Credible Leads to Incredible™
Fang Tian, PhD
Principal Scientist
Head of Cell biology R&D group
November 2020
Advanced drug-resistant cell models for cancer therapeutic resistance studies
2
About ATCC
Founded in 1925, ATCC is a non-profit organization with HQ in Manassas, VA, and an R&D and Services center in Gaithersburg, MD
World’s largest, most diverse biological materials and standards development organization. Information resource for microbes
Innovative R&D company featuring gene editing, microbiome, NGS, advanced models
Multiple accreditations, including ISO 9001 and ISO 13485, cGMP biorepository
Partner with government, industry, and academia
Leading global supplier of authenticated cell lines, viral and microbial standards
Global presence. Sales and distribution in 150 countries, 19 international distributors
Talented team of ~500 employees, over one-third with advanced degrees
About ATCC
3
Therapy resistance in cancer
Therapeutic resistance in cancer is multifactorial and heterogeneous.
Spatial and temporal resistance occurs in tumor cells, within the stroma, or in metastasis.
The underlying mechanisms of drug resistance are diverse and complex, often driven by
• Tumor heterogeneity • Genetic and epigenetic alternations • Drug transporters • Lineage plasticity• Adaptive signaling events• Tumor microenvironment (TME)
Wheel of Resistance
About ATCC
4
Overcoming resistance: new approaches
Applying evolutionary principles• Competitive release of drug-resistant subclone• Intermittent therapy: allow drug-sensitive cells to
outcompete drug-resistant subclones
Modulating the TME• Targeting the tumor vasculature, stroma, and
immune cells
Learning from exceptional responders• Extending exceptional responses to broader
patient populations
Many of the models used in the early stages of research don’t capture cancer’s mechanisms of resistance to therapeutics, which impede progress in drug development and clinical trials.
To overcome this roadblock, ATCC is committed to providing the advanced cell models to push the envelope in cancer research
ATCC drug resistant cell models
Resistant to hormonal therapies
Resistant to chemotherapy
drugs
• CPT • FUdR• doxorubicin • etoposide • dactinomycin • bleomycin • mitoxantrone • thioguanine
• Herceptin• Imatinib
Resistant to targeted therapies
• Tamoxifen
• BRAF inhibitors• MEK inhibitors
Resistant to targeted therapies
Long-term drug selection derived cell lines CRISPR engineered cell lines
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About ATCC
6
Establish drug resistance cell model through gene editing
Drug resistance in melanoma Develop isogenic lines of drug resistance
Ras/Raf/MEK/ERK MAP kinase signaling regulates BRAF inhibitor drug resistance
RAS
Cell survival and proliferation
Wagle N, et al. J Clin Oncol 29(22): 3085-3096, 2011Week 1 Week 15 Week 23
BRAF inhibitor resistance in melanoma patientsAdvanced in vitro cell models that contain defined genetic drug resistance mechanisms are needed to facilitate the development of next-generation therapeutics that can overcome BRAF drug resistance in melanoma.
About ATCC
7
Characterization of A375 isogenic cell lines
Genome sequence
Transcript sequence
Protein expression
Off-target screening
Cell morphology
Cell growth kinetics
Drug response
Cell line stability
Cell line authentication
Sterility test
A375 isogenic lines for 2D drug screening
- 9 . 5 - 9 . 0 - 8 . 5 - 8 . 0 - 7 . 5 - 7 . 0 - 6 . 5 - 6 . 0 - 5 . 5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
D a b r a f e n i b R e s i s t a n c e i n A 3 7 5
I s o g e n i c M e l a n o m a M o d e l s
l o g [ D a b r a f e n i b ] , M
Su
rv
iva
l r
ate
(%
)
K R A S M u t a n t A 3 7 5 I s o g e n i c
A 3 7 5
N R A S M u t a n t A 3 7 5 I s o g e n i c
M E K 1 M u t a n t A 3 7 5 I s o g e n i c
- 8 . 0 - 7 . 5 - 7 . 0 - 6 . 5 - 6 . 0 - 5 . 5
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
N o E t o p o s i d e R e s i s t a n c e i n A 3 7 5
I s o g e n i c M e l a n o m a M o d e l s
l o g [ E t o p o s i d e ] , M
Su
rviv
al
rate
(%
)
K R A S M u t a n t A 3 7 5 I s o g e n i c
A 3 7 5
M E K 1 M u t a n t A 3 7 5 I s o g e n i c
N R A S M u t a n t A 3 7 5 I s o g e n i c
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A375 isogenic lines for 3D culture drug screening
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About ATCC
10
MEK1 isogenic A375 lines for combination therapy study
2D c
ultu
re
3D c
ultu
re
About ATCC
11
KRASG13D A375 isogenic line highly expressing EGFR and PD-L1
KRASG13D A375
PD-L1
IFNγ +, Isotype Control
IFNγ -, Isotype Control
IFNγ -, PD-L1 staining
IFNγ +, PD-L1 staining
Unique features of KRAS isogenic A375 line
About ATCC
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0
20
40
60
PD-L1 mAb concentration (ug/mL)
IFNγ
conc
entr
atio
n (p
g/m
L)
Activated CD8+ T cells + A375-KRAS Co-Culture
PD-L1 mAbIgG1 Isotype
1010.10.01
Isogenic A375 lines for IO and combination therapy study
• KRASG13D A375 Isogenic line harbors BRAF600E, KRASG13D
mutations, highly expresses EGFR and IO checkpoint PD-L1
• Together with it’s parental cell line, KRASG13D A375 Isogenic line can be an ideal drug resistant melanoma model for the studies of commination therapy using BRAF inhibitor and IO checkpoint blockade
About ATCC
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Drug-resistant isogenic A375 lines for in vivo studyGeneration of Luciferase Expressing isogenic A375 Cell Line
Expansion and validation of single clones
Pool evaluation and selectionViral transductionLenti-Luc2 plasmid
80 400
2,00
0
10,0
00
0
1×109
2×109
3×109
Tota
l Flu
x (p
/s)
010
0020
0030
0040
0050
0060
0070
0080
0090
0010
0000
5×104
1×105
1.5×105
2×105
2.5×105
Cell Bioluminescence
Cell Number (cells/well)
Lum
ines
cenc
e (R
LU)
R2 = 0.9997
In vitro Confirmation of Luciferase Expression and Stability
0 10 20 30 400
1×108
2×108
3×108
4×108
5×108
KRAS mutant A-375 Isogenic-Luc2 radiance vs time
Days Post Injection
Rad
ianc
e (p
/s/c
m²/s
r)
KRASG13D A375 Isogenic-Luc2 Tumor Model and in vivo Bioluminescence Imaging
7.0E5
9.3E3
Cou
nts
Day: 3 7 13 21 28
0 1 21×100
1×101
1×102
1×103
1×104
1×105
1×106
KRAS mutant-A375 Isogenic-Luc2
Lum
ines
ence
(RLU
) Blasticidin -Blasticidin +
freezethaw
(months)
Summary
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Cell-based models are critical tools for understanding the mechanisms of drug resistance and developing novel therapeutics.
ATCC has been developing state-of-the-art drug-resistant cancer cell models by using CRISPR-Cas9 gene-editing technology to introduce critical mutations into disease-relevant cell lines.
These novel cell lines have several advantages including cell line homogeneity, stability of the relevant genotype, do not require for continued drug pressure to maintain the cell line, and modeling the acquired resistance to newly developed therapeutics.
Credible Leads to InCredible™
Acknowledgements:
Dedicated R&D and Commercial Teams at ATCC