Therapy of spontaneous pulmonary metastases of a murine mammary carcinoma with anaerobic corynebacteria

Summary Corynebacterium parvum given intravenously or into the tumor 7 or 2 days before surgical removal of primary tumors greatly reduced the number of spontaneous pulmonary metastases of a syngeneic mammary carcinoma of C3Hf/Bu mice. When a single dose of 6000 rads -rays was used to eliminate primary tumors the number of lung metastases significantly increased. Administration of C. parvum before irradiation not only prevented this metastasis — facilitating effect of radiation, but also reduced the number of lung nodules below that in amputated mice. Metastatic spread was not altered by postoperative treatment of mice with single doses of C. parvum or C. granulosum. However, significantly more mice had lung metastases if they were given 2 intraperitoneal injections of C. granulosum.     

A mouse model for immunotherapy of osteosarcoma

Summary Various combinations of the prostaglandins synthetase inhibitors, aspirin or indomethacin, and Corynebacterium parvum were used as adjunctive therapy to surgery in the treatment of the metastasizing Dunn osteosarcoma C3H/HeJ mice. In doses corresponding to those that could be tolerated by humans all three agents given singly reduced the number and estimated weight of metastases whether they were administered pre-operatively or post-operatively. When the two modalities of therapy were combined a significant additive anti-tumour effect was observed with both C. parvum and aspirin and C. parvum and indomethacin, but only if treatment was commenced pre-operatively. Similar results were obtained with two other metastasizing tumours, viz. the B16 melanoma and the Lewis lung carcinoma. In the absence of any evidence for an interaction between the prostaglandin synthetase inhibitors and circulating tumour cells, it was felt that the additive effect could be best explained in terms of interference with prostaglandin-mediated negative feedback of the anti-tumour action of C. parvum.     

Antitumor activity of Corynebacterium parvum administered into the pleural cavity of mice

Summary We investigated the efficacy of intrapleurally (IPl) injected 0.25 mg Corynebacterium parvum (CP) against pulmonary tumor deposits of four syngeneic murine tumors, C3Hf/Bu or CBA fibrosarcoma (FSa) or mammary carcinoma (MCa), and compared its efficacy with that of intravenous (IV) CP. A mode of action of CP given by IPl administration was also studied. When given to mice prior to the IV inoculation of tumor cells, IPl and IV CP reduced the number of metastases of C3Hf/Bu-FSa and CBA-MCa equally well. Intrapleural injection of CP within a week after tumor cell inoculation was more effective against metastases in the lung than IV CP. A combination of IPl and IV administration of CP was more effective in the therapy of lung metastases of CBA-FSa and CBA-MCa than either single treatment alone. Intrapleural and IV injections of CP augmented concomitant immunity to artificially produced lung metastases of C3Hf/Bu-FSa. Intrapleural CP was less effective than IV CP in inducing complete regression of the SC growing C3Hf/Bu-FSa.In contrast to IV CP, IPl CP did not markedly influence the spleen weight and cellularity. It was also less effective in increasing the liver weight. However, CP by either route of injection led to a significant increase in the lung weight. CP injected IPl, but not IV, caused a significant increase in the number of nucleated cells in the pleural cavity of mice. More than 90% of these cells were macrophages, and they were found to be cytotoxic for in vitro cultures of CBA-MCa cells. Activated macrophages also mediated in vivo antitumor resistance, as shown by the abolition of this resistance by treatment of the mice with carrageenan.     

Anti-tumour activity of Lactobacillus casei on lewis lung carcinoma and line-10 hepatoma in syngeneic mice and guinea pigs

Summary The anti-tumour activity of Lactobacillus casei YIT 9018 (LC 9018) on Lewis lung carcinoma (3LL) in C57BL/6 mice and line-10 hepatoma in strain-2 guinea pigs was examined. Intravenous injection of LC 9018 was effective for inhibition of pulmonary metastases in C57BL/6 mice after s.c. inoculation with 3LL tumours. Intralesional (i.l.) injection of LC 9018 was also effective for both prolongation of the survival period and inhibition of pulmonary metastases in 3LL tumour-bearing mice. The combination treatment of i.l. and i.v. injections of LC 9018 before or after surgical excision of the primary tumour remarkably inhibited the pulmonary metastases after inoculation with 3LL tumour. Intralesional injection of LC 9018 was effective for regression of the established tumours of line-10 hepatoma inoculated i.d. and for induction of systemic tumour immunity in strain-2 guinea pigs.     

