2. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
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3. Sample & Assay Technologies
QIAGEN webinars
Access recorded webinars and slides
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4. Sample & Assay Technologies
QIAGEN webinars
Access recorded webinars and slides
Sign up for upcoming webinars
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5. Sample & Assay Technologies
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6. Sample & Assay Technologies
Biology-Focused Solutions for Pathway Analysis
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Company
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7. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
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8. Sample & Assay Technologies
Introduction: Inflammation
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
̣
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
Epigenetic
Changes
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
Title, Location, Date
8
9. Sample & Assay Technologies
‘TLR in Inflammation’ webinar
http://sabiosciences.com/home.php
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10. Sample & Assay Technologies
‘TLR in Inflammation’ webinar
Access previous webinars and slides
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11. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
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11
12. Sample & Assay Technologies
Cancer
“Cancer is the result of uncontrolled growth of a given cell type that occurs together
with the invasion of surrounding tissue and the spread of malignant cells.”
Karin & Greten (2005) Nature Reviews Immunology (5): 749
Three Mechanistic phases
Initiation
Genomic alterations
(DNA Damage)
(DNA mutations)
Promotion
Proliferation & Growth
Progression
Invasion & Metastasis
Inflammation
Title, Location, Date
12
13. Sample & Assay Technologies
Link between Cancer & Inflammation
Bacteria, viruses, chemicals, chronic inflammation
Malignancy
Inflammatory Stimulus
Bladder cancer
Schistosomiasis
Gastric lymphoma
H. pylori-induced gastritis
MALT lymphoma
H. pylori
Hepatocellular carcinoma
HBV, HCV
Kaposi’s sarcoma
HHV8
Bronchial carcinoma
Silica, Asbestos
Mesothelioma
Asbestos
Ovarian cancer
Endometriosis
Colorectal cancer
IBD
Esophageal cancer
Barrett’s metaplasia
Papillary thyroid carcinoma
Thyroiditis
Prostate cancer
Prostatitis
Title, Location, Date
13
14. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
Title, Location, Date
14
15. Sample & Assay Technologies
Inflammation in cancer initiation
Injury
Inflammation
Cytokines
ROS, RNI
(Extracellular)
ROS, RNI
(Intracellular)
Base excision repair
DNA damage
DNA mutations
Tumor Initiation
(DNA damage)
Epigenetic Changes
Adapted from Grivennikov et al, Cell 2010
Title, Location, Date
15
16. Sample & Assay Technologies
Inflammation in cancer initiation
Injury
Inflammation
Cytokines
ROS, RNI
(Extracellular)
ROS, RNI
(Intracellular)
Base excision repair
DNA damage
DNA mutations
Tumor Initiation
(DNA damage)
Epigenetic Changes
Adapted from Grivennikov et al, Cell 2010
Title, Location, Date
16
17. Sample & Assay Technologies
Inflammation in cancer initiation
Title, Location, Date
17
18. Sample & Assay Technologies
Inflammation in cancer initiation
Increased tumor multiplicity in mice experiencing DNA damage
after inflammation
Azoxymethane: colonic carcinogen
Aag+/+
Aag (Alkyladenine DNA glycosylase):
Base excision repair enzyme
Aag-/-
Title, Location, Date
18
19. Sample & Assay Technologies
Inflammation in cancer initiation
Accumulation of Nitrotyrosine in both strains
Nitrotyrosine has been identified as an indicator of cell damage and inflammation,
as well as an indicator of exposure to NO. Repeated cycles of DSS treatment
leads to positive nitrotyrosine staining in colons of treated animals. Similar
staining intensity is detected for both Aag+/+ and Aag-/- colons.
