Failure to boost the increased clearance of embolic tumor cells from the lung with dose scheduling: Corynebacterium parvum studies.

Initial suboptimal, optimal and supraoptimal doses of intraperitoneal corynebacterium parvum were followed by 'booster' doses injected intraperitoneally 5, 10 and 25 days later. Mice were challenged intravenously 4-6 days after the booster doses with [125I]IUrd-labeled B16 tumor cells and the extent of clearance of these cells from the lungs analyzed 4 h later. In a second experiment, mice were given booster doses of C. parvum on days 4, 7 and 10 and then challenged on day 14 with labeled B16 tumor cells. In each experiment, the peak response, in the form of tumor cell clearance, to C. parvum occurred between day 5 and day 10. In no instance was a peak response to any of the initial injections of C. parvum increased by a booster dose. Instead, in some instances, the peak response was abrogated. By day 25 but not day 10, it was possible to restore the response to its peak level with a second optimal dose of C. parvum, but not to increase it.

Heterogeneity of murine mammary adenocarcinoma cell subpopulations. In vitro and in vivo resistance to macrophage cytotoxicity and its association with metastatic capacity.

Properties of Ts, a long-term tissue culture line of T1699 mammary adenocarcinoma of DBA/2 mice, and two of its sublines - TR2 and TLI-1-were comparatively studied. Ts tumors produced no spontaneous metastases, nor did i.v. injection of up to 1 X 10(6) Ts cells produce a lung tumor nodule. Ts cells were susceptible to macrophage cytotoxicity in vitro and i.v. injected cells were rapidly destroyed in the lungs. TLI-1 tumors spontaneously metastasized to the lungs, and i.v. injection of 1 X 10(3) TLI-1 cells produced approximately 15 lung nodules per animal. TLI-1 cells were least susceptible to both macrophage-mediated cytolysis in vitro and in vivo host antitumor mechanisms. TR2 cells were intermediate with respect to all these properties. Differences in their susceptibility to macrophage cytotoxicity were recognized not only by normal peritoneal macrophages but also by murine macrophage lines. On the other hand, all the subpopulations were uniformly resistant to NK activity in vitro. These findings suggest that resistance of tumor cells in vitro to macrophage cytotoxicity corresponds with their in vivo metastatic potential.

Immunological responses to a murine mammary adenocarcinoma. Outgrowth of pulmonary occult metastases induced by immunosuppressive perturbations.

Intravenously (i.v.) or subcutaneously (s.c.) injected T1699 tumor cell subpopulations (Ts, and TLI-1) were destroyed more rapidly in BALB/c nu/nu athymic mice than in syngeneic DBA/2J mice. The number of artificial pulmonary metastases of TLI-1 tumors was significantly lower, and the growth rate of s.c. Ts, and TLI-1 tumors was correspondingly slower in the nude mice. No macroscopic outgrowth of pulmonary metastases of Ts tumors was detected in either group of s.c. tumor-bearers, even though the s.c. tumors progressed and killed the hosts. Unexpectedly, however, s.c. implantation in normal DBA/2J mice of lung homogenates not only from DBA/2J but also from s.c. Ts tumor-bearing BALB/c nu/nu mice produced tumors, suggesting that significant numbers of clonogenic Ts cells were present in the lungs of animals without spontaneous outgrowth of pulmonary metastases. Perturbations of s.c. Ts tumor-bearing DBA/2J or BALB/c nu/nu mice with immunosuppressive drugs or with anti-mouse thymocyte serum, respectively, induced rapid outgrowth of pulmonary metastases.

Mitogenic augmentation of T cells from immunodepressed cancer patients by a murine interleukin-1 (IL-1).

An interleukin-1 (IL-1) lymphocyte-activating factor, with an approximate molecular weight of 13,000-16,000 was isolated from the culture supernatants of a murine macrophage-like cell line, P388D1. Contrary to the general belief that murine mediator is incapable of stimulating human lymphocyte mitogenic responses, leukoagglutinin (LA)-induced mitogenesis of peripheral blood lymphocyte (PBL) were significantly affected by murine IL-1. On one hand, when PBL were obtained from either normal healthy individuals or from cancer patients who still possessed normal levels of general immunocompetence, their mitogenic responses were not augmented any further by the murine mediator. Instead, slight but significant suppression were noted in most cases. On the other hand, the LA-induced responses of PBL from 5 immunodepressed cancer patients were markedly augmented by murine IL-1.

Vascular clearance of embolic tumor cells and colloidal carbon and the levels of serum lysozyme following muramyl dipeptide administration.

