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The present study was designed to test the hypothesis that endogenous prostaglandin E (PGE) promotes the development, growth and metastasis of spontaneous mammary tumors in C3H/HeJ female retired breeder mice. The effect of chronic oral indomethacin (indo) therapy starting at 6 months of age was tested on these parameters as well as on animal survival, in comparison with control mice placed on 0.2% ethanol in drinking water for up to 25 months of age. Indo treatment delayed the initial (up to 27 weeks) development of primary tumors by 11-12 weeks; however, the subsequent rate of tumor appearance was unaffected (totaling 82% in indo-treated vs. 90% in controls by 25 months of age). Spontaneous regression of primary tumors (26% in controls) increased 2-fold (53%) with indo therapy. While the apparent reduction in the growth rate of primary tumors and the overall prolongation of animal survival were not significant, the lifespan of mice bearing multiple tumors was significantly prolonged by therapy. There was also a 2-fold reduction in the incidence of lung metastases in mice bearing detectable primary tumors, and this was more pronounced during the earlier phase of tumor development. Positive immunostaining for cyclooxygenase-2 enzyme (indicative of the cellular source of PGE) was exhibited by tumor cells, stromal cells and macrophages within the primary tumors. Tumors in indo-treated mice exhibited histological evidence of increased differentiation (acinar architecture), significant tumor cell death, mononuclear cell infiltration and reduction in vascularity, indicating that the beneficial effects of indo were due to multiple mechanisms, including improved immune response and reduced angiogenesis.

Experimental work in murine models has shown that, during the development of tumors, prostaglandin E2 produced by host macrophages inactivates natural killer cells and suppresses lymphokine-activated killer (LAK) cell development. Chronic indomethacin therapy when combined with interleukin-2 (IL-2) can totally eradicate experimental lung metastases in these models. A phase II trial was performed to study the clinical efficacy of chronic indomethacin and intermittent IL-2 therapy in patients with advanced renal cell carcinoma. Patients were placed on indomethacin and ranitidine orally at least one week prior to commencing therapy with IL-2. IL-2 was given by continuous infusion for three courses, each consisting of 5 days of treatment with 6 days of rest. Initial dose of IL-2 was 18.0 x 10(6) IU/m2/day for the first course with escalation to 27.0 x 10(6) IU/m2/day for the second and 36.0 x 10(6) IU/m2/day for the third course, if toxicity allowed. Patients were admitted to a general oncology ward for therapy with IL-2, and vasopressor agents were not used. Thirty-two patients were eligible, with 7 patients withdrawing early from the study. Twenty-five patients went on to receive at least one course of IL-2. Two complete and three partial responses were seen for an objective response rate of 5/25 (20%) for eligible and treated patients or 5/32 (16%) for all patients entered onto the study, regardless of treatment status. The response rate to this regimen is comparable with other high dose IL-2 regimens in renal cell carcinoma, including those employing adoptive therapy with lymphokine-activated killer cells.

We had earlier shown that tumor-bearing results in an inactivation of IL-2-dependent effector cells by host macrophage-derived PGE2, and that chronic indomethacin therapy (CIT) aimed at blocking prostaglandin synthesis, combined with multiple rounds of IL-2, can cure experimental metastases of a variety of tumors in mice. We have now tested the efficacy of this therapy on spontaneous as well as experimental metastasis of C3-L5 mammary adenocarcinoma in C3H/HeJ mice. Mice transplanted s.c. with C3-L5 cells (and showing visible spontaneous lung metastases between days 7 and 10) were given CIT starting on day 15, plus 2 5-day rounds of IL-2 or IL-2 alone. Mice injected i.v. with 10(4) C3-L5 cells (and showing lung micrometastases on day 5) were placed on CIT on day 5 and given 3 5-day rounds of IL-2 or treated with IL-2 alone. Control mice received vehicles alone. Results revealed that combined CIT + IL-2 therapy in the spontaneous metastasis model caused a regression of primary tumors, a marked reduction in lung metastases scored on days 25-35 and a marked prolongation of host survival (79% cured). Survivors rechallenged with 10(4) tumor cells i.v. on day 210 resisted tumor growth. In the experimental metastasis model, this therapy also markedly reduced lung metastases and prolonged animal survival (50% cured). In both models, the combination therapy led to the presence of highly active tumoricidal (for C3-L5 and YAC-1 lymphoma targets) lymphocytes with AGM-1+, Lyt-2- and Thy-1 +/- phenotype and macrophages in the spleen and the lungs, and ADCC-promoting activity in the serum. CIT + IL-2 therapy can thus effectively eradicate spontaneous and experimental mammary adenocarcinoma metastasis in mice. It activates natural effector cells in situ, generates ADCC-promoting activity in the serum and results in resistance to tumor take in this moderately immunogenic tumor model.