Title, Location, Date
19
20. Sample & Assay Technologies
Inflammation in cancer initiation
Oncogenic mutations are increased in mice lacking base
excision repair enzymes after inflammation
LCM
PCR Amplification
Sequencing
Gene: Ctnnb1 (β Catenin 1)
Title, Location, Date
20
21. Sample & Assay Technologies
Inflammation in cancer initiation
Conclusions
Inflammatory conditions
RNOS
Base excision repair
Oncogenic mutations
Tumor growth
Title, Location, Date
21
22. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
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22
23. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
qBiomarker Somatic Mutation PCR Arrays & Assays
Analyze Somatic Mutations by Real-time PCR
Profile 85 different
mutations on one array
(372 mutations on one
384 array)
Appropriate controls
for data normalization
and reaction
performance
Focus on your
questions and papers
http://sabiosciences.com/somaticmutation.php
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23
24. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
qBiomarker Somatic Mutation PCR Arrays & Assays
Focused Content
http://sabiosciences.com/somaticmutation.php
Title, Location, Date
24
25. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
qBiomarker Somatic Mutation PCR Arrays & Assays
Focused Content
Title, Location, Date
25
26. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
qBiomarker Somatic Mutation PCR Arrays & Assays
Focused Content
Title, Location, Date
26
27. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
qBiomarker Somatic Mutation PCR Arrays & Assays
Complete Workflow
Sample
Isolation &
Preparation
Assay
Set-up &
Analysis
Customer support group:
1-888-503-3187
support@SABiosciences.com
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28. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
Title, Location, Date
28
29. Sample & Assay Technologies
Inflammation in cancer promotion
Title, Location, Date
29
30. Sample & Assay Technologies
Inflammation in cancer promotion
Mdr2-KO mice develop hepatocellular carcinoma on the
background of chronic hepatitis (inflammatory condition)
Arrowheads indicate tumors
H&E staining
Title, Location, Date
30
31. Sample & Assay Technologies
Inflammation in cancer promotion
NFkB activation is involved in Mdr2-KO hepatocarcinogenesis
NFkB Nuclear staining
TNFa leads to NFkB activation (red = Kupffer cells; yellow = hepatocytes)
NFkB activation is seen predominantly adjacent to
inflamed portal tracts, NOT in all neoplastic
hepatocytes, suggesting an inflamed phenomenon.
(blue = bile epithelium; yellow = hepatocytes)
Title, Location, Date
31
32. Sample & Assay Technologies
Inflammation in cancer promotion
NSAID treatment decreased inflammation
2.5 month old mice
10-day ibuprofen treatment
MPO staining
Decreased neutrophils
Title, Location, Date
32
33. Sample & Assay Technologies
Inflammation in cancer promotion
TNFa drives NFkB activation
Treatment with anti-TNFa decreased
NFkB nuclear staining (activation)
Source of TNFa is the non-hepatocyte fraction of the liver
H: hepatocyte fraction
R: non-hepatocyte fraction
Title, Location, Date
33
34. Sample & Assay Technologies
Inflammation in cancer promotion
NFkB is critical for tumor promotion
Generate DKO mice to directly assess the role of NFkB in hepatocarcinogenesis
∆N-IκBhep
Mdr2 KO
X
NFkB inactivated in hepatocytes
(Dox-activated NFkB)
Double mutant (DM)
Mdr2-/- ∆N-IκBhep
Title, Location, Date
34
35. Sample & Assay Technologies
Inflammation in cancer promotion
NFkB is dispensable for early stages of HCC development
4 month old mice
No change in inflammation
7 month old mice
No change in dysplasia
Title, Location, Date
35
36. Sample & Assay Technologies
Inflammation in cancer promotion
NFkB is critical for tumor promotion
Inactivation of NFkB in
hepatocytes results in decreased
tumor volume
NFkB activated in hepatocytes
Title, Location, Date
36
37. Sample & Assay Technologies
Inflammation in cancer promotion
NFkB inhibition results in suppression of anti-apoptotic factors
Increased hepatocyte apoptosis in DM mice
(DM mice & KO + anti-TNF = inactivation of
NFkB in hepatocytes)
Liver protein extracts from KO mice
Inactivation of NFkB decreased the expression
of anti-apoptotic genes
Title, Location, Date
37
38. Sample & Assay Technologies
Inflammation in cancer promotion
Conclusions
Inflammatory stimulus (Mdr2 KO)
Inflammatory cytokine (TNFa)
NFkB activation
Modulation of gene expression
Tumor growth
Title, Location, Date
Increased survival
38
39. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
Title, Location, Date
39
40. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
RT2 Profiler PCR Arrays & Assays
Analyze gene expression by Real-time PCR
Profile 84 different
genes on one array
(370 genes on one 384
array)
Appropriate controls
for data normalization,
sample quality, and
reaction performance
Focus on your
questions and papers
http://sabiosciences.com/PCRArrayPlate.php
Title, Location, Date
40
41. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
RT2 Profiler PCR Arrays & Assays
Focused Content
Array
Species
NFkB Pathway
Human/Mouse/Rat
NFkB Targets
Human/Mouse/Rat
Apoptosis
Human/Mouse/Rat
TNF Pathway
Human/Mouse/Rat
Others (>140)
Varies
Array
Species
IL6/STAT3 Pathway
Human/Mouse/Rat
JAK/STAT Pathway
Human/Mouse/Rat
Inflammatory responses Human/Mouse/Rat
Title, Location, Date
41
42. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
RT2 Profiler PCR Arrays & Assays
Complete Workflow
Sample
Isolation &
Preparation
Assay
Set-up &
Analysis
Customer support group:
1-888-503-3187
support@SABiosciences.com
Title, Location, Date
42
43. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
Title, Location, Date
43
44. Sample & Assay Technologies
Inflammation in cancer progression
Title, Location, Date
44
45. Sample & Assay Technologies
Inflammation in cancer progression
TAMs and CSF-1
Tumor-associated macrophages (TAMs) populate most solid tumors.
