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pluriBead®
Application Example

Isolation of rare cell types:
Circulating tumor cells

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V 1.0

Isolation of rare cell types:
Circulating tumor cells

Results
Fig. 1a and 1b: Flowcytometric analysis ofCaco-2 cells, staining with
anti-EpCAM-PE.

Introduction
Here we are presenting a new approach to colon carcinoma
circulating tumor cell (CTC) screening using pluriBead® carrying atumor-associated EpCAM antibody.

Fig. 1bFig. 2aFig. 2bFig. 2c2a) Colon carcinoma Caco-2 cells captured by EpCAM-pluriBead®.
2b) Captured colon carcinoma Caco-2 cells, staining with calcein.
2c) Captured colon carcinoma Caco-2 cells spiked in 30 ml whole blood with 0.5 million EpCAMpluriBead®.
wtCaco-2 line
Fig. 3b{2dooble

1inlllceFig. 3aeolnlowhcoThis method is based on a non-magnetic cell separation technology. It does not require any sample pre-treatment. EpCAMpluriBead® can be added directly to a whole blood sample. The
method is also suitable for single cell isolation from different
biological fluids. Moreover, its sensitivity can be additionallyincreased by raising the sample volume. The bound EpCAMpositive colon carcinoma cells can be easily involved in further
molecular-genetic experiments that aim to detect of their mutation status. In case of colon carcinoma, K-ras mutation status isa predictive tool of response anticancer therapy. Thus, the new
method can be considered as a fast and effective instrument forearly cancer diagnostics.

Fig. 1a345

67500 cells 100 cells

Materials and methods

157 bp143 bp143 bpcoofn tsio cst duge ro 1
Di R p TN
PC Bs
bylrontedstge

diUnrontcoed
st trolge
di con
Un ivetgallront

coodraKed
st ctsupred

gestDigedi{114 bp
NeUn114 bp{

157 bpsFig. 3: Screening of colon carcinoma lines for the presence of mutation in 12h codone of K-ras
oncogene by PCR using primers that introduce BsTN1 restriction enzyme sites into PCR products.

A: Labeling

Add EpCAM-pluriBead®
to your sample.

B: Incubation

30 minutes of gentle incubation (recommendedwith pluriPlix®).

C1: Separation

Captured target cells are
separated via
appropriate sieves.

Fig. 4aFig. 4b

Number of spiked cells
1000

500

250

12560

30

10

Caco-2 Negativecontol

EpCAM
157 bp

CK 20Fig. 4a: Sensitivity of PCR method for the detection of K-ras protoncogene in known number ofspiking Caco-2 cells. Fig. 4b: Products of RT-PCR for EpCAM and CK 20 mRNA isolated from theCaco-2 cells, captured by EpCAM-pluriBead® (as a negative control, mRNA from whole blood wasused).
1.5
1.0
0.5

C2: Separation

Target cells bound on
pluriBead® stay on topof the sieve. The rest
runs through.

D: Washing & lysis

Use wash buffer to cleansieve. Lyse cells withTrizol®.

E: Processing

Approaches for the studyof cancer cells:- RNA/DNA isolation- Cell cultureexperiments

Conclusions and perspectives
Here we developed a new approach for the detection of circulating colon tumor cells in blood using EpCAM-pluriBead®. Captured
cells are suitable for further molecular-genetic screening of specific markers, connecting with tumor formation. The developed“in situ immunobeads pcr” method does not require preliminary
RNA/DNA isolation and can effectively save the time of analysis. The further aim of this work is to increase the sensitivity ofmethod raising the sample volume, varying the cell tumor lines
and/or antibody specificity.