Gewählte Publikation:
Hohensee, T.
Isolation of circulating tumor cells from patients with metastatic colorectal cancer.
[ Diplomarbeit/Master Thesis ] Karl-Franzens University of Graz; 2013. pp.97.
- Autor*innen der Med Uni Graz:
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Hohensee Tabea
- Betreuer*innen:
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Haybäck Johannes
-
Kroneis Thomas
- Altmetrics:
- Abstract:
- CRC is one of the most common cancers worldwide. One fourth of them are
diagnosed in an advanced stage, with a shortened survival rate of the effected
patients, even after removal of the primary tumor. Based on these facts a better
understanding of the formation of metastasis is needed. One approach is the
isolation and characterization of circulating tumor cells (CTCs). These cells leave the
primary tumor, circulate through the blood- or lymph system and can then settle in
another part of the body, forming metastasis.
With respect to the OncoTrack project peripheral blood samples of patient suffering
from CRC were collected and further processed. This project focuses on finding a
more accurate in-silico model for colorectal cancer treatment.
There are several techniques available to isolate CTCs focusing on different
characteristics, like size, surface antigens or buoyant density of a cell. In this master
thesis a method based on negative enrichment with immunomagnetic nanobeads
was conducted and improved. While preforming the already established protocol, the
remaining leukocytes were not detectable after the antibody staining. So, different
antibody combinations were looked at until a better setting was established, followed
by whole transcriptome amplification of the isolated single cells or cell pools.
Afterwards, these cells were analyzed on a molecular level with TaqMan© probes,
using multiplex real-time PCR, which I established in this working group.
There, several different tumor markers were tested, containing EpCAM, CK19, CK20,
EGFR, PBGD and CEA, and also eIf3a and eIf4e bp2. CD45 was used as a control
for leukocyte contamination. During establishment of this method two CRC lines,
SW620 and HCT-116 cells were used.
Out of 65 amplified patient samples, two could be proven to express keratin 19,
indicating that theses isolated cells are likely of CRC origin.