Abstract 4984
Background
Circulating tumor cells (CTCs) are shed by the solid tumor into the bloodstream. Consistently, single-cell sequencing of CTCs is a powerful tool to further decipher tumor spatiotemporal heterogeneity, plasticity and to identify new pharmacological targets influencing clinical outcome and response to treatment. The aim is to validate an original workflow allowing the isolation at the single-cell level of mimicking CTCs without interfering with single-cell RNAseq analysis.
Methods
The advances in microfluidic systems and isolation technologies have resulted in the enriched extraction of mimicking CTCs from healthy blood samples. The ClearCell Fx (Biolidics Limited) was used as a label-free microfluidic system for enrichment of wholly intact CTCs, while the cellenONE F1.4 system (Cellenion) was used to isolate single CTCs. The later allowed high-throughput automated isolation and dispensing of single CTCs in 96-well plates. ScRNA libraries were prepared with the NebNext Single Cell/Low Input kit (New England Biolabs).
Results
The h-TERT-shp53/RAS HMEC breast cancer cell line was used as mimicking-CTC cell line to evaluate transcriptomic changes. We compared the transcriptomic profiles at each major step: (1) directly after trypsinization, (2) after spiking in whole blood and enrichment with the ClearCell Fx device, (3) after isolation with the cellenONE instrument in bulk of 200 cells and at the single-cell level. The preparation of the scRNAseq library was successful in over 90% of cases at the single-cell level and 100% for bulk samples. Minor effects of the enrichment and isolation methodology were observed at the single-cell level on the transcriptomic profiles. The quality of the libraries and the sequencing provided reliable scRNAseq analyses.
Conclusions
This protocol could be further improved by adding a pre-selection of CTCs based on negative sorting, using a fluorescent anti-CD45 immune labelling exclusion. Such knowledge of single-cell biology may lead to the development of specific therapies to limit tumor progression and seeding of tumoral cells into secondary healthy organs by blocking newly identified targets.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
Léa Payen-Gay.
Funding
New England Biolabs.
Disclosure
All authors have declared no conflicts of interest.
Resources from the same session
4079 - Triggering anti-GBM immune response with EGFR-mediated photoimmunotherapy
Presenter: Gabriela Kramer-marek
Session: Poster Display session 1
Resources:
Abstract
4364 - Upregulation of sFRP3 circulating expression levels correlates survival outcomes in glioblastoma
Presenter: Gema Bruixola
Session: Poster Display session 1
Resources:
Abstract
2327 - Characterization and pre-clinical modeling of genetic aberrations in pediatric gliomas
Presenter: Itai Moshe
Session: Poster Display session 1
Resources:
Abstract
3154 - Preclinical Study of Novel Tetracyclic Small Molecule, CC12, for Brain Cancer
Presenter: Liyun Fann
Session: Poster Display session 1
Resources:
Abstract
5759 - CHLOROBRAIN phase IB trial: The addition of chloroquine, an autophagy inhibitor, to concurrent radiation and temozolomide for newly diagnosed glioblastoma
Presenter: Inge Compter
Session: Poster Display session 1
Resources:
Abstract
1382 - A Phase II Clinical Trial Evaluating the Efficacy and Safety of Apatinib Combined with dose-dense Temozolomide in Recurrent Glioblastoma
Presenter: Yong Wang
Session: Poster Display session 1
Resources:
Abstract
4407 - Phase 0 Trial of Ceritinib in Brain Metastases and Recurrent Glioblastoma
Presenter: Shwetal Mehta
Session: Poster Display session 1
Resources:
Abstract
1469 - Pembrolizumab (Pem) in recurrent high-grade glioma (HGG) patients with mismatch repair deficiency (MMRd): an observational study
Presenter: Mario Caccese
Session: Poster Display session 1
Resources:
Abstract
4217 - Outcome of high-grade gliomas (HGGs) treated into immunotherapeutic early-phase clinical trials (ieCTs): a single-center experience
Presenter: Matteo Simonelli
Session: Poster Display session 1
Resources:
Abstract
5107 - Tolerability of PCV in Low Grade Glioma: a Real World Experience
Presenter: Razia Aslam
Session: Poster Display session 1
Resources:
Abstract