Volker Schirrmacher

EGF receptor in neoplasia and metastasis.

SummaryEGFR is a member of the tyrosine kinase family of cell surface receptors with a wide range of expression throughout development and in a variety of different cell types. The receptor can transmit signals to cells: i) upon interaction with ligands such as EGF, TGFα, amphiregulin or heparin binding EGF, ii) upon truncation or mutation of extracellular and/or intracellular domains, iii) upon amplification of a basal receptor activity (in the absence of ligand) through cooperation with other cellular signaling pathways or nuclear events (e.g. expression of v-erbA). The activated EGFR can exert pleiotropic functions on cells, depending on their tissue origin and state of differentiation. Under certain conditions it can also contribute to neoplasia and development of metastases. Such conditions can exist upon aberrant receptor/ligand expression and activation (e.g. in the wrong cell; at the wrong time; in the wrong amounts). Aberrant signalling can also occur through constitutive EGFR activation. Oncogenic potential of EGFR has been demonstrated in a wide range of experimental animals. EGFR is also implicated in human cancer, where it may contribute both to the initiation (glioblastoma) and progression (epithelial tumors) of the disease. EGFR may influence key steps in the processes of tumor invasion and dissemination. Involvement of EGFR in tumor spread may indicate a potential use of this receptor as a target for antimetastatic therapy.

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell–T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell–mediated immunity.

Antitumor Vaccination of Patients With Glioblastoma Multiforme: A Pilot Study to Assess Feasibility, Safety, and Clinical Benefit

PURPOSE Prognosis of patients with glioblastoma is poor. Therefore, in glioblastoma patients, we analyzed whether antitumor vaccination with a virus-modified autologous tumor cell vaccine is feasible and safe. Also, we determined the influence on progression-free survival and overall survival and on vaccination-induced antitumor reactivity. PATIENTS AND METHODS In a nonrandomized study, 23 patients were vaccinated and compared with nonvaccinated controls (n = 87). Vaccine was prepared from patients tumor cell cultures by infection of the cells with Newcastle Disease Virus, followed by gamma-irradiation, and applied up to eight times. Antitumor immune reactivity was determined in skin, blood, and relapsed tumor by delayed-type hypersensitivity skin reaction, ELISPOT assay, and immunohistochemistry, respectively. RESULTS Establishment of tumor cell cultures was successful in approximately 90% of patients. After vaccination, we observed no severe side effects. The median progression-free survival of vaccinated patients was 40 weeks (v 26 weeks in controls; log-rank test, P = .024), and the median overall survival of vaccinated patients was 100 weeks (v 49 weeks in controls; log-rank test, P < .001). Forty-five percent of the controls survived 1 year, 11% survived 2 years, and there were no long-term survivors (> or = 3 years). Ninety-one percent of vaccinated patients survived 1 year, 39% survived 2 years, and 4% were long-term survivors. In the vaccinated group, immune monitoring revealed significant increases of delayed-type hypersensitivity reactivity, numbers of tumor-reactive memory T cells, and numbers of CD8(+) tumor-infiltrating T-lymphocytes in secondary tumors. CONCLUSION Postoperative vaccination with virus-modified autologous tumor cells seems to be feasible and safe and to improve the prognosis of patients with glioblastomas. This could be substantiated by the observed antitumor immune response.

Cell surface expression and secretion of heparanase markedly promote tumor angiogenesis and metastasis

The present study emphasizes the importance of cell surface expression and secretion of heparanase (endo-β-d-glucuronidase) in tumor angiogenesis and metastasis. For this purpose, nonmetastatic Eb mouse lymphoma cells were transfected with the predominantly intracellular human heparanase or with a readily secreted chimeric construct composed of the human enzyme and the chicken heparanase signal peptide. Eb cells overexpressing the secreted heparanase invaded a reconstituted basement membrane to a much higher extent than cells overexpressing the intracellular enzyme. Cell invasion was inhibited in the presence of laminaran sulfate, a potent inhibitor of heparanase activity and experimental metastasis. The increased invasiveness in vitro was reflected in vivo by rapid and massive liver colonization and accelerated mortality. In fact, mice inoculated with cells expressing the secreted enzyme succumb because of liver metastasis and dysfunction, as early as 10 days after s.c. inoculation of the cells, when their tumor burden did not exceed 1% of body weight. Cell surface localization and secretion of heparanase markedly stimulated tumor angiogenesis, as demonstrated by a 4–6-fold increase in vessel density and functionality evaluated by MRI of tumors produced by cells expressing the secreted vs. the nonsecreted heparanase, consistent with actual counting of blood vessels. Altogether, our results indicate that the potent proangoigenic and prometastatic properties of heparanase are tightly regulated by its cellular localization and secretion. The increased potency of the secreted enzyme makes it a promising target for anticancer drug development.

