Isolation, identification and characterization of cancer
stem cells from different histopathological grades of
colorectal carcinoma
Aditi Bhattacharya
1
INTRODUCTION
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•
•
According to Globocan reports (2018):
– Colorectal cancer (CRC) – fourth most
common cancer worldwide.
– Second most common cancer in
women.
– Third most common cancer in men.
In India, about 50,982 cases reported in
2015 and the projected numbers for year
2025 is 62,545.
(Asia Pacific J of Cancer Prev, 2015)
Lifestyle and dietary factors – primary
causes for increased incidence of nearly
two-thirds of all CRCs. (Gupta S et al. Colorectal Dis., 2010)
• Symptoms – fever, abdominal pain, altered bowel habits, fecal blood.
• Diagnosis – X-ray, colonoscopy, MRI.
• Treatment – Surgery ± adjuvant chemotherapy/radiotherapy.
(cancernetwork.com)
STAGES & GRADES OF COLORECTAL CANCER
TNM Staging
Histopathological grading
Well differentiated
Poorly differentiated
Staging : Progression or spread
Grading: Cell differentiation
Moderately
differentiated
Normal colon
(Kuepper et al., 2016)
3
Tumor Heterogeneity – Origin of Cancer Stem Cell
Tumor growth
Tumor growth
No tumor
(www.eurostemcell.org)
Cancer stem cell properties
Cancer stem cells share a number of characteristics with normal stem cells.
NORMAL
Stem Cell
(kirshner.bio.purdue.edu)
Cancer stem cell niche
(Anderson E. et al. 2011)
6
Colorectal cancer stem cell markers
MARKER
FUNCTION
ROLE IN TUMORIGENESIS
REFERENCES
CD 44
(H-CAM)
Cell adhesion
Hyaluronic acid receptor
Colony formation, xenograft growth,
Prognostic factor,
Correlation with stage
Barker et al. 2009,
Vaiopoulos et al. 2012
CD 166 (ALCAM)
Cell adhesion
Prognostic factor
Lugli et al.2010
CD 29 (β1- Integrin) Cell adhesion
Colony formation
Vermeulen et al.
2008
Lgr5
Cell signalling
Wnt target gene, crypt base
restriction
Barker et al. 2007
CD 24 (HSA)
Cell adhesion
Clonogenic ability, correlation with
invasiveness & survival.
Vermeulen et al. 2008
Choi et al. 2009
CD 133 (Prominin1)
Cell motility
Prognostic factor
Lugli et al.2010
Elsaba et al. 2010
CD 326 (EpCAM)
Cell adhesion
Tumor budding & metastasis
Gosens et al. 2007
ALDH1
Detoxyfying
enzyme
Tumor initiation in xenografts,
resistance to alkylating agents
Vaiopoulos et al. 2012
Targeting of cancer stem cells
(Todaro et al., 2010)
8
AIM
To isolate, identify, culture and characterise
cancer stem cells from different
histopathological grades of primary untreated
colorectal cancers.
9
OBJECTIVES
1. To isolate, identify and culture CSC from different
histopathological grades of primary untreated fresh
colorectal cancer tissue.
2. To examine genome wide transcriptomic changes in
colorectal cancer stem cells.
3. To validate the putative differential display of genes that help
in characterizing different CSC populations.
10
PATIENT SAMPLE CRITERIA
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•
•
•
•
•
•
•
•
•
PATIENT INCLUSION CRITERIA:
Primary, untreated colorectal cancer cases.
Sporadic cases with confirmed histopathological diagnosis.
Surgically resectable tumors.
No other associated serious medical or surgical conditions.
Patients who are willing to participate with informed consent & Institute Ethics Committee
approval.
PATIENT EXCLUSION CRITERIA:
Patients who have received any form of treatment earlier for colorectal cancer e.g.
neoadjuvant chemotherapy or radiotherapy.