Local and metastatic growth of Lewis lung carcinoma as a function of its transplantation site. Application to the study of the pharmacology of sulphadiazine triazene]

The presence of a local tumour and pulmonary metastases is studied after the administration of the Lewis Lung Carcinoma at different sites of transplantation. The intravenous administration routes, in the tail vein and in the portal vein, injections in the liver and in the muscle of the left hindleg are used. The injection in the tail vein induces pulmonary "metastases". The injection in the portal vein is followed by multiple tumours in the whole liver and pulmonary metastases. An unilobar hepatic tumour and early pulmonary metastases appear after transplantation in the liver. Intra-muscular injection gives a local tumour which can be weighed after amputation of the leg and pulmonary metastases. A test of treatment by Sulphadiazine Triazene points out a weak action on the primary tumour and a larger one on the metastases.     

Effect of systemic administration of mouse recombinant interferon-gamma on the lung tumor metastases in mice

The purpose of this study was to examine the effective anti-metastatic activity by multiple i.v. administrations of mouse recombinant interferon-gamma (IFN-gamma) against pulmonary metastases of 3LL or B16-BL6 melanoma cells after surgical excision of primary tumors. Multiple treatments with IFN-gamma reduced effectively the incidence of pulmonary tumor metastases. Repeated 4 consecutive treatment modalities with IFN-gamma showed remarkable reduction of lung tumor colonies, and also rendered alveolar macrophages (AM) cytotoxic against B16-BL6 cells. In contrast, 14 consecutive administrations of IFN-gamma at any doses (10(2) and 10(3) U/mouse) could not activate macrophages to become cytotoxic, but were effective in regressing metastases. Thus, antimetastatic activity of IFN-gamma may be due to the stimulation of host immune defense systems such as induction of tumoricidal macrophages, presumably the direct antiproliferative action to tumor cells, or both actions under the appropriate administration conditions. We found that the systemic administration of IFN-gamma under appropriate multiple treatment modalities results in the reduction of the lung metastases and can activate AM to become tumor cytotoxic at relatively low doses (10(2) U). High-dose IFN-gamma in the multiple administration schedule was also effective for the reduction of lung tumor colonies, but strongly suppressed the nonspecific immune function and could not activate tumoricidal properties of AM.     

Different metastatic modes of malignant melanoma implanted in the ear of young and old mice

Summary An attempt has been made to determine (a) whether aging plays an important role in resistance against metastasis and (b) whether dithiothreitol, an effective in vitro mitogenic potentiator of splenic cells of young and old mice, can modulate the occurrence of pulmonary metastasis. B16-F10 melanoma cells were injected into the outer ear of young and old female C57BL/6 mice; and the growth of the primary tumor, the palpable size of the cervical lymph node, and the number of lung metastases were then determined at various intervals. The ear was amputated when the primary tumor reached 4 mm in mean diameter. The following results were obtained. (a) The growth rate of the primary tumor in young mice is comparable to that in old mice. (b) Enlargement of the cervical lymph node occurs earlier in old than in young mice. (c) Old mice are more vulnerable to pulmonary metastases, but small metastasized pulmonary colonies are more prominent in old than in young mice. (d) Dithiothreitol (100 g) injected every 2 days after the inoculation of tumor cells is effective in reducing the incidence of pulmonary metastases in old mice.     

Immunotherapy of a murine ovarian carcinoma with Corynebacterium parvum and specific heteroantiserum. I. Activation of peritoneal cells to mediate antibody-dependent cytotoxicity.

Immunotherapy with a combination of Corynebacterium parvum and specific heteroantiserum is significantly more effective than treatment with either single agent in prolonging the survival of mice that have recevied an i.p. injection of syngeneic murine ovarian carcinoma (MOT) cells. Invitro, a combination of C. parvum-activated peritoneal cells and specific heteroantiserum has proven significantly more effective than either single component in destroying 51Cr-labeled MOT cells in the absence of complement. Activation of peritoneal cells to produce lysis of tumor in the presence specific antiserum peaked 3 to 7 days after a single injection of C. parvum and declined to baseline over 3 to 4 weeks. With repeated i.p. injections of C. parvum at appropriate intervals, activation of peritoneal cells could be prolonged and augmented. Among the routes tested, only i.p. administration of C. parvum was effective, although activation of peritoneal cells for cooperation with heteroantiserum was observed over a broad range of i.p. dosage (0.45 to 4.20 mg). These data suggest that the administration of C. parvum by appropriate doses, routes, and schedules can attract and activate a population of peritoneal effectors that mediate antibody-dependent cytotoxicity more effectively than resident peritoneal cells.     

