Identification of Cancer Stem Cells using
Flow Cytometry Analysis
‫تعيين الخاليا الجذعية السرطانية باستخدام تحليل التدفق الخلوي‬
Dr. Ayat Al-Ghafari
Biochemistry Department
Monday 25-5-1436
Outlines
1. Cancer
2. Heterogeneity of cancer
3. Cancer stem cells (CSCs) hypothesis
4. Flow cytometry
• Overview
• Instrument major parts
• How does flow cytometer work
Cancer
• Cancer or malignant neoplasm
disease is a group of diseases
which is characterized by:
1. The uncontrolled growth of cells
(proliferation)
2. Uncontrolled movement (migration
and metastasis)
3. Ability to invade neighboring
tissues (invasion)
4. Resistance to death (necrosis and
apoptosis)
Cancer
• These neoplasms result from the transformation of a
normal cell to a malignant cell through a multistep process
by the alteration of genes and pathways .
www.studyblue.com
Heterogeneity of Cancer
• Tumors are heterogeneous because they often comprise
cytogenetically different clones (Hanahan and Weinberg,
2000).
www.nature.com
Heterogeneity of Cancer
• This heterogeneity results in differences in clinical tumor
behavior and responses to treatment .
• The first evidence of the heterogeneity of tumors was
demonstrated in 1961 by Southam and Brunschwig when
they harvested cancer cells from recurrent tumors and autotransplanted the cells at different sites (Southam and
Brunschwig, 1961).
• This suggested hypothesis that a subset of these cells, not all
of them, had the ability to initiate the tumor.
Heterogeneity of Cancer
• By the early 1990s, it was clear from analysis of cell
surface expressed proteins (CD proteins) that tumor
cells were heterogeneous in terms of their protein
expression.
• Lapidot et al., found that CD34+CD38- cells from
patients with AML were able to initiate tumor when
they injected to NOD-SCID mice while CD34+CD38+
did not initiate the tumor (Lapidot et al., 1994).
Heterogeneity of Cancer
• These findings confirmed that not all the cells in the
tumor are responsible for its progression and lead
to the establishment of a new theory called the
cancer stem cells (CSCs) hypothesis.
• CSCs hypothesis proposes that growth and
progression of many cancers are driven by small
subpopulations of CSCs rather than all the cells in
the tumor (Reya et al., 2001).
Normal stem cells
Rare cells within organs with the ability to selfrenew and differentiate to all types of cells within
the organ to drive organogenesis
Cancer stem cells
Subset of cells within tumors with the ability to
self-renew and differentiate to the phenotypically
diverse drug resistant tumor cell population to
drive tumorigenesis
Cancer Stem Cells (CSCs) Hypothesis
• Several methods are generally used to
characterize CSCs:
1. The isolation of a side population or sub-population (SP)
based on cell surface marker expression by flow cytometry
2. Identification of a small population of cells by their ability to
differentiate
3. Identification of a small population of cells by their ability to
form spheres (colonies)
Flow Cytometry
It is a powerful technique to identify multiple
parameters of a single cell within a heterogeneous
population
Flow cytometry Ploidy analysis
Flow Cytometry Cell Counting
Flow cytometry GFP
expression analysis
Flow Cytometry Overview
• It performs its analysis by passing thousands of cells
/seconds through a laser beam and capturing the light that
emerges from each cell as they passing through.
• Then the data gathered can be analyzed statistically by flow
cytometry software that report cellular characteristics such
as size, complexity, phenotype, and health.
• The flow cytometer contains several parts as shown by the
following image.
Flow Cytometry Instrument Overview
Flow Cytometry Instrument Overview
1. Fluidics system
Which presents the
sample to the
interrogation point and
takes away the waste.
2. Lasers
Which are the light
source for scatter and
fluorescence.
Flow Cytometry Instrument Overview
3. Optical system
Which gather and direct
the light.
4. Detectors
Which received the light .
Flow Cytometry Instrument Overview
5. The external
computer system
Which converts the
signals from the detector
into digital data and
perform the necessary
analyses.
Flow Cytometry Steps
(1) Interrogation point
Flow Cytometry Steps
(2) Hydrodynamic focusing
Flow Cytometry Steps
(3) Forward Scatter
Flow Cytometry Steps
(4) Forward Scatter Detector
Flow Cytometry Steps
(5) Side Scatter Detector
Flow Cytometry Steps
(5) Side Scatter Detector
2-dimensional
dot scatter plot
Flow Cytometry Steps
(5) Side Scatter Detector
There is a correlation
between the data obtained
from both the forward and
the side scatter
Flow Cytometry Steps
(5) Side Scatter Detector
Flow Cytometry Steps
(6) Florescence Detector
It gives more
information about the
structure and function
Example
They wanted to evaluate the effect of two drugs MBO-SC
and MBO-asGCS on the expression of CD44+ in breast
CSCs
References
1. HANAHAN, D. & WEINBERG, R. 2000. The hallmarks of cancer. Cell,
100, 57-70.
2. SOUTHAM, C. & BRUNSCHWIG, A. 1961. Quantitative studies of
autotransplantation of human cancer. Cancer, 14, 971–978.
3. LAPIDOT, T., SIRARD, C., VORMOOR, J., MURDOCH, B., HOANG,
T., CACERES-CORTES, J., MINDEN, M., PATERSON, B., CALIGIURI,
M. A. & DICK, J. E. 1994. A cell initiating human acute myeloid
leukaemia after transplantation into SCID mice. Nature, 367, 645–648.
4. REYA, T., MORRISON, S. J., CLARKE, M. F. & WEISSMAN, I. L.
2001. Stem cells, cancer, and cancer stem cells. Nature, 414, 105-111.
5. https://www.lifetechnologies.com/sa/en/home/brands/invitrogen.html