Manganese superoxide dismutase (SOD2/MnSOD)/catalase and SOD2/GPx1 ratios as biomarkers for tumor progression and metastasis in prostate,colon, and lung cancer

The role of manganese-dependent superoxide dismutase (SOD2/MnSOD) during tumor progression has been studied for several decades with controversial results. While SOD2 downregulation was initially associated with tumor initiation and was proposed as a tumor suppressor gene, recent studies have reported that SOD2 might favor tumor progression and dissemination. To our knowledge this is the first time that changes in SOD2 expression in three different types of tumors, i.e., prostate, lung, and colon cancer, are studied by analyzing both SOD2 mRNA and protein levels in a total of 246 patients׳ samples. In prostate samples, SOD2 protein levels were also increased, especially in middle stage tumors. In the case of colon and lung tumors both mRNA and protein SOD2 levels were increased in malignant tissues compared to those in nontumor samples. More importantly, all metastases analyzed showed increased levels of SOD2 when compared to those of normal primary tissue and healthy adjacent tissue. Together, these results suggest that a common redox imbalance in these three types of tumor occurs at intermediate stages which then might favor migration and invasion, leading to a more aggressive cancer type. Consequently, the ratios SOD2/catalase and SOD2/Gpx1 could be considered as potential markers during progression from tumor growth to metastasis.     

Overexpression of CD39 in Mouse Airways Promotes Bacteria-Induced Inflammation

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases. We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8(+) T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X(1) or P2X(7) receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation.     

Tumor metastasis in an orthotopic murine model of head and neck cancer: possible role of TGF-beta 1 secreted by the tumor cells

In an orthotopic murine model of head and neck cancer, combined subcutaneous and intratumoral vaccination with recombinant vaccinia virus expressing interleukin-2 (rvv-IL-2) induced significant tumor regression early on therapy. However, its efficacy was restricted by recurrent tumor growth and loco-regional metastases. In this study, we explored the mechanism of tumor metastasis. We compared the levels of expression of a number of molecules involved in tumor metastasis, which included transforming growth factor-beta1 (TGF-beta1), E-cadherin, matrix metalloproteinases (MMPs): MT1-MMP, MMP-2, MMP-9, their tissue inhibitors (TIMPs): TIMP-1/TIMP-2, and pro-angiogenic factors CD31, VEGF-R2, and iNOS between primary and metastatic tumors by real-time RT-PCR and immunohistochemistry. We detected spontaneous lymph node and tongue metastasis. Metastasis was delayed in rvv-IL-2 treated mice. Cultured tumor cells expressed negligible amount of TGF-beta1. Untreated or metastatic tumors, on the other hand, expressed high levels of TGF-beta1 and secreted TGF-beta1 in the sera of tumor-bearing mice. Levels of TGF-beta1 in the sera suddenly jumped at the time when tumor metastasis started. In the metastatic tumors, levels of MT1-MMP, MMP-2, and MMP-9 were significantly elevated (P < 0.001), while levels of TIMP-1/TIMP-2 and E-cadherin were decreased (P < 0.001) compared to control or primary tumors. Levels of CD31, VEGF-R2, and iNOS were also significantly elevated in the metastatic lesions (P < 0.001). The concurrence of high levels of TGF-beta1 in the sera, expression of proteins involved in metastasis and initiation of metastasis suggested possible role of TGF-beta1 in on setting the metastatic cascade in this model.     

Defects in the Acquisition of Tumor-Killing Capability of CD8+ Cytotoxic T Cells in Streptozotocin-Induced Diabetic Mice

Emerging evidences have shown that diabetes mellitus not only raises risk but also heightens mortality rate of cancer. It is not clear, however, whether antitumor CD8⁺ cytotoxic T lymphocyte (CTL) response is down-modulated in diabetic hosts. We investigated the impact of hyperglycemia on CTLs'' acquisition of tumor-killing capability by utilizing streptozotocin-induced diabetic (STZ-diabetic) mice. Murine diabetes was induced by intraperitoneal injection of STZ (200 mg/kg) in C57BL/6 mice, 2C-T cell receptor (TCR) transgenic and P14-TCR transgenic mice. The study found that, despite harboring intact proliferative capacity measured with CFSE labeling and MTT assay, STZ-diabetic CD8⁺ CTLs displayed impaired effector functions. After stimulation, STZ-diabetic CD8⁺ CTLs produced less perforin and TNFα assessed by intracellular staining, as well as expressed less CD103 protein. Furthermore, adoptive transfer of STZ-diabetic P14 CD8⁺ effector cells showed an insufficient recruitment to the B16.gp33 melanoma and inadequate production of perforin, granzyme B and TNFα determined by immunohistochemistry in the tumor milieu. As a result, STZ-diabetic CD8⁺ effector cells were neither able to eliminate tumor nor to improve survival of tumor-bearing mice. Taken together, our data suggest that CD8⁺ CTLs are crippled to infiltrate into tumors and thus fail to acquire tumor-killing capability in STZ-diabetic hosts.     

