Fig 2: Presentation of selected CTCs in the peripheral blood of HNC patientsImage gallery presenting CTCs isolated by the ScreenCell® microfiltration system: Cytokeratin, EGFR, DAPI-positive but CD45 negative with a cell size larger than the pore size of the filter (approximately 7µm). Imaged on the Olympus IX3 inverted microscope. Scale bar represents 50µm.

Fig 3: Presentation of selected CTCs in the peripheral blood of HNC patientsImage gallery presenting CTCs captured by the RosetteSep™ system: Cytokeratin, EGFR, DAPI-positive but CD45 negative. Imaged on the Olympus IX3 inverted microscope. Scale bars represent 50 µm.

Fig 4: Representative immunofluorescence analysis of the cellular distribution of (A,C) LPP and (B,D) zyxin in cross sections of the (A,B) mouse femoral and (C,D) mouse mesenteric artery. The isolated blood vessels were fixed with p-formaldehyde overnight, embedded in paraffin, and 3 µm-thin sections were stained with primary antibodies directed against LPP (HPA017342, Sigma-Aldrich, 1:75) or zyxin (HPA004835, Sigma-Aldrich, 1:75) together with primary antibodies directed against a-SMA (F3777, Sigma-Aldrich, 1–200) or CD31 (AF3628, R&D Systems, 1:200). Nuclei were counterstained with DAPI (blue fluorescence). Images were recorded using an Olympus IX3 epifluorescence microscope. The size marker corresponds to 20µm. LPP (red fluorescence) selectively localizes to the medial SMC layer of both arteries (a-SMA-positive area, not shown), whereas zyxin (red fluorescence) is detected both in the intimal layer, that is, in the EC, and in the medial layer of both arteries. In particular in the femoral artery, there is a clear co-detection of zyxin and CD31 (green fluorescence) as indicated by the yellow fluorescence. Since CD31 is present in the EC membrane, while zyxin localizes to the cytoplasm and/or the nucleus, the remainder of the EC reveal positive CD31 immunoreactivity right next to that of zyxin. This also holds true for the mesenteric artery with some yellow fluorescence detectable, too. When compared to LPP, zyxin immunofluorescence in the medial SMC layer of both arteries is clearly weaker.