Janka Held-Feindt

Expression of stem cell markers in human astrocytomas of different WHO grades

According to new hypotheses astrocytomas/gliomas either arise from or attract neural stem cells. Biological markers, particularly antigenic markers, have played a significant role for the characterization of these tumour stem cells (TSCc). Because these studies have been performed with single experimental samples mostly from gliomas, we investigated the expression of the stem cell markers CD133/Prominin, Nestin, Sox-2, Musashi-1, CXCR4, Flt-4/VEGFR-3 and CD105/Endoglin in 72 astrocytomas of different WHO-grades and compared it to normal adult human brain. Expression of their mRNA was quantified by quantitative RT-PCR, of their protein by counting immunopositive cells. In contrast to normal brain, tumour samples showed a high variability for the expression of all markers. However, their mean expression was significantly increased in astrocytomas, but this depended on the WHO grade only for CD133, Nestin, Sox-2 and Musashi-1. Confocal microscopy revealed that these markers mostly could be co-stained with glial fibrillary acidic protein, a marker for astoglial cells, but less frequently with the proliferation marker Ki-67/MIB-1. These markers sometimes, but not necessarily could be co-stained with each other in complex patterns. Our results show that most astrocytomas contain considerable portions of cells expressing stem cell markers. It appears that some of these cells originate from tumour genesis (supporting the stem cell hypothesis) while others are attracted by the tumours. Further functional markers are required to differentiate these TSC-types.

Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases

Purpose Brain metastases are an increasingly common complication in breast cancer patients. The Metastasis Suppressor Genes (MSG) Nm23, KISS1, KAI1, BRMS1, and Mkk4 have been associated with the metastatic potential of breast cancer in vitro and in vivo.Methods The mRNA expression of Nm23, KISS1, KAI1, BRMS1, and Mkk4 in fresh frozen tissue samples of brain metastases from ductal invasive breast cancer specimens was examined in relation to primary tumors. In a first step, mRNA expression screening was carried out using a semi-quantitative RT-PCR approach, in a second step quantitative real-time RT-PCR was performed on selected specimens. By immunohistochemical staining, gene products were visualized on the protein level.Results Semi-quantitative RT-PCR revealed reduced mRNA expression of Nm23, KISS1, KAI1, BRMS, and Mkk4 in brain metastases. Results for KISS1, KAI1, BRMS, and Mkk4 were confirmed by real-time RT-PCR. In detail, mRNA expression reduction in breast cancer brain metastases was tenfold. Expression of MSG could be confirmed by immunohistochemical staining on protein level.Conclusions Our investigations revealed significantly reduced mRNA expression of metastases suppressor genes KISS1, KAI1, BRMS1, and Mkk4 in breast cancer brain metastasis. Particularly, in the case of KISS1 and Mkk4, an important role for future treatment of patients with breast cancer brain metastatic lesions can be assumed.

Enhanced expression and shedding of the transmembrane chemokine CXCL16 by reactive astrocytes and glioma cells.

The transmembrane chemokine CXCL16 is expressed by dendritic and vascular cells and mediates chemotaxis and adhesion of activated T cells via the chemokine receptor CXCR6/Bonzo. Here we describe the expression and shedding of this chemokine by glioma cells in situ and in vitro. By quantitative RT‐PCR and immunohistochemistry, we show that CXCL16 is highly expressed in human gliomas, while expression in normal brain is low and mainly restricted to brain vascular endothelial cells. In cultivated human glioma cells as well as in activated mouse astroglial cells, CXCL16 mRNA and protein is constitutively expressed and further up‐regulated by tumour necrosis factor α (TNFα) and interferon‐γ (IFNγ). CXCL16 is continuously released from glial cells by proteolytic cleavage which is rapidly enhanced by stimulation with phorbol‐12‐myristate‐13‐acetate (PMA). As shown by inhibitor studies, two distinct members of the disintegrin‐like metalloproteinase family ADAM10 and 17 are involved in the constitutive and PMA‐induced shedding of glial CXCL16. In addition to the chemokine, its receptor CXCR6 could be detected by quantitative RT‐PCR in human glioma tissue, cultivated murine astrocytes and at a lower level in microglial cells. Functionally, recombinant soluble CXCL16 enhanced proliferation of CXCR6‐positive murine astroglial and microglial cells. Thus, the transmembrane chemokine CXCL16 is expressed in the brain by malignant and inflamed astroglial cells, shed to a soluble form and targets not only activated T cells but also glial cells themselves.

