SU6668, a multiple tyrosine kinase inhibitor, inhibits progression of human malignant pleural mesothelioma in an orthotopic model

Background and objective: Malignant pleural mesothelioma (MPM) is an aggressive neoplasm of the mesothelium with high chemotherapeutic resistance. In this study, the preclinical therapeutic activity of the multiple tyrosine kinase inhibitor, SU6668, against MPM was examined. Methods: Two human MPM cell lines with different pro‐angiogenic cytokine expression, Y‐MESO‐14 cells that express high levels of vascular endothelial growth factor (VEGF) and MSTO‐211H cells that express high levels of basic fibroblast growth factor (bFGF), were orthotopically inoculated into the thoracic cavities of mice with severe combined immunodeficiency. The mice with MPM were treated or not treated with SU6668 (200 mg/kg/day). Results: SU6668 abrogated the proliferation of endothelial cells stimulated by VEGF or bFGF, but did not directly affect the growth of human MPM cells in vitro. In this orthotopic implantation model, treatment with SU6668 effectively reduced tumour weight and pleural effusion volumes, in association with inhibition of the growth of tumour vasculature. More importantly, treatment with SU6668 significantly prolonged survival time in mice with MPM. Conclusions: These findings suggest that SU6668 has a promising therapeutic effect on the progression of MPM in vivo through its anti‐angiogenic effects.     

The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts

SU5416 and SU6668 are potent antiangiogenic small-molecule inhibitors of receptor tyrosine kinases, including those of the vascular endothelial growth factor and platelet-derived growth factor receptor families. The stem cell factor (SCF) receptor, c-kit, is structurally related to these receptors and, although not expressed on mature peripheral blood cells, is expressed in leukemic blasts derived from 60% to 80% of acute myeloid leukemia (AML) patients. The c-kit kinase inhibitory activity of SU5416 and SU6668 was evaluated in MO7E cells, a human myeloid leukemia cell line. Tyrosine autophosphorylation of the receptor, induced by SCF, was inhibited in these cells by SU5416 and SU6668 in a dose-dependent manner (inhibitory concentration of 50% [IC(50)] 0.1-1 microM). Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, a signaling event downstream of c-kit activation, was also inhibited in a dose-dependent manner. Both compounds also inhibited SCF-induced proliferation of MO7E cells (IC(50) 0.1 microM for SU5416; 0.29 microM for SU6668). Furthermore, both SU5416 and SU6668 induced apoptosis in a dose- and time-dependent manner as measured by the increase in activated caspase-3 and the enhanced cleavage of its substrate poly(ADP-ribose) polymerase. These findings with MO7E cells were extended to leukemic blasts from c-kit(+) patients. In patient blasts, both SU5416 and SU6668 inhibited SCF-induced phosphorylation of c-kit and ERK1/2 and induced apoptosis. These studies indicate that SU5416 and SU6668 inhibit biologic functions of c-kit in addition to exhibiting antiangiogenic properties and suggest that the combination of these activities may provide a novel therapeutic approach for the treatment of AML.     

The combination of chemotherapy and systemic immunotherapy with soluble B7-immunoglobulin G leads to cure of murine leukemia and lymphoma and demonstration of tumor-specific memory responses

