# Isolation of Circulating Tumor Cells from Glioblastoma Patients by Direct Immunomagnetic Targeting
## 3 Results  
### Cell Surface Protein Expression on Glioblastoma Cells  
To identify appropriate cell surface targets for immunomagnetic isolation of GBM cells, a number of cell surface proteins identified from literature searches were screened with appropriate antibodies for the presence on a cohort of GBM cell lines by FACS analysis (Table 2, Table S1). Antibodies that demonstrated limited reactivity to GBM cells (\(\leq\)3% \(\pm\) 2% of the cell population positive) were disregarded for isolation. Those that reacted with GBM cells were further tested for antibody cross-reactivity with healthy donor peripheral blood mononucleated cells (PBMCs); considerable PBMC cross-reactivity suggested unsuitability as potential tools for immunomagnetic GBM-CTC isolation due to expected and undesirable co-capture of PBMCs. The remaining antibodies against promising target proteins were further evaluated by immunocytstaining.  
Since neural cells and melanocytes are embryologically related (both originate developmentally from the neural crest) [14], we included antibodies against the melanoma-associated chondro sulfate proteoglycan (MCSP) and melanoma cell adhesion molecule (MCAM) in our screen. We have previously shown both proteins to be suitable targets for immunomagnetic melanoma CTC isolation [15]. After  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & & \multicolumn{3}{c}{**Detectable [\% of Cells]**} \\ \hline
**Antigen** & **A172** & **LN-229** & **T9 SG** & **U87** & **U251** & **PBMCs** \\ \hline MCAM & 51.0 \(\pm\) 3.6 & 94.6 \(\pm\) 1.7 & 64.1 \(\pm\) 5.4 & 67.8 \(\pm\) 4.9 & 42.7 \(\pm\) 0.1 & - \\ MCSP & 80.4 \(\pm\) 4.1 & 92.1 \(\pm\) 2.6 & - & 89.3 \(\pm\) 13.7 & - & - \\ N-Cadherin & 70.2 \(\pm\) 13.8 & 55.3 \(\pm\) 0.6 & 17.1 \(\pm\) 0.6 & - & 94.3 \(\pm\) 3.3 & - \\ EGFR & 96.8 \(\pm\) 0.3 & 78.1 \(\pm\) 1.3 & 29.2 \(\pm\) 0.4 & 11.2 \(\pm\) 3.2 & 34.5 \(\pm\) 8.3 & - \\ CD271 & 5.9 \(\pm\) 0.1 & 57.3 \(\pm\) 0.4 & 8.6 \(\pm\) 10.5 & - & - & - \\ A2B5 & - & - & - & - & n/d & n/d \\ \hline \hline \end{tabular}
\end{table}
Table 2: Cell surface protein detection on cell lines by flowcytometry.  
the exclusion of other candidates, MCSP and MCAM were identified as the most promising cell surface protein targets for immunomagnetic isolation of GBM cells. MCSP was detected on up to 92% of the cell populations of GBM cell lines tested with high proportions of LN-229 (92%), U87 (89%) and A172 (80%) cells expressing the antigen. However, both T98G and U251 cells were MCSP negative. MCAM was expressed on 43%-95% of the cells of all cell lines. Very high proportions of LN-229 (95%), most U87 (68%) and T98G (64%) cells were positive for MCAM, while around half of A172 (51%) and U251 (43%) cell populations expressed MCAM (Table 2, Figure 1). Importantly, the anti-MCSP (\(\alpha\)MCSP) and anti-MCAM (\(\alpha\)MCAM) antibodies showed negligible reactivity with PBMCs from healthy donors (Table 2).  
Cell surface expression of MCAM (left) and MCSP (right) was screened on the indicated cell lines by immunocytostaining (specific protein detected in green FITC channel, while Hoechst staining detects cell nuclei, merged images included) and flow cytometry. Proportions of cell populations expressing the indicated protein are stated from at least n = 2 independent experiments (as gated on flowcytometry data comparing the light grey protein-specific peak to isotype-matched IgG probing (dark grey peak)). Representative histograms included.