Human monocyte derived dendritic cells express functional P2X and P2Y receptors as well as ecto-nucleotidases

We investigated the expression and function of P2 receptors and ecto-nucleotidases on human monocyte derived dendritic cells (DC). In addition we analyzed the effect of extracellular ATP on the maturation of DC. By RT-PCR, DC were found to express mRNA for several P2X (P2X1, P2X4, P2X5, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y11) receptors. As shown by FURA-2 measurement, triggering of P2 receptors resulted in an increase in free intracellular Ca2+. In combination with Tumor necrosis factor-alpha, ATP increased the expression of the DC surface markers CD80, CD83 and CD86 indicating a maturation promoting effect. DC expressed the ecto-apyrase CD39 and the ecto-5'-nucleotidase CD73 as demonstrated by RT-PCR. Extracellular ATP was rapidly hydrolyzed by these ecto-enzymes as shown by separation of 3H-labeled ATP metabolites using a thin layer technique. These data suggest that ATP acts as a costimulatory factor on DC maturation.     

Reduced expression of neural cell adhesion molecule induces metastatic dissemination of pancreatic beta tumor cells

As in the development of many human cancers, in a transgenic mouse model of beta-cell carcinogenesis (Rip1Tag2), expression of neural cell adhesion molecule (NCAM) changes from the 120-kDa isoform in normal tissue to the 140/180-kDa isoforms in tumors. NCAM-deficient RiplTag2 mice, generated by crossing Rip1Tag2 mice with NCAM knockout mice, develop metastases, a tumor stage that is not seen in normal Rip1Tag2 mice. In contrast, overexpression of NCAM 120 in NCAM-deficient Rip1Tag2 mice prevents tumor metastasis. The results indicate that the loss of NCAM-mediated cell adhesion is one rate-limiting step in the actual metastatic dissemination of beta tumor cells.     

Chemoimmunotherapy for melanoma with dacarbazine and 2,4-dinitrochlorobenzene elicits a specific T cell-dependent immune response

An empirically established chemoimmunotherapy for metastatic melanoma combines the systemic administration of the chemotherapeutic agent dacarbazine (DTIC) with the epifocal application of the contact sensitizer 2,4-dinitrochlorobenzene (DNCB) on cutaneous metastases. Although this therapy yields high response rates resulting in prolonged survival, the mechanisms involved remain unknown. Here, we investigated whether treatment of tumor-bearing mice with DTIC and DNCB resulted in a specific immune response against the tumor. Subcutaneous (s.c.) tumors and lung metastases were induced in C57BL/6 mice by injecting syngeneic B16-melanoma cells s.c. or into the lateral tail vein, respectively. Mice were treated with intraperitoneal injections of DTIC followed by epifocal application of DNCB. This therapeutic approach significantly reduced the growth of s.c. tumors as well as lung metastases. Our data showed that the effector mechanisms involved are dependent on T cells. No therapeutic effect was observed in immunodeficient RAG-1(-/-) mice, or when the contact sensitizer DNCB was replaced by skin irritants (croton oil or tributyltin). Splenic lymphocytes obtained from treated mice displayed a three-fold higher specific cytolytic activity against B16 cells than in tumor-bearing controls. Both CD8(+) and CD4(+) T cells were able to lyse B16 cells. No changes were observed in natural killer (NK) cell activity. Likewise, tumor-infiltrating lymphocytes (TIL) of treated mice showed higher cytolytic activity than that of controls. Analysis of cytokine expression in s.c. tumors revealed increased mRNA levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) in treated tumors. Together, these findings demonstrate the ability of DTIC/DNCB treatment to induce an effective T cell-dependent host immune response against a syngeneic tumor.     

Augmentation of cutaneous immune responses by ATP gamma S: purinergic agonists define a novel class of immunologic adjuvants

Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPgammaS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPgammaS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPgammaS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1beta, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X(1), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(9), and P2Y(11) receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.     

