Download Primary Cells: Optimizing Cancer Research and Drug

Pharma&Biotech
BioResearch
Primary Cells: Optimizing Cancer Research
and Drug Discovery
18 March 2014 / Speaker: Sean Fuerst
19 March 2014 / Speaker: Dr. Heiko Bueth
Lonza Walkersville, Inc., Walkersville, MD 21793 / 2014© Lonza
Clonetics™ Primary Cells & Media
Topics

What Is a Primary Cell Culture?

Primary Cells in Cancer Research & Drug Discovery

Introduction to Clonetics™ Primary Cells

Poietics™ Stem Cells

Nucleofector™ Transfection

Cancer Research Product Portfolio
2
Cancer Research Market

Approximately 12.5 mn new cases of cancer are diagnosed worldwide
each year

The global market for Biomarkers is forecast to reach $34 bn by the year 20171

Considerable research is in progress for drug discovery for cancer1



Innovative cancer therapies based on cancer molecular biology
Antisense oligonucleotides in clinical trials for cancer
Small interfering RNAs (siRNAs) can be targeted to tumors

NCI’s Developmental Therapeutics Program (DTP) has over 400,000 drugs
that have gone through screening process

Half a million lives saved in 30 years thanks to cancer research2
1.
2.
PRWEB
Cancer Research UK
3
Clonetics™ Primary Cells & Media
Topics

What Is a Primary Cell Culture?

Primary Cells in Cancer Research & Drug Discovery

Introduction to Clonetics™ Primary Cells

Poietics™ Stem Cells

Nucleofector™ Transfection

Cancer Research Product Portfolio
4
What Are Primary Cells?

Isolated from an animal or human tissue

Limited Lifespan (with the exception of some derived
from tumors)

Senescence

Do not contain any man-made changes to their
structure or genetic make-up

Most physiologically relevant in vitro tool without
in vivo studies

Primary cells are ideal models for cancer biomarker
discovery, drug screening, cancer development,
cancer stem cells
5
Primary Cells vs. Cell Lines
Cell Lines
Primary Cells

Permanently established culture

Limited lifespan

High mutation/modification rate

Low mutation/modification rate

Different genetic makeup from
original tissue

Isolated directly from human or
animal tissue

Extensive pre-characterization
required

Minimal pre-characterization needed

Cheaper to work upfront but > 3
cell lines required at minimum for
comprehensive study

Priced slightly higher but limited
samples needed

Not as physiologically relevant

Stepping stone to in vivo model
6
Concerns About Cell Lines

NIH highlights authentication of cultured cell lines as critical for grant
applications to be considered of the highest quality1

Some cell lines may not be the cell type they have historically been reported
In a study of over 500 leukemia-lymphoma cell lines, 15% of the cell lines were
misidentified2
18-36% of cell lines are misidentified or cross-contaminated3



Cell lines in cancer research do not provide complete or accurate data
Nature News: Some argue tumor cells obtained directly from patients are the best
way to study cancer 4
Review Breast Cancer Cell Lines: An alternative method is to use cultures from
primary breast tumors5


1.
2.
3.
4.
5.
Notice Number: NOT-OD-08-017 <grants.nih.gov/grants/guide/notice-files/NOT-OD-08-017.html>
Drexler et al., (2003)
Hughes P et al. (2007)
Brendan Borrell. Nature News: How accurate are cancer cell lines?. Vol 463.18 February 2010.
Burdall et al., Breast Cancer Cell Lines: friend or foe?. (2003). Review
7
Primary Cells
Provide More Relevant Data
D M Peehl. Review: Primary cell cultures as models of prostate
cancer development. Endocr Relat Cancer. 2005 Mar;12(1):19-47
“Recent studies revealed that many of these [prostate cancer cell
lines] are in fact derivatives of the three aforementioned cell lines
[PC-3, DU145 and LNCaP] or other non-prostatic lines (van
Bokhoven et al. 2003). The few bona fide cell lines, almost all derived
from metastases, do not span the range of prostate cancer
phenotypes, and in particular are not representative of primary
adenocarcinomas of the prostate. Furthermore, the question of how
extensively long-term culture alters the biological properties of cell
lines is always lurking. For these and other reasons, primary cultures
of malignant prostatic cells and their normal epithelial counterparts
are sought.
During the past 20 years, many of the technical hurdles involved in
growing primary cultures of human prostatic epithelial cells have
been overcome, and a variety of more or less similar methods have
been reported”
8
Clonetics™ Primary Cells & Media
Topics

What Is a Primary Cell Culture?