Histamine type-2 receptor antagonists (H-2RA) have been used chronically to prevent dyspepsia in cancer patients subjected to immunotherapy with chronic indomethacin (Indo) and intermittent IL-2 in our cancer centre. We tested the effects of these agents during immunotherapy of C3H/HeJ mice transplanted s.c. with 5 x 10(5) C3L5 mammary adenocarcinoma cells. Tumor-transplanted mice were divided into groups receiving: (1) Indo (14 micrograms/ml); (2) H-2RA, i.e. (a) ranitidine at 28.6 micrograms/ml (Ran-lo) or 143 micrograms/ml (Ran-hi), or (b) famotidine (Fam) at 4.3 micrograms/ml, or (c) cimetidine (Cim) at 107 micrograms/ml, all in the drinking water on days 5-24; (3) IL-2 (1.5 x 10(3) Cetus U i.p. every 8 h on days 10-14 and 20-24); (4) combinations of H-2RA + Indo; or (5) combinations of H-2RA + Indo + IL-2. Animals were killed on day 24 for examination of primary s.c. tumor growth, secondary lung metastasis and splenocyte cytotoxicity against YAC-1 lymphoma cells (51Cr release assay). Results revealed: (1) primary tumor growth was reduced in mice treated with Fam + Indo, Indo + IL-2 and any of the H-2RA + Indo + IL-2 (no difference observed within the last two groups); (2) lung metastases decreased in mice treated with IL-2 alone, Indo + IL-2, and Indo + IL-2 + Ran-hi; (3) splenic cytotoxicity was suppressed in tumor-bearing controls, with partial restoration seen in Ran (both doses), Ran-lo + Indo, Ran-lo + Indo + IL-2, and Cim + Indo + IL-2 treated groups. Nearly complete restoration was seen in Cim, Cim + Indo, Indo + IL-2, Ran-hi + Indo + IL-2, and Fam + Indo + IL-2 groups. Thus, addition of H-2RA did not alter the overall therapeutic efficacy of the standard Indo + IL-2 tumor immunotherapy.

Experimental work has shown that, during the development of tumours, host macrophages are triggered to produce high levels of prostaglandin E2 which inactivates natural killer cells and suppresses lymphokine activated killer cell development. Sustained, uninterrupted indomethacin treatment, when combined with interleukin-2 (IL-2), can totally eradicate experimental metastases. Most trials utilizing high dose IL-2 employ indomethacin and ranitidine in order to alleviate or prevent IL-2 toxicity, but only administer these medications concurrently with IL-2 therapy. A Phase II trial was conducted in patients with advanced melanoma. Patients received 50-75 mg indomethacin three times daily and 150 mg ranitidine twice daily, starting at least 1 week prior to IL-2 and continuing until intolerance or disease progression. Continuous venous infusion IL-2 was administered for three courses, each consisting of 5 days of treatment at 18 x 10(6) iu/m2/day for the first course with escalation of dose for the subsequent courses if toxicity allowed. Twenty-one patients were eligible to receive all components of therapy. Three patients achieved an objective response (one complete and two partial), giving a response rate of 14%. However, two of these objective responses (one complete and one partial) were achieved on indomethacin and ranitidine alone, prior to the commencement of IL-2 therapy. In this study, indomethacin and ranitidine, without IL-2, have been shown to have antitumour activity in advanced melanoma; continuous infusion IL-2 appeared to add little to the response seen with these two agents.