Evaluation of human carcinoembryonic-antigen (CEA)-transduced and non-transduced murine tumors as potential targets for anti-CEA therapies

The MC-38 C57BL/6 mouse colon adenocarcinoma cell line has been transduced with a retroviral construct containing cDNA encoding the human carcinoembryonic antigen (CEA) gene [Robbins PF, Kantor JA, Salgaller M, Horan Hand P, Fernsten PD, Schlom J (1991) Cancer Res 51: 3657]. Two clones, MC-38-ceal and MC-38-cea2, expressed high levels of CEA on their cell surface. A third CEA-expressing cell line, MCA-102-cea3, was similarly derived by transduction of the MCA-102 C57BL/6 mouse fibrosarcoma cell line and is described here. In this study, the three CEA-transduced murine tumor cell lines (MC-38-cea1, MC-38-cea2, MCA-102-cea3) were evaluated for their tumorigenic potential, as well as their ability to serve as in vivo model systems for active and passive immunotherapy studies. Parameters that were investigated include tumor growth rate, the antibody response of the host to CEA, and the CEA content of the tumors. The MC-38-cea2 model appeared to be the most appropriate for immunotherapy studies. Biodistribution studies, using an¹²⁵I-labeled anti-CEA mAb, demonstrated efficient tumor targeting of MC-38-cea2 tumors in C57BL/6 and athymic mice.     

Early role of CD4+ Th1 cells and antibodies in HER-2 adenovirus vaccine protection against autochthonous mammary carcinomas

HER-2 is an oncogenic tumor-associated Ag that is overexpressed in several human tumors including breast and ovarian cancer. The efficacy and mechanism of a HER-2-expressing recombinant adenoviral vaccine to protect against tumorigenesis was examined using HER-2 transgenic (BALB-neuT) mice, which develop spontaneous breast tumors in all 10 mammary glands, and also using a transplantable mouse tumor model. Vaccination beginning at 6-8 wk of age (through 19 wk of age) prevented development of spontaneous mammary tumors even after 50 wk, whereas the animals in the control groups had tumors in all mammary glands by 25 wk. Such long-term protection after the last boost has not been achieved previously in this transgenic mouse in which the oncogene is continuously spawning tumorigenesis. Using beta(2)-microglobulin-knockout, IFN-gamma-knockout, and B cell-deficient mice, CD4(+) and CD8(+) cell depletion, and Ab transfer studies, we show that induction of anti-HER-2/neu Abs are both necessary and sufficient for protection, and the IgG2a isotype is most effective. In contrast, CD8(+) T cells are not necessary at all, and CD4(+) T cells are necessary for only 36-48 h after immunization to provide help for B cells but not as effector cells. Equal protection in immunized mice deficient in FcgammaRI/III excluded an FcR-mediated mechanism. Anti-HER-2 serum not only inhibited growth of mammary tumor cell lines expressing HER-2 in vitro but also protected mice from tumors in vivo, suggesting a direct action of Ab on the tumor cells. Such a vaccine may provide Ab-mediated protection against HER-2-expressing breast cancers in humans.     

Green fluorescent protein (GFP)-expressing tumor model derived from a spontaneous osteosarcoma in a vascular endothelial growth factor (VEGF)-GFP transgenic mouse

Vascular endothelial growth factor (VEGF) mediates tumor angiogenesis, growth, and metastasis. Murine models of metastatic tumors in which green fluorescent protein (GFP) expression is driven by the VEGF promoter can be imaged both intravitally and externally and thus offer many possibilities for real-time studies of tumor angiogenesis, metastasis, and treatment in vivo. In our defined-flora animal facility, an 11-month-old female transgenic mouse with a C3H background (VEGF(P)-GFP/C3H) developed a spontaneous tumor that expressed GFP under the control of VEGF. Necropsy and histopathologic examination revealed an osteosarcoma with lung metastases. Fresh tumor fragments were transplanted successfully into other VEGF(P)-GFP/C3H transgenic mice. During the first five generations, the tumor "take rate" was 100% (25 of 25 animals), with a latent period of 8 days and an average tumor volume of 1500 mm3 at 36 days. Transplanted tumors have maintained their original histopathologic characteristics and metastatic behavior. In addition, the tumor grows in wild-type C3H mice with an 83% take rate (10 of 12 animals) and as monolayer cells in vitro. GFP was expressed strongly in tumor tissue, lung metastatic foci, and cultured tumor cells. Real-time growth of tumors grown in dorsal skin chambers in C3H mice could be visualized using GFP fluorescence. In addition, GFP fluorescence of metastatic lesions in lungs of C3H mice was clearly visible by multiphoton laser scanning microscopy. This in vitro and in vivo transplantable and metastatic osteosarcoma (Os-P0107) is an attractive model for further study of tumor pathophysiology and treatment efficiency affecting VEGF expression.     

Behavior of metastatic and nonmetastatic breast tumors in old mice

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.     