Primary Cells in Cancer Research & Drug Discovery

Introduction to Clonetics™ Primary Cells

Poietics™ Stem Cells

Nucleofector™ Transfection

Cancer Research Product Portfolio
9
Cell Lines Do Not Always Behave the
Same As Primary Cells
Effect of Camptothecin on primary and continuous renal
cells measured using ViaLight™ Plus
% maximal response

110
100
90
80
70
60
50
40
30
20
10
0
HEK 293
primary HRE
primary HRCE
Control
10 1
10 2
10 3
10 4
[Camptothecin] nM
EC 50
HEK 293
HRE
HRCE
68.10
537.5
557.4
10
Some Pathways Only Work in
Primary Cells
Localization of hMUNC13-4/RFP in cytolytic
T lymphocytes (CTLs)

CTLs recognize foreign cells and lyse them via the
“immunological synapse”

At the immunological synapse CTLs secrete cytolytic
granules containing Perforin

hMUNC13-4 is involved in an activation step
preceeding vesicle membrane fusion in cytolytic
granule secretion

Nucleofection™ of a plasmid expressing
hMUNC13-4/RFP fusion

Upon target cell contact, (L, M) hMUNC13-4 polarizes
and extensively co-localizes with Perforin-containing
cytolytic granules at the immunological synapse
Isolated CTL
CTL docked to
target cell
Reprinted from: Feldmann J et al. (2003) Cell 115(4):461-73; with permission of Elsevier.
11
Primary Endothelial Cells Diversity
Cannot Be Captured with Cell Lines

Endothelial cell diversity revealed by
global expression profiling





Gene expression analysis
Endothelial cells
14 distinct locations
53 different lots
Conclusions



Different expression based on vessel size
Different expression between veins and
arteries
Differences are conserved over some
passages
Chi JT. et al. (2003) PNAS 100:10623-8. By permission of the author. Copyright 2003 National Academy of Sciences.
12
Primary Cells and
Cancer Biomarker Discovery

Cancer Biomarkers: Biological molecules predictive of tumor
development

Challenges



Incomplete or misleading profile: Cell lines have altered profile than
actual tumor tissue. Mutations, copy number variations,
chromosome aberrations, lack of characterization
High costs: 3-10 cell lines required at minimum from same origin for
comprehensive study
Resolution


Primary cells as controls for complete and validated profile
Cost-effective: No pre-characterization, minimal samples required
13
Clonetics™ Human Umbilical Vein Endothelial
Cells (HUVECs) – Cancer Biomarker Discovery

NGAL has been proposed as an early
biomarker in pancreatic cancer (PaCa)

Clonetics™ HUVEC cells were assayed for
tube formation in presence of high and low
levels of NGAL. The tube formation was
reduced by 69% with high levels of NGAL

Additional data suggested NGAL potently
blocks pancreatic cancer angiogenesis in vitro
in part by reducing VEGF secretion
Zhimin Tong et al. Neutrophil Gelatinase–Associated Lipocalin: A Novel
Suppressor of Invasion and Angiogenesis in Pancreatic Cancer. Cancer
Res 2008;68:6100-6108
HUVEC diluted in conditioned medium (1:1
dilution) from NGAL overexpression or shNGAL
clones were seeded onto 96-well plates that were
precoated with Matrigel. After 16 h, the total
number of vascular tubes were counted.
The levels of VEGF in the culture medium were
assayed by using a commercially available VEGF
ELISA kit
14
Primary Cells and Drug Screening

Applications


Challenges



New drug targets, Drug resistance assays, chemotherapy drugs
Assays developed with cell lines cannot easily be replicated in vivo
In vivo models are expensive for prelim drug screens
Resolution


Biological relevance: Assays with primary cells are more
representative of in vivo models
Cost-effective: Use primary cells for refinement studies before
stepping into animal work
15
Clonetics™ Human Prostate Epithelial
Cells – Drug Screening

HTS of 4910 drugs to determine efficacy for prostate cancer

Clonetics PrECs (Cat. No. CC-2555) utilized as control with malignant and non-malignant
cell lines

3 drug compounds (see below) were identified to inhibit prostate cancer cell proliferation,
but had little effect on control cells
Kristiina Iljin et al. High-Throughput Cell-Based Screening of 4910 Known Drugs and Drug-like Small Molecules Identifies Disulfiram
as an Inhibitor of Prostate Cancer Cell Growth. Clin Cancer Res October 1, 2009 15; 6070
16
Primary Cells and Cancer Formation

