Novel strategies for circulating tumor cell analysis in glioblastoma

Glioblastoma (GBM) is an aggressive brain tumor characterized by marked intra- and inter-tumoral heterogeneity and inevitable recurrence. Although extracranial metastasis is rare, circulating tumor cells (CTCs) are consistently detected in GBM patients, suggesting their potential as liquid biopsy biomarkers.

Lessi's team established and optimized a workflow that combines physical enrichment with the Parsortix system and single-cell isolation using the DEPArray NxT platform, enabling the genomic and phenotypic characterization of both bulk and single CTCs. In a cohort of 27 GBM patients, whole-exome sequencing (WES) confirmed that CTCs share somatic mutations and copy number alterations (CNAs) with matched primary and recurrent tumors, proving their tumor origin. CTCs were significantly more abundant in primary compared to recurrent GBM, and absent in radionecrosis and lymphoma cases, establishing a diagnostic cutoff of 4 CTCs per 5 mL of blood. Single-cell analyses revealed striking heterogeneity, including CTCs lacking detectable CNAs. Similar CNA-negative populations were also observed in scRNA-seq data from GBM cell lines, suggesting the coexistence of malignant and tumor microenvironment–derived cells in circulation.

Beyond GBM, the team applied the same Parsortix–DEPArray pipeline to pediatric osteosarcoma (OS) patients. Here, they identified both mesenchymal-like and epithelial-like CTC subpopulations, with longitudinal tracking of epithelial-like CTCs correlating with chemotherapy response. These results underscore the versatility and translational relevance of their workflow across distinct tumor types, paving the way for the development of personalized liquid biopsy strategies.

Why join this webinar?

• Discover how circulating tumor cells (CTCs) can serve as powerful liquid biopsy biomarkers in glioblastoma.
• Learn about an innovative workflow combining Parsortix® and DEPArray technologies for CTC analysis.
• Understand how CTCs reflect tumor mutations and heterogeneity at the single-cell level.
• See the importance of analyzing bulk CTCs enriched with the Parsortix system to obtain comprehensive tumor information (e.g., whole-exome data) without the need for whole genome amplification.
• Explore how the same applications are extended to pediatric osteosarcoma, a highly aggressive tumor

Cristina Ciccioli, PhD

Head of Assay Development, ANGLE

BIOGRAPHY

Blood speaks: decoding glioblastoma with liquid biopsy multi-omics

Liquid biopsy is widely used in systemic cancers, offering a minimally invasive approach to monitor tumor progression through blood-based biomarkers such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and extracellular vesicles (EVs). However, its application in glioblastoma (GBM) remains limited. This is partly due to the low levels of ctDNA shedding and the absence of epithelial CTCs, which are the main targets of many existing CTC platforms. These challenges have hindered the full potential of liquid biopsy in neuro-oncology. 

ANGLE established a method for the comprehensive evaluation of circulating biomarkers in the blood of patients with GBM. Whole blood was collected from 15 treatment-naïve patients with Grade 4 GBM, using three blood collection tubes (BCTs) per subject. Plasma was separated from each sample for ctDNA analysis, and the remaining blood processed for the enrichment of CTCs and large EVs on the Parsortix® device. Material harvested from one BCT was analyzed using a research use only immunofluorescence assay targeting epithelial and mesenchymal markers. The harvests from the remaining tubes, together with DNA isolated from plasma, were subjected to sequencing of specific genes such as IDH1, IDH2, BRAF, EGFR, and detection of low-frequency mutations, CNVs, and fusions for genomic profiling. 

This study demonstrated that the developed workflow can effectively isolate CTCs and EVs from the blood of GBM patients. CTCs were detected in 60% (9/15) of patients, all displaying a mesenchymal phenotype (mean: 7; median: 6; range: 2–17), while EVs were identified in 73% (11/15) of patients (range: 1–300). Ongoing molecular analyses of CTCs and ctDNA aim to determine their utility as informative biomarkers, with the goal of enabling dynamic testing strategies to support precision cancer therapies in neuro-oncology.