Presence of secondaries in colon or rectum.
Patients with family history of colorectal cancers or any hereditary or genetic associations
Patients with any other serious medical or surgical condition.
Patients who are not willing to give consent for participation in this study.
11
WORK PLAN
FRESH NORMAL COLORECTAL TISSUE
[from adjoining area]
FRESH COLORECTAL CANCER TISSUE
[n=6 for each histopathological grade]
SINGLE CELL SUSPENSION using Collagenase IV
FACS
CANCER STEM
CELLS
TUMOR SPHERE
ASSAY
BULK TUMOR
CELLS
NORMAL COLON
STEM CELLS
NORMAL COLON
CELLS
RNA ISOLATION
MICROARRAY
REAL TIME PCR
IMMUNOHISTOCHEMISTRY
12
RESULTS
Objective 1
To isolate, identify and culture CSC from different
histopathological grades of primary untreated fresh
colorectal cancer tissue.
13
Clinico-pathological characteristics of patients
Age (in years)
≥60
33%
Sex
≤30
9%
a.
Mucinous
6%
PD
6%
d.
Others
12%
b.
Not known
17%
T1
1%
M-Stage
Not known
17%
N2
14%
T3
54%
Left colon
16%
N-Stage
T2
18%
T4
10%
e.
Right colon
54%
c.
T-Stage
WD
13%
MD
59%
Transverse
colon
9%
Male
61%
Tumor Grade
SRC
4%
Rectum
9%
Female
39%
31-59
58%
Tumor location
Both colon &
rectum
12%
f.
Not known
17%
M0
31%
N0
51%
N1
18%
g.
Mx
44%
M1
8%
1A. SINGLE CELL SUSPENSION
Tumor fragment minced into multiple fragments
200 U Collagenase IV per ml of HBSS
Incubation for 30-60 min at 37oC
Tissue Cell suspension in HBSS
Pass through 70 µm filters
Pass through 40 µm filters
Single cell suspension
Cell Counting and staining
15
1B. FLOW CYTOMETRY AND CELL
SORTING DATA
Colorectal cancer : CD44-PE & CD166-Alexa Fluor647
Normal colon
: CD29-PE & Lgr5-Alexa Fluor647
16
Representative flow cytometry analysis of moderately differentiated (MD) CRC and
its adjacent normal tissue.
1.3
74.2
21.0
CD44 - PE
3.5
CD166 – AF647
Colon cancer stem cells (DP) – 1.3 %; Bulk tumor cells (DN) – 74.2%
6.6
45.3
0.3
CD29 - PE
47.8
Lgr5 – AF647
Normal colon stem cells (DP) – 6.6%; Bulk normal colon cells (DN) – 45.3 %
Representative flow cytometry analysis of well differentiated (WD) CRC and its
adjacent normal tissue.
0.7
82.7
4.0
CD44 - PE
12.6
CD166 – AF647
Colon cancer stem cells (DP) – 0.7 %; Bulk tumor cells (DN) – 82.7%
1.3
52.7
0.1
CD29 - PE
46.0
Lgr5 – AF647
Normal colon stem cells (DP) – 1.3 %; Bulk normal colon cells (DN) – 52.7 %
Representative flow cytometry analysis of poorly differentiated (PD) CRC and its
adjacent normal tissue.
4.9
55.2
1.6
CD44 - PE
38.3
CD166 – AF647
Colon cancer stem cells (DP) – 4.9 %; Bulk Tumor cells (DN) – 55.2 %
1.2
24.2
0.1
CD29 - PE
74.6
Lgr5 – AF647
Normal colon stem cells (DP) – 1.2 %; Bulk normal colon cells (DN) – 24.2 %
n=3
*** -- P-value < 0.0001
Dual positive cells isolated from CRC (MD) formed larger and more number of
spheres as compared to the dual positive cells isolated from adjacent normal
colon tissue.