We examined: (a) whether in vitro-generated lymphocyte-activated killer (LAK) cells from normal mice and splenic killer cells from tumor-bearing mice subjected to interleukin-2 (IL-2) therapy alone or in combination with chronic indomethacin therapy have any detrimental effects on the spleen colony-forming units (CFU-S) of the normal bone marrow (BM); and (b) the effects of these immunotherapy protocols on CFU-S numbers in host hemopoietic organs. Effects of in vitro-generated LAK cells (normal C3H/HeN mouse splenocytes cultured with 1000 units IL-2/10(6) cells for 72 h) on BM CFU-S were examined by incubating macrophage-depleted BM cells with LAK cells at 1:2.5 and 1:5 BM:LAK cell ratios or with LAK cell supernatant for 4 h. The cells were washed and subsequently injected into irradiated mice. Irradiated mice were also reconstituted with BM cells or LAK cells incubated alone. Spleen colonies were scored macroscopically and microscopically on day 7 after reconstitution of lethally irradiated mice with the various cell combinations. A comparison of colony numbers produced by LAK and BM cell mixture revealed that LAK cells at either dose had no suppressive effect on the colony-forming ability of BM at the macroscopic and microscopic levels of analysis. The supernatant of cultured LAK cells had a minor suppressive effect on colony formation at the macroscopic but not the microscopic level of analysis, indicating the presence of one or more suppressive factors capable of mediating a short-term inhibitory effect. In the immunotherapy experiment, C3H/HeN mice transplanted s.c. with 5 x 10(5) C3L5 mammary adenocarcinoma cells received either vehicle alone (controls), IL-2 (1.5 x 10(4) Cetus units i.p. every 8 h on days 10-14 and days 20-25), or chronic indomethacin therapy (10 micrograms/ml in drinking water from day 5 onwards) plus IL-2 as above. Animals were killed 24-25 days after tumor transplantation to examine: (a) the number of metastatic lung nodules; (b) the effects of co-incubating therapy-generated splenic effector cells with normal BM cells for 4 h on BM CFU-S, and (c) the CFU-S content of host BM and spleen. Results revealed a drop in spontaneous lung metastases from a mean of 50 in control mice to 18 with IL-2 therapy alone, and to 5 with chronic indomethacin therapy plus IL-2 therapy. Splenocytes from normal and tumor-bearing control or treated mice, when incubated with normal BM, had no effect on spleen colony formation at the macroscopic level.(ABSTRACT TRUNCATED AT 400 WORDS)

Preclinical models of advanced melanoma have shown that chronic indomethacin therapy combined with interleukin 2 (IL-2) can eradicate experimental metastases. A phase II trial was done in patients with advanced melanoma. Indomethacin and ranitidine were begun at least one week before IL-2. Of the objective responses in 3 patients, 2 were achieved on ranitidine and indomethacin alone, before start of IL-2. Indomethacin and ranitidine may be responsible for some responses in melanoma patients previously attributed to IL-2.

