M. Jane Ehrke

Immunomodulation in cancer therapeutics

Clinical verification is being obtained, with a variety of different therapeutic approaches, for the concept that anticancer treatments based on exploiting the hosts own antitumor defense mechanism can be beneficial. Nevertheless, as was seen with both chemotherapeutic and radiation treatments, the benefit of single agent treatments is not great. It is anticipated that, in attempting to realize the maximal potential of anticancer treatments based on exploiting the hosts own antitumor defense mechanism, it will be necessary to utilize combination therapies. For medical-ethical reasons, the ability to effectively combine such treatments with ones with proven clinical efficacy should increase the enthusiasm for the initiation of clinical trials. Chemotherapeutic agents may serve this purpose, since, contrary to the generally held tenet that anticancer chemotherapeutic agents are merely immunosuppressive, there is considerable literature describing their ability to augment antitumor host defenses. This review attempts to collate this information derived by numerous investigators employing diverse experimental approaches with a number of the most widely used anticancer chemotherapeutic agents.

Evidence for the involvement of ecto-5′-nucleotidase (CD73) in drug resistance

Increased ecto‐5′‐nucleotidase (ecto‐5′NT) protein expression in several multidrug‐resistant (MDR) cell lines, documented previously by our group, suggests that this enzyme is involved in drug resistance. Here, Northern blot analysis of selected cell lines and their MDR variants positively correlated ecto‐5′NT protein with its mRNA expression. An inhibitor of ecto‐5′NT enzymatic activity, α,β‐methyleneadenosine 5′‐diphosphate (AMP‐CP), was used to determine if functionally active enzyme had a role in drug resistance. AMP‐CP (0.3 mM) reversed the resistance of ecto‐5′NT‐positive MDR cells (MCF7/A6, L1210/A) to doxorubicin, whereas it did not affect the doxorubicin sensitivity of the ecto‐5′NT‐negative parental cell lines or that of 2 ecto‐5′NT‐negative MDR cell lines (HL60/VCR and A2780/DX5). Furthermore, AMP‐CP increased rhodamine uptake and inhibited rhodamine efflux from ecto‐5′NT‐positive MDR cells without affecting ecto‐5′NT‐negative MDR cells. The presence of exogenous adenosine (0.5 μM) circumvented AMP‐CP‐induced inhibition of rhodamine efflux from EL4/ADM cells. AMP‐CP inhibited the growth of the ecto‐5′NT‐positive L1210/A MDR cells but had no effect on the growth of the parental cell line. Determination of intracellular ATP levels indicated that MDR cells which had increased ecto‐5′NT expression also had a lower intracellular ATP level than their parental cells. Our results suggest that, in certain MDR cell lines, ecto‐5′NT serves as a required accessory molecule in resistance mediated by ATP‐dependent mechanisms and that growth‐sustaining nucleosides are provided by this salvage pathway.

Selective effects of adriamycin on murine host defense systems.

The clinical value of cancer chemotherapy is limited by the fact that the , anticancer agents available to date are not sufficiently selective in their antitumor action. Because of this, it may not be possible to overcome even relatively moderate degrees of tumor resistance and still avoid unacceptable toxicity. As discussed elsewhere (Mihich 1978), current approaches in cancer chemotherapy are aimed at reducing these limitations through the development of new selective treatments. Measurable differences in antigenicity have been found in tumor cells in animals (see Mihich 1978) and in man (Reinherz & Schlossman 1981, Ritzet al. 1981). Although in humans the differences seem to be primarily quantitative in nature, their existence has led to the hope that responses of the host to tumorassociated antigens might be exploited in designing more selective treatments of neoplastic disease. Difficulties that may have to be overcome in order to exploit tumor immunity therapeuticaily include those related to the fact that anticancer drugs may suppress host defenses through the very antiproliferative action which is at the basis of their antitumor effects (Mihich 1971, 1975). Furthermore, augmented tumor growth may be a consequence of modifications of immunoregulation possibly caused by tumor, drug or immunomanipulations. Thus, an understanding of the interactions between chemotherapeutic treatments and host

Doxorubicin induces specific immune functions and cytokine expression in peritoneal cells

