A specific vaccine effective against stage I and stage II malignant disease in guinea pigs effect of variations in preparations and storage

Summary Guinea pigs, each with an established, syngeneic dermal line 10 tumor and lymph node metastases, were immunized by intradermal injection of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG. Immunization eradicated 7- or 10-day-old dermal tumors (about 10 or 12 mm in diameter, respectively) and prevented growth of microscopic lymph node metastases. Fourteen-day-old dermal tumors (about 15 mm in diameter) were not rejected by immunization.Guinea pigs with stage II disease (21-day-old dermal tumors and palpable metastases in the first draining lymph node) were treated by excision of the dermal tumor and the first draining lymph node, and by specific immunization. This treatment eliminated tumor cells remaining in the second draining lymph nodes. The surgical treatment alone was not curative, palpable metastases in the second draining lymph nodes progressed and the animals died (some with visible lung metastases).Emulsions containing killed BCG were good adjuvants even after prolonged storage at 4° C, but lost most of their adjuvant activity after autoclaving or freezing.     

Eradication of microscopic lymph node metastases of the guinea pig line-10 tumor after intradermal injection of endotoxin plus mycobacterial components

Summary Non-viable microbial agents were used to treat lymph node metastases of the line-10 hepatocarcinoma in strain two guinea pigs. Oil droplet vaccines were administered by intradermal injection adjacent to the site of dermal tumors. The primary tumors were removed surgically before or after immunotherapy. Control animals, treated with surgery alone, died of metastatic tumor growth. The mycobacterial glycolipid, P₃, plus polysaccharide deficient endotoxin (Re Et) eliminated lymph node metastases when the primary tumors were excised 7 days or 1 day after immunotherapy. The combination of P₃, BCG cell wall skeleton and Re Et was also effective when there was an interval of 1 or 7 days between immunotherapy and surgery. In addition, this combination retarded, and in some experiments, eliminated metastatic tumor growth in animals given immunotherapy immediately prior to surgery and in animals given immunotherapy 2 days after surgery.     

Specific tumor memory induced by polyethylene-glycol-modified interleukin-2 requires both helper and cytotoxic T cells

Local polyethylene-glycol (PEG)-modified interleukin-2 (IL-2) immunotherapy of the guinea pig Line 10 (L10) tumor was previously demonstrated to evoke long-lasting systemic immunity after cure of the tumor and metastases. T cells, most likely the helper T cell subpopulation, were demonstrated to be crucial to the antitumor effects. Here we show that systemic immunity is induced within 7 days after the start of PEG-IL-2 therapy, indicating a rapid systemic priming of L10-specific T cells. No in vitro cytotoxic activity was detected in cell suspensions obtained from the primary tumor site, the regional lymph node or the spleen when isolated during (days 21 and 28) intratumoral treatment with 200 000 IU PEG-IL-2. These data confirm our carlier results obtained with 60 000 IU PEG-IL-2. Moreover, no cytolytic activity was observed in the chromium-release assay after in vitro restimulation with irradiated tumor cells. Specific L10 immunity can be transferred using spleen cell suspensions. Depletion of such a suspension of helper T cells resulted in rejection of the primary tumor in two out of four animals, but all the guinea pigs developed lymph node metastases. Removal of the cytotoxic/suppressor phenotype caused rejection of the dermal tumor in four of eight guinea pigs, but the capacity to prevent lymph node metastases was retained in all animals. Thus, depletion of either subtype reduces, but does not abrogate, the capacity to transfer L10 immunity with spleen cells. In conclusion, our data suggest that tumor cell killing through direct T cell cytotoxicity is not the main mode of action in PEG-IL-2-induced L10 tumor regression, PEG-IL-2 therapy induces early systemic immunity, resulting in rejection of a distant tumor, and the transfer of this immunity depends mainly on the presence of helper T cells, although cytotoxic T cells may also play a role.     

