Isolation and characterisation of putative colorectal cancer stem cells
Author(s)
du Potet, Elodie
Type
Thesis
Abstract
The isolation and characterisation of cancer stem cells (CSCs) remain a major
challenge. The ‘traditional’ (or ‘stochastic’) model of cancer suggests that cancer
cells progress through clonal evolution and therefore, all cancer cells must be
destroyed. The alternative model, the ‘hierarchical’ (or CSC) model proposes that a
subpopulation of cancer cells, referred to as CSCs, initiates and sustains the
continuous expansion of cancer growth. Studies have shown that CSCs are similar to
normal stem cells as they are able to self-renew and to differentiate into a non-CSC
progeny. However, their proliferation pathways are deregulated, they can form
metastasis, trigger recurrences after chemotherapy and are tumourigenic. In the CSC
model, the CSCs essentially need to be targeted and eradicated.
Although it is recognised that CSCs do exist, there is still a large gap in defining their
molecular and functional characteristics. In this study, we have explored different
approaches to isolate colorectal CSCs.
In the first approach, CSCs were isolated using a putative CSC marker, CD133.
Cancer cell suspensions were obtained from patient tumour specimens. Several
conditions were used to isolate CD133+ cells by immunoaffinity. Technical
difficulties were encountered throughout the procedure that prevented the isolation of
CSCs using the CD133 antibody. In the second approach, we determined whether
CSCs could be isolated using chemotherapeutic drugs since it has been shown that
CSCs are resistant to such treatments. Cancer cells resistant to a short-term exposure
with the chemotherapy drug oxaliplatin were isolated from two colorectal cancer cell
lines. Data from in vivo tumourigenicity, expression of putative CSC markers and
quiescence indicated that the intrinsically resistant cells did not exhibit CSC
properties when compared with the untreated population. In the third approach, CSCs
were isolated based on the aldehyde dehydrogenase 1 (ALDH1) activity. A high
ALDH1 activity has been shown to be a marker of stem cells/CSCs.
Immunohistochemistry revealed that ALDH1hi cells expressed more putative CSC
markers CD44, CD166, ABCG2 and Lgr-5 than ALDH1lo cells. However, both
populations were similarly clonogenic in vitro, they were equally invasive, as
resistant to chemotherapeutic regimens and their cell cycle status was similar.
In conclusion, the approaches taken to isolate CSCs from cancer tissue samples or
cell lines generated limited success. The data suggest that more refined techniques are
required to isolate CSCs. On the other hand, they highlight the techniques that should
be avoided in future studies. The results also question several concepts of the CSC
theory, such as the intrinsic resistance of CSCs, and therefore emphasize on the need
of gathering more evidence to validate the CSC model.
challenge. The ‘traditional’ (or ‘stochastic’) model of cancer suggests that cancer
cells progress through clonal evolution and therefore, all cancer cells must be
destroyed. The alternative model, the ‘hierarchical’ (or CSC) model proposes that a
subpopulation of cancer cells, referred to as CSCs, initiates and sustains the
continuous expansion of cancer growth. Studies have shown that CSCs are similar to
normal stem cells as they are able to self-renew and to differentiate into a non-CSC
progeny. However, their proliferation pathways are deregulated, they can form
metastasis, trigger recurrences after chemotherapy and are tumourigenic. In the CSC
model, the CSCs essentially need to be targeted and eradicated.
Although it is recognised that CSCs do exist, there is still a large gap in defining their
molecular and functional characteristics. In this study, we have explored different
approaches to isolate colorectal CSCs.
In the first approach, CSCs were isolated using a putative CSC marker, CD133.
Cancer cell suspensions were obtained from patient tumour specimens. Several
conditions were used to isolate CD133+ cells by immunoaffinity. Technical
difficulties were encountered throughout the procedure that prevented the isolation of
CSCs using the CD133 antibody. In the second approach, we determined whether
CSCs could be isolated using chemotherapeutic drugs since it has been shown that
CSCs are resistant to such treatments. Cancer cells resistant to a short-term exposure
with the chemotherapy drug oxaliplatin were isolated from two colorectal cancer cell
lines. Data from in vivo tumourigenicity, expression of putative CSC markers and
quiescence indicated that the intrinsically resistant cells did not exhibit CSC
properties when compared with the untreated population. In the third approach, CSCs
were isolated based on the aldehyde dehydrogenase 1 (ALDH1) activity. A high
ALDH1 activity has been shown to be a marker of stem cells/CSCs.
Immunohistochemistry revealed that ALDH1hi cells expressed more putative CSC
markers CD44, CD166, ABCG2 and Lgr-5 than ALDH1lo cells. However, both
populations were similarly clonogenic in vitro, they were equally invasive, as
resistant to chemotherapeutic regimens and their cell cycle status was similar.
In conclusion, the approaches taken to isolate CSCs from cancer tissue samples or
cell lines generated limited success. The data suggest that more refined techniques are
required to isolate CSCs. On the other hand, they highlight the techniques that should
be avoided in future studies. The results also question several concepts of the CSC
theory, such as the intrinsic resistance of CSCs, and therefore emphasize on the need
of gathering more evidence to validate the CSC model.
Date Issued
2010
Date Awarded
2010-10
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Levicar, Natasa
Creator
du Potet, Elodie
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)