In this study the efficacy of treatment of two cyclo-oxygenase inhibitors, ibuprofen (Ibu) and indomethacin (Indo), are compared in the immunotherapy of metastasis designed to reverse prostaglandin E2 (PGE2)-mediated inactivation of interleukin-2 (IL-2)-dependent host killer cell lineages. These agents were tested either alone for the prevention of metastasis or in combination with IL-2 for the eradication of established metastasis. C3H/HeN mice were placed on chronic oral Ibu (CIbT; 200 and 600 micrograms/ml of water) or Indo (CIT; 10 micrograms/ml) 5 days after s.c. transplantation of 5 x 10(5) metastatic C3L5 mammary carcinoma for the prevention of spontaneous lung metastases. They showed intolerance to Indo at a dosage of 14 micrograms/ml, which was well tolerated by other mouse strains in previous studies, but tolerated the Ibu dosages used. Control and treated mice were killed on day 30 to score metastatic lung colonies, to evaluate killer activity in splenocytes against natural killer (NK)-sensitive YAC-1 lymphoma or NK-resistant C3L5 adenocarcinoma and 8911 lymphoma targets, and to phenotype the surface markers of killer cells. CIbT and CIT alone at the above dosage significantly reduced the number of lung colonies, retarded local tumor growth and restored NK activity of splenic killer cells expressing AGM-1+, Thy-1-, Lyt-2- phenotype. To treat established lung metastasis, mice bearing 15-day C3L5 transplants were given CIbT or CIT alone or in combination with two 4-day rounds (days 20-23, 31-34) of IL-2 (15,000 Cetus units, i.p. every 8 h) and were killed on day 35 to score lung colonies and characterize splenic killer cells. CIbT or CIT alone reduced the number of spontaneous lung metastases and restored anti-YAC-1 killer function of splenocytes with NK-like phenotype (AGM-1+, Thy-1-, Lyt-2-); some anti-C3L5 killer function was also generated in the high dose Ibu group and the killer cell showed AGM-1+, Thy-1+ and Lyt-2+ phenotype. Combined therapies with CIbT or CIT plus IL-2 were more effective in reducing metastases and promoting killer cell function, the best results being achieved with high dose Ibu+IL-2. All killer cells expressed AGM-1 and Thy-1. In addition, C3L5 killer cells also expressed Lyt-2, suggesting T-cell stimulation. PGE2 synthesis in the host was inhibited by at least 50% in mice subjected to CIbT or CIT.(ABSTRACT TRUNCATED AT 400 WORDS)

We have shown that prostaglandin E2-(PGE2) mediated inactivation of all killer lineage cells is a common event in the tumor-bearing host, so that chronic indomethacin therapy (CIT) combined with multiple rounds of IL-2 cures spontaneous and experimental metastases of a variety of murine tumors by activating killer cells in situ. Ehrlich ascites tumor (EAT) is fatal for any mouse strain even with a small i.p. inoculum. We compared the therapeutic efficacy of chronic indomethacin therapy (CIT), CIT + interleukin-2 (IL-2), IL-2 + lymphokine activated killer (LAK) cells and CIT + IL-2 + LAK cells on EAT grown in CBA mice. CIT alone retarded tumor growth and stimulated cytotoxic activity in splenocytes as well as tumor-infiltrating lymphocytes against YAC-1 lymphoma and EAT targets but resulted in no cure. The therapeutic efficacy in prolonging the median survival improved in the following order: CIT less than IL-2 + LAK cells less than CIT + IL-2 less than CIT + IL-2 + LAK cells. The last regimen cured 90% of mice and resulted in a short-lasting immunity against a second tumor challenge in some of the animals. Thus, this triple combination therapy may hold promise for eradicating carcinomatous ascites in the human.

