Adult thymus transplantation with allogeneic intra-bone marrow-bone marrow transplantation from same donor induces high thymopoiesis, mild graft-versus-host reaction and strong graft-versus-tumour effects.

Although allogeneic bone marrow transplantation (BMT) plus donor lymphocyte infusion (DLI) is performed for solid tumours to enhance graft-versus-tumour (GVT) effects, a graft-versus-host reaction (GVHR) is also elicited. We carried out intra-bone marrow-bone marrow transplantation (IBM-BMT) plus adult thymus transplantation (ATT) from the same donor to supply alloreactive T cells continually. Normal mice treated with IBM-BMT + ATT survived for a long time with high donor-derived thymopoiesis and mild GVHR. The percentage of CD4(+) FoxP3(+) regulatory T cells in the spleen of the mice treated with IBM-BMT + ATT was lower than in normal B6 mice or mice treated with IBM-BMT alone, but higher than in mice treated with IBM-BMT + DLI; the mice treated with IBM-BMT + DLI showed severe GVHR. In tumour-bearing mice, tumour growth was more strongly inhibited by IBM-BMT + ATT than by IBM-BMT alone. Mice treated with IBM-BMT + a high dose of DLI also showed tumour regression comparable to that of mice treated with IBM-BMT + ATT but died early of GVHD. By contrast, mice treated with IBM-BMT + a low dose of DLI showed longer survival but less tumour regression than the mice treated with IBM-BMT + ATT. Histologically, significant numbers of CD8(+) T cells were found to have infiltrated the tumour in the mice treated with IBM-BMT + ATT. The number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL)-positive apoptotic tumour cells also significantly increased in the mice treated with IBM-BMT + ATT. Allogeneic IBM-BMT + ATT thus can induce high thymopoiesis, preserving strong GVT effects without severe GVHR.

Synergistic antitumor effect of CXCL10 with hyperthermia.

PURPOSE: IFN-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) has been described as an antiangiogenic chemokine and displays a potent antitumor activity in vivo. In the present study, we try to investigate whether the combination therapy of hyperthermia, a physical antiangiogenic modality, with CXCL10 would completely eradicate the established solid tumors. METHODS: Immunocompetent BALB/c mice bearing Meth A fibrosarcoma were established. Mice were treated with either CXCL10 at 25 microg/kg once a day for 20 days, hyperthermia was given twice (at 42 degrees C for 1 h, on day 6 and 12 after the initiation of CXCL10), or together. Tumor volume and survival time were observed. The microvessel density was determined by CD31 immunofluorescence. Histologic analysis and assessment of apoptotic cells were also conducted in tumor tissues. RESULTS: The results showed that CXCL10 and hyperthermia inhibited the growth of Meth A fibrosarcoma and interestingly, the combination therapy enhanced the antiangiogenic effects and completely eradicated the established solid tumors. Histological examination revealed that CXCL10 + hyperthermia led to increased induction of apoptosis, tumor necrosis, and elevated lymphocyte infiltration compared with the controls. Moreover, the tumor eradicated animals developed a protective T-cell-dependent antitumor memory response against Meth A tumor cells rechallenge. CONCLUSIONS: Our finding is that the combination therapy can achieve a synergistic antitumor efficacy, supporting the idea that the combination of two antiangiogenic agents may lead to improved clinical outcome. These findings could open new perspectives in clinical antitumor therapy.

Enhancement of antitumor response to sarcoma 45 in rats by combination of whole-body hyperthermia and interleukin-2.

AIM: To evaluate the summarized effect of hyperthermia and interleukin-2 (IL-2) administration on antitumor defense in tumor-bearing rats. METHODS: Nonbred rats after subcutaneous inoculation of sarcoma 45 cells were treated with whole-body hyperthermia (WBH, +42.5 degrees C, 60 min) and interleukin-2 (IL-2, 10,000 U/kg of body weight). Parameters of tumor growth and survival of animals were monitored till day 33 after tumor cell inoculation. RESULTS: Combined application of WBH and IL-2 at 5th day after tumor cell transplantation resulted in a delay of tumor growth and improvement of survival parameters in comparison with control group or animals that received separate treatment. Therapeutic efficacy of WBH combined with IL-2 was analogous to a single-dose chemotherapy with cyclophosphamide. CONCLUSION: Combined application of WBH and IL-2 is the useful approach for cancer immunotherapy.