Major mechanisms underlying poor immune responses to autologous tumor-associated antigens are overwhelming tumor kinetics and the absence of effective T-cell costimulation by antigen-presenting cells. To address these issues, leukemia and lymphoma mice were treated with the combination of chemotherapy and systemic immunotherapy with recombinant soluble murine B7-immunoglobulin G (IgG) molecules. In this report, 3 murine models were used, a radiation-induced SJL acute myeloid leukemia, a transplantable spontaneous SJL lymphoma, and the C57BL/6 EL-4 thymic lymphoma. Various treatment modalities were evaluated: single treatments with either B7-IgG or chemotherapy as well as combination therapies. The results demonstrate the following: (1) in all tumor models, the combination of chemotherapy and soluble B7-IgGs is more potent than either therapy alone, leading to cure of tumor-bearing animals; (2) the therapeutic responses are T-cell-dependent, because combined therapy is not efficacious in severe combined immunodeficient mice; (3) the rejection of tumor cells leads to the development of tumor-specific immunity, because cured mice are immune to the rejected tumor but not to a different syngeneic tumor; and (4) (51)Cr release assays show that rejection of tumor cells leads to the development of very potent tumor-specific cytotoxic T-lymphocyte activity. On the basis of these results, it is proposed that chemotherapy-mediated tumor reduction, together with consequent augmented tumor-antigen presentation to activated T cells, are primary mechanisms leading to curative responses. The safety profile of the B7-IgG fusion proteins and their synergy with chemotherapy strongly suggest that the combination regimen is a promising strategy in cancer treatment.     

Combining antiangiogenic therapy with immunotherapy exerts better therapeutical effects on large tumors in a woodchuck hepatoma model

Cytokine and antiangiogenic gene therapies have proved effective in implanted hepatocellular carcinoma (HCC) models in which small tumor burdens were established in small rodents. These models, however, may not reflect human HCCs, which are frequently detected at a stage when tumors are large and multifocal. In addition, HCC in patients is often associated with viral hepatitis. To investigate the effectiveness of a mixture type of gene therapy strategy on large tumor burdens, we used the woodchuck model in which woodchuck hepatitis virus-induced HCCs are large and multifocal, simulating the conditions in humans. Adenoviruses encoding antiangiogenic factors (pigment epithelium-derived factor and endostatin) or cytokines (GM-CSF and IL-12) were delivered via the hepatic artery separately or in combination into woodchuck livers bearing HCCs. Our results showed that the mixture type of strategy, which contained two cytokines and two antiangiogenic factors, had better antitumor effects on large tumors as compared with monotherapy either with antiangiogenic or cytokine genes. The immunotherapy recruited significant levels of CD3⁺ T cells that infiltrated the tumors, whereas the antiangiogenesis-based therapy significantly reduced tumor vasculature. The mixture type of gene therapy achieved both effects. In addition, it induced high levels of natural killer cells and apoptotic cells and reduced the levels of immunosuppressive effectors in the tumor regions. Hence, antiangiogenic therapy may provide the advantage of reducing immune tolerance in large tumors, making them more vulnerable to the immune reactions. Our study implies that in the future, the combination therapy may prove effective for the treatment of patients with advanced HCC.     

A gene therapy for cancer using intramuscular injection of plasmid DNA encoding interferon α

A cancer treatment is described in which i.m. injection of plasmid DNA (pDNA) encoding murine interferon α (mIFN-α) leads to potent antitumor effects on primary and metastatic tumors in mice. Mice bearing s.c. B16F10 melanoma, Cloudman melanoma, or glioma 261 tumors were injected i.m. with mIFN-α pDNA. In all three tumor models, a significant reduction in tumor volume and enhancement of survival was found after IFN pDNA therapy. The mIFN-α pDNA could be injected as infrequently as once every other week and still produce a significant antitumor effect, and, in a metastatic tumor model, the therapy markedly reduced the number of lung tumor metastases. Depletion of immune cell subsets indicated that CD8⁺ T cells were required for the antitumor response. These studies demonstrate that primary and metastatic tumors can be treated systemically by i.m. injection of a plasmid encoding a cytokine gene.     