Eicosanoids and metastasis: experimental aspects in Lewis lung carcinoma

Lewis lung primary carcinomas have been extracted for eicosanoids, and the findings examined in relation to lung metastases. The order of the 5 compounds measured was PGE2 greater than PGE1 greater than PGF2 alpha greater than 6-keto-PGF1 alpha greater than TXB2. On the basis of the observation that the balance of PGI2 and TXA2 is altered in metastasis (Honn et al., 1983), the effects of Nafazatrom, a PGI2 enhancing agent, and imidazole, a thromboxane synthetase inhibitor, were tested. The experimental approach taken was to study spontaneous lung metastases after removal of the primary tumour at 13 days after tumour cell inoculation. Both Nafazatrom and imidazole decreased the lung weight when given during the period either before or after the excision of the primary tumour. There was a general trend toward an increase in the number of small lung nodules (greater than 2 mm) and a decrease in large lung nodules (greater than 2 mm) as a result of the chemotherapy. Mean survival time of the mice was significantly different among the five groups, with the mice surviving the longest in the group treated with Nafazatrom after the excision of the primary tumour.     

The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo

We showed previously that adoptive immunotherapy with the combination of LAK cells and recombinant IL 2 (RIL 2) can markedly reduce pulmonary micrometastases from multiple sarcomas established 3 days after the i.v. injection of syngeneic tumor cells in C57BL/6 mice. In this report, we analyzed the factors required for successful therapy. Titration analysis in vivo revealed an inverse relationship between the number of pulmonary metastases remaining after treatment and both the number of LAK cells and the amount of RIL 2 administered. Fresh or unstimulated splenocytes had no anti-tumor effect; a 2- to 3-day incubation of splenocytes in RIL 2 was required. LAK cells generated from allogeneic DBA (H-2d) splenocytes were as effective in vivo as syngeneic, C57BL/6 (H-2b) LAK cells. The anti-metastatic capacity of LAK cells was significantly reduced or eliminated when irradiated with 3000 rad before adoptive transfer. The combined therapy of LAK cells plus RIL 2 was shown to be highly effective in mice immunosuppressed by 500 rad total body irradiation and in treating macrometastases established in the lung 10 days after the i.v. injection of sarcoma cells. Further, reduction of both micrometastases and macrometastases could also be achieved by RIL 2 alone when administered at higher levels than were required with LAK cells. The value of LAK cell transfer and of IL 2 administration for the treatment of tumors established at other sites is currently under investigation.     

Treatment with agmatine inhibits Cryptosporidium parvum infection in infant mice

Cryptosporidium parvum is an intracellular protozoan parasite that causes enteric infection and diarrhea in a wide range of mammalian hosts, including humans and economically important livestock species. There are no effective vaccines or drug treatments available for cryptosporidiosis. Cryptosporidium parvum utilizes a unique metabolic pathway for the synthesis of polyamines, forming agmatine as an intermediary metabolite. We treated infant mice with oral doses of agmatine for 2 days before, the day of, and 5 days following experimental infection with C. parvum. Mice treated with agmatine were significantly less infected with C. parvum than were control mice receiving phosphate-buffered saline. Mice treated with agmatine only on the day of experimental infection with C. parvum were also significantly less infected than were control mice. These data suggest that exogenous agmatine alters the metabolism of C. parvum sufficient to interfere with its ability to colonize the mammalian intestine.     

Development of a bioassay for the antitumor activity of biological response modifiers of the reticuloendothelial stimulant class

Summary Following intravenous administration of various doses of DiLuzio glucan, Wellcome C. parvum, or BCG, the increase in clearance of intravenous ¹²⁵IUdR-radiolabeled B16 tumor cells from the lung correlated with a subsequent reduction in the outgrowth of pulmonary tumor nodules. The ranges of values for tumor cell clearance were very much narrower for most groups of animals than the ranges obtained for the number of pulmonary tumor nodules in similar groups, allowing 2–3 times the definition of data with the former method.     

