Generation of a permanent cell line from tumour explants.

<p>(<b>A</b>) Matched sample (upper left panel) and single cells (lower left panel) obtained from explants of a larynx carcinoma were stained for the expression of EpCAM. Harvested cells were passaged to establish a permanently growing cell line. PiCa cells grew in adherent manner and displayed heterogeneity in morphology (right panel). (<b>B</b>) PiCa cells were monitored for the cell surface expression of EpCAM, EGF-R, and CD44 using specific antibodies. Cells represented one determined population (FSC) and were vital (PI) as shown in the dot plot graph (upper left). (<b>C</b>) PiCa cells were transiently transfected with control or EpCAM-specific siRNA. After 24 hrs, cells were analysed for the cell surface expression of EpCAM upon flow cytometry. Isotype control staining is depicted as solid, purple curve, control siRNA as green line, and EpCAM-siRNA as dashed pink line. (<b>D</b>) PiCa cells were transiently transfected with the eGFP-C1 expression plasmid. After 24 hrs, expression of GFP was assessed upon flow cytometry. Dot plots graphs represent control transfectants (upper panel) and GFP transfectants (lower panel). Quadrants were set according to control transfectants and statistics are given below. (<b>E</b>) PiCa cells (1×10⁴, 1×10⁵, 1×10⁶) were subcutaneously injected into the left and right flanks of immunocompromised NOD-SCID mice. After 21 days, tumours were surgically removed, cryo-preserved and stained for the expression of EpCAM. Matched primary specimens were stained in parallel (left panels) to xenotransplants (right panels) and are depicted in 100× and 200× magnification. Injected cell numbers and tumour weights with mean and standard deviations are given in tabular manner.