Human tumor cell modification by virus infection: an efficient and safe way to produce cancer vaccine with pleiotropic immune stimulatory properties when using Newcastle disease virus

Direct infection of tumor cells with viruses transferring protective or therapeutic genes, a frequently used procedure for production of tumor vaccines in human gene therapy, is an approach which is often limited by the number of tumor cells that can reliably be infected as well as by issues of selectivity and safety. We report an efficient, selective and safe way of infecting human tumor cells with a natural virus with interesting pleiotropic immune stimulatory properties, the avian paramyxovirus Newcastle disease virus (NDV). Two of the six viral genes (HN and F) modify the tumor cell surface by introduction of new adhesion molecules for lymphocyte interactions and other viral genes stimulate host cell genes and local production of cytokines and chemokines which can recruit a broad antitumor response in vivo. A large variety of human tumor cells is shown to be efficiently infected by NDV with viral replication being independent of tumor cell proliferation. Such properties make NDV a suitable agent for modification of noncultured freshly isolated and γ-irradiated patient-derived tumor cells. For the apathogenic non-lytic strain NDV-Ulster which is used in our clinical vaccine trials, we demonstrate selective replication in tumor cells as compared with corresponding normal cells. Furthermore, we present evidence that new virions produced by infected tumor cells are non-infectious using three different quantitative test methods. Our results demonstrate feasibility and broad applicability of this strategy of human tumor vaccine modification. Post-operative vaccination with the autologous virus-modified vaccine ATV-NDV thus provides a reasonable potential for pleiotropic modifications of the immune response of cancer patients against their own tumor.

Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients

Previous studies with animal tumors showed that bone marrow (BM) is a privileged site where potentially lethal tumor cells are controlled in a dormant state by the immune system. Here, we investigated BM of breast cancer patients with respect to tumor cell content, immune activation status and memory T‐cell content. BM‐derived cells from primary operated breast cancer patients (n = 90) were compared with those from healthy donors (n = 10) and also with cells from respective blood samples. Cytokeratin 19‐positive tumor cells were detected by nested polymerase chain reaction. Three‐color flow cytometry was used to identify numbers and activation state of T cells, natural killer (NK) cells, monocytes/macrophages and subsets by a panel of monoclonal antibodies (mAbs). The proportion of memory T cells among the CD4 and CD8 T cells was much higher in BM of cancer patients than in healthy donors (p < 0.001). The extent of memory T‐cell increase was related to the size of the primary tumor. Patient‐derived BM memory CD8 T cells could be shown to contain specific HLA‐A2/Her‐2/neu369–377 tetramer binding cells. Patients with disseminated tumor cells in their BM had more memory CD4 T cells and more CD56+ CD8+ cells than patients with tumor cell‐negative BM. Only some of the immunological changes seen in BM samples of cancer patients were also detectable in peripheral blood samples. Our hypothesis that BM is a special compartment for immunological memory and tumor dormancy is supported by the above findings. The overall results reveal that BM is a valuable additional compartment for immune diagnosis in pathological conditions and possibly for follow‐up treatment strategies.

Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

ABSTRACT The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3ζ lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.