Lymphocytes generated by in vivo priming and in vitro sensitization demonstrate therapeutic efficacy against a murine tumor that lacks apparent immunogenicity

The adoptive transfer of sensitized lymphocytes is an effective means to mediate the regression of established tumors. However, successful therapy can only be demonstrated in animal models where tumors are intrinsically immunogenic, capable of eliciting systemic immunity. To explore the potential of this therapeutic approach to tumors of less immunogenicity, we have selected and used a murine tumor, MCA 102, for the current study because all attempts to immunize syngeneic mice failed. We report here that inoculation of mice with a mixture of tumor cells and a bacterial adjuvant, Corynebacterium parvum led to the production of sensitized, but not fully functional, lymphocytes in the draining lymph nodes (LN). These cells, termed pre-effector cells, could nevertheless further differentiate to acquire full immunologic function by an established in vitro sensitization culture method. In adoptive immunotherapy experiments, transfer of as few as 1.5 X 10(7) in vitro sensitized cells not only reduced established pulmonary MCA 102 metastases but also prolonged survival and cured tumors in a majority of the treated animals. In order to elicit pre-effector cells in vivo, inoculation with both tumor cells and C. parvum was essential. Although a broad range of numbers of MCA 102 tumor cells appeared to be effective, generation of pre-effector cells was dependent on the dose of C. parvum. We have found that a C. parvum dose of 25 micrograms was optimal, whereas higher doses of the adjuvant had suppressive effects. Analysis of the kinetics of their appearance revealed that the generation of pre-effector cells was transient. They were detectable 7 days after in vivo priming followed by a rapid decline. Furthermore, pre-effector cells were detected only in the regional draining LN. No reactivity was demonstrable in the spleen, mesenteric LN, PBL, or bone marrow. Taken together, these results expand the scope of immunotherapy by demonstrating the feasibility of manipulating a limited and obscure immune response to the MCA 102 tumor for therapeutic efficacy.     

Immunogenicity, macrophage sensitivity, and therapeutic response to C. parvum of fibrosarcomas induced in C. parvum-treated and untreated mice

Summary No correlation has been observed between the therapeutic response of isogeneic transplants of methylcholanthrene-induced murine fibrosarcomas to Corynebacterium parvum (CP) and the sensitivity of the tumour cells to the cytotoxic action of CP-activated macrophages (Mø) in vitro. Neither of these parameters appeared to be influenced by repeated administration of CP during induction of the primary tumour or by whether the carcinogen was administered in solution or as crystals on a Millipore disc. On the other hand, tumours which developed in CP-treated mice after injection of a small dose (0.1 mg) of methylcholanthrene (MC) were more immunogenic than those which developed after the same dose of MC in untreated mice, as judged by the capacity of an injection of an irradiated tumour cell suspension to protect isogeneic mice against subsequent challenge with viable cells. Further work is needed to explain these findings.     

Evidence for granulocyte-mediated macrophage activation after C. parvum immunization

It has been previously demonstrated that at the peak of the peritoneal response to Corynebacterium parvum (Day 4), cytolytic macrophages can be characterized by the presence of intracellular bacteria. In the present study, the role of neutrophils in the activation of peritoneal macrophages by C. parvum was investigated. Inflammatory neutrophils isolated 5 hr after ip administration of C. parvum were transferred to normal, syngeneic mice and the peritoneal macrophages of recipients harvested 4 days later were tested for cytoxicity against HeLa cells. Neutrophils isolated from mice 5 hr after C. parvum immunization were effective in inducing cytolytic macrophages. Less than 100-fold as much bacteria was needed to induce comparable levels of cytotoxic activity when introduced inside granulocytes. Neutrophils obtained from mice 48 hr after C. parvum injection or mononuclear cells were not good macrophage activators. Viable neutrophils were not required as freeze-thawed cells were able to activate macrophages in recipient mice. The intracellular distribution of C. parvum changed dramatically with time. Initially almost all bacteria were found within neutrophils. By 24 hr, many macrophages contained either bacteria or granulocytes which had ingested C. parvum. Pyridine extracts of C. parvum, which do not activate peritoneal macrophages when injected directly into mice, did not induce neutrophils capable of activating macrophages. The residue of pyridine-extracted C. parvum did induce neutrophils that could activate macrophages when transferred. The results suggest that processing of the bacteria by inflammatory granulocytes may be an obligatory step in macrophage activation by this agent. The peak response occurred earlier than T-cell immunity is usually observed and it is suggested that direct activation of macrophages via ingestion of neutrophils may represent the earliest stage of macrophage activation by C. parvum.     

