Cancer and Inflammation - 2013

Sample & Assay Technologies
Cancer & Inflammation

Raed Samara, Ph.D. R&D Scientist
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
Title, Location, Date
QIAGEN webinars
Access recorded webinars and slides
http://sabiosciences.com/home.php
QIAGEN webinars
Access recorded webinars and slides
Sign up for upcoming webinars Access previous webinars and slides
Keep up to date: Follow Pathway focused biology on Facebook
Latest information on pathway and disease research, resources and demos.
Biology-Focused Solutions for Pathway Analysis

SABiosciences is now a Company
Title, Location, Date 6
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
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
Epigenetic Changes mRNA Changes
Acute Inflammation
Cytokines & Chemokines Signaling Pathways Immune system composition
Infection Clearance Tissue Homeostasis
Chronic Inflammation
Pre-cancer & Cancer Chronic Inflammatory Diseases

TLR in Inflammation webinar
TLR in Inflammation webinar
Access previous webinars and slides
10
11
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
12
Link between Cancer & Inflammation

Bacteria, viruses, chemicals, chronic inflammation
Malignancy Bladder cancer Gastric lymphoma MALT lymphoma Hepatocellular carcinoma Kaposis sarcoma Bronchial carcinoma Mesothelioma Ovarian cancer Colorectal cancer Esophageal cancer Papillary thyroid carcinoma Prostate cancer
Inflammatory Stimulus Schistosomiasis H. pylori-induced gastritis H. pylori HBV, HCV HHV8 Silica, Asbestos Asbestos Endometriosis IBD Barretts metaplasia Thyroiditis Prostatitis
13
14
Inflammation in cancer initiation
Injury Inflammation ROS, RNI (Extracellular) Cytokines
ROS, RNI (Intracellular)
Base excision repair
DNA damage DNA mutations Tumor Initiation (DNA damage) Epigenetic Changes
Adapted from Grivennikov et al, Cell 2010

Injury Inflammation ROS, RNI (Extracellular) Cytokines
ROS, RNI (Intracellular)
DNA damage DNA mutations Tumor Initiation (DNA damage) Epigenetic Changes
Adapted from Grivennikov et al, Cell 2010

17

Increased tumor multiplicity in mice experiencing DNA damage after inflammation
Azoxymethane: colonic carcinogen
Aag+/+ Aag (Alkyladenine DNA glycosylase): Base excision repair enzyme Aag-/-
18

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.

Oncogenic mutations are increased in mice lacking base excision repair enzymes after inflammation
LCM
PCR Amplification
Sequencing
Gene: Ctnnb1 ( Catenin 1)
20

Conclusions
Inflammatory conditions
RNOS
Oncogenic mutations
Tumor growth
21
22
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
23

qBiomarker Somatic Mutation PCR Arrays & Assays Focused Content
http://sabiosciences.com/somaticmutation.php

25

26

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
28
Inflammation in cancer promotion
29

Mdr2-KO mice develop hepatocellular carcinoma on the background of chronic hepatitis (inflammatory condition)
Arrowheads indicate tumors
H&E staining
30

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)
31

NSAID treatment decreased inflammation
2.5 month old mice 10-day ibuprofen treatment
MPO staining Decreased neutrophils
32

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
33

NFkB is critical for tumor promotion
Generate DKO mice to directly assess the role of NFkB in hepatocarcinogenesis Mdr2 KO N-IBhep
X
NFkB inactivated in hepatocytes (Dox-activated NFkB) Double mutant (DM) Mdr2-/- N-IBhep
34

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
35

NFkB is critical for tumor promotion
Inactivation of NFkB in hepatocytes results in decreased tumor volume
NFkB activated in hepatocytes
36

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
37

Conclusions
Inflammatory stimulus (Mdr2 KO)
Inflammatory cytokine (TNFa)
NFkB activation
Modulation of gene expression
Tumor growth
Increased survival
38
39

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
http://sabiosciences.com/PCRArrayPlate.php

RT2 Profiler PCR Arrays & Assays Focused Content
Array NFkB Pathway NFkB Targets Apoptosis TNF Pathway Others (>140) Array IL6/STAT3 Pathway JAK/STAT Pathway
Species Human/Mouse/Rat Human/Mouse/Rat Human/Mouse/Rat Human/Mouse/Rat Varies Species Human/Mouse/Rat Human/Mouse/Rat
Inflammatory responses Human/Mouse/Rat
41

RT2 Profiler PCR Arrays & Assays Complete Workflow

43
Inflammation in cancer progression
44

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.
45
Generate DM mice to directly assess the role of CSF-1 in cancer progression MMTV-PyMT CSF1op
X
CSF1 null recessive mutation (CSF-1 not expressed) Double mutant (DM) PyMT-CSF1op
46

Growth and size of primary mammary tumors are not altered in absence of CSF-1
CSF-1 No CSF-1
47