Co-stimulatory effect of nitric oxide on endothelial NF-κB implies a physiological self-amplifying mechanism

Here we investigated the effects of the second messenger molecule NO at various concentrations on the activation of transcription factor NF‐κB, IκB‐α kinase (IKK‐α), Jun N‐terminal kinase (JNK) and apoptosis in murine endothelial cells. Low concentrations of NO alone failed to activate NF‐κB, IKK‐α and JNK. When NF‐κB was prestimulated by TNF‐α or phorbol 12‐myristate 13‐acetate, the addition of NO at low concentrations enhanced the activation of NF‐κB. This provides a mechanism for a self‐amplifying signal in the inflammatory response, since the inducible NO synthase in endothelial cells is regulated by NF‐κB. The co‐stimulatory effect of NO on NF‐κB activation was also evident from IKK‐α kinase assays and reporter gene experiments in endothelial cells. High doses of NO impaired the TNF‐α‐induced DNA‐binding activity of NF‐κB. Accordingly, these high amounts of NO also repressed the TNF‐α‐induced transactivation by NF‐κB as efficient as dexamethasone. The doses of NO required for the inhibition of NF‐κB are not cytotoxic for the endothelial cells, enabling the establishment of an autoregulatory loop for NF‐κB signaling.

Clinical trials of antitumor vaccination with an autologous tumor cell vaccine modified by virus infection: improvement of patient survival based on improved antitumor immune memory

For active specific immunotherapy of cancer patients, we designed the autologous virus–modified tumor cell vaccine ATV-NDV. The rationale of this vaccine is to link multiple tumor-associated antigens (TAAs) from individual patient-derived tumor cells with multiple danger signals (DS) derived from virus infection (dsRNA, HN, IFN-α). This allows activation of multiple innate immune responses (monocytes, dendritic cells, and NK cells) as well as adaptive immune responses (CD4 and CD8 memory T cells). Preexisting antitumor memory T cells from cancer patients could be activated by antitumor vaccination with ATV-NDV as seen by augmentation of antitumor memory delayed-type hypersensitivity (DTH) responses. In a variety of phase II vaccination studies, an optimal formulation of this vaccine could improve long-term survival beyond what is seen in conventional standard therapies. A new concept is presented which proposes that a certain threshold of antitumor immune memory plays an important role (1) in the control of residual tumor cells which remain after most therapies and (2) for long-term survival of treated cancer patients. This immune memory is T-cell based and most likely maintained by persisting TAAs from residual dormant tumor cells. Such immune memory was prominent in the bone marrow in animal tumor models as well as in cancer patients. Immunization with a tumor vaccine in which individual TAAs are combined with DS from virus infection appears to have a positive effect on antitumor immune memory and on patient survival.

Tumor Infiltrating T Lymphocytes in Colorectal Cancer: Tumor-Selective Activation and Cytotoxic Activity In Situ

Objective:To examine whether tumor-selective infiltration, activation, and cytotoxic activity of tumor infiltrating T lymphocytes (TIL) can be demonstrated in situ in colorectal cancer samples. Summary Background Data:Recent studies indicated a correlation between the presence of TIL and an improved prognosis in colorectal cancer. However, tumor-selective activation and cytotoxic activity of CD8+ TIL in situ in colorectal cancer patients have not yet been examined. Methods:Tumor samples from 49 patients and corresponding normal mucosa samples from 23 patients with colorectal cancer (UICC stages II–IV) were examined for TIL. Two-color fluorescence immunohistochemistry and multicolor flowcytometric (FACS) analysis were used for quantification of CD8+ T cells and measurement of their activation status (CD69-expression) and cytotoxic activity (CD107a-expression) in situ. Presence of tumor antigen-reactive T cells in tumor, blood, and bone marrow was evaluated by IFN-γ Elispot analysis. Results:While absolute numbers of CD8+ T cells were similar, CD4+ T helper cells were significantly increased in tumor tissue compared with normal mucosa. There was a significantly higher proportion of activated and cytotoxically active CD8+ TIL in colorectal cancer compared with normal mucosa. Increased activation, cytotoxic activity, and functional reactivity of TIL were correlated with the presence of functional tumor antigen-reactive T cells in the blood and bone marrow. The proportion of activated TIL decreased significantly with higher tumor stage. Conclusions:Tumor-selective activation and cytotoxic activity of CD8+ TIL and tumor-selective migration of CD4+ T helper cells were demonstrated in colorectal cancer for the first time. Our data support the immunogenicity of colorectal cancer and suggest clinical significance of tumor-specific immune responses.