We have previously shown that NK lineage cells migrate to the murine decidua of pregnancy; but with advancing gestation, they are progressively inactivated in situ by prostaglandins of the E series (PGE2) secreted by decidual cells and decidual macrophages. We have also shown that the same mechanism inactivates all killer lineage cells in the human decidua, and that this inactivation is at least in part due to a down-regulation of IL-2 receptors and an inhibition of IL-2 production in situ. We examined whether chronic indomethacin therapy (to block prostaglandin synthesis), or a systemic administration of a high dose of IL-2, or a combination of both agents administered to pregnant mice could activate killer cells in situ and interfere with the progress of pregnancy; and if so, whether there was a causal relationship between the two events. Pregnant CD1 mice (Day 5 of gestation) were subjected to chronic indomethacin therapy (14 micrograms/ml in drinking water up to Day 15, or 50 micrograms twice daily sc or ip up to Day 10), high dose IL-2 therapy (25,000 Cetus U of human recombinant IL-2, ip every 8 or 12 hr for 3-5 days), or a combination of the two. These treatments led to pregnancy loss in 89-100% of mice, in contrast to 1% loss in control, vehicle-treated mice. Uterine mononuclear cells isolated from the embryo resorption sites exhibited high killer activity against YAC-1 lymphoma as well as murine trophoblast targets, with NK-like phenotype (Asialo GM-1+, Thy-1-) after indomethacin therapy and LAK-like phenotype (AGM-1+, Thy-1+) after IL-2 or indomethacin + IL-2 therapy. That AGM-1+ killer cells resulted in the pregnancy loss was suggested by the findings that in two of three separate experiments, iv injections of AGM-1 ab into pregnant indomethacin + IL-2-treated mice nearly completely prevented the fetoplacental demise (reducing it to 7.7% from 100%). These results reveal that PGE2-mediated inactivation of killer lineage cells in the decidua in situ is conducive to the survival of the conceptus.

We have shown that macrophage-derived prostaglandin (PG)E2 inactivates all interleukin 2 (IL-2) dependent killer cell lineages in the tumor-bearing host, so that chronic indomethacin therapy (CIT) combined with multiple rounds of IL-2 can cure experimental and spontaneous metastases of a variety of murine tumors. We tested the efficacy of this therapy on experimental human melanoma metastasis in nude mice and characterized the killer cells generated in situ. BALB/c nude mice were injected i.v. with 2 x 10(6) human lung-metastasizing line P52, MeWo melanoma cells. After 5 weeks, when lung nodules were well established, mice received vehicles alone (control) or were given (a) CIT (14 micrograms/ml in drinking water); (b) three rounds of IL-2, 25,000 Cetus U, 8 hourly i.p. (days 40-44, 50-54, 60-64); (c) CIT + three rounds of IL-2; (d) CIT + four rounds of IL-2 (round 4 on days 70-74); and (e) CIT + five rounds of IL-2 (round 5 on days 80-84). Control and experimental mice were killed on day 71 to score lung colonies and evaluate killer activity in splenic and lung lymphocytes and macrophages against murine YAC-1 lymphoma and B16F10 melanoma, human P52 melanoma, K562 erythroleukemia, and Raji lymphoma targets. Killer cells for P52 were phenotyped for Thy-1, Lyt-2, and asialo-GM-1 markers by ab + C'-mediated deletion of killer function. Mice in all groups were also kept for survival. CIT alone improved splenic NK activity but marginally reduced the lung colony counts or prolonged the survival time. Three rounds of IL-2 alone reduced the median colony counts by 50% and prolonged the survival by 2 weeks, but resulted in no long-term, disease-free survival, in spite of significant activation of LAK cells with Thy-1-, Lyt-2-, AGM-1+ phenotype in the spleen. CIT + 3 rounds of IL-2 reduced the median colony counts from 40 to 0 and improved the survival from a median of 66 (control) to 120 days (40% surviving 260 + days). CIT + four or five rounds of IL-2 caused long-term (260 + days) survival of 80% mice, most surviving 400 + days. The combination therapy activated killer lymphocytes (Thy-1-, Lyt-2-, AGM-1+) and, to a smaller extent, macrophages (AGM-1 +/-) in the spleen and the lungs, showing a high cytocidal ability for all the targets.(ABSTRACT TRUNCATED AT 400 WORDS)

