Fig 2: Specificity of EpCAM ElISA. (A) Dot Blot analysis of used detection antibodies to react with spiked native recombinant EpEX standard (10 ng) in assay buffer (PBS/1%BSA), serum (pool of 6 healthy donors) and ascites (pool of 12 negative patients). Capture and detection antibodies specifically react with EpEX in all biological matrices used and gave low background in serum and ascites (B) Detection antibodies bind specifically to EpCAM and EpEX-TM in established HEK-293FT cell lines expressing different variants of EpCAM or the control protein YFP (Western Blot). Results were confirmed by sandwich ELISA in cell extracts and respective supernatants. Concentrations are calculated for 1 µg total cellular protein extract. sEpCAM levels in supernatants of the different HEK cell lines were calculated with regard to cell number, culture volume and time. (C) Western Blot analysis of EpCAM expression in different human cancer cell lines under standard culture conditions (D) Concentrations of sEpCAM in supernatants were calculated with regard to cell number, culture volume and time. HRT-18 Apo and HRT-18 Nec indicate supernatants of HRT-18 cells that underwent apoptosis (100 µM A23187) or necrosis (100 mM H2O2).

Fig 3: EpCAM standard and stable control cells lines for ELISA. (A) Coomassie brilliant Blue staining of PAGE gel in the presence of two different standard proteins (GRP78 and BAP). Purity of the EpEX was approx. 93% as determined by Page Blue staining and densitrometric analysis. Molecular weight marker indicates reference proteins in KDa. EpEX-FLAG (293 aa) has an estimated size of 35 KDa. (B) Real-Time confocal microscopy pictures of living HEK293FT cell lines expressing different EpCAM isoforms fused to YFP: EpCAM-YFP shows expression only on cell membrane, EpEX-TM-YFP is detectable in the endoplasmatic reticulum and on cell membrane. Due to the lack of transmembrane region EpICD-YFP is found in cytosol and nucleus of living cells.

Fig 4: Measurement of sEPCAM in ascites. (A) Linear functions with recombinant EpCAM standard (156 pg - 5,000 pg/mL) spiked in ascites as biological matrix. (B) Scatter dot blot analysis with 95% confidence interval of the three ascites etiologies (C pos., C neg., LC). Soluble EpCAM concentrations were significantly different between C pos. and C neg. (*p < 0.05) and between C pos. and LC (**p < 0.05).

Fig 5: Analysis of ADCC in control cell lines. Effects of sEpCAM on catumaxomab-dependent cell mediated cytotoxicity (A) HEK EpCAM-YFP, and HEK EpICD-YFP cells were incubated without or with 1 ng/mL catumaxomab together with a 10-fold excess of human PBMNCs. PBMNCs with catumaxomab were efficiently killing EpCAM-YFP cells, but did not attack EpICD-YFP cells. Living cells are displayed by fluorescence microscopy (left picture). Incubation with 1 ng/mL EpEX already inhibited catumaxomab -dependent cell mediated cytotoxicity on EpCAM-YFP overexpressing HEK cells. Bars indicate 100 µm. (B) HEK EpCAM-YFP, and HEK EpICD-YFP cells were incubated without or with 1 ng/mL catumaxomab together with a 10-fold excess of human PBMNCs and increasing concentrations of EpEX (0, 0.2, 1, 5 ng/mL). YFP positive events were counted by flow cytometry (488 nm) and normalized to control cells without catumaxomab/PBMNC incubation (100%). ADCC was repeated three times (Mean ± SEM).