To examine the basis of the immune modulation induced by the anticancer agent doxorubicin (DOX), the immunophenotype, tumoricidal activity, cytokine protein and mRNA expression were determined using peritoneal exudate cells (PEC) from saline-treated (untreated) and DOX-treated mice. A greater percentage of PEC from DOX-treated mice than from untreated mice were adherent to plastic, had characteristics of granulocytes, and were positive for the NK1.1, CD11b/Mac-1, and CD3 markers. DOX decreased the percentage of CD45R/B220+ cells. PEC from DOX-treated mice had greater tumoricidal potential than those from untreated mice since IL2, LPS, or IFNγ alone increased the cytolytic activity of PEC from DOX-treated mice, whereas PEC from untreated mice required both LPS and IFNγ to become cytolytic. DOX treatment modulated the expression of specific cytokines. Following stimulation in culture, PEC from DOX-treated mice produced more TNF, IL1, and IFNγ than PEC from untreated mice. DOX treatment increased the levels of TNF, but not IL1, mRNA and decreased the levels of IL6 mRNA and protein. These data demonstrate that a single DOX injection induces specific effects in PEC and, as a consequence, increases the tumoricidal potential of cells of the macrophage and natural killer types.

Species-specific TNF induction of thymocyte proliferation

SummaryRecombinant murine (rMu) tumor necrosis factor (TNF), in a standard comitogenic assay with phytohemagglutinin, induced murine thymocyte proliferation, while up to 10,000-fold higher concentrations of recombinant human TNF did not. The induction of thymocyte proliferation was dependent upon TNF concentration in a biphasic manner. Thus, 100 to 1000 units/ml TNF were near optimal while concentrations ≥1,000 units/ml caused apparent down regulation. The effect was abrogated by neutralizing antibody to rMu-TNF but not by neutralizing antibody to rMu-interleukin 1α or β. The rMu-TNF did not induce proliferation of the mature murine T-helper cell line, D10.G4.1, in the presence of mitogen. Taken together the results indicate that TNF, in a strictly species-specific manner, can regulate thymocyte proliferation independently of interleukin 1.

Protective specific immunity induced by doxorubicin plus TNF-α combination treatment of EL4 lymphoma-bearing C57BL/6 mice

The therapeutic efficacy of a single (day 8), moderate dose (4 mg/kg, i.v.) of doxorubicin (DOX, Adriamycin) combined with recombinant human TNF‐α (3 different doses and 5 different schedules, i.v.) was evaluated in C57BL/6 mice bearing an implant (s.c.) of the DOX‐sensitive, TNF‐α‐resistant EL4 lymphoma. In parallel to monitoring survival, the levels of several host anti‐tumor cytolytic effector functions of splenocytes and thymocytes were evaluated throughout the treatment period and in long‐term survivors (LTS). DOX treatment alone resulted in a moderate (approx. 20%) increase in life span but no cures. TNF‐α alone, at any tested dose or schedule, had little or no positive effect on survival. The combinations of DOX and TNF‐α were only slightly better than DOX alone with respect to the time to death of mice that died (approx. 29% increase); however, each of the combinations involving 1,000 U TNF‐α/injection produced a fraction (20% to 80%) of LTS. The host defense activities examined included those of splenic and thymic cytolytic T lymphocytes (CTL) and lymphokine‐activated killer cells as well as splenic tumoricidal macrophages. Although most activities were modulated by tumor growth and/or treatment, only CTL responsiveness appeared to correlate with survival. CTL activity in the treated groups with LTS was significantly higher than in control groups late in the treatment period. Finally, ex vivo analyses of splenocytes and thymocytes together with the rejection of implanted tumor at 17 months established that LTS displayed specific long‐term immune memory. Int. J. Cancer 87:101–109, 2000.

Adriamycin induced immunomodulation: Dependence upon time of administration

The immunomodulating capabilities of the anti-neoplastic agent, Adriamycin, were investigated. The day of Adriamycin administration to mice was varied from -15 to -1, day 0 being when mice were either immunized or sacrificed and their spleen cells sensitized in culture. Humoral and cellular immune responses against allogeneic or xenogeneic cellular antigens in mice and in culture, as well as phagocytic and ADCC activities were evaluated using spleen cell populations. The cellular responses and phagocytic activities were affected in a cyclical manner with time after Adriamycins administration. Peaks of increased activity were seen subsequent to day -5 and day -11, administration and low activities following day -1, -3 and day -7, -9 administration. The humoral responses were not affected in a biphasic manner but single peaks of increased activity were seen which corresponded to the times of low cellular cytolytic and phagocytic activities. The ADCC was independent of time of Adriamycin administration. The significance of these findings to the design of therapeutic protocols is discussed.

