Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein

Abstract

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.

Isolation of Circulating Tumor Cells from Glioblastoma Patients by Direct Immunomagnetic Targeting

“…VAR2CSA has a high affinity to chondroitin 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 chondroitin 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.…”

Optimization of rVAR2-Based Isolation of Cancer Cells in Blood for Building a Robust Assay for Clinical Detection of Circulating Tumor Cells

“…This study therefore set out to explore and optimize different technical parameters and approaches for rVAR2-based cancer cell isolation. We have previously shown that rVAR2-coated paramagnetic beads can isolate CTCs from clinical cancer patient blood samples [12,26]. However, in this study, the aim was to further optimize the technical parameters to obtain the highest level of assay performance and the required reproducibility, as well as paving the way for a standardized optimization method of bead coupling to rVAR2, which could also inspire development strategies of other bead-coupled CTC-targeting moiety technologies.…”

“…VAR2CSA was produced in two recombinant versions (rVAR2), DBL1-ID2a and ID1-ID2a, in SHuffle®T7 Competent E. coli as previously described [12]. Both constructs include a C-terminal hexa His-tag and V5 tag, as well as an N-terminal SpyTag.…”

“…SpyCatcher (SpyC) was produced in the E. coli BL21 strain and multibiotinylated using NHS biotin as previously described [12]. Furthermore, the SpyCatcher was mutated to carry a cysteine at a single position (S8C) and was similarly produced in E. coli BL21 strain.…”

“Circulating Tumor Cells: Finding Rare Events for a Huge Knowledge of Cancer Dissemination”

“…Although extracranial metastases in glioma patients are rarely observed, recent studies have shown the presence of CTCs in the bloodstream. Bang-Christensen et al demonstrated how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines and identified a panel of proteoglycans, known to be essential for glioma progression [7]. Cerebrospinal fluid (CSF) is one of the promising diagnostic targets because CSF passes through the central nervous system, harvests tumor-related markers from brain tissue and then, delivers them into peripheral parts of the human body where CSF can be sampled using minimally invasive and routine clinical procedure.…”