The rate of the CD8-dependent initial reduction in tumor volume is not limited by contact-dependent perforin, Fas ligand, or TNF-mediated cytolysis

Established EG7 tumors expressing OVA and growing at an intradermal site become rapidly reduced in size following adoptive therapy with in vitro-generated type I CD8 T cell (Tc1) effectors generated from naive CD8 T cells from transgenic TCR OVA-specific mice. Tc1 effectors kill EG7 target cells in vitro by a perforin-dependent mechanism. However, we show that there is no quantitative diminution of the initial phase of antitumor activity in vivo, whether the Tc1 effectors are derived from perforin-, Fas ligand-, or TNF-deficient transgenic TCR mice or whether the recipients are perforin deficient. Tumors are also equally well controlled whether the Tc1 effectors come from mice deficient in perforin plus Fas ligand or perforin plus TNF. Control of tumor growth is diminished when Tc1 effectors generated from IFN-gamma-deficient mice are used. We conclude that control of tumor growth is not in any way affected by loss of contact-mediated lytic mechanisms, and conclude that the CD8 effectors must act by recruiting host effector mechanisms to control tumor growth.     

Preclinical models for the evaluation of targeted therapies of metastatic disease

It has been estimated that approx 60–70% of cancer patients harbor overt or subclinical metastases at diagnosis, and it is the eradication of such systemic disease that largely determines survival. Preclinical tumor model systems employed to evaluate potential new treatment strategies should aim to represent the process and patterns of metastasis of their clinical counterparts as closely as possible. Severe combined immune-deficient (SCID) andnu/nu mice have been extensively used as hosts for the growth of human tumor cell lines and in some cases fresh tumor material. However, in most instances the resulting neoplasms fail to metastasize, and the aberrant immune systems of such animals has limited their use mainly to passive therapies of localized disease. Recently, the development of specially selected tumor variants and the use of appropriate orthotopic sites for implantation has provided several models in which dissemination can be demonstrated. Where the gene coding for a potential target antigen has been cloned, and where its overexpression or mutation is associated with malignancy (e.g., c-erbB-2, H-ras), transgenic mice may yield tumors that will develop in these immunocompetent hosts. In some cases such tumors exhibit metastasis. A third approach is to transfect human genes of interest into appropriate rodent tumors expressing the desired metastatic phenotype. These various approaches are compared with particular reference to mammary carcinoma biology.     

P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice

Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.     

Lymphangiogenesis and tumor metastasis

The lymphatic system transports interstitial fluid and macromolecules from tissues back to the blood circulation, and plays an important role in the immune response by directing the traffic of lymphocytes and antigen-presenting cells. The lymphatic system also constitutes one of the most important pathways of tumor dissemination. In many human cancers, increased expression of vascular endothelial growth factor-C (VEGF-C) is correlated with regional lymph node metastases. Experimental studies using transgenic mice overexpressing VEGF-C or xenotransplantation of VEGF-C-expressing tumor cells into immunodeficient mice have demonstrated a role for VEGF-C in tumor lymphangiogenesis and the subsequent formation of lymph node metastases. However, there is at present little evidence for lymphangiogenesis in human tumors and the relative importance of preexisting vs. newly formed lymphatics for metastasis in humans remains to be determined. Nonetheless, the striking correlation between the levels of VEGF-C in primary human tumors and lymph node metastases predicts its importance in cancer spread. Aside from promoting lymphangiogenesis, VEGF-C may also activate lymphatics to promote tumor cell chemotaxis, lymphatic intravasation and hence tumor cell dissemination.Work in the authors' laboratories was supported by grants from the Swiss National Science Foundation (no. 3100–064037.00) (to M.S.P), the Speaker's Fund for Biomedical Research (to M.S.) and the Peter Sharp Foundation (to M.S.). Parts of this review will be published in abbreviated form in Thrombosis and Haemostasis     

A model for CD8+ CTL tumor immunosurveillance and regulation of tumor escape by CD4 T cells through an effect on quality of CTL.

Understanding immune mechanisms influencing cancer regression, recurrence, and metastasis may be critical to developing effective immunotherapy. Using a tumor expressing HIV gp160 as a model viral tumor Ag, we found a growth-regression-recurrence pattern, and used this to investigate mechanisms of immunosurveillance. Regression was dependent on CD8 T cells, and recurrent tumors were resistant to CTL, had substantially reduced expression of epitope mRNA, but retained the gp160 gene, MHC, and processing apparatus. Increasing CTL numbers by advance priming with vaccinia virus expressing gp160 prevented only the initial tumor growth but not the later appearance of escape variants. Unexpectedly, CD4 cell depletion protected mice from tumor recurrence, whereas IL-4 knockout mice, deficient in Th2 cells, did not show this protection, and IFN-gamma knockout mice were more susceptible. Purified CD8 T cells from CD4-depleted mice following tumor regression had more IFN-gamma mRNA and lysed tumor cells without stimulation ex vivo, in contrast to CD4-intact mice. Thus, the quality as well as quantity of CD8+ CTL determines the completeness of immunosurveillance and is controlled by CD4 T cells but not solely Th2 cytokines. This model of immunosurveillance may indicate ways to enhance the efficacy of surveillance and improve immunotherapy.