Synergistic effects of active specific immunotherapy and chemotherapy in guinea pigs with disseminated cancer

Sewall Wright strain 2 guinea pigs bearing pulmonary metastases of the syngeneic line 10 (L10) hepatocarcinoma were treated with a vaccine composed of 10(7) bacillus Calmette-Guérin admixed with 10(7) x-irradiated L10 tumor cells beginning 10 days after tumor inoculation. Although this treatment failed to cure most of the guinea pigs of their metastatic disease, histologic examination of the pulmonary tumors in the vaccinated guinea pigs provided evidence of a cell-mediated hypersensitivity response that disrupted the normally compact architecture seen in control tumors. When a monoclonal antibody against the L10 tumor was injected i.v. to evaluate the vascular permeability of the tumors, significantly more antibody localized in tumors of vaccinated guinea pigs than in tumors of untreated controls. These results suggested that blood-borne substances could be delivered more efficiently to L10 metastases after the tumor-bearing guinea pigs had been treated with vaccine. To determine whether such increased vascular permeability would enhance the antitumor effects of chemotherapeutic agents, combined immunotherapy and chemotherapy studies were performed. Although cyclophosphamide treatment by itself did not cure L10-bearing guinea pigs, cyclophosphamide used in conjunction with prior immunotherapy increased the survival rate of animals to more than twice that of animals treated with immunotherapy alone (74 vs 33%). These results suggest that one mechanism by which active specific immunotherapy enhances chemotherapy of disseminated tumors is by rendering tumor foci more permeable to subsequently administered cytotoxic drugs.     

Augmentation of immune response to syngeneic fibrosarcoma T241 with in vivo protection

Summary Lewis T241 fibrosarcoma, a syngeneic tumor in C57 BL/6J mice, was found to be poorly immunogenic. When tumor-bearing animals (TBA) were challenged with tumor cells either concomitantly or after excision of a growing tumor no protection was observed. In vivo (Winn) neutralization assays also showed a lack of tumor immunogenicity. However, in vitro studies showed that a significant proliferative response could be elicited from the spleen cells of TBA when these cells were cultured with either mitomycin-C-treated tumor cells or KCl tumor extract. Similarly, macrophage migration inhibition factor (MIF) was produced by TBA spleen cells upon incubation with KCl tumor extract, but no cell-mediated cytotoxicity to T241 target cells was observed with various lymphoid cell populations at any stage of tumor growth. Immunization of syngeneic animals with Vibrio cholerae neuraminidase(VCN)-treated, irradiated tumor cells alone or admixed with Freund's complete adjuvant (FCA) resulted in decreased tumor growth and fewer pulmonary metastases following challenge with 10⁶ tumor cells. No complete tumor rejection was observed. In contrast, 13 of 16 animals immunized with irradiated tumor cells admixed with FCA rejected 10⁵ tumor cells. Animals that grew tumors had significantly reduced tumor growths and pulmonary metastases. Lymph node and peritoneal exudate cells (PEC) of immunized animals showed significant cytotoxicity to T241 cells.     

The efficacy of BCG-induced tumor immunity in guinea pigs with regional and systemic malignancy

Summary It has been previously demonstrated that transplanted syngeneic line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2) regressed and regional lymph node metastases were eliminated after intratumoral injection of viable Mycobacterium bovis strain BCG. During the course of this reaction there is the development of systemic tumor immunity. The purpose of this study was to determine the relative efficacy of the induced tumor immunity to eliminate regional as well as systemic tumor burden. The approach to evaluate the efficacy of BCG-induced systemic tumor immunity in vivo, for regional as well as systemic tumor, was to develop a competition assay using increasing doses of intravascular disseminated line-10 tumor cells in animals with established regional tumors. The results clearly show that the efficacy of intratumoral BCG injection in producing regression of regional tumor is abrogated by initial intravascular doses of 10³–10⁶ line-10 cells. That the vascular systemic tumor burden diminished the effective systemic tumor immunity was demonstrated by the inability of animals with systemic tumor burdens to reject contralateral challenge of line-10 tumor cells. The capability of BCG-treated animals to reject contralateral line-10 challenge was inversely proportional to the initial intravascular tumor dose. Survival studies clearly demonstrate that a significant therapeutic effect could be achieved in guinea pigs with regional skin tumors and limited vascular metastases when the modality of therapy included BCG-intratumoral injection, followed 6 weeks later by surgery of the established skin tumor and regional lymph node. These results suggest that the development of tumor immunity after BCG-intratumoral injection is not impaired by the systemic tumor burden, but rather that it is preempted at distant sites. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; i.a., intraarterially; i.d., intradermally; i.v., intravenously; SDA, superficial distal axillary.     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