Various doses of coded samples of the parent muramyl dipeptide entity and eight synthetic analogues were injected intravenously into C57BL/6J mice, and the extent of clearance of intravenously injected 5-[125I]iodo-2-deoxyuridine (125I-dUrd) radioactively labeled B16 tumor cells from the lung was measured 3 days later. The results of between one and four experiments with each compound demonstrated that five compounds, including the parent compound, produced dose-dependent increases in the loss of tumor cells from the lung. The same five compounds also caused an increase in the clearance of intravascular carbon clearance. Monitoring of serum lysozyme levels revealed no significant increases compared to controls at doses of up to 1 mg/mouse following muramyl dipeptide (MDP) administration i.v., i.p. or s.c. at any time up to 30 days after administration. Increased pulmonary tumor cell clearance did not occur after s.c. MDP administration and peaked 3 days after i.p. or i.v. MDP, reaching near normal levels by Day 7.

An interactive computer graphic system for 3-d stereoscopic reconstruction from serial sections: analysis of metastatic growth.

An interactive computer graphic system has been developed for 3-D reconstruction of stained serial sections. The system has been used for 3-D reconstruction of portions of mouse lung containing melanoma nodules. A stereoscopic color picture of a wire model of the reconstructed object is produced. The volume, surface area, and sphericity index of structures such as tumor nodules can be computed.

Dose, route, and time dependence of serum lysozyme and antitumor activity following administration of glucan, Corynebacterium parvum, pyran, or lipopolysaccharide to mice.

Following the administration of Di Luzio particulate glucan, Corynebacterium parvum, pyran (maleic anhydride vinyl ether 6), and lipopolysaccharide (Shigella) to inbred C57BL/6J mice, dose, route, and time-dependent studies were undertaken on antitumor activity and serum lysozyme levels to explore the possible relevance of serum lysozyme as a useful index of antitumor activity. Antitumor activity was assessed by measurement of the extent of loss of iv injected 125I-labeled 5-iodo-2'-deoxyuridine-labeled B16 tumor cells. Increases in serum lysozyme levels were dose-, route-, and time-dependent and varied greatly from one agent to another. The peak levels of antitumor activity were similar for all agents but were also critically dose- and time-dependent. Correlations of serum lysozyme levels and antitumor activity were inexact. The doses for peak lysozyme level increases were higher than those for peak antitumor activity. Antitumor activity peaked earlier and lasted longer than serum lysozyme level increases.

Further studies on endocrine factors and the growth and spread of B16 melanoma.

An intramuscularly inoculated B16 melanoma line grew or slowly in normal female mice than in either those following oophorectomy and/or estrogen therapy, or in male syngenic C57BL6J mice following orchidectomy and/or estrogen therapy. The number of metastases in estrogen-treated mice was higher than in the other groups.

Development of a bioassay for anti tumor activity of biological response modifiers of the reticuloendothelial stimulant class: reproducibility of the bioassay.

Intravenously administered DiLuzio glucan, Wellcome C. parvum, and Glaxo BCG caused dose dependent increases in the tumor cell loss from the lungs of C57BL/6J and DBA/2J mice challenged respectively with intravenous 125IuDR labelled B16 or T 1699 mammary carcinoma cells. The results were reproducible with respect to the optimal doses and the magnitudes and shapes of the dose response curves.

Three dimensional reconstruction of disseminated cancer modules.

A computer system which can be used to perform three dimensional reconstruction of dispersed cancer growth from serial sections is described. The system was used to reconstruct and analyze melanoma nodules growing in the left upper lobes of the lungs of seven mice following intravenous inoculation of a single cell suspension of melanoma cells. The analysis shows a great variability in the tumor size within a single mouse, and also in different mice. The preponderance of tumors (40 out of 50) were subpleural and extended to the surface of the lung at some point. An analysis of the spatial distribution of the tumors was performed which indicates that the tumors may not be randomly dispersed in the lungs. The mechanisms which could lead to the observed results are discussed.

Differences in mitogenic responses of murine T cells to two distinct phytohemagglutinin (PHA) subcomponents.

Two distinct mitogenic subcomponents of phytohemagglutinin (PHA)--leucoagglutinin (LA) and "purified" PHA--apparently stimulated different subpopulations of murine T cells. In the DBA/2J strain, the mitogenic responses of splenic lymphocytes to LA reached maximal levels after 24 to 36 hr exposure and almost completely disappeared by 48 hr, whereas maximal responses to PHA were maintained after 48 hr incubation. The levels of LA-responding T cells were highest in DBA/2J spleens at 5 weeks of age but markedly declined by 9 weeks of age, whereas thymic levels of LA-responding T cells reached a maximum at 9 weeks of age and remained maximal past 15 weeks of age. PHA-responding cells, in contrast, reached maximal levels in both the spleen and thymus of DBA/2J mice at 9 weeks of age. In C57BL/6J mice, splenic and thymic lymphocytes responded similarly to both components, except that the response of splenic lymphocytes to PHA reached a maximum after shorter incubation time and declined sooner than their response to LA. The mitogenic responses of C57BL/6J thymocytes to both components were already at their peak by 5 weeks of age and almost totally disappeared by 9 weeks, whereas the responses of splenic lymphocytes were maximal at 9-15 weeks of age. The responses of DBA/2J splenocytes to LA was significantly augmented by PHA, but LA markedly suppressed the proliferative responses to PHA.

