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
4883 - A New Population Model Validated Pharmacokinetic Similarity of HLX01 and Rituximab in B-Cell Lymphoma
Presenter: Yuankai Shi
Session: Poster Display session 1
Resources:
Abstract
4908 - Efficacy of salvage therapy in the treatment of Helicobacter pylori-positive gastric low-grade mucosa-associated lymphoid tissue lymphoma
Presenter: Sung-Nam Lim
Session: Poster Display session 1
Resources:
Abstract
2360 - Mutational analysis of extranodal marginal zone lymphoma using next generation sequencing
Presenter: Seok Jae Huh
Session: Poster Display session 1
Resources:
Abstract
2430 - Clinical features, treatment and outcomes of colon and rectum mucosa-associated lymphoid tissue (MALT) lymphoma: Literature reviews published in English between 1993 and 2017
Presenter: Jeong Yeon Kim
Session: Poster Display session 1
Resources:
Abstract
4654 - Splenic marginal zone lymphoma: clinical characteristics and prognostic factors in a series of 52 patients
Presenter: Guldane Cengiz Seval
Session: Poster Display session 1
Resources:
Abstract
1732 - Safety and efficacy of Bendamustine and Rituximab (BR) regimen in Indian Chronic Lymphocytic Leukemia patients
Presenter: Ajay Gogia
Session: Poster Display session 1
Resources:
Abstract
5784 - N-terminal B-type natriuretic peptide (NT-proBNP) as an independed prognostic marker for patients with newly diagnosed multiple myeloma complicated by dialysis-dependent renal failure
Presenter: Sergey Semochkin
Session: Poster Display session 1
Resources:
Abstract
836 - The first-line effect of Bortezomib-based Therapy on Clinical Outcomes for Taiwanese Patients with multiple myeloma
Presenter: Ching-Liang Ho
Session: Poster Display session 1
Resources:
Abstract
2085 - Impact of Donor Lymphocyte Infusion in Relapsing Myeloid Neoplasms Post Allogeneic Hematopoietic Stem Cell Transplantation
Presenter: Hanafy Hafez
Session: Poster Display session 1
Resources:
Abstract
6079 - Invasive fungal diseases in patients with Hodgkin’s lymphoma before and after allogeneic hematopoietic stem cell transplantation
Presenter: Marina Popova
Session: Poster Display session 1
Resources:
Abstract