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Objective 2
To examine genome wide transcriptomic
changes in colorectal cancer stem cells.
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QC of RNA – Integrity of RNA
Samples
loaded
Gel
loaded
into chip
Ladder
Representative Electropherogram obtained
by a Bioanalyzer
RIN score calculated by Agilent 2100 Expert software
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2A. RIN SCORE REPORT
Well differentiated Tumor
P49 – Tumor DP
P57 – Tumor DN
* RIN score threshold for microarray is ≥ 7.0
P49 – Tumor DN
P58 – Tumor DN
Moderately differentiated Tumor
P45 – Tumor DP
P28 – Tumor DP
P53 – Tumor DP
P45 – Tumor DN
P28 – Tumor DN
P53 – Tumor DN
Adjacent normal tissue
P25 – Normal DP
P34 – Normal DP
P37 – Normal DP
P30 – Normal DN
P34 – Normal DN
P37 – Normal DN
2B. MICROARRAY
Isolation of total RNA from tissues/cell samples
Preparation of cDNA
Transcription, Incorporation of fluorescent label (Cy3) and Amplification of cRNA
Fragmentation and charging on the slide (4x44k)
Overnight Hybridization
Washing of Slide
Scanning of slides
Feature Extraction
Import of data into Analysis Software in txt Format
Data Analysis and Derivation of Biological Meaning
Principle Component Analysis based on differentially expressed genes
between CSCs and NSCs (MD-DP vs N-DP)
GeneSpring 14.9.1
*n= 3
NSC
CSC
Volcano Plot of Differential Expression Analysis
Cut off parameters:
• Fold change > 2
• p-value < 0.05
Cancer Stem Cells (CSCs) versus Normal Stem Cells (NSCs)
Hierarchical Clustering Analysis based on differential
expression of genes
NSC
CSC
Differentially regulated signaling pathways
S. No.
Pathways
Targets
1.
NFkB pathway
5
2.
Chemokine-mediated signaling
pathway
7
3.
BMP pathway
5
4.
Non-canonical Wnt pathway
3
5.
Tyrosine phosphorylation
4
6.
Positive regulation of PI3K
5
7.
Canonical Wnt pathway
8
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Differential Expression Analysis after Gene Ontology
NSC
CSC
Network Analysis of target genes using GeneGO MetaCore
Network analysis uses a complex algorithm which computes the putative interactions
between different molecules on the basis of statistical probability.
Network Analysis of target genes using GO MetaCore
Network analysis confirms that our target genes conform to the signaling pathway.
Pathway analysis of differentially regulated genes using GeneGO MetaCore
Target genes interact
with each other and
directly or indirectly
contribute
towards
colorectal
cancer
pathogenesis.
Objective 3
To validate the putative differential display of genes
that help in characterizing different CSC populations.
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Wnt canonical and non-canonical signaling pathway
Wnt canonical
Wnt non-canonical
(Chae et al., 2018)
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KEGG database
Canonical Pathway
Planar cell polarity
pathway (PCP)
Non canonical Pathway
3A. Real-Time PCR
Isolation of total RNA from sorted cells from patients
Quantify amount of RNA isolated
Prepare cDNA using random hexamers
Run real time PCR by loading 10 ng cDNA/well
Calculate relative gene expression (RGE) by ΔCt method
Validation of gene expression by real-time PCR
n=15/13
p-value=0.1285
n=15/13
p-value=0.0099
Wnt2
Frizzled10
Β-Catenin
LEF1
Wisp1
n=15/10
p-value=0.0073
n=15/9
*Gene expression normalized to 18s rRNA expression
*Mann-Whitney U-test
p-value=0.0736
(** -- p-value<0.01)
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Validation of gene expression by real-time PCR
p-value=0.1796
n=15/12
n=15/13
p-value=0.3109
Wnt5a
Frizzled10
NfatC3
n=15/9
p-value=0.6764
Gene expression of Wnt canonical pathway associated genes (Wnt2, β-catenin, LEF1, Wisp1) and
non-canonical pathway associated genes (Wnt5a, NfatC3, Fzd10) were found to be higher in CSC as
compared to NSC but only LEF1 and β-catenin showed statistically significant increase.