Matrix-degrading proteases ADAMTS4 and ADAMTS5 (disintegrins and metalloproteinases with thrombospondin motifs 4 and 5) are expressed in human glioblastomas

Brain tumors, in particular glioblastomas, have a high morbidity and mortality, mainly due to their invasive nature. A prerequisite for this invasiveness is cell migration based on increased expression of proteases digesting the extracellular matrix. Brevican, an important extracellular proteoglycan that is upregulated in glioblastomas, can be degraded by certain proteases. We demonstrate that in human glioblastomas secretory proteases like ADAMTS4 and ADAMTS5 (aggrecanases 1 and 2; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs) are expressed on the mRNA and protein levels in considerable amounts. Real‐time RT‐PCR shows a higher levels of ADAMTS4 and 5 expressions in glioblastomas in situ, compared to cultured human glioblastoma cells. The upregulation of these proteases in vivo by cytokines may explain this difference. In vitro, transforming growth factor‐β induces ADAMTS4, but less ADAMTS5, and interleukin‐1β ADAMTS5, but not ADAMTS4. As demonstrated by immunohistochemistry and confocal microscopy in situ, ADAMTS5 expression is confined to proliferating glioblastoma cells of surgical tumor sections and with lower intensity to astroglial cells in normal brain sections, as opposed to brevican. In vitro, glioblastoma‐derived ADAMTS5 degrades recombinant human brevican to several smaller fragments. Our results show that ADAMTS4 and 5 are upregulated on proliferating glioblastoma cells and these proteases may contribute to their invasive potential.

Somatostatin inhibits the production of vascular endothelial growth factor in human glioma cells

In various cell types, the neuro‐ and endocrine peptide somatostatin induces inhibitory and anti‐secretory effects. Since somatostatin receptors, especially of the subtype sst2A, are constantly over‐expressed in gliomas, we investigated the influence of somatostatin and the receptor subtype‐selective peptide/non‐peptide agonists octreotide and L‐054,522 on the secretion of the most important angiogenesis factor produced by gliomas, vascular endothelial growth factor (VEGF). Cultivated cells from solid human gliomas of different stages and glioma cell lines secreted variable amounts of VEGF, which could be lowered to 25% to 80% by co‐incubation with somatostatin or sst2‐selective agonists (octreotide and L‐054,522). These effects were dose‐dependent at nanomolar concentrations. Stimulation with different growth factors (EGF, bFGF) or hypoxia considerably increased VEGF production over basal levels. Growth factor–induced VEGF synthesis could be suppressed to <50% by co‐incubation with somatostatin or an sst2‐selective agonist; this was less pronounced in hypoxia‐induced VEGF synthesis. The effects were detected at the protein and mRNA levels. These experiments indicate a potent anti‐secretory action of somatostatin or sst2 agonists on human glioma cells that may be useful for inhibiting angiogenesis in these tumors.

Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism

The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling complex (DISC). Here we report that adenovirus 14.7K protein inhibits ligand-induced TNFR1 internalization. Analysis of purified magnetically labeled TNFR1 complexes from murine and human cells stably transduced with 14.7K revealed that prevention of TNFR1 internalization resulted in inhibition of DISC formation. In contrast, 14.7K did not affect TNF-induced NF-kappaB activation via recruitment of receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor 2 (TRAF-2). Inhibition of endocytosis by 14.7K was effected by failure of coordinated temporal and spatial assembly of essential components of the endocytic machinery such as Rab5 and dynamin 2 at the site of the activated TNFR1. Furthermore, we found that the same TNF defense mechanisms were instrumental in protecting wild-type adenovirus-infected human cells expressing 14.7K. This study describes a new molecular mechanism implemented by a virus to escape immunosurveillance by selectively targeting TNFR1 endocytosis to prevent TNF-induced DISC formation.

Expression of VEGF and its receptors in different brain tumors

Abstract Introduction: Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and -2 are considered to play a major in tumor angiogenesis, which is a prerequisite for growth of solid tumors. Glioblastoma multiforme is a prominent example of VEGF-induced tumor vascularization; however, little is known about VEGF and in particular VEGFR expression in other types of brain tumors. Methods: VEGFR mRNA was quantified by real time RT-PCR in 12 different types of brain tumors and compared to VEGF protein content measured by ELISA. VEGF splice variants were determined by an RT-PCR method. Results: VEGF protein was highest in glioblastoma and metastatic kidney tumors. In all types of tumors the diffusible splice forms VEGF121 and VEGF165 were expressed; VEGF189 was minor in a few tumors. Expression of VEGF receptors did not necessarily correlate with VEGF content. Both were highly expressed in glioblastomas, but in meningiomas VEGF was low and VEGFR high, and in metastatic tumors the reverse. With few exceptions, in particular oligodendrogliomas, VEGFR-1 expression was parallel to VEGFR-2 expression. Interestingly, for the astrocytic gliomas, the expression of VEGFR correlated well to the tumor malignancy, even better than VEGF content. Conclusions: These results show that VEGF and VEGFR expression in various types of brain tumors differ and are not necessarily parallel.

