Browsing by Author "Wicklein, Daniel"

Although survival rates of colon cancer patients diagnosed at an early stage (T1-2N0M0; Dukes A) vary considerably according to the studies cited, several studies indicate development of distant metastases already occurring in a considerable percentage of these patients leading to the death of the patients. This particular high risk group cannot be identified properly as no marker exists to identify these patients. As the Wnt/Win pathway plays a crucial role in metastasis formation in colorectal carcinoma, we analysed whether the transcription factor brachyury critically involved in this pathway may predict metastasis formation in these patients. The expression of brachyury-homologous (T) was immunohistochemically analysed in 748 patients and the data were correlated with classical and newer prognostic markers in colorectal cancer. Early stages colorectal cancer patients (T1-2N0M0, Dukes A) showed a significantly decreased survival when brachyury was expressed in the tumour tissue while no correlation was observed in later tumour stages. Hence a subset of colorectal cancers exists in which the ability to metastasise is already present at early stages of tumour growth and this high risk group can now be detected by immunohistochemistry. (C) 2010 Elsevier Ltd. All rights reserved.

Purpose: The aim of this study was to establish co-labeling of mesenchymal stromal cells (MSC) for the detection of single MSC in-vivo by MRI and histological validation. Materials and Methods: Mouse MSC were co-labeled with fluorescent iron oxide micro-particles and carboxyfluorescein succinimidyl ester (CFSE). The cellular iron content was determined by atomic absorption spectrometry. Cell proliferation and expression of characteristic surface markers were determined by flow cytometry. The chondrogenic differentiation capacity was assessed. Different amounts of cells (n1=5000, n2= 15000, n3=50000) were injected into the left heart ventricle of 12 mice. The animals underwent sequential MRI on a clinical 3.0T scanner (Intera, Philips Medical Systems, Best, The Netherlands). For histological validation cryosections were examined by fluorescent microscopy. Results: Magnetic and fluorescent labeling of MSC was established (mean cellular iron content 23.6 3pg). Flow cytometry showed similar cell proliferation and receptor expression of labeled and unlabeled MSC. Chondrogenic differentiation of labeled MSC was verified. After cell injection MRI revealed multiple signal voids in the brain and fewer signal voids in the kidneys. In the brain, an average of 4.61.2 (n1), 9.03.6 (n2) and 25.0 1.0 (n3) signal voids were detected per MRI slice. An average of 8.73.1 (n1), 22.06.1 (n2) and 89.86.5 (n3) labeled cells per corresponding stack of adjacent cryosections could be detected in the brain. Statistical correlation of the numbers of MRI signal voids in the brain and single MSC found by histology revealed a correlation coefficient of r=0.91. Conclusion: The study demonstrates efficient magnetic and fluorescent co-labeling of MSC and their detection on a single cell level in mice by in-vivo MRI and histology. The described techniques may broaden the methods for in-vivo tracking of MSC. Key Points: center dot Detection of single magnetically labeled MSC in-vivo using a clinical 3.0T MRI is possible. center dot Fluorescent and magnetic co-labeling does not affect cell vitality. center dot The number of cells detected by MRI and histology has a high correlation. Citation Format: center dot Salamon J, Wicklein D, Didie M etal. Magnetic Resonance Imaging of Single Co-Labeled Mesenchymal Stromal Cells after Intracardial Injection in Mice. Fortschr Rontgenstr 2014; 186: 367-376 Zusammenfassung Ziel: Etablierung einer Doppelmarkierung mesenchymaler Stromazellen (MSZ) zur in-vivo Detektion einzelner MSZ mittels MRT und zur histologischen Validierung. Material und Methoden: Murine MSZ wurden mit fluoreszierenden Eisenmikropartikeln und Carboxyfluorescein Succinimidyl Ester (CFSE) markiert. Der zellulare Eisengehalt wurde mittels Atomabsorptionsspektrometrie bestimmt. Zellprolieferation und Expression charakteristischer Oberflachenmarker wurden mittels Durchflusszytometrie bestimmt. Die chondrogene Differenzierungskapazitat wurde uberpruft. Verschiedene Zellanzahlen (n1=5000, n2=15000, n3=50000) wurden bei 12 Mausen in den linken Herzventrikel injiziert. Es erfolgte die sequenzielle MRT der Tiere an einem klinischen 3.0T MRT. Zur histologischen Validierung wurden Kryostatschnitte fluoreszensmikroskopisch untersucht. Ergebnisse: Die magnetische und fluoreszierende Doppelmarkierung von MSZ wurde etabliert (mittlerer zellularer Eisengehalt 23,6 +/- 4,3pg). Durchflusszytometrisch zeigten sichahnliche Zellprolieferationsraten und Rezeptorexpressionsprofile von markierten und unmarkierten MSZ. Die chondrogene Differenzierung der doppelt markierten MSZ wurde verifiziert. Nach Zellinjektion zeigten sich im MRT multiple Signalausloschungen im Hirn und geringer in der Niere. Im Hirn fanden sich durchschnittlich 4,6 +/- 1,2 (n1), 9,0 +/- 3,6 (n2) und 25,0 +/- 1,0 (n3) Signalausloschungen pro Schicht. Durchschnittlich fanden sich 8,7 +/- 3,1 (n1), 22,0 +/- 6,1 (n2) und 89,8 +/- 6,5 (n3) Zellen pro korrespondierendem Kryostatschnitt. Die statistische Korrelation der mittels MRT detektierten Signalausloschungen und der histologisch nachgewiesenen Zellen ergab einen Korrelationskoeffizienten von r=0,91. Schlussfolgerung: Die Studie zeigt die erfolgreiche magnetische und fluoreszierende Doppelmarkierung von MSZ und deren Detektion auf Einzelzellniveau mittels in vivo MRT und Histologie. Die beschriebenen Techniken tragen zur Erweiterung der Methoden fur das in vivo Monitoring von MSZ bei. Kernaussagen: center dot Die Detektion einzelner magnetisch markierter Zellen in vivo im 3,0T MRT ist moglich. center dot Die magnetische und Fluoreszenzmarkierung haben keinen negativen Einfluss auf die Zellvitalitat. center dot Die Anzahl mittels MRT und Histologie detektierter Zellen zeigt eine hohe Korrelation.

Abstract Background The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin β4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. Methods We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. Results We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b + Gr-1 Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. Conclusions These findings suggest a distinct vulnerability of ITGB4 Lo tumors for MDSC-directed immunotherapies. Graphical Abstract