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Wnt non-canonical signaling pathway
•
Wnt5A (Wnt family member 5A)
– Regulates osteogenesis in MSCs
– Promotes EMT via β-catenin pathway in NSCLC,
bladder cancer, breast cancer, prostate cancer,
colorectal etc.
– Upregulates migration and invasion in
osteosarcoma via SRC/ERK/MMP-14 pathway
•
NfatC3 (Nuclear Factor Of Activated T Cells 3)
– Heterodimeric transcription factor
– Critical for human postnatal myogenesis, acts
via Calcineurin pathway
– Upregulated under hypoxic conditions via Orai1Notch pathway in breast cancer.
– Involved in SFRP2-induced angiogenesis in
endothelial cells.
– Associated with high β-catenin expression in
human acute infarction tissues, mediates
hypertrophy via MAPK pathway
(Chae et al., 2018)
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3B. Immunohistochemistry
Deparaffinization of tissue sections
Antigen retrieval for 20 mins in citrate buffer
Blocking in Peroxide Block
Superblock (kit) incubation for 10 mins
Primary antibody incubation overnight at 4⁰C
Secondary antibody incubation for 1 hour at RT
DAB staining and hematoxylin counterstain
Coverslip mounting with DPX
Analysis: % positivity and H score
Wnt5a (1:1000)
NfatC3 (1:100)
CD44 (1:600)
Lgr5 (1:400)
Immunohistochemical analysis of Wnt5a expression in CRC (MD) and normal
colon tissue sections
TUMOR
a.
b.
NORMAL
COLON
n=31
c.
d
.
Intra-tumoral heterogeneity in Wnt5a expression in CRC (MD) tissue section
TUMOR
a.
b.
c.
d.
TUMOR
Immunohistochemical comparison of Wnt5a expression in Epithelium of CRC (MD)
and normal colon tissue sections
**
WNt5a_Epi_H score
WNt5a_Epi_%positivity
**
Tumor
Normal
Tumor
Normal
n=31
(** -- p-value<0.01)
Wnt5a expression was significantly higher in the epithelial region of CRC (MD) tissue
as compared to normal colon tissue.
Immunohistochemical comparison of Wnt5a expression in intra-tumoral/stromal
region of CRC (MD) and normal colon tissue sections
ns
WNt5a_IT_H score
WNt5a_IT_%positivity
ns
Tumor
Normal
Tumor
Normal
n=31
There was no statistically significant difference in Wnt5a expression in the intratumoral region of CRC (MD) tissue as compared to normal colon tissue.
ROC curves to understand the sensitivity and specificity of Wnt5a expression in
CRC (MD) and normal colon tissue sections
•Positivity: AUC 0.613, p-value 0.127, For a
positivity of 82.5% or more, sensitivity = 61.3%,
specificity = 51.6%
•H Score: AUC 0.609, p-value 0.139, For H score of
9.5 or more, sensitivity = 80.6%, specificity =
41.9%
•Positivity: AUC 0.631, p-value 0.071, For a positivity
of 82.5% or more, sensitivity = 64.5%, specificity =
48.4%
•H Score: AUC 0.535, p-value 0.074, For H score of
9.5 or more, sensitivity = 32.3%, specificity = 74.2%
47
Immunohistochemical analysis of NfatC3 (nuclear) expression in CRC (MD) and normal
colon tissue sections
TUMOR
a.
b.
NORMAL
COLON
n=31
c.
d.
Immunohistochemical comparison of NfatC3 (nuclear) expression in epithelium of
CRC (MD) and normal colon tissue sections
***
a.