Modulation of the development of cell-mediated immunity: Possible role of the products of the cyclo-oxygenase and the lipoxygenase pathways of arachidonic acid metabolism

Abstract Various inhibitors of arachidonic acid (AA) metabolism were investigated for their effects on the development of cell-mediated immunity (CMI) by murine lymphocytes against allogeneic P815 mastocytoma cells in culture. These effects were measured by measuring cellular proliferation and generation of cytoxicity. Indomethacin, aspirin, octadeca-9, 12-diynoic acid (Ro 3-1314) and d , 1-6-chloro-α-methylcarbazole-2-acetic acid (Ro 20-5720), inhibitors of cyclo-oxygenase, caused augmentation of CMI response development. Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, caused 50% inhibition at 30 μM. 5,8,11,14-Eicosatetraynoic acid, an inhibitor of lipoxygenase and cyclo-oxygenase, enhanced CMI at 8 μM, but inhibited these responses by 50% at 60 μM. Imidazole and 1-methylimidazole, inhibitors of thromboxane synthetase, inhibited CMI by 50% at about 3 mM. Tranylcypromine (tranyl), an inhibitor of prostacyclin synthetase, enhanced CMI. Indomethacin, imidazole or tranylcypromine must be added to the lymphocyte cultures within 24 h after antigen in order to have an effect on CMI. The combination of indomethacin and imidazole showed that they are antagonistic in their effects on CMI. The combination of indomethacin and tranyl showed that the stimulatory effects of indomethacin and tranyl are additive. The combination of indomethacin and NDGA showed that the stimulatory effect of indomethacin is prevented by NDGA. Taken together the data suggest that the presence of various inhibitors of AA metabolism affect the regulation of CMI, apparently by changing the relative levels of the metabolites of AA. These findings imply that the products of both the lipoxygenase and the cyclo-oxygenase pathways may play important roles in the modulation of CMI and that these products may have opposing properties in terms of effects on CMI. The dependence of the effects upon the time of inhibitor addition to CMI cultures suggests that the modulation occurs early during the inductive phase of CMI.

Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor α combination treatment of EL4-lymphoma-bearing C57BL/6 mice

Fig. 5 Thymic effector lytic activity — the effect of tumor rechallenge. Conditions were the same as those given in Fig. 2, except the dose of Cy used was 150 mg/kg. Mice were sacrificed on day 128 and thymocyte suspensions prepared. R mice rechallenged on day 60 by i.p. implantation of 5x 104 viable EL4 tumor cells: VR mice not rechallenged. Evaluation of cytolytic activity after stimulation culture was against both EL4 or C1498 radiolabeled tumor target cells and in both 6-h and 18-h 51 Cr-release assays. R thymocytes were also examined for cytolytic activity on day 128. Immediately after the thymus had been obtained from the mice and single-cell suspensions prepared. These fresh R thymocytes (F-R) were plated with IL-2 (2.4 ng/ml) and radiolabeled target cells and 51 Cr release was assessed 18 h later Fig. 7 Effect of combination treatment compared to CY alone on the thymic effector lytic activity. Conditions are the same as those given in Fig. 5, where the dose of CY was 150 mg/kg. Data using cells obtained on day 128 from rechallenged mice are shown

Indomethacin modulation of Adriamycin-induced effects on multiple cytolytic effector functions*

SummaryThe anticancer agent, Adriamycin (ADM), in addition to being a potent cytotoxic drug has been shown to be an effective immunomodulator. This study was undertaken to determine whether ADM-induced changes in the production of prostaglandins (particularly PGE2) are involved in ADM-associated modifications of selected host defenses. Spleen cells from normal or ADM-treated (5 mg/kg; day −5) C57BL/6 mice were assessed for the following activities: fresh (day 0) and cultured natural killer (NK), cytotoxic T lymphocyte, lymphokine-activated killer (LAK), Fc-dependent phagocytosis and tumoricidal macrophage. All activities were assessed with and without the addition of indomethacin, an inhibitor of the first step of the cyclo-oxygenase pathway of prostaglandin synthesis. Depending on culture conditions, the cytotoxic T lymphocyte and splenic tumoricidal macrophage activities were either unaffected or were augmented by ADM treatment of the spleen donor mice or by addition of indomethacin to the culture, and these effects were apparently independent of one another. In contrast, ADM treatment generally resulted in reduced NK and LAK activities relative to control and elevated Fc-dependent phagocytosis. The addition of indomethacin to the culture effectively reversed these effects. Furthermore, spleen cells from ADM-treated mice were found to produce twice the amount of PGE2 in culture compared to cells from untreated mice. Finally, the direct addition of PGE2 to NK cultures resulted in a dose-dependent inhibition of NK activity and the dose required was comparable to the amount of PGE2 produced by cultured spleen cells from ADM-treated mice. Taken together, these results indicate that at least some of the immunomodulatory effects of ADM are an indirect result of ADM-induced changes in PGE2 production.