Standardized mistletoe extract augments immune response and down-regulates local and metastatic tumor growth in murine models.

The immunomodulatory and antimetastatic activity of standardized aqueous mistletoe extract (sME) was evaluated in BALB/c-mice. Regular subcutaneous (s.c.) applications (three times per week for 14 consecutive days; 2, 20, 100 and 500 micrograms per injection and mouse) up-regulated thymocyte and peripheral blood leukocyte counts in tumor-bearing mice. Tumor weight and tumor volume were significantly down-regulated after application of sME doses greater than 20 micrograms per injection. To check the influence of sME treatment on growth of experimental metastases, RAW 117 H 10 lymphosarcoma cells and L-1 sarcoma cells were intravenously inoculated into BALB/c-mice to establish liver and lung colonization, respectively. sME was regularly administered starting 24 hours after tumor cell challenge. Organ colonization was investigated on day 14 after tumor cell inoculation and demonstrated statistically significant (p < 0.05) reductions of experimental liver and lung metastases for sME-treated mice.

Anticancer activity of rViscumin (recombinant mistletoe lectin) in tumor colonization models with immunocompetent mice.

The antitumoral and immunostimulating properties of rViscumin (recombinant mistletoe lectin) were investigated in two mouse tumor models. After intravenous inoculation with RAW-117-P or L-1 sarcoma cells in Balb/c mice, rViscumin was given s.c. at non-toxic doses ranging from 0.3 to 150 ng rViscumin/kg. One set of experiments was performed to investigate the survival of rViscumin-treated animals. Another set was carried out to analyze the effect of rViscumin treatment on the number of tumor colonies in infiltrated lungs (RAW-117P) or liver (L-1) and the activation of immune cell subsets, respectively. An overall prolonged survival time after treatment with rViscumin and a reduction in the number of tumor colonies after administration of certain rViscumin doses was observed. Immunophenotyping of the peripheral leukocytes of treated mice revealed increased numbers of T-lymphocytes, pan-NK cells and activated monocytes. The results indicate that rViscumin has antineoplastic properties and might therefore be a promising candidate in cancer therapy.

The B subunit of Shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens.

Immunization with peptide or recombinant proteins generally fails to elicit CTL, which are thought to play a key role in the control of virus-infected cells and tumor growth. In this study we show that the nontoxic B subunit of Shiga toxin fused to a tumor peptide derived from the mouse mastocytoma P815 can induce specific CTL in mice without the use of adjuvant. The Shiga B subunit acts as a vector rather than as an adjuvant, because coinjection of the tumor peptide and the B subunit as separate entities does not lead to CTL induction. We also demonstrated that in vitro the B subunit mediates the delivery of various exogenous CD8 T cell epitopes into the conventional MHC class I-restricted pathway, as this process is inhibited by brefeldin A and lactacystin and requires a functional TAP system. In contrast to other nonviral methods for transport of exogenous Ags into the endogenous MHC class I pathway that involve macropinocytosis or phagocytosis, the Shiga B subunit targets this pathway in a receptor-dependent manner, namely via binding to the glycolipid Gb3. Because this receptor is highly expressed on various dendritic cells, it should allow preferential targeting of the Shiga B subunit to these professional APCs. Therefore, the Shiga B subunit appears to represent an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL without the need for adjuvant.

Synergistic action of fms-like tyrosine kinase 3 ligand and CD40 ligand in the induction of dendritic cells and generation of antitumor immunity in vivo.

Daily treatment of mice with fms-like tyrosine kinase 3 ligand (Flt3L) leads to a significant increase in the number of dendritic cells and induces antitumor immunity. Here, we show that Flt3L and CD40 ligand (CD40L) synergize in the generation of immune responses against two poorly immunogenic tumors, leading to complete tumor rejection in a high proportion of mice. Rechallenge of the Flt3L + CD40L-treated mice with the immunizing tumor resulted in complete inhibition of tumor growth, indicating that these animals had developed long-lasting antitumor immunity. In addition, we demonstrate that endogenous CD40L plays a critical role in antitumor immunity, since blockade of CD40-CD40L interactions in vivo prevents the generation of antitumor immunity in therapeutic and vaccination protocols. Dendritic cells generated in mice treated with Flt3L alone or in combination with CD40L were equally potent in stimulating allogeneic T cells and expressed similar levels of MHC class II, CD80, and CD86. However, mice treated with Flt3L + CD40L had significantly more dendritic cells than mice treated with either of the cytokines alone, suggesting that CD40L promotes the proliferation and/or survival of dendritic cells generated by Flt3L treatment. Dendritic cells generated in this manner are likely to be involved in the priming of antitumor immune responses.