NfatC3_Epi_H score
NfatC3_Epi_%positivity
**
Tumor
Normal
b.
Tumor
Normal
n=31
(** -- p-value<0.01)
(*** -- p-value<0.001)
NfatC3 expression was significantly higher in the epithelial region of normal colon as
compared to CRC (MD).
Immunohistochemical comparison of NfatC3 (nuclear) expression in intratumoral/stromal region of CRC (MD) and normal colon tissue sections
nsns
NfatC3_IT_H score
NfatC3_IT_%positivity
nsns
Tumor
Normal
Tumor
Normal
n=31
There was no statistically significant difference in NfatC3 (nuclear) expression in the
intra-tumoral region of CRC (MD) tissue as compared to normal colon tissue.
ROC curves to understand the sensitivity and specificity of NfatC3 expression in
CRC (MD) and normal colon tissue sections
•Positivity: AUC 0.728, p-value 0.009, For a positivity
of 55% or more, sensitivity = 65.2%, specificity =
77.3%
•H Score: AUC 0.769, p-value 0.002, For H score of 7 or
more, sensitivity = 65.2%, specificity = 77.3%
•Positivity: AUC 0.653, p-value 0.078, For a positivity
of 72.5% or more, sensitivity = 65.2%, specificity =
68.2%
•H Score: AUC 0.631, p-value 0.131, For H score of 9.5
or more, sensitivity = 65.2%, specificity = 68.2%
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Immunohistochemical analysis of Lgr5 protein expression in normal colon tissue sections
NORMAL
COLON
a.
b.
NORMAL
COLON
n=31
c.
d.
52
Immunohistochemical analysis of CD44 protein expression in CRC (MD) tissue sections
TUMOR
a.
b.
TUMOR
n=31
c.
d.
Correlation between expression of Wnt5a with NfatC3, CD44 and Lgr5
Correlation coefficient, r= 0.043
p-value=0.37
Correlation coefficient, r= -0.092
p-value=0.311
Correlation coefficient, r= 0.077
p-value=0.341
• There was no correlation between Wnt5a and NfatC3 expression in tumor
and normal colon sections.
• No correlation was found between Wnt5a and Lgr5 expression in normal
colon and Wnt5a and CD44 expression in CRC (MD) sections.
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Correlation between expression of NfatC3 with Lgr5 and CD44
Correlation coefficient, r= -0.109
p-value=0.28
Correlation coefficient, r= 0.001
p-value=0.497
No correlation was found between NfatC3 and Lgr5 expression in normal
colon and NfatC3 and CD44 expression in CRC (MD) section.
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Summary
➢ The proportion of CSCs present in CRC is variable and differs from patient to
patient, even with those having the same histopathological stage and grade.
➢ The percentage of NSCs in normal colon tissue is less variable and varies between
1.0 to 2.5%.
➢ CSCs exhibit a higher proliferation rate as compared to NSCs.
➢ NSCs are potentially quiescent in nature and a much less proportion of cells show
sphere-forming ability as compared to CSCs.
➢ Protein localization by IHC of Wnt5a showed positive expression across both
epithelium and intra-tumor/mucosal stroma. However, the intensity of Wnt5a
expression in tumor was significantly higher than in normal colon sections.
➢ IHC of NfatC3 revealed significant decrease in its nuclear localization and
expression, in the epithelium as well as intra-tumor stroma, as compared to
normal colon.
➢ Differential gene expression of CSCs derived from moderately differentiated CRCs
and their adjacent NSCs reveal regulation of some interesting signaling pathways
associated with tumorigenesis like PI3K signaling and BMP signaling pathway.
➢ No correlation could be established between the Wnt5a and NfatC3 expression as
well as with established CSC (CD44) and NSC markers (Lgr5).
56
Conclusion
NfatC3 acts a potential negative regulator of
colorectal cancer. Further study of the gene
function will shed light on its role in cancer
and cancer stem cells biology.
57