Impairment of systemic concomitant antitumor immunity by excess soluble tumor antigen

Summary Animals bearing a 3-methylcholanthrene induced sarcoma called MC1 rejected substantial numbers of a suspension of the same tumor cells injected IV in comparison with normal rats. The factors that protected the host against lung metastases were impaired by the administration of tumor antigen in the form of irradiated tumor cells or soluble tumor antigen. Animals bearing an MC1 tumor which received either unrelated MC11 irradiated tumor cells or soluble tumor antigen had more lung metastasis than the animals not given any tumor products. However, a statistically significant increase in the number of lung tumor nodules was observed in the rats treated with MC1, compared with those treated with MC11 tumor antigen (soluble tumor antigen or irradiated tumor cells) or no tumor antigen. The increase in the outgrowth of lung tumor nodules in the tumor-bearing host given an excess of tumor materials was produced by a dual mechanism of inhibition of the concomitant immune resistance and nonspecific resistance. The present study shows that soluble tumor antigen similar to material shed from a primary tumor is able to impair concomitant immune resistance to tumor cells within the lungs.     

Antimetastatic effect of Lactobacillus casei YIT9018 (LC 9018) on a highly metastatic variant of B16 melanoma in C57BL/6J mice

Summary The effect of Lactobacillus casei YIT9018 (LC 9018) on a highly metastatic variant of B16 melanoma, B16-BL6, was determined in C57BL/6 mice. Intralesional (i.l.) injection of LC 9018 inhibited tumor growth and prolonged the survival after s.c. inoculation of B16-BL6 into C57BL/6 mice. Injection of LC 9018 i.v. protected the mice against pulmonary metastasis after i.v. inoculation of B16-BL6. Injection of LC 9018 i.l. before surgical excision of the primary tumor inhibited axillary lymph node metastasis and i.v. injection of LC 9018 after surgical excision of the primary tumor inhibited both axillary lymph node and lung metastases. On the other hand, the combination of i.l. and i.v. injections of LC 9018 markedly inhibited both lymph node and lung metastases. Natural killer cell activity of axillary lymph node cells was augmented by the injection of LC 9018 into a front footpad, while the cytolytic activity of axillary lymph node cells was significantly enhanced. However, the cytolytic activity was diminished by depleting whole lymph node cells of the plastic adherent cells. Furthermore, alveolar macrophage-mediated cytotoxic activity was augmented by the i.v. injection of LC 9018.     

Eradication of microscopic hepatic metastases by active specific immunization

Strain-2 guinea pigs, each with microscopic deposits of line 10 hepatocarcinoma in the liver, were treated by ID immunization with a mixture of irradiated tumor cells and an oil-in-water emulsion containing cell walls of Mycobacterium bovis strain Bacillus Calmette-Guérin (BCG CWE). Injection of line 10 hepatoma cells into the hepatic portal vein led to the development of tumor foci in the liver, metastasis in the hepatic lymph node, malignant ascites, and death. Active immunization using irradiated line 10 cells and BCG CWE was effective therapy when administered 1, 7, and 14 days after intraportal injection of line 10 cells. Effective immunization required both irradiated line 10 tumor cells and the BCG cell wall emulsion. Immunization with BCG CWE admixed with irradiated line 1 tumor cells, a hepatoma antigenically distinct from line 10, did not prevent outgrowth of line 10 deposits in the liver. Animals rendered free of disease could reject a challenge of line 10 tumor cells but not of line 1 tumor cells.     

Model for mediastinal lymph node metastasis produced by orthotopic intrapulmonary implantation of lung cancer cells in mice

This study is designed to establish a pulmonary tumor model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma cell (LLC) suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 sec per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph nodes metastasis were observed in all mice that were succeeded to form a lung nodule after intrapulmonary implantation. The size of tumor nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21 +/- 2 days (range; 19-24 days). Histopathological analysis revealed that no metastatic tumor was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumor cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of CDDP on day 1 after tumor implantation tended to suppress the primary tumor nodule and significantly inhibited the lymph node metastasis. Thus, a solitary pulmonary tumor nodule model with lymph node metastasis approximates clinical lung cancer, and may provide a useful basis for lung cancer research.     

