## 3 Results
### Immunostaining of Selective Protein Markers for Ovarian Cancer  
We also documented protein markers that were previously known to be found in ovarian cancer cells. These proteins were CA125, cytokeratin-18, E-cadherin, N-cadherin and vimentin. Again, we confirmed the immunological specificity using OVCAR-5 and SKOV-3, before using these antibodies with ascitie fluid-derived cells.  
The immunostaining for CA125, cytokeratin-18, E-cadherin and N-cadherin was positively observed in the OVCAR-5 cell lines. However, vimentin was less prominent in this cell line (Figure 3C). In contrast, the SKOV-3 cell line showed positive immunostaining of N-cadherin, vimentin, CA-125 and cytokerain-18. There was a low but detectable level of E-cadherin expression in this cell line (Figure 3D).  
Table 2 and Figure 4 summarise all relevant characteristics of ascitie fluid-derived cell samples. In Table 2, four of the 20 samples formed compact colonies and 16 showed loose colonies when they were cultured in cell monolayers. When cultured in a non-adherent condition, 12 (60%) of the samples formed compact aggregates, 7 (35%) of the samples showed small clusters, and 1 (5%) had a single-cell appearance.  
Figure 3: The expression of selected protein markers in ovarian cancer cell lines, OVCAR-5 and SKOV-3, and as ascitie fluid derived cells. OVCAR-5 shows a membrane positive staining for EGFR and negative staining for HER-2. SKOV-3 cell aggregates show membrane positive expression of EGFR and HER-2 (**A**). Ascitie fluid-derived cells show positive immunostaining for EGFR and HER-2 (**B**). OVCAR-5 shows positive staining for CA125, cytokeratin-18 and E-cadherin, but low expression of mesenchymal protein markers, N-cadherin and vimentin (**C**). SKOV-3 cell aggregates are positive for the staining of CA125, cytokeratin-18, N-cadherin, and vimentin, but very low for E-cadherin (**D**).  
Figure 4A,B show the characteristics of growth patterns in cell monolayers and non-adherent culture conditions and the immunostaining of selective proteins in selective asciti-derived cell samples. In the Table 2, nine (45%) of the 20 patient cells showed a detectable level of EGFR. Eight (40%) showed both expression of EGFR and HER-2 in the same cells. Three (15%) showed a low but detectable expression of EGFR and HER-2. There was no detection of HER-2 alone in any patient cells. Five (25%) of the 20 showed E-cadherin staining associated with the plasma membrane. All were positive for CA-125 and N-cadherin, whilst 19 (95%) expressed vimentin and cytokeratin-18.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Characteristic** & **Number** & **(\%)** \\ \hline
**Ovarian cancer** & 20 & \\
**Patients** & & \\
**Cell morphology** & & \\
**Cell monolayer** & & \\ Compact colony & 4 & 20 \\ Loose colony & 16 & 80 \\
**Floating condition** & & \\ Compact aggregates & 12 & 60 \\ Small cluster & 7 & 35 \\ Single cells & 1 & 5 \\
**Protein expression** & & \\ CA125 & 20 & 100 \\ Cytokeratin-18 & 19 & 95 \\ E-cadherin & 5 & 25 \\ N-cadherin & 20 & 100 \\ Vimentin & 19 & 95 \\ High EGFR only & 9 & 45 \\ High HER-2 only & 0 & \\ High EGFR/HER-2 & 8 & 40 \\ Low EGFR/HER-2 & 3 & 15 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Summary of characteristics of ascitie fluid-derived cells that are cultured in in vitro condition and selective protein markers for epithelial cells.  
Figure 4: _Cont_.