Deborah J. Cameron

Relationship of the suppression of macrophage mediated tumor cytotoxicity in conjunction with secretion of prostaglandin from the macrophages of breast cancer patients

Peripheral blood monocyte derived macrophages obtained from breast cancer patients are non-cytotoxic towards human tumor cells. However, when indomethacin, a prostaglandin synthetase inhibitor, was added to the macrophage tumor cell incubation mixture, the breast cancer patients macrophages became capable of killing the tumor cells. Furthermore, the macrophage preparations obtained from breast cancer patients demonstrated a 64% increase in the secretion of PGE2, when compared with macrophages obtained from normal donors. It is already known that prostaglandin can inhibit both natural lymphocyte cytotoxicity and antibody dependent cell-mediated cytotoxicity against human tumor cells in vitro. These results indicate that increased synthesis of prostaglandin can also inhibit macrophage mediated cytotoxicity. Therefore, it is possible that manipulation of this mechanism might enhance the effectiveness of the macrophage response and should be a consideration in assessing macrophage-tumor cell interaction in vitro and in vivo.

A comparison of the cytotoxic potential in polymorphonuclear leukocytes obtained from normal donors and cancer patients

Human polymorphonuclear leukocytes (PMNs) obtained from both normal donors and cancer patients kill human tumor cells in vitro. Furthermore, the PMNs were shown to exert a greater cytotoxic effect on malignant targets than they did on nonmalignant targets. In addition, colon and breast cancer patients with stage I disease were found to possess PMN cells which were less effective in killing the tumor cells than were the PMN cells from normal donors. In contrast, the colon and breast cancer patients with stage IV disease possessed PMN cells which were more effective than normal cells in killing the tumor targets. No clear cut trend was found for PMN cells obtained from breast and colon cancer patients diagnosed with stage II and III disease. However, it must also be noted that individual patients did not always conform to the group pattern.

Cytotoxicity of cancer patient's macrophages for tumor cells.

Monocyte derived macrophages were isolated from the peripheral blood of 66 patients with either breast, colon, gynecological or hematological cancers. The macrophages from the breast and gynecologic cancer patients generally did not acquire enhanced cytotoxicity for human tumor cells after incubation with bacterial lipopolysaccharide (LPS). However, when the macrophages isolated from colon and hematologic cancer patients were studied, more than 50% of these patients possessed cytotoxic macrophages. LPS induced macrophage mediated cytotoxicity was also found to be inhibited by factors present in many cancer patients plasma. Twenty‐three of the 50 cancer patients studied possessed a plasma inhibitor capable of supressing macrophage mediated cytotoxicity by more than 50%. Furthermore, of these 50 patients, 47 were incapable of killing the tumor cells in vitro either because they possessed nonresponsive macrophages and/or they possessed a plasma inhibitory factor. Thus, although macrophage cytotoxic function may be normal in some patients with cancer, cytotoxicity may be inhibited in some patients by factors in autologous plasma thereby rendering the macrophages ineffective in vivo.

Ability of cancer patients' macrophages to kill autologous tumor targets: Effect of prostaglandin inhibitors on cytotoxicity

Monocyte‐derived macrophages isolated from the peripheral blood of 17 patients with cancer were studied for their cytotoxic effects on a sensitive allogeneic tumor target as well as autologous tumor cells. Macrophages from four of the cancer patients were cytotoxic towards the allogeneic tumor target. Five patients had macrophages which were cytotoxic towards the autologous targets. When indomethacin, a prostaglandin inhibitor, was added to the noncytotoxic macrophages during the cytotoxicity assay, macrophages from 5 of the 11 cancer patients studied became cytotoxic for the allogeneic target, and macrophages from 4 of the 11 patients became cytotoxic for the autologous tumor cells. However, it was also noted that those cancer patients who possessed cytotoxic macrophages in the absence of indomethacin, became noncytotoxic towards the tumor cells after addition of indomethacin. These results indicate that addition of indomethacin to cytotoxic macrophages suppresses macrophage mediated cytotoxicity whereas addition of indomethacin to noncytotoxic macrophages greatly enhances macrophage mediated cytotoxicity. This theory was further substantiated when it was observed that the cytotoxic macrophages obtained from normal donors also became noncytotoxic for a sensitive tumor cell line after the addition of indomethacin during the cytotoxicity assay.

Inhibitory factors derived from human tumors: Isolation of factors which suppress macrophage mediated cytotoxicity

Previously, we had demonstrated that plasma inhibitory factors obtained from colon cancer patients suppressed macrophage mediated cytotoxicity. When this inhibitor was further purified, four fractions (A, B, C and D) with inhibitory activity were isolated. In this study, it was found that these four purified inhibitory factors also dramatically decreased the extent of macrophage mediated cytotoxicity induced by LPS when added during the activation phase of the assay. When the secretions from a variety of fresh tumors as well as several established tumor cell lines were purified utilizing immunoadsorbent techniques, those same inhibitory factors were recovered. It was found that 8 of the 11 secretions obtained from a variety of fresh tumors and established tumor cell lines secreted inhibitor A; 6 secreted inhibitor B and 5 secreted inhibitor C. (The presence of inhibitor D was not studied.) Furthermore, one of the 11 tumors studied secreted none of these inhibitory factors. These findings lend support to the theory that these inhibitory factors which are initially produced by the tumor and subsequently shed into the sera ultimately lead to in vivo suppression of macrophage mediated cytotoxicity.