Modulation of adoptive cell transfers in experimental allergic encephalomyelitis by antigen treatment of donors

Experimental allergic encephalomyelitis has been adoptively transferred using lymph node cells from Strain 13 guinea pig donors sensitized with purified encephalitogenic myelin basic protein. Adoptive cell transfer was used to examine the immunocompetence of lymph node cells obtained from guinea pigs protected from disease development by treatment with MBP. Lymph node cells from guinea pigs unresponsive to EAE challenge do not adoptively transfer disease. Cells obtained from guinea pigs treated with MBP following encephalitogenic challenge are competent in adoptive transfer with respect to pathologic lesions, but not clinical disease. The clinical and pathologic responses of recipients of the histocompatible lymphocyte populations are similar to those seen in the treatment-matched donor controls, suggesting that under these circumstances lymphoid cells, rather than circulating soluble factors, are responsible for disease induction and suppression.     

The use of immunologically competent cells from pig mesenteric lymph nodes to treat pulmonary tumors in mice

Summary Pulmonary tumors were produced in A strain mice by intravenous injection of 1×10⁶ A strain mammary carcinoma cells. The mice were killed on day 14, their lungs fixed in Bouin's fluid, and the number of tumors counted.The mesenteric lymph node chains of pigs were immunized by implantation of tissue into the mesentery. In all animals the middle segment of node chain was excised. The remaining segments of node proximal and distal to the resected segment were, in separate pigs, nonimmunized or immunized against mouse tumor, immunized against mouse tumor or mouse skin, or immunized against human tumor or mouse tumor. All segments of node chain were removed 7 days after immunization for preparation of cell suspensions.When tumor cells were combined in a ratio of 1 : 10 or 1 : 60 with mouse tumor-immune pig cells, there was a significant reduction in tumor formation compared to that in mice receiving tumor cells alone.Injection of mouse tumor-immune pig cells on day 7, to treat tumors inoculated on day 0, was ineffective. However, when the mice received, in addition, 200 rad thoracic irradiation on day 3, immune pig cells reduced the number of tumors compared to that in animals receiving irradiation alone, or irradiation and nonimmune pig cells.In further experiments, in order to increase the number of pig cells reaching the lungs, a splenectomy was performed on day 6, prior to intravenous injection of immune cells on day 7. A comparison was made of the antitumor effect of pig cells immunized against mouse tumor, mouse skin, or human tumor. Cells immunized against either mouse tissue were equally effective in reducing the number of tumors compared to the number in animals receiving tumor cells alone. However, cells immune to human tumor were ineffective.     

Feasibility and limits of an orthotopic human colon cancer model in nude mice

We sought to develop an accurate colorectal cancer model in nude mice with stable local growth, tumor cell dissemination, and reproducible metastatic capacity. To this end, we orthotopically transplanted histologically intact human colorectal cancer tissue from 10 human patients into nude mice. After successful local tumor growth, tumor tissues were retransplanted as many as 9 times in serial passage. All specimens were transplanted using microsurgical techniques. Histologic, immunohistochemical, and polymerase chain reaction techniques were used to determine tumor growth rates and kinetics, development of regional lymph node and distant hepatic metastases, and the induction of minimal residual disease (MRD). Stable local tumor growth rates with variable growth kinetics were detected in 73.4% of all mice. The lymph node and hepatic metastasis rates were low, at 18.4% and 4.9%, respectively. MRD, as reflected by CK20 positivity of the bone marrow in animals with lymph node and hepatic metastases, was present in 22.2%. The orthotopic colorectal cancer model described here is feasible for the induction of reproducible local tumor growth but is limited by variable growth kinetics and the low rate of lymph node and hepatic metastases. Cytokeratin-positive cells indicative of MRD could be detected in the bone marrow of approximately 25% of the nude mice with metastases. The observed induction of MRD after orthotopic implantation of intact human colon cancer in animals with lymph node and hepatic metastases might be improved if established colon cancer cell lines were used.     