A2A adenosine receptor protects tumors from antitumor T cells

The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here we confirm this hypothesis by showing that genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in approximately 60% of A2AR-deficient mice with no rejection observed in control WT mice. The use of antagonists, including caffeine, or targeting the A2 receptors by siRNA pretreatment of T cells improved the inhibition of tumor growth, destruction of metastases, and prevention of neovascularization by antitumor T cells. The data suggest that effects of A2AR are T cell autonomous. The inhibition of antitumor T cells via their A2AR in the adenosine-rich tumor microenvironment may explain the paradoxical coexistence of tumors and antitumor immune cells in some cancer patients (the "Hellstrom paradox"). We propose to target the hypoxia-->adenosine-->A2AR pathway as a cancer immunotherapy strategy to prevent the inhibition of antitumor T cells in the tumor microenvironment. The same strategy may prevent the premature termination of immune response and improve the vaccine-induced development of antitumor and antiviral T cells. The observations of autoimmunity during melanoma rejection in A2AR-deficient mice suggest that A2AR in T cells is also important in preventing autoimmunity. Thus, although using the hypoxia-->adenosine-->A2AR pathway inhibitors may improve antitumor immunity, the recruitment of this pathway by selective drugs is expected to attenuate the autoimmune tissue damage.     

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 μg/kg once a day for 20 days, hyperthermia was given twice (at 42°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. Ping Chen, Ling-lin Yang, Huo-zhen Hu, and Yu-quan Wei contributed equally to this work.     

The potent antitumor effects of combined p16 gene and GM-CSF gene therapy through efficient induction of antitumor immunity

Purpose: Tumor suppressor gene therapy and cytokine gene therapy have limited antitumor effects when used alone. Thus, in the present study, we investigated the antitumor potentials of the combined transfer of the p16 tumor suppressor gene and the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene. Methods: The adenovirus-harboring p16 gene (Adp16) and adenovirus-harboring GM-CSF (AdGMCSF) gene were utilized for the treatment of established tumors in vivo. The mice were inoculated s.c. with Renca renal carcinoma cells and 3 days later received an intratumoral injection of Adp16 in combination with AdGMCSF. Results: The results demonstrated that tumor-bearing mice treated with Adp16 and AdGMCSF showed more potent inhibition of tumor growth and a prolonged survival period than mice treated with Adp16, AdGMCSF, adenovirus-expressing β-galactosidase or PBS (P < 0.01). Treatments of the mice with Adp16 alone or AdGMCSF alone also showed obvious antitumor effects as compared with those mice treated with PBS (P < 0.05). After combined p16 and AdGMCSF gene therapy, the expression of H-2K^d and Fas molecules on freshly isolated tumor cells increased markedly, and more CD₄ ⁺ T cells and CD₈ ⁺ T cells infiltrated in the tumor sites. The cytotoxicity of natural killer cells and specific cytotoxic T lymphocytes increased more significantly after the combined therapy. Conclusions: Our results demonstrated that combination p16 gene and GM-CSF gene therapy could inhibit the growth of established tumors in mice more significantly through efficient induction of antitumor immunity. Received: 11 April 2000 / Accepted: 19 July 2000     

Adenovirus-mediated combined suicide gene and interleukin-2 gene therapy for the treatment of established tumor and induction of antitumor immunity

The antitumor effect of the combined transfer of a suicide gene and a cytokine gene was evaluated in the present study. Adenoviruses expressing Escherichia coli cytosine deaminase (AdCD) and adenoviruses expressing murine interleukin-2 (AdIL-2) were utilized for the treatment of established tumors. The mice were inoculated s.c. with FBL-3 erythroleukemia cells and 3 days later received an intratumoral injection of AdCD in the presence or absence of AdIL-2 followed by intraperitoneal 5-fluorocytosine (5-FC) administration. The results demonstrated that tumor-bearing mice treated with AdCD/5-FC in combination with AdIL-2 showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdCD/5-FC, AdIL-2, adenovirus expressing β-galactosidase/5-FC or phosphate-buffered saline. The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD4⁺ and CD8⁺ T cells infiltrating the tumor after combined therapy. The splenic natural killer and cytotoxic T lymphocyte activities increased significantly in the mice after combined therapy with AdCD/5-FC/AdIL-2. Our results demonstrate that therapy combining a suicide gene and IL-2 gene can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently. Received: 4 May 1998 / Accepted: 4 August 1998     