Understand how cancer forms


Challenges



Primary cells can be transformed into cancer cells through genetic
mutations and introduction of oncogenes
Cell lines cannot offer same application as they are not
representative of actual human tissue
Well-characterized primary cells needed
Resolution

Primary cells from Lonza are extensively characterized and
publication validated
17
Clonetics™ Human Mammary Epithelial
Cells (HMECs) – Cancer Formation

Clonetics™ HMECs were infected with retrovirus and
express LT, hTERT and H-rasV12

Introduced in mice by injection

HMECs have successfully been transformed to
tumorigenicity through the introduction of a limited set
of oncogenes
Brian Elenbaas, Lisa Spirio et.al. Human breast cancer cells generated by oncogenic transformation of primary
mammary epithelial. Genes Dev. 2001 Jan 1;15(1):50-65
18
Primary Cells and Stem Cell Biology

Cancer Stem Cells (CSCs) Hypothesis



Challenges


Cancer stem cells originate from normal tissue stem cells
CSCs share several properties with normal stem cells, such as their
capacity for self-renewal and their ability to differentiate
Cell lines are difficult to re-program as they may not contain stemcell like components so CSC study becomes difficult
Resolution


Primary cells contain subpopulations of stem cell markers
Primary cells can be re-programmed into iPSCs
19
Clonetics™ Human Prostate Epithelial Cells
(PrECs) and Media – Cancer Stem Cells

Prostate cancer arises within the epithelial
compartment and generally believed to begin within
the stem-cells

In the adult human prostate, CD133/ABCG2
expression is indicative of small population of
putative stem cells that suppress differentiation

Clonetics™ PrECs (Cat. No. CC-2555) utilized
alongside in-house isolated PrEC’s to assess stemcell like properties. Both cell types were cultured in
Clonetics™ PrEGM Growth Media BulletKit (Cat. No.
CC-3166). Both exhibit high expression of stem-cell
markers CD133/ABCG2
Expression of stem cell markers in PrEC
cultures. A: ABCG2-FITC and CD133-PE
(AC141) expression in PrECs. B: dual-label
flow cytometry showing that CD133+ cells
are also ABCG2+
Donald J. Vander Griend et.al. The Role of CD133 in Normal Human Prostate Stem
Cells and Malignant Cancer-Initiating Cells. Cancer Res 2008;68:9703-9711
20
Clonetics™ Primary Cells & Media
Topics

What Is a Primary Cell Culture?

Primary Cells in Cancer Research & Drug Discovery

Introduction to Clonetics™ Primary Cells

Poietics™ Stem Cells

Nucleofector™ Transfection

Cancer Research Product Portfolio
21
Why Use Clonetics™ Primary Cells?
Customer Needs
Lonza Solutions

Product quality


Product availability


Reliability


Cost-effective


Easy to culture


Convenience

Superior to cell lines, Minimum specs
guarantee, ISO-certified, extensive QC
& consistency
100+ cell types (human and animal),
donor variety, steady supply schedule
40+ years experience, market leader,
10X more publications than competitors
No pre-characterization needed; No
repeat analyses; Refine experiments
and reduce animal work
Specialized support team to assist,
optimized protocols guaranteed to work
Cryopreserved amp or proliferating in
flasks/plates
22
Clonetics Growth Media Systems

Developed for each cell type and tested against our primary cells

BulletKit™ Media: Clonetics™ SingleQuots™ Kits and basal media sold
together as a “package deal” but can be bought separately as well

Optimized protocols supplied with all media systems
Basal Media
(Water, Amino Acids, Vitamins, Sugar)
+
SingleQuots™ Kit
(Growth Factors: Serum, Insulin,
Antibacterial Agents, etc)
=
BulletKit™ Media
23
Clonetics™ Primary Normal Human Cells

Breast Cancer: Mammary Epithelial Cells (130 unique
publications for breast cancer research)

Prostate Cancer: Epithelial, Smooth Muscle and
Stromal cells

Pancreatic Cancer: Pancreatic Islets and Acinar Tissue

Lung Cancer: airway fibroblasts, epithelial and smooth
muscle cells from various tissues

Skin Cancer: Endothelial cells, fibroblasts, keratinocytes,
melanocytes

All related growth media kits available for each cell type
with optimized protocols
Additional cell types available.
Visit www.lonza.com/primary for complete listing.
24
Clonetics™ Primary Cells & Media
Topics

What Is a Primary Cell Culture?