Collaboration between specific anti-tumor immunity and chemotherapeutic agents.

Treatment of DBA/2J mice bearing a T1699 syngeneic mammary adenocarcinoma (Ts) with a single intraperitoneal (i.p.) injection of 100 ug melphalan produced complete tumor regression in about 65% of the animals treated; however, tumors recurred in about 85% of these regressors after 15-25 days' remission. The drug regimen was ineffective against Ts tumors growing in immunosuppressed or immunodeficient animals. Stimulation of immunologically intact Ts tumor-bearers with bacterial lipopolysaccharide (LPS) or with phytohemagglutinin (PHA) 3 days prior to melphalan therapy, on the other hand, produced not only higher rates of tumor regression but also significant increases in the number of permanent cures. A tumor induced by T1699 subline TR2 was resistant to the same regimen, although Ts and TR2 cells were equally susceptible to the cytotoxic and growth-inhibiting activities of the drug in vitro. In contrast, the combination of specifically armed monocytes and melphalan in vitro produced enhanced killing of Ts cells but not of TR2 cells. Analysis of the collaborative cytotoxicity between immune effector cells and melphalan indicated that exposure of tumor cells to killer cells increased the drug susceptibility of the tumor cells, but not the reverse. These results suggest a possible mechanism for in vivo resistance of tumors to chemotherapeutic agents that is not directly associated with the drug resistance of the tumor cells in vitro.

Variations in the level of haematogenous antitumour immunity during progressive tumour growth and spontaneous blood-borne metastatic spread.

Inbred DBA2 mice bearing the syngeneic 1699 mammary tumour in the hind limb were challenged intravenously with 125IUdR-labelled single 1699 cell suspensions, and the amount of radioactivity in the lungs compared 20--24 h later with that in the lungs of normal mice or in those of mice bearing the antigenically unrelated syngeneic SaD2 tumour. An immunologically specific decrease in radioactivity was evident at variable periods of time after tumour induction, depending on the number of cells used to induce the leg tumours, but fell below that in normal mice as the leg tumours progressed beyond a weight of approximately 1 g. As assessed by microscopic scanning of serial histological sections of the same lungs the incidence of spontaneous metastases rose to between 80 and 100% immediately the amount of cell loss from the lungs of the tumour-bearing mice reached that of the normal controls. This extremely rapid series of events does not allow a definitive conclusion as to whether immunity failed and led to metastatic spread or vice versa, but does underline the strong association of immunity with the blood-borne dissemination of tumour cells in this tumour system. Following excision of tumours, in no instance was immunity detected 14 days later, and in a single experiment did not reach detectable limits until 25 days after excision, at a time when the lung metastases were observed mostly to have regressed spontaneously.

Effect of glucan and other adjuvants on the clearance of radiolabeled tumor cells from mouse lungs.

The iv injection of two forms of glucan (DiLuzio and Yamamura), Corynebacterium parvum, or bacillus Calmette-Guérin (BCG) caused a significant increase in the rate and extent of clearance of subsequently injected radiolabeled T1699 carcinoma cells from the lungs of syngeneic DBA2 mice. Dose-response curves were obtained for each agent, the relative rates of clearance of radiolabeled cells were established, and preliminary sequential comparative studies were undertaken. The optimum dose for C. parvum appeared to be lower than for DiLuzio glucan which appeared to be lower than for Yamamura glucan or BCG. The results indicated that at optimum doses one form of glucan (DiLuzio) was as effective as C. parvum and more effective than BCG or a second form of glucan (Yamamura), with respect to the time for the genesis, the magnitude, and the length of time of the response. Very preliminary experiments with a highly malignant and poorly immunogenic cell line of B16 melanoma in syngeneic C57BL/6J mice demonstrated a correlation between an increased level of clearance of radiolabeled cells and a reduced number of subsequent macroscopic lung tumor nodules.

Rat sarcoma model supports both "soil seed" and "mechanical" theories of metastatic spread.

Following injection into the portal venous or vena caval systems, tumour cells are held up almost exclusively in the liver or lung respectively, and subsequent outgrowth of tumour only occurs in these organs. Following systemic arterial injection, cells are distributed, and subsequently grow, in a variety of organs. However, the adrenal gland supports tumour growth from much fewer cells than the lung, and this is partly due to the fact the rate of tumour cell loss in the initial 48 h is very high in the latter compared to the former organ.