Standardization of cancer stem cell enumeration and isolation
methods allows cross-comparison among different tumor entities
David Agorku1, Sandy Reiss1, Melanie Jungblut1, Jürgen Dittmer2, Georg Daeschlein3, Andreas Bosio1, and Olaf Hardt1
1
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany | 2 Clinic of Gynecology, University of Halle, Halle (Saale), Germany | 3 Clinic of Dermatology, University of Greifswald, Greifswald, Germany
A
Automated tissue dissociation
Single-cell suspensions from tumor tissue are prepared by
using the gentleMACS Dissociator and C Tubes.
Magnetic labeling of CD24+ and
CD45+ cells
Cells to be depleted from the suspension are labeled
with MACS MicroBeads, i.e., antibodies coupled to superparamagnetic particles, in a short 15-min incubation step.
Quantification of melanoma-specific CSCs and TILs
A
Results
A
B
CD235a-PE
Positive selection of CD44+ cells
Magnetically labeled CD44+ cells are retained within the
column. The flow-through contains the non-labeled CD44–
cells.
B
PI
– RBC
PI
CD45-FITC
CD271 (LNGFR)-APC
1
Semi-automated dissociation of human tumor tissue
Labeling of CD44+ cells
CD44+ cells within the flow-through fraction are labeled
with MicroBeads.
Elution of CD44+ cells
The separation column is removed from the magnetic
field and the magnetically labeled CSCs are flushed out.
Removal of MicroBeads is not required. The cells are
immediately ready for further experiments.
CD45-FITC
CD235a-PE
D
Target cell
labeled with
MACS MicroBead
Figure 2
showing dissociated melanoma tissue, RBC amounted
to 16%. The presence of RBC hampers the accurate
determination of cell viability as well as absolute cell
counts for TILs, CSCs, and other tumor-resident cells.
Common methods of eliminating RBC from cell
suspensions include RBC lysis and density gradient
Melanoma-specific TILs are CD45-positive, whereas CSCs
in melanoma express CD271 (LNGFR)⁴. Eliminating RBC
as well as dead cells from flow cytometric analysis led
to identification of 6% TILs and 94% tumor cells in the
shown example of dissociated melanoma tissue (fig. 2A).
Without exclusion, RBC would affect the accurate
CSC-enriched fraction
Original fraction
FSC
FSC
Figure 4
Glioblastoma as well as medulloblastoma tumors were
shown to contain CSCs as identified by CD133 expression⁶,⁷.
In particular, it was demonstrated that the AC133 epitope,
but not the entire CD133 protein expression is lost upon
CSC differentiation². Using an AC133-specific antibody
coupled to superparamagnetic MicroBeads, CD133+
glioblastoma CSCs were enriched from 0.5% in the
original fraction to a purity of 91% (fig. 4).
determination of the TIL fraction within the total tumor
cell population as RBC increase the number of CD45– cells.
Using an equivalent gating strategy for another melanoma
sample, an accurate quantif ication of CD271
(LNGFR)+MSCA-1–CD45– CSCs and CD271 (LNGFR)+MSCA1+CD45– tumor-infiltrating MSCs was performed (fig. 2B).
Figure 3
CD44-APC
CSC-enriched fraction
CD24-FITC
CD24-FITC
Original fraction
CSC-depleted fraction
CSC-enriched fraction
CD45-PE
CD45-PE
We have developed a standardized platform for CSC
enumeration and isolation. This platform is based on
automated, user-independent procedures for the
dissociation of human and implanted mouse tumor tissue.
Dissociation yields high amounts of single cells at viability
rates of around 90%. The integrity of cell surface epitopes
is preserved. Furthermore, we generated conjugates for
antibodies recognizing epitopes that are relevant for CSC
analysis. The conjugates were tested on cell lines and
primary tumor tissue for the quantification and isolation
of CSCs. As a proof of principle, populations of CD133+
and CD45–CD24 –CD44+ CSCs were efficiently isolated
from glioblastoma at 91% purity and from an invasive
ductal mamma carcinoma at 94% purity. In addition, we
present optimized gating and exclusion strategies that
avoid analytical bias in the flow cytometric quantification
of CSCs from primary tissue, e.g., human melanoma tissue.
