CTC isolation & analysis

Deciphering CTC heterogeneity at single cell level

Circulating tumor cells (CTCs) are cells that have been shed from a tumor and entered the blood circulation. Following adaptation and colonization of the microenvironment of a secondary site they form metastases which are responsible for over 90% of tumor-related deaths. CTCs, obtained through a simple blood draw, can serve as a “liquid biopsy” to monitor tumor characteristics in real-time, including inter- and intra-tumor heterogeneity. However, CTC isolation and subsequent characterization are technically challenging due to the low CTC cell numbers among an abundance of white and red blood cells. A wide range of analytical methods for CTC detection, enrichment and isolation has been developed. They exploit CTC-specific properties such as surface marker expression or e.g. size, density or deformability.

ALS has developed a unique and complete solution for detection and isolation of CTCs after enrichment and staining steps. Cells isolated can then be used for DNA, RNA or proteome analysis. This solution is centered around the ALS CellCelector™ system utilizing specifically developed consumables (e. g. CellCelector Nanowell arrays, MagnetPick™ slides) and validated protocols.

Single cell CTC isolation and analysis workflow using the ALS CellCelector™

The ALS CellCelector™ used within a typical CTC isolation and analysis workflow. * Red blood cell (RBC) lysis is optional and depends on the used enrichment technology; **Certain enrichment technologies allow in-situ staining after enrichment; in other cases the staining should be done after retrieving the sample from the enrichment device.

Full compatibility with existing enrichment technologies

As demonstrated in numerous publications the ALS CellCelector™ platform is compatible with a variety of upstream enrichment technologies:

Positive immuno-magnetic enrichment (based on EpCAM surface markers and others): e.g. CellSearch^®, Isoflux™, MagSweeper™

Negative depletion of white blood cells (WBCs) based on CD45 surface markers: e.g. Dynabeads™, RosetteSep™

Label-free separation based on microfluidics or filters: e.g. Parsortix™, Clearbridge™, Vortex™, ScreenCell™, RareCells™, Circulogix™ etc.

ALS has developed workflows and consumables which allow handling of up to several hundred thousand of blood cells per sample without need for sample volume reduction.

Enrichment technologies	References
Positive immuno magnetic
CellSearch® (EpCAM)	Neumann et al., 2016
Isoflux™ (EpCAM)	Ma et al., 2016; Cabezas-Camarero et al., 2019; Nimir et al., 2019; Ding et al., 2019
MagSweeper™ (EpCAM)	Lohr et al., 2014
Dynabeads and hand-held magnet (rVAR2)	Agerbæk et al., 2018; Bang-Christensen et al., 2019
*CD45 negative depletion*
Dynabeads® CD45/Dynal® MPC®-S	Blassl et al., 2016
RosetteSep™ Human CD45	Yao et al., 2014
*Size-based separation*
Parsortix™	Lampignano et al., 2017; Szczerba et al., 2019; Gkountela et al., 2019; Reinhardt et al., 2019; Donato et al., 2019
*Densitiy gradient centrifugation*
OncoQuick™	Heidary et al., 2014

CTC enrichment technologies used prior to the ALS CellCelector™ single cell isolation and corresponding published papers (full article references can be found on the Publications page).

CTC isolation from various cancer types

The ALS CellCelector™ technology is capable to isolate pure single tumor cells from liquid biopsies of any cancer type. Leading cancer centres throughout the world using the CellCelector™ system are working on developments in numerous cancer entities and application areas. Full article references can be found on the Publications page.

Cancer type	References
Breast	Heidary et al., 2014; Schneck et al., 2015; Neumann et al., 2016; Lampignano et al., 2017; Szczerba et al., 2019; Gkountela et al., 2019; Reinhardt et al., 2019; Sprouse et al., 2019
Prostate	Lohr et al., 2014; Ma et al., 2016; Nimir et al., 2019
Colorectal	Adalsteinsson et al., 2013; Cabezas-Camarero et al., 2019
Pancreatic	Kim et al., 2019
Ovarian	Blassl et al., 2016
Lung	Yao et al., 2014; Ding et al., 2019
Brain	Agerbæk et al., 2018; Bang-Christensen et al., 2019
Melanoma	Sprouse et al., 2019

Benefits of CellCelector platform for CTC isolation

Reliable

Very low cell loss (no dead volume)
Single cell picking efficiency up to 100 % (depending on workflow)
Automated picking control (Picked/Failed)

Fast

20 individual single CTCs picked within just 10 min making the CellCelector™ compatible with RNA analysis and live cell picking
Process for enumeration and isolation of CTCs takes around 1 hour per sample (starting from an enriched and stained sample)

Easy to use

No complex sample preparation necessary
User-friendly yet powerful software

Highly versatile

Isolation of individual single CTCs and clusters
Isolation of fixed and live cells
Direct compatibility with various upstream enrichment methods
Automated CTC detection based on cell morphology, pathological stains and/or up to 6 fluorescence labels
Automated, semi-automated or manual cell selection for picking
Picking of either individual cells or cell pools
Compatible with most labware: well plates, Petri dishes, microscope slides, PCR plates/tubes, nanowell arrays, custom consumables etc.

Fully compatible with various downstream analysis and cultivation workflows

Compatibility with a wide range of single cell molecular analysis methods including NGS, RNAseq etc.
Low aspiration and dispensing volumes for seamless compatibility with all single cell WGA and qRT-PCR kits
Temperature-controlled destination positions (4 to 37 °C) for preventing RNA degradation or facilitate live cell survival
Low shear stress and fast picking enabling live cell expansion

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