TAMs are associated with poor prognosis.
Colony-stimulating factor (CSF-1) is a regulator of the proliferation, differentiation, and
survival of macrophages.
CSF-1 expression is prevalent in invasive tumor cells.
Title, Location, Date
45
46. Sample & Assay Technologies
Inflammation in cancer progression
Generate DM mice to directly assess the role of CSF-1 in cancer progression
CSF1op
MMTV-PyMT
X
CSF1 null recessive mutation
(CSF-1 not expressed)
Double mutant (DM)
PyMT-CSF1op
Title, Location, Date
46
47. Sample & Assay Technologies
Inflammation in cancer progression
Growth and size of primary mammary tumors are not altered in
absence of CSF-1
CSF-1
No CSF-1
Title, Location, Date
47
48. Sample & Assay Technologies
Inflammation in cancer progression
Metastasis of mammary tumors is decreased in absence of
CSF-1
CSF-1
No CSF-1
Title, Location, Date
48
49. Sample & Assay Technologies
Inflammation in cancer progression
Progression of mammary tumors is decreased in absence of
CSF-1
CSF-1
Title, Location, Date
No CSF-1
49
50. Sample & Assay Technologies
Inflammation in cancer progression
Presence of TAMs is decreased in absence of CSF-1
CSF-1
No CSF-1
+/op
op/op
7-wk mice
(F4/80 staining)
19-20-wk mice
(F4/80 staining)
Title, Location, Date
50
51. Sample & Assay Technologies
Inflammation in cancer progression
Conclusions
Development of primary tumor
Growth of primary tumor
Recruitment of TAMs to primary tumor
Metastasis of primary tumor to distant sites
Title, Location, Date
51
52. Sample & Assay Technologies
Inflammation in cancer progression
There is a correlation between TAM abundance and poor prognosis.
