Measurement of leukocyte-platelet aggregates (LPA) by FACS: a comparative analysis.

In addition to their role in hemostasis and coagulation platelets bear critical roles in modulation of the innate and adaptive immune system. Upon platelet activation in response to tissue injury, bacterial or viral infections, they secrete many soluble factors or directly interact with leukocytes. An increase of leukocyte-platelet aggregates (LPA) has been described for many pathological conditions. Nevertheless, a standardized method for the reliable measurement of PLAs is not securely established. This methodical study provides a comparison of four different protocols from the literature and summarizes major pitfalls of measuring and interpreting leukocyte-platelet aggregates. The different techniques vary in the workup of the blood samples, applying variable washing and centrifugation steps or the use of erythrocyte lysis. All samples were finally analyzed by flow cytometry. Leukocyte subsets were stained with specific antibodies and platelet aggregates were identified by additional expression of CD41. The different procedures generated very heterogeneous data from the same blood sample which highlight the abundance of error measuring LPA. The most reproducible technique turned out to be a two-color whole blood flow cytometry assay with erythrocyte lysis and without washing or centrifugation steps avoiding platelet activation and artificial aggregate formation to achieve data mirroring the true situation in peripheral blood.

Altered expression of E-Cadherin-related transcription factors indicates partial epithelial-mesenchymal transition in aggressive meningiomas.

E-Cadherin has been suggested to be involved in meningioma progression but is also known as a key player of epithelial to mesenchymal transition (EMT). We wondered whether the adherens junction protein E-Cadherin, the tight junction protein Zo-1, and transcription factors suppressing E-Cadherin expression (Slug, Snail, Twist, Zeb-1) are differentially expressed between histopathological subtypes of meningioma, and if the expression of these factors is related to biological features of meningiomas. Analyzing 85 meningiomas of various histopathological subtypes and grades of malignancy by immunohistochemistry and 50 of them in addition by real-Time-PCR, we observed significantly reduced expression of Zeb-1, Twist and Slug, together with slightly increased expression levels for E-Cadherin and Zo- 1 in fibroblastic WHO-grade I tumors compared to meningothelial WHO grade I tumors, contradicting the hypothesis of EMT in the fibroblastic meningiomas characterized by mesenchymal appearance. However, comparing aggressive WHO grade II or III meningiomas with WHO-grade I tumors, we observed altered expression levels (loss of E-Cadherin and Zo-1, increased expression of Zeb-1 and Slug) indicating molecular features of EMT in aggressive meningiomas. This was supported by reduced E-Cadherin and increased Slug levels in recurrent compared to non-recurrent meningiomas. The expression levels of E-cadherin and Zo-1 were positively correlated with expression of NF2 mRNA. In primary meningioma cultures and IOMM-Lee meningioma cells, EMT induction by TGF-ß resulted in altered morphology and increased expression of EMT associated transcription factors. Meningioma cells with allelic losses of NF2 showed generally higher levels of various EMT relevant proteins, but were unresponsive to TGF-ß treatment. Our data indicate that aggressive meningiomas of WHO grade II/III are characterized by molecular alterations indicating partial EMT. This might contribute to the aggressive biology of these tumors.

Re-evaluation of cytostatic therapies for meningiomas in vitro.

PURPOSE: The purpose was to re-evaluate in cell culture models the therapeutic usefulness of some discussed chemotherapies or targeted therapies for meningiomas with a special emphasis on the role of the neurofibromatosis type 2 (NF2) tumor suppressor, which had been neglected so far. In addition, the study intended to evaluate a potential benefit from a treatment with drugs which are well established in other fields of medicine and have been linked recently with tumor disease by epidemiological studies. METHODS: Meningioma cell lines corresponding to various subtypes and pairs of syngenic meningioma cell lines with or without shRNA-induced NF2 knockdown were analyzed for their dose-dependent response to the drugs in microtiter tetrazolium assays, BrdU assays and for selected cases in ELISAs measuring nucleosome liberation to specifically separate cell death from pure inhibition of cell proliferation. RESULTS: We confirmed a moderate efficacy of hydroxyurea (HU) in clinically relevant concentrations. Under appropriate dosing, we neither detected major responses to the alkylating compound temozolomide nor to various drugs targeting membrane receptors or enzymes (tamoxifen, erlotinib, mifepristone, losartan, metformin and verapamil). Only concentrations far beyond achievable serum levels generated significant effects with the exception of losartan, which showed no effects at all. Chemosensitivity varied markedly among meningioma cell lines. Importantly, cells with NF2 loss exhibited a significantly higher induction of cell death by HU. CONCLUSIONS: Alternative chemotherapeutic or targeted approaches besides HU have still to be evaluated in further studies, and the role of NF2 must be taken into account.