number of tumor foci in the lungs. Administration of carageenan or silica followed by injection of 7T8OG caused a significant increase (P less than 0.01) in the number of pulmonary tumor nodules compared to treatment with 7T8OG only. These findings indicate that activated macrophages or perhaps their cytokine (tumor necrosis factor) also contribute to the host tumor defense by 7T8OG.

Biomechanical destruction of cancer cells in skeletal muscle: a rate-regulator for hematogenous metastasis.

Following left ventricular injection into mice, B16 melanoma and Ehrlich ascites tumor cells are arrested in the microvasculature of the quadriceps femoris muscles. Within 5 min of delivery the effects of variation in surrounding tissue pressure on the survival of the arrested cells have been determined by bioassays of denervated (relaxed), non-contracting or contracting muscle. The results show that cancer cell survival is greatest in denervated, then non-contracting muscle, and least in electrically stimulated muscle. The results are in accord with the hypothesis that the rapid death of most cancer cells after delivery to at least some target organs is a consequence of their mechanical interactions within the microvasculature.

Interaction of tumor and surrounding tissue of mice inoculated B16 melanoma variants in terms of enzyme activity

1. 1. The interactions of B16-F1 and B16-F10 tumors with their surrounding tissues in terms of enzyme activities such as cathepsin B, hemoglobin(Hb)-hydrolase, acid phosphatase, β-glucuronidase and plasminogen activator were investigated when said tumors proliferated locally and at secondary sites throughout the host's circulatory system. 2. 2. In the case of B16-F1 and B16-F10 tumor cells proliferating under the skin, statistical differences were not detected between the enzyme activities of the skin surrounding the tumors and control skin, nor between B16-F1 and B16-F10 tumors, except for β-glucuronidase. 3. 3. In the case of B16-F1 and B16-F10 tumor cells metastasizing to lung, statistical differences were detected between numerous enzyme activities of the lung tissues surrounding the tumors and control lung tissue, and also between B16-F1 and B16-F10 tumors. 4. 4. The activities of cathepsin B and acid phosphatase of lung tissue surrounding B16-F1 tumor were lower than those of the control lung. 5. 5. β-Glucuronidase activity of lung tissue surrounding B16-F10 tumor was higher than that of the control lung. 6. 6. The activities of cathepsin B, Hb-hydrolase and β-glucuronidase of the B16-F10 tumor were higher than those of the B16-F1 tumor. 7. 7. Results indicate that metastasized B16 melanoma tumor cells interact with surrounding lung tissues, and that cathepsin B, Hb-hydrolase and β-glucuronidase might play important roles in the metastasis of the malignant tumor.

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destruction of B16 melanoma cells mediated by bystander cytolysis in vitro.

Two Lyt-1+, L3T4a+ autoreactive T cell clones specific for self-class II major histocompatibility complex (MHC) gene products were established from lymph node cells and spleen cells of C57BL/6J mice, respectively, by different methods. They were stimulated to proliferate in culture in response to I-Ab antigen-bearing syngeneic spleen cells in a class II MHC-restricted manner. This stimulation was inhibited completely by the addition of anti-L3T4a (GK1.5) or anti-I-Ab (3JP) monoclonal antibodies. The autoreactive T cell clones lysed syngeneic I-Ab+ target cells such as lipopolysaccharide (LPS) blasts. They also lysed I-A- bystander cells such as Cloudman and B16 melanoma and lymphoid tumor cells in the presence of I-Ab+ stimulator cells but not I-Ad+ cells. This bystander killing was most likely mediated by soluble factors released from the autoreactive T cells in response to I-Ab antigens, because culture supernatants from activated autoreactive T cells inhibited the proliferation of B16 melanoma cells in vitro and also had significant cytolytic activity. Both lymphotoxin and interferon-gamma were released from activated autoreactive T cells, suggesting that these cytotoxic lymphokines were responsible for autoreactive T cell-mediated cytolysis. The finding that the two clones, established independently and by different methods, show self-class II MHC antigen-restricted cytolysis, and bystander cytolysis suggests that these properties are not restricted to a unique population of autoreactive T cells. These results favor the concept that in vivo, autoreactive T cells may express not only regulatory activity in regard to antibody responses, but also anti-tumor activity via bystander cytolysis.