CX3CR1 promotes recruitment of human glioma-infiltrating microglia/macrophages (GIMs)

The transmembrane chemokine CX3CL1 and its receptor CX3CR1 are thought to be involved in the trafficking of immune cells during an immune response and in the pathology of various human diseases including cancer. However, little is known about the expression and function of CX3CR1 in human glioma-infiltrating microglia/macrophages (GIMs), representing the major cellular stroma component of highly malignant gliomas. Here, we show that CX3CR1 is overexpressed at both the mRNA and protein level in solid human astrocytomas of different malignancy grades and in glioblastomas. CX3CR1 was localized in ionized calcium-binding adapter molecule 1 (Iba1) and CD11b/c positive GIMs in situ as shown by fluorescence microscopy. In accordance with this, freshly isolated human GIM-enriched fractions separated by CD11b MACS technology displayed high Iba1 and CX3CR1 mRNA expression levels in vitro. Moreover, cultured human GIMs responded to CX3CL1-triggered activation of CX3CR1 with adhesion and migration in vitro. Besides an increase in motility, CX3CL1 also enhanced expression of matrix metalloproteases 2, 9, and 14 in GIM fractions in vitro. These data indicate that the CX3CL1/CX3CR1 system has a crucial tumor-promoting role in human glioblastomas via its impact on glioma-infiltrating immune subsets.

Force-Dependent Development of Neuropathic Central Pain and Time-Related CCL2/CCR2 Expression after Graded Spinal Cord Contusion Injuries of the Rat

Spinal cord injury (SCI) often results in intractable chronic central pain syndromes. Recently chemokines such as CCL2 were identified as possible key integrators of neuropathic pain and inflammation after peripheral nerve lesion. The focus of the current study was the investigation of time-dependent CCL2 and CCR2 expression in relation to central neuropathic pain development after spinal cord impact lesions of 100, 150, or 200 kdyn force on spinal cord level T9 in adult rats. Below-level pain was monitored with weekly sensory testing for 42 days after SCI. In parallel expression of CCL2/CCR2 on cervical, thoracic, and lumbar levels was investigated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry early (7 days [7d]), intermediate (15d), and late (42d) after lesion. Cellular source and anatomical pain related expression was determined by double-immunohistochemistry. Force-defined SCI led to acute mechanical hypersensitivity in all lesion groups, and to persistent below-level pain in severely injured animals. While in the early post-operative time course, CCL2 and CCR2 were expressed in astroglia and granulocytes only on level T9; there was additional astroglial CCL2 expression in dorsal columns and dorsal horns above and below T9 of severely injured animals 42d after lesion. In dorsal horns (level L3-L5) of animals exhibiting chronic below-level pain CCL2 was co-expressed with transmitters and receptors that are involved in nociceptive processing like calcitonin gene-related peptide (CGRP), Substance-P, vanilloid-receptor-1, and its activated phosphorylated form. These data demonstrate lesion grade dependence of below-level pain development and suggest chemokines as potential candidates for integrating inflammation and central neuropathic pain after SCI.

Surgical treatment for brain metastases: prognostic factors and survival in 177 patients

Brain metastases are an increasingly frequent complication of cancer. Advances in diagnosis and treatment have led to wider indications for surgery. We present a single-institution series of 177 patients and discuss our results with regard to the literature. Special focus is on patients with advanced age, multiple brain metastases, extracranial metastases, and brain metastasis recurrence. All patients underwent craniotomy for the resection of solid tumor brain metastases between 1994 and 2001 in our department. Perioperative morbidity and mortality as well as survival were evaluated. The median patient age was 59 years (range 32–86 years). In 177 patients, 348 brain metastases were detected, of which 68.0% were supratentorial and 32.0% were infratentorial. According to univariate analysis, the following parameters were significantly associated with prolonged patient survival: (1) age <70 years, (2) one to three intracranial metastases, (3) favorable postoperative performance, (4) resection of all intracranial lesions, and (5) recraniotomy for brain metastasis recurrence. In contrast, the presence of extracranial metastases, metachronous diagnosis, and solitary brain metastases had no influence on survival. As expected, younger age and limited number of brain metastases (up to three) are favorable prognostic factors. Remarkably, the presence of extracranial metastases had no influence on patient survival.