In vivo macrophage-mediated tumor cytotoxicity in the mouse

Tumor-bearing animals injected with either LPS-activated or control macrophages were capable of suppressing tumor growth in vivo. Tumor growth rate and survival times were assessed for each group of animals. At day 12 after injection of the P815 tumor cells, no difference in tumor size could be demonstrated in any of the groups. However, by day 17 the tumors in the animals treated with 2.5 X 10(6) or 5 X 10(6) control as well as LPS-activated macrophages did not continue to increase in size as was seen in the case of the control animals. When examining survival times, it appeared that the animals treated with control or activated macrophages generally survived approximately 10 days longer than did the animals receiving no treatment (30-day vs 20 day survival). Thus, it appears that tumor cell growth can be slowed down in vivo when control or activated macrophages are injected into the site of the tumor mass.

Specificity of macrophage-mediated cytotoxicity: Role of target and effector cell fucose

In this study, we have examined the role cell surface carbohydrates play in the activation of macrophages to kill tumor cells as well as the effect removal of tumor cell surface fucose has on the susceptibility of the treated target cells to the cytotoxic macrophages. After incubation of human monocyte-derived macrophages with alpha-L-fucosidase, a glycosidase which splits terminal alpha-L-fucose from oligosaccharides, the macrophages were no longer able to respond to lipopolysaccharide (LPS). These experiments strongly suggest that alpha-L-fucose probably comprises an essential part of the macrophage membrane receptor for LPS. In another series of experiments tumor cells were treated with alpha-L-fucosidase prior to co-cultivation with macrophages to determine whether the presence of alpha-L-fucose on the tumor cell surface had any effect on macrophage mediated cytotoxicity. It was found that the MA-160 sensitive tumor target, after alpha-L-fucosidase treatment, became resistant to the effects of the cyto-toxic macrophages whereas the effect of the alpha-L-fucosidase treatment was blocked by addition of alpha-L-fucose to the incubation mixture.

Cytotoxicity of cancer patients' macrophages for tumor cells: Purification and characterization of plasma inhibitory factors obtained from colon cancer patients

Monocyte derived macrophages from breast and gynecologic cancer patients generally do not acquire enhanced cytotoxicity for human tumor cells after incubation with bacterial lipopolysaccharide (LPS) whereas the macrophages isolated from colon and hematologic cancer patients are cytotoxic. However, it was also found that 75% of the patients possessing cytotoxic macrophages also had a plasma factor which suppressed macrophage mediated cytotoxicity. The plasma inhibitory factor obtained from a colon cancer patient was purified utilizing Sephadex G-200 column chromatography and 4 fractions (A, B, C and D) with inhibitory activity, were isolated. When a plasma sample obtained from a colon cancer patient found to be lacking the inhibitor of macrophage cytotoxicity was fractionated, 2 fractions were isolated with inhibitory activity. These fractions corresponded to fractions A and C of the inhibitory sample. Pooled AB+ serum was also fractionated and no inhibitory fractions could be isolated. The inhibitory factors were further characterized and it was found that fractions A and B appear to be inhibitors of lysosomal enzyme activity and fraction C appears to be an inhibitor of protease activity. When the plasma from cancer patients known to possess an inhibitor of macrophage mediated cytotoxicity was examined for the presence of fraction A, B, C and D, it was found that every colon cancer patient studied possessed inhibitors A, B, C and D. Breast cancer patients possessed some combination of A, B and C but all lacked fraction D and the gynecologic cancer patients possessed some combination of factors A, B and D but they all lacked inhibitor C.

Isolation and characterization of anti-neoplastic sturgeon factors

Sephadex G-200 column chromatography was utilized to isolate three anti-neoplastic compounds (A, B and C) from sturgeon sera which were cytotoxic for human tumor cells but would not kill normal cells in vitro. Furthermore, it was found that most of the sturgeons studied produced one or more of these same compounds. Immunoadsorbent techniques were utilized to further purify and isolate the three factors in larger quantities. When these immunoadsorbent purified factors were studied by SDS-polyacrylamide gel electrophoresis, factors A and C consisted of only 2 protein bands with molecular weights slightly greater than 68,000 and factor B consisted of 8 - 10 protein bands with molecular weights ranging from 30,000 to approximately 70,000. Factor A was not destroyed by heating at 56 degrees C for 30 min and appears to kill tumor cells by binding to the target cells. Factor B also was not destroyed by heating at 56 degrees C for 30 min. However, this factor differs from factor A in that it did not kill by binding to the tumor cell. Instead, it appears that a soluble factor is responsible for the cytotoxic effects observed with factor B. Factor C is destroyed by heating at 56 degrees C for 30 min and this factor kills the tumor cells by binding to the target cells. These factors were also examined for their ability to enhance macrophage mediated cytotoxicity. Sturgeon factors A and B enhance macrophage mediated cytotoxicity whereas factor C has no effect on the ability of normal human macrophages to kill human tumor cells.