Role of NK cells in the control of metastatic spread and growth of tumor cells in mice

The ability of BALB/c nude and C57BL/6 mice to eliminate tumor cells from the blood stream was severely impaired after a single inoculation of 0.2 ml of anti-asialo BMI (asGMI) serum, diluted 1:40 to 1:320. The number of i.v.-inoculated YAC-I cells surviving in the lungs of BALB/c nude mice pretreated with anti-asGMI serum was 28 times higher than in the control nude mice. In this respect, nude mice treated with anti-asGMI behaved similarly to beige mice. The increase in the initial survival of tumor cells in the mice that was induced by pre-treatment with anti-asGMI resulted in a substantial increase in the number of artificial lung metastases that developed. In C57BL/6 +/+ mice treated with anti-asGMI and in C57BL/6 beige mice, i.v. inoculation of B16 melanoma cells induced 10 times more metastatic foci in the lungs than in the control C57BL/6 +/+ mice. In contrast, in nude mice which possess higher levels of NK reactivity, metastatic growth was suppressed 7-fold in comparison with intact C57BL/6 +/+ mice. In beige mice and in C57BL/6 +/+ mice treated with anti-asGMI, multiple metastatic foci developed in the liver, whereas in control C57BL/6 +/+ and nude mice, no extrapulmonary metastases were found. These data indicate that B16 melanoma cells are able to grow in the liver, but their growth is ordinarily prevented by NK cells. The antimetastatic defense of C57BL/6 mice treated by anti-asGMI could be restored by transplantation of 40 X 10(6) normal spleen cells. This antimetastatic effect of transplanted spleen cells was mediated by asGMI-bearing cells, since after in vitro pre-treatment of normal spleen cells with anti-asGMI and complement, they lost their ability to inhibit the development of artificial metastases in the lungs of C57BL/6 mice. Suppression of NK reactivity by multiple injections of anti-asGMI (every 4 to 5 days), in C57BL/6 mice inoculated intrafootpad (i.f.p.) with B16 melanoma or 3LL tumor cells, did not influence the growth of local tumors, but dramatically accelerated the development of spontaneous pulmonary metastases. These data demonstrate that NK cells may play an important role in resistance to the dissemination of tumor cells, and therefore contribute to the control of metastasis formation in mice.

Augmentation of metastasis formation by thioglycollate-elicited macrophages.

Inoculation of thioglycollate-elicited peritoneal exudate cells (PEC) into C57BL/6 mice reduced the rate of lung clearance of several intravenously (i.v.) injected murine tumor cells, and increased by up to 100-fold the number of artificially-induced metastatic lung nodules produced by the i.v. injection of B16 melanoma or Lewis lung carcinoma (3LL) tumor cells. Maximum effects were observed when PEC were injected either before, or shortly after, tumor cells. Modulation of lung clearance or metastasis formation was observed only with PEC and not with a variety of other cells, such as splenocytes, thymocytes P815 mastocytoma cells, or several macrophage-like cell lines (PU5-1.8 and IC-21). Lysates of PEC were as efficient in reducing lung clearance and augmenting metastasis formation as were intact viable PEC. Lysates of other cell types, including P815 and the macrophage-like cell lines, were unable to produce these effects. PEC populations, enriched for macrophages by adherence to plastic or by percoll density gradient sedimentation, also increased the number of B16-induced artificial metastasis, implicating the macrophage as the cell responsible for these observations.

Relationship of host immune status to tumor cell arrest, distribution, and survival in experimental metastasis.

The kinetics of initial arrest in organs, distribution, survival, and fate of 125I-iododeoxyuridine-labeled B16 melanoma tumor cells injected intravenously into normal, tumor-sensitized and immune manipulated syngeneic and allogeneic mice were investigated. Groups of animals were killed at intervals ranging from two minutes to 14 days after intravenous tumor cell injection. Lungs, liver, spleen and blood were collected from each animal and processed so that radioactivity associated with DNA of tumor cells viable at the time of sacrifice could be monitored. The following conclusions can be made: initial tumor cell arrest in organs is influenced by the host immune status but it does not correlate with the survival kinetics or development into tumors. The same tumor, which was rejected in mice after a subcutaneous tumor challenge, grew in the lungs after intravenous injection. Therefore, rejection of a subcutaneous challenge as the sole criterion of host immunity to neoplasms should be questioned. Allogeneic animals are not appropriate for use as a model system for the study of experimental metastasis. Animals sensitized to a tumor exhibit kinetic patterns of tumor cell arrest and survival that differ from normal syngeneic hosts.