Enrichment of EpCAM^pos/neg cells with Dynabeads.

<p>EpCAM^pos (upper panel; MCF7, SKBR3, T47D, HCC1500 and ZR-75-1) and EpCAM^low/neg (lower panel; MDA-MB-231) cells were captured with Dynabeads coupled with antibodies for EpCAM, Trop2 and CD49f (1 μg ab/2.5x10⁵ cells/1x10⁷ beads). No cells bound to uncoated Dynabeads (NT = no target). Cells were imaged after a DAPI stain and are depicted as brightfield/DAPI merge; 10x magnification.</p

Representative images of potential CTCs (CK^pos/CD45^neg) and double positive (CK^pos/CD45^pos) events enriched from EpCAM-depleted patient samples.

<p>Immunofluorescence staining of (A) CTCs (top: #8, Adem-c-Met; bottom: #25, Dyna-CD49f) and (B) CK^pos/CD45^pos events (top: #8, Adem-c-Met; bottom: #13, Adem-Trop2) enriched after EpCAM-depletion are shown. Adem-/Dynabeads captured cells were stained for DAPI (blue), pan-CK (green) and CD45 (yellow); 40x magnification.</p

Adhesion of EpCAM^pos/neg cells on manually spotted arrays.

<p>2.5x10⁴ EpCAM^pos cells (pool of MCF7, SKBR3, HCC1500 and ZR-75-1; upper panel) and EpCAM^low/_neg cells (MDA-MB-231; lower panel) were incubated for cell adhesion experiments on glass substrates (NEXTERION slides AL) manually coated with anti-EpCAM [Ber-EP4], anti-Trop2, anti-CD49f (0.1 mg/ml each), collagen I (Col), hyaluronic acid (HA) and laminin (Lam) (0.2 mg/ml each). Cell adhesion was visualized by Coomassie; 20x magnification.</p

Genomic profiling of CellCelector identified and isolated whole genome amplified EpCAM^neg single cells confirms their malignant nature.

<p>(A) One EpCAM^pos CTC was selected and identified via CellSearch and re-identified with the CellCelector for a positive DAPI and CK-PE (displayed in the TRITC channel), and a negative CD45 (Cy5 channel) stain. Three EpCAM^neg CTCs (I, II, III) from the CellSearch EpCAM-depleted fraction of the same patient were enriched with CD44-Adembeads and stained for DAPI/pan-CK-FITC/CD45-AF647. Single cells were isolated via the CellCelector, the whole genomic material was amplified and (B) genome wide aCGH profiles were obtained confirming that EpCAM-independent enrichment captures malignant cells. Chromosomal regions highlighted in grey show common somatic copy number alterations, light blue areas represent different chromosomal aberrations between both CTC populations.</p

Number of EpCAM-enriched CTCs and potential CTCs/double positive events (CK^pos/CD45^pos) within the EpCAM-depleted fractions in blood of breast cancer patients.

<p>(A) CTC count determined by EpCAM-enrichment and subsequent CK/DAPI stain in 29 blood samples of 25 patients (DIII and DIV). (B) Number of potential CTCs (top) and double positive events (bottom) within the respective EpCAM-depleted supernatants of the same blood samples; total event numbers (CK^pos/CD45^neg and CK^pos/CD45^pos) represent the sum of all events identified after 2–6 immunomagnetic enrichments for each blood/patient sample.</p

Expression of surface markers on EpCAM^pos and EpCAM^low/neg cells.

<p>Differential protein expression of EpCAM (green), Trop2, CD49f, CK8, CD146 and ADAM8 (red) in EpCAM^pos (MCF7, ER⁺PR^-HER2^-, top; and SKBR3, ER^-PR^-HER2⁺, middle) and EpCAM^low/neg (MDA-MB-231, ER^-PR^-HER2^-, bottom) cell lines is displayed by immunofluorescence staining of cytospins; blue = DAPI, 40x magnification.</p

Adhesion of EpCAM^pos/neg cells on multi-marker arrays.

<p>2x10⁵ EpCAM^pos (cell pool of MCF7, SKBR3, HCC1500, ZR-75-1, TMX2-28) (A) and EpCAM^low/neg cells (MDA-MB-231) (B) were either stained with 1 μM MitoTracker Green FM (A) or MitoTracker Orange CM (B) and incubated for cell adhesion experiments on NEXTERION slides AL, coated with different antibodies and ECM molecules, alone and in combination (0.1 mg/ml each). The labeling above the single spots indicates respective capture molecules (Iso = isotype control, ms = mouse, Lam = laminin, Col = collagen, HA = hyaluronic acid, rt = rat, T = Trop2, EpC = EpCAM, 49f = CD49f, rb = rabbit). (C) Overlay image of (A) and (B); scale bars (white) = 500 μm, 20x magnification. (D) Array layout (5x5 mm) with 36 spots (spot diameter = 500 μm; pitch = 800 μm) printed on NEXTERION slides AL.</p

Medium Cut-Off (MCO) Membranes Reduce Inflammation in Chronic Dialysis Patients—A Randomized Controlled Clinical Trial

<div><p>Background</p><p>To increase the removal of middle-sized uremic toxins a new membrane with enhanced permeability and selectivity, called Medium Cut-Off membrane (MCO-Ci) has been developed that at the same time ensures the retention of albumin. Because many middle-sized substances may contribute to micro-inflammation we hypothesized that the use of MCO-Ci influences the inflammatory state in hemodialysis patients.</p><p>Methods</p><p>The randomized crossover trial in 48 patients compared MCO-Ci dialysis to High-flux dialysis of 4 weeks duration each plus 8 weeks extension phase. Primary endpoint was the gene expression of TNF-α and IL-6 in peripheral blood mononuclear cells (PBMCs), secondary endpoints were plasma levels of specified inflammatory mediators and cytokines.</p><p>Results</p><p>After four weeks of MCO-Ci the expression of TNF-α mRNA (Relative quantification (RQ) from 0.92 ± 0.34 to 0.75 ± 0.31, -18.5%, p<0.001)-α and IL-6 mRNA (RQ from 0.78 ± 0.80 to 0.60 ± 0.43, -23.1%, p<0.01) was reduced to a significantly greater extent than with High-flux dialyzers (TNF mRNA-RQ: -14.3%; IL-6 mRNA-RQ: -3.5%). After retransformation of logarithmically transformed data, measurements after MCO were reduced to 82% of those after HF (95% CI 74%–91%). 4 weeks use of MCO-Ci resulted in long-lasting change in plasma levels of several cytokines and other substances with a significant decrease for sTNFR1, kappa and lambda free light chains, urea and an increase for Lp-PLA2 (PLA2G7) compared to High-flux. Albumin levels dropped significantly after 4 weeks of MCO dialysis but increased after additional 8 weeks of MCO dialysis. Twelve weeks treatment with MCO-Ci was well tolerated regarding the number of (S)AEs. In the extension period levels of CRP, TNF-α-mRNA and IL-6 mRNA remained stable in High-flux as well as in MCO-Ci.</p><p>Conclusions</p><p>MCO-Ci dialyzers modulate inflammation in chronic HD patients to a greater extent compared to High-flux dialyzers. Transcription of pro-inflammatory cytokines in peripheral leukocytes is markedly reduced and removal of soluble mediators is enhanced with MCO dialysis. Serum albumin concentrations stabilize after an initial drop. These results encourage further trials with longer treatment periods and clinical endpoints.</p></div

Patient Flow Chart.

<p>Patient Flow Chart.</p

Demography and Clinical Patient Data ITT Population.

<p>Demography and Clinical Patient Data ITT Population.</p