# Isolation of Circulating Tumor Cells from Glioblastoma Patients by Direct Immunomagnetic Targeting
## 4 Discussion  
The aim of this study was to develop a positive immunomagnetic enrichment strategy for GBM-CTCs and improve identification markers for GBM-CTCs. A cohort of neurological cell-specific, cell surface markers were considered less useful for CTC isolation since there was no, the low or variable expression on cultured GBM cells or when the expression on common blood cells was detected with the validated antibodies used in our study. While most of the markers we screened had previously been detected by others on cultured GBM cells or tissue (Tables S1 and S2), our inability to replicate some of these findings in our cultured GBM cells may be due to factors such as different antibodies used, different protein levels between cultured cells and GBM tissue or increasing heterogeneity of long term established cell cultures used in the various laboratories. The most promising markers, MCAM and MCSP, have been successfully used by us and others for the isolation of melanoma CTCs previously [15, 16]. Since melanocytes originate from the neural crest [14], the detection of GBM cells is not surprising. It is important to note however that MCAM and MCSP were not equally well expressed on all tested GBM cell lines (proportion of positive cell population, Figure 1). If such heterogeneity translates to patient-derived GBM-CTCs, GBM-CTCs expressing low antigen would be captured less efficiently. While immunomagnetic targeting of MCAM and MCSP proved independently successful for the capture of LN-229 cells, the combination of both antibodies was superior for cell isolation. Additionally, our previous data clearly show that the strategy of targeting both proteins together improved melanoma CTC capture from patient blood [15]. Therefore, we used the same strategy for successful GBM-CTCs enrichment from patient samples. CTC counts for our study were low, which is consistent with reported GBM-CTC counts in other studies [8, 9, 10, 13] and our immunomagnetic targeted CTC isolation produces better CTC enrichment (ratio CTCs: residual blood cells) than some previous studies likely to make certain down-stream analyses easier. This is the first method positively immune-targeting GBM-CTCs for enrichment. Recently, another study did achieve GBM-CTC enrichment by conjugating a malaria protein, VAR2 CSA, to magnetic beads. VAR2 CSA has a high affinity to chondro sulfate proteoglycans proposed to be uniquely expressed on cancer cells. The study isolated CTCs in comparable numbers to our study [17]. Of note, MCSP belongs to the chondro sulfate proteoglycan protein family and our data confirm high expression on some GBM cell lines but total lack on others, highlighting that targeting more than one cell surface protein should be considered advantageous for GBM-CTC isolation. In our hands, markers such as nestin, previously suggested as suitable for GBM-CTC detection, were less satisfactory [9]; this may have been due to a different anti-nestin antibody (raised in rabbit) used here, due to compatibility with our isolation antibodies. Since our isolation antibodies were raised in mice, a mouse-anti-nestin antibody would have produced strong cross-reactivity with magnetic beads. Our nestin probing showed positivity in GBM cells but also PBMCs (Figure S1). Figure 5: CTC status and survival. Nevertheless, we identified one marker to better distinguish GBM-CTCs from residual lymphocytes. GLAST, supposedly present on all brain cells, is a transporter protein, and is normally cell-membrane localized, but can be internalized and predominantly found in the nucleus in GBM tissue and cultured GBM cells [18]. Our GBM cell staining confirmed nuclear GLAST for all but LN-229 cells (Figure 2). The recognition of anti-GLAST as an additional identification antibody is important, since the commonly used GFAP identification marker protein is not present in all GBM cells [12]. Interestingly, in our hands, immunocytostaining did not detect GFAP in any of the GBM cell lines tested, whereas GLAST was readily detected in all five GBM cell lines.