MACS Column
CD44-APC
C
CSC-depleted fraction
CD44-APC
CD44-APC
Anti-MSCA-1-PE
MACS Separator
CD24-FITC
CD44-APC
The recently developed Tumor Dissociation Kit in
combination with the gentleMACS™ Dissociator³ allowed
for the semi-automated preparation of single-cell
suspensions from human tumor tissue. The resulting cell
samples contained significant amounts of red blood cells
(RBC) in most of the cases. In this experiment (fig. 1A),
Original fraction
CD44-APC
Anti-MSCA-1-PE
Figure 1
B
CD45-FITC
CD271 (LNGFR)-APC
C
B
Conclusion
approx. 16% RBC
CD235a-PE
Isolation of glioblastoma CSCs
A
Depletion of CD24+ and CD45+ cells
Labeled and non-labeled cells are separated in a MACS
Column placed in the magnetic field of a MACS Separator,
a strong permanent magnet. The flow-through is collected
as the non-labeled CD24–CD45– fraction and is used for
further cell isolation.
– RBC
– PI+ cells
4
was dissociated using a standardized, semiautomated
procedure based on the gentleMACS Dissociator. Breast
CSCs were isolated by depletion of CD24+ and CD45+
cells and subsequent enrichment of CD44+ cells. All
populations were checked for expression of CD24 and
CD44 (fig. 3B) as well as CD45 and CD44 (fig. 3C). Using
thismethod, CSCs were enriched from 5% in the original
fraction to a purity of 94%.
CD133
2
3
Isolation of breast cancer stem cells
Human mammary carcinoma stem cells are defined
by the expression of CD44 and the absence of CD24⁵.
Analysis of further surface markers showed that CD45
has to be used as exclusion marker since the majority
of CD44+ cells are CD45+CD44+ TILs. Based on these
results, a magnetic cell sorting strategy was established,
allowing for the isolation of CD45–CD24–CD44+ breast
CSCs (fig. 3A). Human mammary carcinoma tissue
CD133
which cleave off several CSC surface markers, including
ABCB5 and CD44, may cause incorrect results with regard
to the differential expression of protease-sensitive
markers¹. A further factor causing inconsistencies among
results is the use of antibodies that are specific to certain
subforms of cell surface markers. Glycosylation- and
splice-isoform–dependent epitopes, as found for, e.g.,
CD24 and CD133, are differentially expressed among
various tissues and cell states. In particular, it was shown
that the AC133 epitope is lost upon CSC differentiation,
although CD133 expression persists².
To circumvent these pitfalls, we developed a standardized platform that allows fast and highly reproducible
CSC enumeration and isolation from various tumor
tissues.
PI
As solid tumors are inherently heterogeneous entities, it
is of great importance to analyze the various tumorresident cell and stem cell subpopulations. In particular,
cancer stem cells (CSCs), also called tumor-initiating cells,
have gained substantial interest over the past few years.
CSCs have been isolated from multiple tumor entities
and were shown to play a crucial role during tumor
growth and metastasis.
However, isolation and analysis of CSCs, even from single
tumor entities such as melanoma, showed contradictory
results among different research groups. This can partially
be explained by variations in the dissociation and isolation
methods used. When analyzing putative CSC markers,
one has to keep in mind that the procedure used for
dissociation of primary tissue prior to staining may cause
a technical bias. Aggressive proteases, such as trypsin,
these parameters were determined after excluding RBC
by gating off CD235a+ cells. In the shown example (fig. 1B)
viability amounted to 86% at a yield of 5.8×10⁷ cells per
gram of tissue.
The optimized tumor dissociation procedure preserves
cell surface epitopes and yields single cells at high
viabilities around 90%, which allowed us to culture the
tumor cells (fig. 1C: day 2, fig. 1D: day 10 in vitro).
PI
Introduction
centrifugation, which, however, are very time-consuming
and prone to cell loss. To avoid these drawbacks, the
resulting single-cell suspensions were labeled with a
CD235a (glycophorin A) antibody prior to flow cytometric
analysis. This method allowed for the fast and simple
discrimination and exclusion of RBC for subsequent
analysis (fig. 1A). Without exclusion, RBC would appear
in the PI– fraction, and both overall cell viability and
absolute cell count would be overestimated. Therefore,
Permanent Abstract Number: 5340
CD45-PE
Outlook
By using reliable methods for the analysis and isolation
of CSCs from various sources, a major cause of inconsistent
results in this field can be avoided. Thus, the comparison
of CSC populations among distinct tumor entities will
be possible.
References
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