Cancer and Inflammation_2013 (1)

Sample & Assay Technologies

Cancer & InflammationRaed Samara, Ph.D.R&D Scientist

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

Title, Location, Date 2

Sample & Assay Technologies QIAGEN webinars

Access recorded webinars and slides


http://sabiosciences.com/home.php

Sample & Assay Technologies QIAGEN webinars

Access recorded webinars and slides


Sign up for upcoming webinarsAccess previous webinars and slides

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

SABiosciences is now a Company








�ELISArrays


� Promotions


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/InfectionTissue Damage

Acute Inflammation

Chronic InflammationInfection ClearanceTissue Homeostasis

Pre-cancer & CancerChronic Inflammatory Diseases

Cytokines & ChemokinesSignaling PathwaysImmune system composition

EpigeneticChanges

mRNAChanges

Sample & Assay Technologies ‘TLR in Inflammation’ webinar



Sample & Assay Technologies ‘TLR in Inflammation’ webinar


Access previous webinars and slides






�ELISArrays


� Promotions


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

Initiation Promotion ProgressionGenomic alterations Proliferation & Growth Invasion & Metastasis(DNA Damage)(DNA mutations)

Three Mechanistic phases

Inflammation

Sample & Assay Technologies Link between Cancer & 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

Bacteria, viruses, chemicals, chronic inflammation







�ELISArrays


� Promotions


Sample & Assay Technologies Inflammation in cancer initiation


DNA damageDNA mutations

Epigenetic ChangesTumor Initiation(DNA damage)

ROS, RNI(Intracellular)

Cytokines

ROS, RNI(Extracellular)

Inflammation

Injury

Adapted from Grivennikov et al, Cell 2010

Base excision repair



DNA damageDNA mutations

Epigenetic ChangesTumor Initiation(DNA damage)

ROS, RNI(Intracellular)

Cytokines

ROS, RNI(Extracellular)

Inflammation

Injury

Adapted from Grivennikov et al, Cell 2010





Increased tumor multiplicity in mice experiencing DNA damage after inflammation


Aag+/+

Aag-/-

Aag (Alkyladenine DNA glycosylase): Base excision repair enzyme

Azoxymethane: colonic carcinogen


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


Gene: Ctnnb1 (β Catenin 1)

LCM PCR Amplification Sequencing


Conclusions


Oncogenic mutations

Tumor growth

RNOS

Inflammatory conditions







�ELISArrays


� Promotions


Sample & Assay Technologies QIAGEN tools for cancer & inflammation

qBiomarker Somatic Mutation PCR Arrays & Assays Analyze Somatic Mutations by Real-time PCR


http://sabiosciences.com/somaticmutation.php

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


qBiomarker Somatic Mutation PCR Arrays & Assays Focused Content


http://sabiosciences.com/somaticmutation.php








qBiomarker Somatic Mutation PCR Arrays & Assays Complete Workflow


SampleIsolation & Preparation

Assay Set-up & Analysis

Customer support group:1-888-503-3187

[email protected]






�ELISArrays


� Promotions


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


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)


NSAID treatment decreased inflammation


2.5 month old mice10-day ibuprofen treatment

MPO stainingDecreased neutrophils


TNFa drives NFkB activation


Treatment with anti-TNFa decreased NFkB nuclear staining (activation)

Source of TNFa is the non-hepatocyte fraction of the liverH: hepatocyte fractionR: non-hepatocyte fraction


NFkB is critical for tumor promotion


Generate DKO mice to directly assess the role of NFkB in hepatocarcinogenesis

Mdr2 KO ∆N-IκBhep

NFkB inactivated in hepatocytes(Dox-activated NFkB)

Double mutant (DM) Mdr2-/- ∆N-IκBhep

X


NFkB is dispensable for early stages of HCC development


4 month old miceNo change in inflammation

7 month old miceNo change in dysplasia


NFkB is critical for tumor promotion


NFkB activated in hepatocytes

Inactivation of NFkB in hepatocytes results in decreased tumor volume


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)

Inactivation of NFkB decreased the expression of anti-apoptotic genes

Liver protein extracts from KO mice


Conclusions


NFkB activation

Tumor growth

Inflammatory cytokine (TNFa)

Inflammatory stimulus (Mdr2 KO)

Increased survival

Modulation of gene expression






�ELISArrays


� Promotions



RT2 Profiler PCR Arrays & Assays Analyze gene expression by Real-time PCR


http://sabiosciences.com/PCRArrayPlate.php

Profile 84 different genes on one array(370 genes on one 384 array)

Appropriate controls for data normalization, sample quality, and reaction performance



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


RT2 Profiler PCR Arrays & Assays Complete Workflow





[email protected]






�ELISArrays


� Promotions


Sample & Assay Technologies Inflammation in cancer progression



� 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.