Metastasis of mammary tumors is decreased in absence of CSF-1
CSF-1 No CSF-1
48

Progression of mammary tumors is decreased in absence of CSF-1
CSF-1
No CSF-1
49

Presence of TAMs is decreased in absence of CSF-1
+/op
CSF-1
No CSF-1
op/op
7-wk mice (F4/80 staining)
19-20-wk mice (F4/80 staining)
50

Conclusions
Development of primary tumor
Growth of primary tumor
Recruitment of TAMs to primary tumor
Metastasis of primary tumor to distant sites
51
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

53

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
http://sabiosciences.com/elisarray.php

ELISArray kits Focused Content
http://sabiosciences.com/elisarray.php Customer support group: 1-888-503-3187 support@SABiosciences.com
55
56
miRNA in cancer & inflammation
57
miRNA webinars
58
miRNA webinars
Access previous webinars and slides
59
60

Src activation induces transformation of normal cells
MCF10A cells (immortalized normal mammary epithelial cells)
Tamoxifen treatment
Activation of Src
Transformation
61

Src-induced transformation is accompanied by an inflammatory response mediated by NFkB
62

Src-induced transformation is accompanied by increased LIN28B and decreased Let-7a expression
Real-time PCR Extract mRNA Reverse transcribe to cDNA Screen 365 miRNAs
SRC activation
NFkB activation
Increase LIN28B Decrease Let-7a
63

Let-7a and LIN28B regulate IL6 expression and activity during Src-induced transformation
Increasing Let-7a levels decreases IL6 levels
Decreasing LIN28B levels decreases IL6 levels
SRC activation
NFkB activation
Increase IL6
64

The IL6 signaling pathway, mediated through STAT3, regulates Src-induced transformation
SRC activation
NFkB activation
Increase IL6
Transformation

Conclusions
Oncogene activation (Src)
NFkB activation
Modulation of miRNA expression
Inflammatory cytokine (IL6)
Transformation
67

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
http://sabiosciences.com/mirna.php
68

miScript miRNA PCR Arrays & Assays Focused Content Screen ENTIRE miRNome (ALL documented miRNAs)
http://sabiosciences.com/mirna_pcr_array.php

miScript miRNA PCR Arrays & Assays Complete Workflow

70
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
Keep up to date: Follow Pathway focused biology on Facebook
Latest information on pathway and disease research, resources and demos.
73
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 oRT Profiler (mRNA): FDK-PAFAS12 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? QIAWEBINARS@QIAGEN.COM


Cancer and Inflammation - 2013

Diunggah oleh

Informasi Dokumen

Judul Asli

Hak Cipta

Format Tersedia

Bagikan dokumen Ini

Bagikan atau Tanam Dokumen

Opsi Berbagi

Apakah menurut Anda dokumen ini bermanfaat?

Apakah konten ini tidak pantas?

Hak Cipta:

Format Tersedia

Cancer and Inflammation - 2013

Diunggah oleh

Hak Cipta:

Format Tersedia

Sample & Assay Technologies

Cancer & Inflammation

Sample & Assay Technologies

Cancer & Inflammation Webinar Outline

Title, Location, Date

Sample & Assay Technologies

Title, Location, Date

Sample & Assay Technologies

Sign up for upcoming webinars Access previous webinars and slides

Title, Location, Date

Sample & Assay Technologies

Keep up to date: Follow Pathway focused biology on Facebook

Latest information on pathway and disease research, resources and demos.

Sample & Assay Technologies

Biology-Focused Solutions for Pathway Analysis

Sample & Assay Technologies

Cancer & Inflammation Webinar Outline

Title, Location, Date

Sample & Assay Technologies

Microbes/Infection Tissue Damage

Epigenetic Changes mRNA Changes

Cytokines & Chemokines Signaling Pathways Immune system composition

Infection Clearance Tissue Homeostasis

Pre-cancer & Cancer Chronic Inflammatory Diseases

Sample & Assay Technologies

TLR in Inflammation webinar

Title, Location, Date

Sample & Assay Technologies

TLR in Inflammation webinar

Access previous webinars and slides

Title, Location, Date

Sample & Assay Technologies

Cancer & Inflammation Webinar Outline

Title, Location, Date

Sample & Assay Technologies

Title, Location, Date

Sample & Assay Technologies

Link between Cancer & Inflammation

Title, Location, Date

Sample & Assay Technologies

Cancer & Inflammation Webinar Outline

Title, Location, Date

Sample & Assay Technologies

Inflammation in cancer initiation

Injury Inflammation ROS, RNI (Extracellular) Cytokines

ROS, RNI (Intracellular)

Base excision repair

Adapted from Grivennikov et al, Cell 2010

Sample & Assay Technologies

Inflammation in cancer initiation

Injury Inflammation ROS, RNI (Extracellular) Cytokines

ROS, RNI (Intracellular)

Base excision repair

Adapted from Grivennikov et al, Cell 2010

Sample & Assay Technologies

Inflammation in cancer initiation

Title, Location, Date

Sample & Assay Technologies

Inflammation in cancer initiation

Azoxymethane: colonic carcinogen

Title, Location, Date

Sample & Assay Technologies

Inflammation in cancer initiation