However, it is not only the number of TAMs but also the cytokine-expression
profile of TAMs that might link inflammation with tumor progression
Balkwell et al. Cancer Cell 7; Mar 2005; 211
Title, Location, Date
52
53. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
Title, Location, Date
53
54. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
ELISArray kits
Analyze Protein expression
Profile 12 different
analytes from 6
samples on one array
Appropriate controls
for reaction
performance
Also:
Mix-N-Match
Single-Analyte
Focus on your
questions and papers
http://sabiosciences.com/elisarray.php
Title, Location, Date
54
55. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
ELISArray kits
Focused Content
http://sabiosciences.com/elisarray.php
Customer support group:
1-888-503-3187
support@SABiosciences.com
Title, Location, Date
55
56. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
Title, Location, Date
56
57. Sample & Assay Technologies
miRNA in cancer & inflammation
Title, Location, Date
57
61. Sample & Assay Technologies
miRNA in cancer & inflammation
Src activation induces transformation of normal cells
MCF10A cells (immortalized normal mammary epithelial cells)
Tamoxifen treatment
Activation of Src
Title, Location, Date
Transformation
61
62. Sample & Assay Technologies
miRNA in cancer & inflammation
Src-induced transformation is accompanied by an inflammatory
response mediated by NFkB
Title, Location, Date
62
63. Sample & Assay Technologies
miRNA in cancer & inflammation
Src-induced transformation is accompanied by increased
LIN28B and decreased Let-7a expression
Real-time PCR
Extract mRNA
SRC activation
Reverse transcribe to cDNA
NFkB activation
Screen 365 miRNAs
Increase LIN28B
Decrease Let-7a
Title, Location, Date
63
64. Sample & Assay Technologies
miRNA in cancer & inflammation
Let-7a and LIN28B regulate IL6 expression and activity during
Src-induced transformation
Increasing Let-7a levels decreases
IL6 levels
SRC activation
NFkB activation
Decreasing LIN28B levels decreases
IL6 levels
Increase LIN28B
Increase IL6
Decrease Let-7a
Title, Location, Date
64
65. Sample & Assay Technologies
miRNA in cancer & inflammation
The IL6 signaling pathway, mediated through STAT3, regulates
Src-induced transformation
SRC activation
NFkB activation
Increase LIN28B
Increase IL6
Decrease Let-7a
Transformation
Title, Location, Date
65
66. Sample & Assay Technologies
miRNA in cancer & inflammation
Conclusions
Oncogene activation (Src)
NFkB activation
Modulation of miRNA expression
Inflammatory cytokine (IL6)
Transformation
Title, Location, Date
66
67. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
Title, Location, Date
67
68. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
miScript miRNA PCR Arrays & Assays
Analyze miRNA expression by Real-time PCR
Profile 84 different
miRNAs on one array
(370 miRNAs on one
384 array)
Appropriate controls
for data normalization,
sample quality, and
reaction performance
Focus on your
questions and papers
http://sabiosciences.com/mirna.php
Title, Location, Date
68
69. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
miScript miRNA PCR Arrays & Assays
Focused Content
Screen ENTIRE miRNome
(ALL documented miRNAs)
http://sabiosciences.com/mirna_pcr_array.php
Title, Location, Date
69
70. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
miScript miRNA PCR Arrays & Assays
Complete Workflow
Sample
Isolation &
Preparation
Assay
Set-up &
Analysis
Customer support group:
1-888-503-3187
support@SABiosciences.com
Title, Location, Date
70
71. Sample & Assay Technologies
QIAGEN tools for cancer & inflammation
Gene Expression
RT-PCR
Somatic mutations
Copy Number Variations & Alterations
Epigenetics
miRNA
DNA methylation
Histone modifications
Functional Studies
Reporter assays
siRNA/shRNA
Protein Expression
Cytokine & Chemokine levels
̣
̣
̣
̣
http://sabiosciences.com/home.php
http://sabiosciences.com/seminarlist.php
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71
72. Sample & Assay Technologies
Keep up to date: Follow Pathway focused biology on Facebook
Latest
information on
pathway and
disease
research,
resources and
demos.
73. Sample & Assay Technologies
Cancer & Inflammation Webinar Outline
Inflammation – brief overview
Cancer
Inflammation in Cancer Initiation
qBiomarker Somatic Mutations System
Inflammation in Cancer Promotion
RT2 Profiler System
Inflammation in Cancer Progression
ELISArrays
miRNA in Cancer & Inflammation
miScript miRNA System
Promotions
Title, Location, Date
73
74. Sample & Assay Technologies
Thank you for attending
Would you like to try a Cancer & Inflammation-related PCR Array?
PCR Array Starter Pack Promotion
PCR Arrays of any Pathway (FREE)
• 2 96-well/100-well (2 samples) OR
• 1 384-well (4 samples)
2 Profiler (mRNA): FDK-PAFAS12
oRT
omiScript (miRNA): FDK-MIFAS12
oqBiomarker (Copy Number): FDK-CNVINT12
oEpiTect ChIP (Chromatin modifications): FDK-CPFAS12
Required Reagents (w/ Purchase)
• RT2 First-Strand cDNA Synthesis Kit, OR
• RT2 SYBR Green Mastermix (2-Pack)
Call 1-888-503-3187 to order
Email: support@SABiosciences.com
(US and Canada only)
Webinar-related question?
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