Mechanisms involved in the expression of Jones-Mote hypersensitivity: II. Lymph node morphology and in vitro correlates

Lymph node morphology, in vitro lymphocyte transformation, and inhibition of macrophage migration were studied at varying intervals after sensitization for Jones-Mote hypersensitivity (JMH) with ovalbumin (OA) in Freund's incomplete adjuvant (FIA). The effect of cyclophosphamide (CY, 300 mg/kg), given 3 days before sensitization with OA in FIA, was also studied in an attempt to clarify further its role in increasing the intensity of skin reactions and its effect on the passive transfer of skin reactivity described in the preceding paper. There were increased numbers of large pyroninophilic cells in paracortical areas of draining lymph nodes and increased in vitro DNA synthesis, by lymph node cells, in animals treated with CY 3 days before sensitization with OA in FIA. There was no inhibition of macrophage migration of PEC from animals sensitized with OA in FIA, whether or not these guinea pigs had been treated with CY before sensitization.     

Hereditary melanoblastoma in miniature pigs and its successful therapy by devitalization technique.

Applying selective breeding for 10 years we established the MeLiM (Melanoblastoma-bearing Libechov Minipigs) strain. Melanoblastoma (MB) in this strain shows a hereditary occurrence. Cutaneous tumours are usually nodular, multiple and distributed on various parts of body. They appear in darkly pigmented animals already at the birth or during two months thereafter (57% of all animals). Numerous organ metastases mainly into the spleen, lymph nodes and lungs are regularly ascertained in animals with cutaneous MB. Tumour cells were surprisingly found also in the inner organs of phenotypically healthy minipigs in which no cutaneous MBs were observed visually (27% of all animals). About 34% of all affected piglets die during the first 2 months of age. These features document a malignancy of this tumour in the MeLiM strain. Original surgical technique was applied in more than 40 affected minipigs at 1-2 months of age. It consists in a devitalization (ischemization) of one of cutaneous tumours by the mattress sutures conducted around the tumour base without any excision of tumour tissue. This simple procedure causes a total destruction of MB cells in all cutaneous tumours as well as in all organ metastases during 4-6 months. Animals treated by this technique were fully healed of tumour cells and no relapses were observed. This technique could bring similar positive results also in therapy of human MB.     

Antigen-bearing Langerhans cells in skin,dermal lymphatics and in lymph nodes

Ferritin-challenged skin sites and draining lymph nodes were studied in normal guinea pigs and in guinea pigs which had been passively sensitized to ferritin or peroxidase by lymphoid cell transfer to ascertain whether Langerhans cells can bind antigen in skin and carry it to lymph nodes. After intradermal challenge with amounts of ferritin as small at 5 μg, ferritin-containing Langerhans cells were seen by electron microscopy in the marginal sinus and cortex of draining lymph nodes in ferritinscnsitized animals and, to an apparently lesser degree, in control animals. Lymph nodes from unchallenged normal guinea pigs contained rare Langerhans cells, none of which had ferritin. The findings indicate that Langerhans cells may pick up antigen in skin and from there circulate to draining lymph nodes, thus carrying out a function analogous to macrophages. In this way they may exhibit antigen to lymphocytes both in skin and in lymph nodes.     

Experimental allergic orchitis and aspermatogenesis. VI. Transfer of allergic orchitis with immune cells.

Typical experimental allergic orchitis (EAO) and aspermatogenesis were successfully transferred to strain 13 guinea pigs with peritoneal exudate and lymph node cells from male and female donor guinea pigs (lacking detectable antibody) previously sensitized with 9 mug of highly purified GP1 glucoprotein isolated from the sperm acrosome. Attempts to transfer the disease with circulating antibody from hyperimmunized animals were not successful. These studies support a cell-mediated basis for the immunopathologic events in EAO.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

Active specific immunotherapy of guinea pigs with visceral tumor implants

Summary Guinea pigs, each with established, 7-day-old, syngeneic visceral micrometastases of line 10 tumor implanted intravenously, were immunized by intradermal inoculations into several sites of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG or Mycobacterium phlei bacilli. This treatment led to survival of 72 of 80 treated animals (90%). Therapeutic effectiveness depended on the dose of mycobacteria and on that of irradiated tumor cells. Animals treated by intradermal injection of mycobacteria attached to oil droplets alone or with irradiated tumor cells alone, all died with multiple foci of pulmonary tumor.