TAMs and CSF-1




Generate DM mice to directly assess the role of CSF-1 in cancer progression

MMTV-PyMT CSF1op

CSF1 null recessive mutation (CSF-1 not expressed)

Double mutant (DM) PyMT-CSF1op

X


Growth and size of primary mammary tumors are not altered in absence of CSF-1


CSF-1No CSF-1


Metastasis of mammary tumors is decreased in absence of CSF-1


CSF-1No CSF-1


Progression of mammary tumors is decreased in absence of CSF-1


CSF-1 No CSF-1


Presence of TAMs is decreased in absence of CSF-1


+/op op/op

7-wk mice(F4/80 staining)

19-20-wk mice(F4/80 staining)

CSF-1 No CSF-1


Conclusions


Recruitment of TAMs to primary tumor

Growth of primary tumor

Development of primary tumor

Metastasis of primary tumor to distant sites



� 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






�ELISArrays


� Promotions



ELISArray kits Analyze Protein expression


Also:Mix-N-MatchSingle-Analyte

http://sabiosciences.com/elisarray.php

Profile 12 different analytes from 6 samples on one array

Appropriate controls for reaction performance



ELISArray kits Focused Content


http://sabiosciences.com/elisarray.php


[email protected]






�ELISArrays


� Promotions


Sample & Assay Technologies miRNA in cancer & inflammation


Sample & Assay Technologies miRNA webinars



Sample & Assay Technologies miRNA webinars


Access previous webinars and slides

Sample & Assay Technologies



Src activation induces transformation of normal cells


Tamoxifen treatment Activation of Src

MCF10A cells (immortalized normal mammary epithelial cells)

Transformation


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



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 activationIncrease LIN28B

Decrease Let-7a


Let-7a and LIN28B regulate IL6 expression and activity during Src-induced transformation



Decrease Let-7aIncrease IL6

Increasing Let-7a levels decreases IL6 levels

Decreasing LIN28B levels decreases IL6 levels


The IL6 signaling pathway, mediated through STAT3, regulates Src-induced transformation



Decrease Let-7aIncrease IL6

Transformation


Conclusions


Modulation of miRNA expression

NFkB activation

Oncogene activation (Src)

Transformation

Inflammatory cytokine (IL6)






�ELISArrays


� Promotions



miScript miRNA PCR Arrays & Assays Analyze miRNA expression by Real-time PCR


http://sabiosciences.com/mirna.php

Profile 84 different miRNAs on one array(370 miRNAs on one 384 array)

Appropriate controls for data normalization, sample quality, and reaction performance



miScript miRNA PCR Arrays & Assays Focused Content


http://sabiosciences.com/mirna_pcr_array.php

Screen ENTIRE miRNome (ALL documented miRNAs)


miScript miRNA PCR Arrays & Assays Complete Workflow





[email protected]


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.phphttp://sabiosciences.com/seminarlist.php

Sample & Assay Technologies Keep up to date: Follow Pathway focused biology on Facebook

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






�ELISArrays


� Promotions


Sample & Assay Technologies Thank you for attending

Would you like to try a Cancer & Inflammation-related PCR Array?


PCR Array Starter Pack PromotionPCR Arrays of any Pathway (FREE)

• 2 96-well/100-well (2 samples) OR• 1 384-well (4 samples)

oRT2 Profiler (mRNA): FDK-PAFAS12omiScript (miRNA): FDK-MIFAS12oqBiomarker (Copy Number): FDK-CNVINT12oEpiTect ChIP (Chromatin modifications): FDK-CPFAS12Required Reagents (w/ Purchase)

• RT2 First-Strand cDNA Synthesis Kit, OR• RT2 SYBR Green Mastermix (2-Pack)

Webinar-related question?

[email protected]

Call 1-888-503-3187 to order

Email: [email protected](US and Canada only)

Documents

Cancer and Inflammation_2013 (1)