# Isolation of Circulating Tumor Cells from Glioblastoma Patients by Direct Immunomagnetic Targeting
## 1 Introduction  
Glioblastoma (GBM) is the most common form of primary brain cancer in adults and one of the most lethal forms of cancer; only 5% of patients survive five years [1]. Diagnosis requires surgery to obtain tissue for standard histopathological processing, but the location of some tumors makes access challenging [2]. Moreover, follow-up MRI scans can be difficult to interpret as to whether patients have chemoradiation-related treatment changes such as pseudo-progression versus recurrent disease, and timely clinical decision making may thus be hampered. Therefore, there is a critical need for new methods to determine prognosis and identify biomarkers that guide therapy decisions in GBM.  
Circulating tumor cells (CTCs) are cancer cells that detach from the main tumor mass and enter the bloodstream which may be associated with the metastatic process. Over the past 15 years, clinical studies suggest that capture and enumeration of CTCs can provide important prognostic information [3; 4; 5]; moreover, these cells can be isolated and analyzed for molecular biomarkers [6]. For many years, it had been argued that CTCs were not present in GBM patients, due to the rarity of the development of the distant metastatic disease, and the belief that the inability of the cells to egress across the blood-brain barrier created an insurmountable difficulty. However, in 2014, CTCs were discovered in the bloodstream of GBM patients by three research teams. The proportion of CTC-positive patients ranged from 21% to 72% (29/141, 13/33 and 8/11), generally with low CTC counts. On this basis, it was proposed that CTCs could provide an alternative method of accessing GBM cells, compared to surgery, for analysis of relevant biomarkers [7].  
The methods for detecting GBM-CTCs in these seminal studies involved separation from red blood cells by gradient centrifugation, alone or followed by CD45-targeted cell depletion to exclude white blood cells, combined with immunocytostaining for GBM specific markers, mainly glial fibrillary acidic protein (GFAP), or fluorescence in situ hybridization (FISH) probing of the chromosome 8 centromere to determine aneuploidy. Alternatively, viral transduction of green fluorescent protein expressed under the regulation of the telomerase (hTERT) promoter to identify cancer cells was used for CTC detection [8; 9; 10; 11]. However, these methods of CTC detection may not be ideal, as millions of cells need to be screened for GBM markers by microscopy, or negative enrichment may cause loss of already rare CTCs due to potential antibody cross-reactions or physical co-segregation of CTCs amongst blood cells in the process. Further, current markers used to identify GBM-CTCs lack specificity, and clearly distinguishing CTCs from residual rare or common blood cells may either be associated with lack of specificity causing relatively high background staining, or potential of false-positive detection [10]; or lack of sensitivity due to marker heterogeneity in GBM cells, meaning some CTCs may be missed [8; 12]. A more recent study used size exclusion to successfully detect GBM-CTCs and less frequently GBM-CTC clusters [13]; size exclusion CTC enrichment relies on tumor cells being larger than residual blood cells which is common, but not always the case.  
Here, we tested a range of antibodies against candidate GBM cell surface proteins to develop a positive immunomagnetic GBM-CTC isolation protocol. We further screened for other GBM-CTC identification markers which established one additional marker useful for brain cancer CTC identification. Using this novel method, we have successfully isolated GBM-CTCs from GBM patient blood samples.