Active Immunotherapy with Corynebacterium parvum and Chemotherapy in Murine Fibrosarcomas

Corynebacterium parvum used alone to enhance immunological reactivity produced transient inhibition of the growth of chemically induced isogenic mouse tumours. Attempts were made to combine C. Parvum with cyclophosphamide to see whether this would increase the latter''s effectiveness in inhibiting early but established tumours. Of the various regimens tested, the administration of the C. parvum 12 days after a single dose of chemotherapy produced dramatic inhibition of tumour growth and resulted in complete and lasting regressions in up to 70% of tumour-bearing animals. The most important variable in this regimen is the time between the chemotherapy and the subsequent immunotherapy.It is possible that non-specific active immunotherapy with agents such as C. parvum may be a valuable adjunct to the conventional cyto-reductive treatments of cancer, but the time of administration of such therapy is probably critical for each tumour and for each chemotherapeutic regimen.     

Trial of the cysteine cathepsin inhibitor JPM-OEt on early and advanced mammary cancer stages in the MMTV-PyMT-transgenic mouse model

Abstract Recent data suggest proteases of the papain-like cysteine cathepsin family as molecular targets for cancer therapy. Here, we report the treatment of polyoma middle T oncogene-induced breast cancers in mice with the cell-permeable broad-spectrum cysteine cathepsin inhibitor JPM-OEt. Up to 100 mg/kg inhibitor was intraperitoneally injected once per day in two trials on early and advanced cancers. In both trials, transient delays in tumour growth were observed. However, at the endpoint of both experiments no significant differences in tumour weights, histopathology and lung metastasis were found between the inhibitor and the control group. The invasive strand formation of collagen I-embedded tumour cell spheroids generated from primary tumours of inhibitor-treated mice in the early cancer trial could be inhibited in vitro by JPM-OEt; a result arguing against induction of resistance to the inhibitor. Measurement of cysteine cathepsin activities in tissue extracts after intraperitoneal injection of JPM-OEt revealed effective inhibition of cysteine cathepsins in pancreas, kidneys and liver, while activities in mammary cancers and in lungs were not significantly affected. We conclude that the pharmacokinetic properties of JPM-OEt, which result in poor bioavailability, may prohibit its use for stand-alone treatment of solid mammary cancers and their lung metastases.     

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.     

Inhibitory effect of Boerhaavia diffusa on experimental metastasis by B16F10 melanoma in C57BL/6 mice

Administration of the aqueous methanol (3:7) extract of B.diffusa was found to be effective in reducing the metastases formation by B16F10 melanoma cells. Prophylactic administration of the extract (0.5 mg/dose) inhibited the metastases formation by about 95% as compared to untreated control animals. There was 87% of inhibition in the lung metastases formation in syngenic C57BL/6 mice, when the extract was administered simultaneously with tumour challenge. Biochemical parameters such as lung collagen hydroxyproline, hexosamines and uronic acid levels were also reduced significantly (P < 0.001) in the treated animals. Levels of serum sialic acids and gamma-glutamyltranspeptidase that are markers of neoplastic proliferation were also reduced in the tumour plus extract treated animals. More over treatment with the extract enhanced the survival of the animals more than double that of untreated control animals. When a non-toxic concentration of the extract was treated directly to the B16F10 cells in vitro, it inhibited the cell proliferation as estimated by the 3H - thymidine uptake assay. From the Zymogram analysis using culture supernatant from the extract treated cells it became evident that the components of the extract inhibited the expression or activity of gelatinases A and B (MMP-2 and MMP-9). Since the MMPs are intimately associated with cell invasion and angiogenesis, inhibition of these functions along with the anti-proliferative activity (cytostatic) may be contributing to the antimetastatic property shown by B. diffusa.     

Inhibition of spontaneous pulmonary metastases of Lewis lung carcinoma by oral treatment with Respivax and Broncho-Vaxom

Summary The antimetastatic activity of orally administered polybacterial vaccines, Broncho-Vaxom (BV) and Respivax (RV) was examined in C57BL/6 mice, bearing implants of Lewis lung carcinoma (3LL) in the footpad. The oral administration of BV or RV for 10 consecutive days before or after surgery caused significant reduction of the number and volume of lung metastases. In addition, the therapeutic potential of BV and RV was examined in combination with chemotherapy to determine if there is additive activity. In animals bearing pulmonary micrometastases, treatment with a combination of cyclophosphamide at 50–150 mg/kg with BV or RV was found to be more effective than each of these treatments alone. In immune function studies it was established that the oral administration of BV and RV induced an increase in the number of cells, recovered by broncho-alveolar lavage, and alveolar macrophages were dominant in these cell populations. Furthermore, oral treatment of mice with these vaccines rendered their alveolar macrophages tumoricidal for syngeneic metastatic 3LL cells in vitro. These results show that pulmonary macrophages induced by oral administration of BV and RV played a key role in the inhibition of metastasis in 3LL-bearing mice.