Host's immune response in electrotherapy of murine tumors by direct current.

Electrotherapy by low level direct current has been demonstrated to have antitumor effects in different murine tumor models and in clinics. Electrotherapy in "field" configuration, where electrodes are placed subcutaneously outside of the tumor in a way that tumor lies in between the electrodes, was performed in immunodeficient nude and immunocompetent mice. Electrotherapy was much more effective in immunocompetent mice based on the observed tumor growth retardation, thus demonstrating that antitumor effectiveness of electrotherapy greatly depends on host's immune response. Further experiments were conducted by combining electrotherapy with concomitant immunotherapy in order to potentiate the antitumor effect of electrotherapy. Immunotherapy consisted of local delivery of genetically engineered cells selected for IL-2 secretion. This combined treatment was much more effective than any of the treatments alone.

Mutated mitogen-activated protein kinase: a tumor rejection antigen of mouse sarcoma.

The molecular basis of the polymorphic tumor rejection antigens of chemically induced sarcomas of inbred mice remains a mystery, despite the discovery of these antigens over 40 years ago and their critical importance to the foundation of tumor immunology. In an analysis of a panel of BALB/c 3-methylcholanthrene-induced tumors, we identified one tumor, CMS5, that elicited a strong cytotoxic T cell response with exquisite specificity for CMS5. A stable cloned line of T cells with this specificity (C18) was used to screen a CMS5 cDNA expression library. The gene encoding the C18-defined antigen was identified as a mutated form of a mouse mitogen-activated protein kinase, ERK2, and a peptide incorporating the resulting amino acid substitution (lysine to glutamine) was efficiently recognized by C18. Vaccination with this peptide elicited specific resistance to CMS5 challenge. Extensive efforts to isolate antigen-loss variants of CMS5 were unsuccessful, suggesting that the mutated mitogen-activated protein kinase is essential for maintenance of the malignant phenotype.

Activation of the anti-tumor effector cells by Radix bupleuri.

Radix bupleuri, the root of Bupleuri spp., Chinese medicinal herbs used for the treatment of influenza, malaria and menstrual disorders, were extracted with hot water and separated into five different fractions (RB, RBI, RBII, RBIII and RBIV) by stepwise alcohol precipitation. One of these fractions, RBI, was then fractionated into RBIa and RBIb by gel filtration using G-100 Sephadex. These two fractions were further purified into RBIai, RBIaii and RBIbi, RBIbii fractions respectively by ion-exchange chromatography using DEAE-Sephadex. Each of these fractions is a heteropolymer consisting mainly of carbohydrate and varying proportions of protein and uronic acid. RBIaii was found to show strong anti-tumor activities in sarcoma-bearing mice. Mechanistic studies showed that RBIaii exhibited a potent activating effect on the cytotoxic activity of macrophages, NK and LAK cells against tumor cells. In addition, RBIaii could increase the number of tumor infiltrating lymphocytes (TILs) in the tumor site of WEHI-164-bearing mice. Furthermore, RBIaii could induce the release of interferon-gamma by lymphocytes in vitro.

Systemic antitumor effects of electrochemotherapy combined with histoincompatible cells secreting interleukin-2.

Electrochemotherapy is an antitumor treatment that combines a cytotoxic drug with the local administration of electric pulses delivered at the tumor site. We previously found that in mice the cure rate of subcutaneous transplanted tumors treated by electrochemotherapy is increased by repeated systemic interleukin-2 (IL-2) injections. Moreover, histoincompatible cells engineered to secrete IL-2 allow the rejection of syngeneic tumor cells when both cells are inoculated together. In this study of preestablished tumors in mice we show that after electrochemotherapy, delayed peritumoral injections of histoincompatible IL-2-producing cells result in the cure of almost all the tumors. Moreover, this combined local treatment leads to cures of untreated, contralaterally transplanted tumors. This systemic antitumor immunity also resulted in complete protection of the cured mice against further inocula of the tumor cells. These results, which were obtained using allogeneic as well as xenogeneic IL-2-secreting cells, suggest that electrochemotherapy combined with such cellular immunotherapy might be a useful approach for the treatment of metastasizing cancers.

gamma-Interferon plays a key role in T-cell-induced tumor regression.