Colorectal cancer and immunity:what we know and perspectives

Strong evidence supports the concept of immunosurveillance and immunoediting in colorectal cancer.In particular,the density of T CD8+and CD45+lymphocyte infiltration was recently shown to have a better prognostic value than the classic tumor node metastasis classification factor.Other immune subsets,as macrophages,natural killer cells or unconventionnal lymphocytes,seem to play an important role.Induction of regulatory T cells(Tregs)or immunosuppressive molecules such as PD-1 or CTLA-4 and downregulation of antigen-presenting molecules are major escape mechanisms to antitumor immune response.The development of these mechanisms is a major obstacle to the establishment of an effective immune response,but also to the use of immunotherapy.Although im-munotherapy is not yet routinely used in colorectal cancer,we now know that most treatments used(chemotherapy and biotherapy)have immunomodulatory effects,such as induction of immunogenic cell death by chemotherapy,inhibition of immunosuppression by antiangiogenic agents,and antibody-dependent cytotoxicity induced by cetuximab.Finally,many immunotherapy strategies are being developed and tested in phaseⅠtoⅢclinical trials.The most promising strategies are boosting the immune system with cytokines,inhibition of immunoregulatory checkpoints,vaccination with vectorized antigens,and adoptive cell therapy.Comprehension of antitumor immune response and combination of the different approaches of immunotherapy may allow the use of effective immunotherapy for treatment of colorectal cancer in the near future.     

Metronomic chemotherapy in combination with antiangiogenic treatment induces mosaic vascular reduction and tumor growth inhibition in hepatocellular carcinoma xenografts

Background

In addition to sprouting angiogenesis, other mechanisms, such as mosaic tumor vessel formation, have been recognized to contribute to tumor vascularization. We sought to examine vascular alteration as well as tumor growth inhibition after treatment with antiangiogenic therapy, chemotherapy alone or in combination.

Methods

Hepatocellular carcinoma cells (Hep3B) expressed green fluorescent protein were utilized to establish orthotopic xenograft model in nude mice. The formation and distribution of mosaic vessels was analyzed quantitatively by immunolabeling. Next, changes in tumor microcirculation and therapeutic effects on tumor growth were evaluated in several different treatment groups: control, conventional doxorubicin, metronomic doxorubicin, bevacizumab, bevacizumab plus conventional doxorubicin, and bevacizumab plus metronomic doxorubicin. In addition, we examined the effects of combined regimens on lung metastasis using a highly metastatic human hepatocellular carcinoma (HCCLM3) mouse model.

Results

Approximately 62?% of the vessels were present in the central part or near the midsection of the tumor and were mosaic. Only the combined antiangiogenic treatment and chemotherapy (metronomic schedule, P?=?0.00; conventional schedule, P?=?0.02) had a significant effect on the degree of mosaic vasculature. Metronomic doxorubicin in combination with bevacizumab had an even more profound effect than bevacizumab plus conventional doxorubicin (P?<?0.05) on tumor growth inhibition and survival. However, bevacizumab plus metronomic doxorubicin failed to inhibit lung metastasis compared with antiangiogenic monotherapy.

Conclusions

Metronomic chemotherapy in combination with antiangiogenic treatment results in the reduction of mosaic tumor vasculature, inhibition of tumor growth, and enhanced survival of mice. Further investigation of drug scheduling is required to optimize antitumor activity.     