We had earlier shown that a single round of interleukin 2 (IL-2) in chronically indomethacin treated mice can totally or nearly totally ameliorate established, experimental lung metastases and reactivate natural killer (NK) and lymphokine activated killer-like cells in the spleen. The present study examined whether a lasting cure of metastasis is obtainable by chronic indomethacin therapy (CIT) combined with single or multiple rounds of IL-2, and if so, what are the morphological, phenotypic, and functional properties of tumoricidal cells generated in situ. Experimental lung metastasis was produced in B6 mice by an i.v. injection of 10(6) B16F10 melanoma cells to compare therapeutic effects of six protocols: (a) CIT (14 micrograms/ml via drinking water) starting on day 5 when pulmonary micrometastases are well established; (b) CIT combined with a single round of IL-2 (25,000 units, i.p., every 8 h for 5 days on days 10 through 14); (c) CIT combined with two rounds of IL-2 (days 10-14 and 20-24); (d) two rounds of IL-2 alone; (e) two rounds of IL-2 plus indomethacin given only during the IL-2 therapy; and (f) which was similar to (c), but in addition, followed by repeated injections of IL-2 (25,000 units twice a day on Mondays and Fridays for 8 consecutive weeks). Results revealed that chronic indomethacin therapy alone or two rounds of IL-2 alone, or two rounds of IL-2 plus discontinuous indomethacin therapy reduced the lung metastases (examined at 21-25 days) by about two-thirds. In contrast, both single and multiple rounds of IL-2 in chronically indomethacin treated mice totally or nearly totally eradicated the lung metastases. However, long-term disease-free survival (greater than 13-16 months) resulted only with multiple rounds of IL-2. With chronic indomethacin therapy alone, NK-like (AGM-1+, Thy-1-,Lyt-2-) killer lymphocytes (capable of killing NK sensitive YAC-1 lymphoma and B16F10 melanoma targets) appeared in the spleen, but not lungs; no killer activity was generated in macrophages at either site. Addition of a single round of IL-2 generated lymphokine activated killer-like killer lymphocytes (also capable of killing an NK resistant target) of the same phenotype, but of higher activity in the spleen; some lymphokine activated killer-like killer function was generated among pulmonary lymphocytes which were AGM-1+, Thy-1+,Lyt-2-, as well as among splenic but not pulmonary macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

We have shown earlier that a decline in splenic natural killer (NK) activity during the development of transplanted or spontaneous tumors in mice results from an inactivation of NK lineage cells, mediated by prostaglandins (primarily PGE2) secreted by NK suppressor cells of the monocyte-macrophage lineage. In the present study we have used a C3H mouse mammary carcinoma model to examine whether this mechanism of NK suppression is conducive to tumor metastasis in vivo and whether a reversal of this suppression by a chronic indomethacin therapy can prevent metastatic spread from the primary tumor site. Three mammary tumor lines, all derived in our laboratory from a spontaneous C3H mammary tumor were employed: T-58 (uncloned parental line, having weak lung metastasizing ability from the subcutaneous site), C3 (a clone of T-58, showing high metastatic ability), and C10 (a nonmetastatic clone of T-58). Although the degree of NK susceptibility of these lines varied inversely with their metastatic potential, none was NK resistant. A chronic administration of indomethacin in the drinking water (14 micrograms/ml) to mice beginning on Day 4 after subcutaneous transplantation of 10(6) tumor cells resulted in a significant reduction in the growth rate of primary tumors in all hosts and led to a complete or nearly complete abrogation of lung metastasis in T-58- or C3-transplanted hosts examined at 1 month after tumor transplantation; C10-transplanted mice showed no metastasis in the control or the treated group. Concomitantly, there was a substantial restoration of splenic NK activity in all indomethacin-treated hosts. Plastic-adherent cells (greater than 95% macrophages) isolated from tumors growing in control mice, when coincubated for 20 hr with normal splenic effector cells caused a suppression of NK activity, reversible in the presence of indomethacin (10(-5) M) in vitro. Similar cells recovered from the residual primary tumors in indomethacin-treated mice had no suppressor ability. Chemically pure PGE2 (at concentrations of 0.5 to 1 X 10(-6) M, but not 0.25 X 10(-6) to 10(-8) M) also caused a suppression of NK activity of normal splenic effector cells, when added during the 4-hr 51Cr-release assay or allowed to interact with effector cells alone for a 20-hr incubation period; a removal of the cell-free PGE2 in the latter case prior to the NK assay did not relieve the suppression.(ABSTRACT TRUNCATED AT 400 WORDS)