Recent studies have demonstrated that noncytolytic T-cells can mediate regression of murine tumors. In this report, we demonstrate that MCA-105 tumor-draining lymph node cells (DLN) activated with the protein kinase C activator, bryostatin 1, plus a calcium ionophore are capable of inducing specific tumor regression in vivo when adoptively transferred to mice with established metastases. However, these activated DLN cells lack in vitro cytotoxicity against autologous tumor. Antibody against gamma-interferon (IFN-gamma) markedly inhibited the therapeutic efficacy of these activated DLN cells. Anti-tumor necrosis factor produced a statistically significant but weaker inhibition of tumor regression. IFN-gamma, but not tumor necrosis factor alpha, could be shown to be secreted by activated DLN cells in vitro in response to specific tumor. Secretion of IFN-gamma was primarily a function of CD8+ T-cells. IFN-gamma was not directly cytotoxic to sarcoma cells in vitro. Moreover, tumor cells incubated with IFN-gamma were not more susceptible to lysis by activated DLN cells. However, recombinant murine IFN-gamma had a significant antiproliferative effect against MCA-105 tumor cells when tested in a [3H]thymidine uptake assay. Similarly, supernatants obtained from DLN/autologous tumor cocultures markedly inhibited MCA-105 proliferation; this antiproliferative effect was abrogated by the addition of anti-IFN-gamma antibody to the cultures. These results suggest that secretion of IFN-gamma by adoptively transferred DLN cells plays an essential role in tumor rejection. The dominant effect of IFN-gamma may be its demonstrated antiproliferative activity.

Stimulation of host resistance against tumors by glycyrrhizin, an active component of licorice roots.

The effect of glycyrrhizin (GR), a Chinese herbal drug extracted from licorice roots, on the host resistance to tumors was investigated in a murine system. Administration of GR to BALB/c mice, which were inoculated s.c. with 1 x 10(6) cells/mouse of Meth A tumors, resulted in either no antitumor effect (8/20, 40%), a delay in tumor growth (9/20, 45%), or elimination of tumor growth (3/20, 15%) in these mice. In addition, the incidence of Meth A solid tumors was inhibited by GR when mice were inoculated with 1.5 x 10(4) cells/mouse or less of Meth A tumor cells, but not 7.5 x 10(4) cells/mouse or more. These results indicate that in this murine tumor system GR has a very weak antitumor effect. When GR at a dose of 20 mg/kg was administered to mice immunized with allogeneic lymphocytes 1 to 9 days after the immunization, the generation of suppressor macrophages (S-M phi) was clearly reduced as compared with that of S-M phi generated in immunized controls. In addition, when allospecific CTL (allo-CTL), which were generated in alloimmunized mice treated with GR followed by in vitro stimulation with allogeneic lymphocytes in a mixed lymphocyte reaction, were adoptively transferred to tumor-bearing mice treated with GR, the antitumor activity of allo-CTL derived from immunized mice treated with GR was markedly enhanced as compared with that of allo-CTL from immunized mice. Furthermore, established solid tumors were completely eliminated when interleukin-2 immunotherapy was performed in these mice in combination with GR treatments, but not interleukin-2 or GR alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor activity of polysaccharide from a Chinese medicinal herb, Acanthopanax giraldii Harms.

The results of experiments with Acanthopanax giraldii polysaccharide (AGP) demonstrated that it inhibited the growth of solid Sarcoma 180 and prolonged the survival time significantly. In tumor-bearing mice, AGP enhanced the phagocytosis and chemiluminescence of macrophages. By the immunofluorescent method, binding of the third component of complement (C3) cleavage product to macrophages and proportion of C3 positive cells were increased. In crossed immunoelectrophoresis, human serum C3 was converted by AGP and appeared as the 3rd peak. The height of the 3rd peak was directly proportional to doses of AGP. The residual CH50 units of human serum decreased dose-dependently. These results suggest that the antitumor activity of AGP is related to the enhancement of immune responses.

Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses.

We have recently described a new antitumor treatment, electrochemotherapy (ECT), based on the large local potentiation of the effects of the chemotherapeutic agent bleomycin (BLM) by electric pulses (EP) delivered at the tumor site. We demonstrate here that the host's immune response participates in cure achievement. We obtain an increase of the rate of completely cured animals by injecting mice with interleukin-2 (IL-2).