α‐Galactosylceramide activates antitumor immunity against liver tumor

Aim: α‐Galactosylceramide (α‐GalCer) has been attracting attention as a novel approach to treat metastatic liver cancer. We investigated the detailed process of activating liver dendritic cells (DC) and immune cells after α‐GalCer treatment in the mouse liver tumor model. Methods: BALB/c mice bearing CMS4 liver tumor (p53 peptide‐expressing tumor) were treated by α‐GalCer. We evaluated the activation of liver DC and immune cells after α‐GalCer treatment. Interferon (IFN)‐γ enzyme‐linked immunosorbent spot (ELISPOT) assay was performed to detect p53 peptide‐specific cytotoxic T lymphocytes (CTL). To assess the impact of systemic acquired immunity by α‐GalCer treatment, 28 days after liver tumor treatment, CMS4 cells or Colon26 cells were re‐challenged s.c. Results: The liver weights of α‐GalCer‐treated mice were significantly lighter than those of vehicle‐treated mice. Depletion experiments revealed that natural killer (NK) cells were essential for the antitumor effect of α‐GalCer. α‐GalCer treatment significantly increased the population of DC and NK cells in the liver. The expressions of co‐stimulatory molecules on liver DC significantly increased with the peak at 1 day after α‐GalCer administration. IFN‐γ ELISPOT assay demonstrated that p53 peptide‐specific CTL was generated efficiently in α‐GalCer‐treated mice. ⁵¹Cr‐release assay revealed that CD8⁺, not CD4⁺, CTL against CMS4 cells were generated in α‐GalCer‐treated mice. The mice that had been protected from CMS4 liver tumor by α‐GalCer injection became resistant against s.c. CMS4 re‐challenge, but not against Colon26 re‐challenge. Conclusion: These results demonstrated the therapeutic potential of α‐GalCer against liver cancer through activating liver DC and immune cells in the liver.     

The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine

Please cite this paper as: Svindland et al. The mucosal and systemic immune responses elicited by a chitosan‐adjuvanted intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses DOI:10.1111/j.1750‐2659.2011.00271.x. Background Development of influenza vaccines that induce mucosal immunity has been highlighted by the World Health Organisation as a priority (Vaccine 2005;23:1529). Dose‐sparing strategies and an efficient mass‐vaccination regime will be paramount to reduce the morbidity and mortality of a future H5N1 pandemic. Objectives This study has investigated the immune response and the dose‐sparing potential of a chitosan‐adjuvanted intranasal H5N1 (RG‐14) subunit (SU) vaccine in a mouse model. Methods Groups of mice were intranasally immunised once or twice with a chitosan (5 mg/ml)‐adjuvanted SU vaccine [7·5, 15 or 30 μg haemagglutinin (HA)] or with a non‐adjuvanted SU vaccine (30 μg HA). For comparison, another group of mice were intranasally immunised with a whole H5N1 (RG‐14) virus (WV) vaccine (15 μg HA), and the control group consisted of unimmunised mice. Results The chitosan‐adjuvanted SU vaccine induced an immune response superior to that of the non‐adjuvanted SU vaccine. Compared with the non‐adjuvanted SU group, the chitosan‐adjuvanted SU vaccine elicited higher numbers of influenza‐specific antibody‐secreting cells (ASCs), higher concentrations of local and systemic antibodies and correspondingly an improved haemagglutination inhibition (HI) and single radial haemolysis (SRH) response against both the homologous vaccine strain and drifted H5 strains. We measured a mixed T‐helper 1/T‐helper 2 cytokine response in the chitosan‐adjuvanted SU groups, and these groups had an increased percentage of virus‐specific CD4⁺ T cells producing two Thelper 1 (Th1) cytokines simultaneously compared with the non‐adjuvanted SU group. Overall, the WV vaccine induced higher antibody concentrations in sera and an HI and SRH response similar to that of the chitosan‐adjuvanted SU vaccine. Furthermore, the WV vaccine formulation showed a stronger bias towards a T‐helper 1 profile than the SU vaccine and elicited the highest frequencies of CD4⁺ Th1 cells simultaneously secreting three different cytokines (INFγ⁺, IL2⁺ and INFα⁺). As expected, two immunisations gave a better immune response than one in all groups. The control group had very low or not detectable results in the performed immunoassays. Conclusion The cross‐clade serum reactivity, improved B‐ and T‐cell responses and dose‐sparing potential of chitosan show that a chitosan‐adjuvanted intranasal influenza vaccine is a promising candidate vaccine for further preclinical development.     

