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neCTC-BC-Kit:
EpCAM-independent isolation of circulating tumor cells in breast cancer

Optimize isolation of 100% pure breast cancer cells from liquid biopsies by combining our new isolation kit with our well-proven CellCelector™ technology

High-precision isolation of (rare) single circulating tumor cells (CTCs) from complex cell populations derived from patient samples is now readily available. However most antibody-based isolation solutions target CTCs which express the epithelial cell adhesion molecule (EpCAM). This approach does neglect EpCAM-low or negative cells which may represent a CTC subpopulation generated by epithelial-mesenchymal transition (EMT). Published data suggest that EMT may contribute to tumor resistance 1,2,3 and may also be related to tumor cell stemness 4,5.


1Fischer KR, Durrans A, Lee S, et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature. 2015;527:472–476.
2Zhuo W, Wang Y, Zhuo X, et al. Knockdown of Snail, a novel zinc finger transcription factor, via RNA interference increases A549 cell sensitivity to cisplatin via JNK/mitochondrial pathway. Lung Cancer. 2008;62:8–14.
3Zheng X, Carstens JL, Kim J, et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature. 2015;527:525–530.
4Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133:704–715.
5Liu S, Cong Y, Wang D, et al. Breast cancer stem cells transition between epithelial and mesenchymal states reflective of their normal counterparts. Stem Cell Reports. 2014;2:78–91.
In combination with the ALS CellCelector, an automated single cell screening and picking system, the neCTC-BC-Kit will provide a great way to isolate breast cancer CTCs with all EpCAM-expression levels either in your own lab or as service offered by our state-of-the-art cellular and molecular biology laboratory partners.

The neCTC-BC-Kit is based on magnetic beads + ferro fluid and utilizes the combination of two antibodies specific for cell surface proteins.  Its performance has been tested and validated in our partner laboratories and shows the following key advantages:



1) The antibody mix captures both EpCAM-positive and EpCAM-negative tumor cells

Figure 1 shows that the two antibodies used in the neCTC-BC-Kit can bind both EpCAM-positive (T47d, Sk-Br-3, MCF-7) and EpCAM-negative (MDA-MB-231) tumor cells .
Fig.1: The selected antibodies detect both EpCAM-positive and EpCAM-negative breast cancer cell lines
Fig. 2: The selected antibodies in combination detect synergistically more CTCs than used as single capture reagent


2) The two antibodies capture tumor cells in synergistic action

The performance of the neCTC-BC-Kit with blood samples from breast cancer patients is important. For this purpose the kit was tested  with 7.5 ml of peripheral blood of three breast cancer patients. Both antibodies can capture CTCs but the cell yield is significantly greater if both antibodies are used simultaneously. The combination of antibodies has a synergistic effect.



3) The enriched CTCs show a wide spectrum in terms of EpCAM expression

The neCTC-BC-Kit was tested to detect both EpCAM-positive and EpCAM-negative CTCs. Figure 3 depicts that CTCs detected with neCTC-BC-Kit express EpCAM in the full range from “negative” to “high”.
Fig.3: The selected antibodies in combination detect CTCs with a broad continuum of EpCAM-expression
Fig. 4: The selected antibodies in combination detect CTCs, verified by low pass sequening



4) Using low pass sequencing it can be demonstrated that the enriched cells are tumor cells.

Individual CTCs were isolated with the ALS CellCelector and amplified. Genomic DNA was sequenced with low resolution (low pass).

Figure 4 shows the aberrations of the genomic DNA providing the evidence that these cells are derived from breast cancer.

Summary


The neCTC-BC-Kit is able to synergistically detect and capture CTCs in blood of breast cancer patients with a wide range of EpCAM expression and enables the isolation and analysis of CTCs with different EpCAM-expression levels from the same patient’s blood sample.

The neCTC-BC-Kit can be provided utilizing various sorts of magnetic beads or ferrofluids enabling its use for applications using hand held magnets or in automated CTC isolation systems. The antibodies will be provided as separate mix in order to enable direct and indirect cell capture . Optionally control samples with exact numbers of EpCAM-positive and EpCAM-negative breast cancer cell lines can be provided in order to verify capture efficiency.

With the neCTC-BC-Kit you’ll now be able to isolate CTCs from breast cancer samples independently from their EpCAM expression level with ease and without requiring expensive equipment. Using just a hand-held magnet you’ll be able to enrich your samples to a point where they are ready for purification by micromanipulation. This can be done manually or fully automatically using our ALS CellCelector technology.


Do you have any questions? Wanna give it a try?
                 
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