Primary Cells in Cancer Research & Drug Discovery

Introduction to Clonetics™ Primary Cells

Poietics™ Stem Cells

Nucleofector™ Transfection

Cancer Research Product Portfolio
25
Poietics™ Stem Cells and Media

Human Mesenchymal Stem Cells with Growth and Differentiation
Media Kits


Human Hematopoietic Stem Cells with Growth Media and
EasyDiff™ Kits


Study cancer progression in bone marrow, blood and lymph tissues
Isolated from cord blood and bone marrow for blood cancer and
transplantation studies
Osteoclast Precursors, Preadipocytes, Adipose-derived Stem Cells and
Media Kits

Study various bone and lipoma tumors
26
Clonetics™ Primary Cells & Media
Topics

What Is a Primary Cell Culture?

Primary Cells in Cancer Research & Drug Discovery

Introduction to Clonetics™ Primary Cells

Poietics™ Stem Cells

Nucleofector™ Transfection

Cancer Research Product Portfolio
27
Nucleofector™ Technology
Benefits for Cancer Research

Non-viral transfection of primary cells and
difficult-to-transfect cancer cell lines with up
to 90% efficiency

High cell viability and preservation of cell
functionality

High flexibility in cell types, substrates or
sample throughput

Option of adherent Nucleofection™ adding
more convenience

More than 100 ready-to-use protocols and
more than 800 publications with cancer cells
28
Nucleofector™ Technology
Proven DNA Transport Into the Nucleus
Transport
DNA

Expression
GFP
Nucleus
DAPI
Merge
Primary NHDF-neo (Neonatal normal human dermal fibroblasts) were
transfected with labeled plasmid DNA encoding GFP, fixed after 2h in
3.5% PFA and analyzed by confocal microscopy
29
Transfection of Primary Cells or
Cancer Cells Lines by Nucleofection™
Blood
Breast
Colon
Liver
Lung
Neurological
Prostate
Other
30
Clonetics™ Primary Cells & Media
Topics

What Is a Primary Cell Culture?

Primary Cells in Cancer Research & Drug Discovery

Introduction to Clonetics™ Primary Cells

Poietics™ Stem Cells

Nucleofector™ Transfection

Cancer Research Product Portfolio
31
Lonza Cancer Product Portfolio


Blood & Lymph

Blood & lymphatic micro-vascular
endothelial cells

Hematopoietic stem cells from
cord blood

Mesenchymal stem cells

Fresh bone marrow





Pulmonary

Bronchial epithelial & smooth
muscle cells

Fibroblasts

Micro-vascular endothelial cells

Pulmonary artery endothelial and
smooth muscle cells

Small airway epithelial cells

Related Clonetics™ and Poietics™
Media tested and guaranteed to perform
www.lonza.com/primary

Nucleofector™ Transfection for primary
cells and cancer cell lines

Clonetics™ Conditionally Immortalized
Cell Lines (CCICs)
Bone



Osteoblasts
Osteoclast precursors
Hematopoietic stem cells from
bone marrow
Fresh bone marrow
Cell Function assays for bone
mineralization and resorption
Mammary

Mammary epithelial
Pancreatic

Fresh Human Pancreatic Islets

Acinar tissue (custom isolation)


Reproductive

Prostate epithelial, smooth muscle 
and stromal cells

Uterine endothelial and smooth
muscle cells

Bladder endothelial and smooth 
muscle cells
Biowhittaker™ liquid and powder media
for cancer research and therapeutic
applications
Nucleic Acid and Protein
Electrophoresis products
Skin






Blood & lymphatic microvascular
endothelial cells
Adult and neonatal MV endothelial
cells
Fibroblasts
Keratinocytes
Melanocytes
Pre-adipocytes and Adiposederived stem cells
32
How to Order or Pose Questions

Email order to: [email protected] or [email protected]

For order-related questions, contact Customer Service



For scientific support please contact



U.S : 800 638 8174 (toll free) or [email protected]
Europe: +32 87 321 611 or [email protected]
U.S: +1 800 521 0390 or [email protected]
Europe: +32 87 321 611 or [email protected]
Visit our website at www.lonza.com/cancer for more information
33
Interested to Learn More?

Join our upcoming webinar:
Clonetics™ Cells in Pancreatic Cancer Research
Slot 1: Tuesday, 1 April 2014
2PM EDT (New York) / 11 AM PDT (Los Angeles)
Slot 2: Wednesday, 2 April 2014
10 AM CEST (Berlin) / 9 AM BST (London) / 5 PM JST (Tokyo)

Register at: www.lonza.com/webinar14
34
Thank You for Your Kind Attention