Statin-AE: a novel angiostatin-endostatin fusion protein with enhanced antiangiogenic and antitumor activity

The combination of angiostatin and endostatin has been shown to have synergistic antiangiogenic and antitumor effects when the genes for these proteins are delivered to tumor cells by retroviral gene transfer. Here we report the construction of a murine angiostatin–endostatin fusion gene (Statin-AE) which shows enhanced antiangiogenic activity on human umbilical vein endothelial cell (HUVEC) tube formation in vitro compared with angiostatin or endostatin alone. Similarly, the fusion gene demonstrates antiangiogenic effects in vivo and antitumor activity in a B16F10 melanoma model when co-delivered by retroviral packaging cell inoculation in mice. The fusion gene demonstrates significantly greater inhibition of tumor growth compared with angiostatin, endostatin or the combination of genes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Vaccination with a recombinant vaccinia virus encoding a "self" antigen induces autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4(+) T lymphocytes

Many human and mouse tumor antigens are normal, nonmutated tissue differentiation antigens. Consequently, immunization with these "self" antigens could induce autoimmunity. When we tried to induce immune responses to five mouse melanocyte differentiation antigens, gp100, MART-1, tyrosinase, and tyrosinase-related proteins (TRP) 1 and TRP-2, we observed striking depigmentation and melanocyte destruction only in the skin of mice inoculated with a vaccinia virus encoding mouse TRP-1. These mice rejected a lethal challenge of B16 melanoma, indicating the immune response against TRP-1 could destroy both normal and malignant melanocytes. Cytotoxic T lymphocytes specific for TRP-1 could not be detected in depigmented mice, but high titers of IgG anti-TRP-1 antibodies were present. Experiments with knockout mice revealed an absolute dependence on major histocompatibility complex class II, but not major histocompatibility complex class I, for the induction of both vitiligo and tumor protection. Together, these results suggest that the deliberate induction of self-reactivity using a recombinant viral vector can lead to tumor destruction, and that in this model, CD4(+) T lymphocytes are an integral part of this process. Vaccine strategies targeting tissue differentiation antigens may be valuable in cancers arising from nonessential cells and organs such as melanocytes, prostate, testis, breast, and ovary.     

Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis

Tumor-derived immune suppression is considered to be a major mechanism of tumor evasion from the immune system destruction, however, little is known regarding the induction of T-cell functional suppression by tumor-derived exosomes. Herein, we investigate tumor-derived exosomes involved in normal immunological communications as means of inhibiting an antitumor T-cell response. Exosomes derived from LNCaP, a human prostate cancer cell line, were visualized by FACS and identified based on size (80-200 nm) in comparison to marker beads. Exosomes from tumor cell line inhibited T-cell proliferation. Dose-dependent apoptosis of T cells was induced by co-culture with tumor exosomes. Addition of anti-FasL antibody blocked the apoptosis induction by tumor exosomes. This study suggests that induction of T-cell apoptosis by tumor-derived exosomes appears to be a novel mechanism of tumor immune evasion.     

Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo.