Decreased mortality of Norman murine sarcoma in mice treated with the immunomodulator, Acemannan.

An extract from the parenchyma of Aloe barbadensis Miller shown to contain long chain polydispersed beta (1,4)-linked mannan polymers with random O-acetyl groups (acemannan, Carrisyn) was found to initiate the phagocyte production of monokines that supported antibody dependent cellular cytotoxicity and stimulated blastogenesis in thymocytes. Acemannan, in both enriched and highly purified forms, was administered intraperitoneally to female CFW mice into which murine sarcoma cells had been subcutaneously implanted. The rapidly growing, highly malignant and invasive sarcoma grew in 100% of implanted control animals, resulting in mortality in 20 to 46 days, dependent on the number of cells implanted. Approximately 40% of animals treated with acemannan at the time of tumor cell implantation (1.5 x 10(6) cells) survived. Tumors in acemannan-treated animals exhibited vascular congestion, edema, polymorphonuclear leukocyte infiltration, and central necrosing foci with hemorrhage and peripheral fibrosis. The data indicate that in vivo treatment of peritoneal macrophages stimulates the macrophage production of monokines, including interleukin-1 and tumor necrosis factor. The data further indicate that sarcomas in animals treated i.p. with acemannan at the time of tumor cell implantation were infiltrated by immune system cells, became necrotic, and regressed. The combined data suggest that acemannan-stimulated synthesis of monokines resulted in the initiation of immune attack, necrosis, and regression of implanted sarcomas in mice.

Whole-body hyperthermia maintains the secondary immune response of specific antitumour immune T cells.

The effect of hyperthermia on the spleen cells of Meth A-hyperimmunized BALB/c mice (Meth A-Im-SPL) was examined. Three kinds of heating were employed: (a) a cell suspension of Meth A-Im-SPL was directly heated for 1 h at 41 degrees C; (b) the whole body of Meth A-hyperimmunized mice was heated in the same manner--the antitumour activity of Meth A-Im-SPL was examined by Winn assay after heating; (c) the whole body of BALB/c mice intradermally inoculated with a mixture of Meth A-Im-SPL and Meth A cells (Winn assay) was heated in the same manner. The results showed that the antitumour activity of Meth A-Im-SPL was not affected by heating in vivo (Experiments B and C), although it was affected in vitro (Experiment A). Furthermore, slight augmentation of the antitumour activity of Meth A-Im-SPL was observed on heating in vivo. This shows the possibility of combination therapy involving adoptive transfer and whole-body hyperthermia.

Anti-tumor protection induced in mice by fatty acid conjugates: alkyl butyrates and poly(ethylene glycol) dibutyrates.

Simple fatty acids, especially butyrate salts, have interesting biological properties, since they are able to down-regulate cell growth and promote various differentiated cellular functions. Their use for anti-tumor treatment is, however, hampered by their over-rapid diffusion in the blood, followed by a short-lived biological action. We have therefore devised conjugates linking butyrate with either (i) aliphatic alcohols of increasing carbon numbers ranging from C4 to C12 or (ii) poly(ethylene glycols) of increasing molecular weights. In both cases, the resulting butyric esters can be hydrolysed by esterases which can release biologically active subunits from the synthetic compounds. As shown in the present study, only one conjugate in each series gave satisfactory anti-tumor protection: namely, I-octyl butyrate and poly(ethylene glycol 1000) dibutyrate respectively. A single immune-stimulatory injection of purified Corynebacterium parvum extract prior to administration of the conjugates significantly increased the anti-tumor potency.

Enhanced thermal response of a rat sarcoma by Corynebacterium parvum.

Corynebacterium parvum was investigated in the response of rat Mc7 sarcoma to local waterbath hyperthermia. Heat treatment of 1-1.5 cm3 foot tumors at 43 degrees C for 2 h resulted in complete regression of 71% of the tumors. The Mc7 cure was reduced to 31% when the tumors were heated at 43 degrees C for 1.5 h. C. parvum (700 micrograms, i.v.) when given 1-3 days before tumor heating at 43 degrees C for 1.5 h increased the host phagocytic activity, and the tumor regression from 31% to 65% (P less than 0.05). C. parvum by itself had no curative effect on the tumor, and it did not enhance the thermal response of normal rat foot to hyperthermia. These findings suggest that host response to tumor heating may be 'non-specific' in nature involving phagocytes of the reticulo-endothelial system.