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
5939 - Matrix metalloproteinases and their tissue inhibitors genes abnormal DNA methylation in breast cancer
Presenter: Olga Simonova
Session: Poster Display session 1
Resources:
Abstract
2703 - Uveal melanoma cell lines depend on multiple signaling pathways for survival
Presenter: John Park
Session: Poster Display session 1
Resources:
Abstract
4849 - XAF1 and ZNF313 complex stimulates ER stress-induced apoptosis via direct GRP78 inhibition.
Presenter: Sungchan Jang
Session: Poster Display session 1
Resources:
Abstract
4801 - XAF1 assembles a destructive complex to induce BRCA1-mediated apoptosis via suppressing ERa and switching estrogen function
Presenter: Seung-hun Jang
Session: Poster Display session 1
Resources:
Abstract
3416 - Cancer associated fibroblasts promote cancer progression via Wnt2 secretion in colorectal cancer
Presenter: Hideaki Karasawa
Session: Poster Display session 1
Resources:
Abstract
4273 - Paired-related homeobox 1 overexpression promotes invasion and metastasis and is a prognostic factor for worse disease-free survival in patients with lung cancer
Presenter: Jung-jyh Hung
Session: Poster Display session 1
Resources:
Abstract
4241 - LncRNA-GC1 contributes to gastric cancer chemo-resistance through inhibition of miR-551b-3p and the overexpression of dysbindin
Presenter: Xin Guo
Session: Poster Display session 1
Resources:
Abstract
5388 - GLPG 1790, a new selective EPHA2 inhibitor, is active in colorectal cancer cell lines belonging to the CMS4/mesenchymal-like subtype
Presenter: Pietro Paolo Vitiello
Session: Poster Display session 1
Resources:
Abstract
5208 - Characterisation of growth hormone signal transduction in primary melanoma cell lines
Presenter: Karla Sousa
Session: Poster Display session 1
Resources:
Abstract
3156 - LAPTM5 protein can regulate TGF-β mediated MAPK and Smad signaling pathways in ovarian cancer cell
Presenter: Yan Gao
Session: Poster Display session 1
Resources:
Abstract