Recruitment of antigen-specific tumor-infiltrating lymphocytes (TILs) is a major goal for immunotherapy of malignant tumours. We now describe that T-cell-activating superantigens targeted to a tumor by monoclonal antibodies induced large numbers of pseudospecific TILs and eradication of micrometastases. As a model for tumor micrometastases, syngeneic B16 melanoma cells transfected with the human colon carcinoma antigen C215 were injected intravenously into C57BL/6 mice and therapy with an anti-C215 Fab fragment-staphylococcal enterotoxin A (C215Fab-SEA) fusion protein reacting with the C215 antigen was initiated when visible lung metastases were established. More than 90% reduction of the number of lung metastases was observed when mice carrying 5-day-old established lung metastases were treated with C215Fab-SEA. The antitumor effect of C215Fab-SEA was shown to be T-cell-dependent since no therapeutic effect was seen in T-cell-deficient nude mice. Depletion of T-cell subsets by injection of monoclonal antibody demonstrated that CD8+ cells were the most prominent effector cells although some contribution from CD4+ cells was also noted. C215Fab-SEA treatment induced massive tumor infiltration of CD4+ and CD8+ T cells, while only scattered T cells were observed in untreated tumors. SEA treatment alone induced a slight general inflammatory response in the lung parenchyme, but no specific accumulation of T cells was seen in the tumor. TILs induced by C215Fab-SEA were mainly CD8+ but a substantial number of CD4+ cells were also present. Immunohistochemical analysis showed strong production of the tumoricidal cytokines tumor necrosis factor alpha and interferon gamma in the tumor. Thus, the C215Fab-SEA fusion protein targets effector T lymphocytes to established tumors in vivo and provokes a strong local antitumor immune response.     

前列腺癌树突细胞瘤苗的制备及其应用

目的 构建前列腺癌树突细胞（DC）瘤苗,并探讨其体内外抗前列腺癌的作用。方法 分离C57BL／6小鼠骨髓前体细胞制备DC,光镜下观察DC的形态学特征,混合淋巴细胞试验、辣根过氧化物酶（HRP）吞噬试验观察DC的生物学特性。经RM-1前列腺癌细胞裂解产物致敏构建DC瘤苗,将DC瘤苗皮下注射于18只前列腺癌模型小鼠。结果成熟DC突起多而长,胞内囊泡少,刺激T细胞增殖能力强;DC瘤苗分泌IL-12能力增强;小鼠应用DC瘤苗后,其脾脏T细胞对RM-1细胞具有特异性杀伤作用;荷瘤小鼠肿瘤生长缓慢,坏死明显,瘤体内及肿瘤周围有大量炎细胞浸润。结论小鼠骨髓单核细胞在粒细胞／巨细胞集落刺激因子（GM-CSF）和IL-4诱导下可转化为DC。RM-1前列腺癌细胞裂解物致敏构建的DC瘤苗能诱导T细胞对RM-1细胞产生特异性杀伤作用,且能分泌更多的IL-12,可用于前列腺癌的免疫治疗。     

Increased induction of antitumor response by exosomes derived from interleukin-2 gene-modified tumor cells

Purpose Tumor-derived exosomes (TEX) have been proposed as a new kind of cancer vaccine; however, their in vivo antitumor effects are not satisfactory. In order to further improve the efficacy of vaccination with TEX, we investigated whether interleukin-2 (IL-2) genetic modification of tumor cells can make IL-2 presence in the exosomes, thus increasing antitumor effects of the TEX. Methods E.G7-OVA tumor cells expressing Ovalbumin (OVA) as a tumor model antigen were used to prepare TEX by serial centrifugation and sucrose gradients ultracentrifugation. To demonstrate their antitumor effects, IL-2-containing exosomes (Exo/IL-2) were injected subcutaneously into C57BL/C mice: either bearing tumor or followed by tumor inoculation. Results We found IL-2 within those exosomes as detected by both ELISA and Western blot. Vaccination with these Exo/IL-2 could induce antigen-specific Th1-polarized immune response and Cytotoxic T lymphocytes (CTL) more efficiently, resulting in more significant inhibition of tumor growth. CD8⁺ T cells are the main effector cells, however, CD4⁺ T cells, and NK cells are also involved in the induction of antitumor response of this approach. Conclusions Our results demonstrate that IL-2 genetic modification of tumor cells can make the TEX contain IL-2 with the increased antitumor effects, representing a promising way of exosome-based tumor vaccine. The first two authors contributed equally to this work.