My applications of in vitro imaging in disease mechanism research, drug modes of action study, and drug discovery, with my collaborators and friends. (Second version)

1. Tackling Diseases with
In Vitro Imaging
Mei Zhang, MD, PhD
July 10, 2009
Mitochondria in live CV-1 cells
The power of cellular imaging
• Reveal disease mechanism
• Tracking intracellular drug targets
• Determine drug MoA
• Optimize HTS hits
• Screen library compounds
Lysosomal contents in fused CT60 cells
1

2. Elucidation of cellular mechanisms for human
disease
1. Niemann-Pick C disease (NPC) (late endosomal tubular trafficking
defects) (@NIH)
2. Mucolipidosis IV (ML4) (protein trafficking defects and calcium
accumulation in lysosomes) (@NIH)
3. Alzheimer’s disease (AD) (reduction of early endocytic recycling,
accumulation of g-secretase substrates in ERC) (@MGH)
4. Cancer metastasis, angiogenesis, microenvironment (SDF1 and
CXCR4 axel in metastasis) (@Synta)
5. Cancer cell stress (ROS and HSP70 in cancer) (@Synta)
6. Tumor vasculature (Microtubule and vascular structure) (@Synta)
7. Inflammation (TLR9 activation by CpG and cRel trafficking;
CRACM1 and Stim1 trafficking) (@Synta)
Lysosomes Lysosomes
c-Rel
c-Rel
Niemann-Pick C disease (NPC) Mucolipidosis IV (ML4)
(Trafficking defect) (Protein location and Ca++ defects)
Endosomes
Inflammation
(TLR9, c-Rel trafficking) Reveal
Disease
Mechanism
Alzheimer’s disease (AD)
Pseudopodia (APP and g-secretase)
ROS
VE-Cadherin Viability
Cancer metastasis, angiogenesis,
microenvironment Tumor vasculature Cancer cell stress
(CXCR4; HSP90) (VDA) (Elesclomol)
2

3. Localization of membrane and soluble proteins
in intracellular organelles
1. Plasma membrane (CXCR4, S1P1, CRACM1) (@Synta)
2. Early endosomes (APP and APP-C99) (@MGH)
3. Late endosomes (NPC1, NPC2, MLN64, ML4, and activated CXCR4)
(@NIH and Synta)
4. Lysosomes (NPC1, NPC2, MLN64, Mucolipin-1, and activated CXCR4)
(@NIH and Synta)
5. Secretory granules (Insulin) (@Synta)
6. Lipid droplets (NPC defect, hexaminidase C short form) (@NIH)
7. ER (Mutant NPC1 and ML4) (@NIH)
8. Golgi apparatus (FcER) (@NIH)
9. Mitochondria (Ceramide, MLN64-cholesterol acceptor) (@NIH and Synta)
10. Microtubules (Alpha-tubulin, Spag6) (@NIH and Synta)
11. Actin filaments (16Q) (@NIH)
12. Nuclei (16Q) (@NIH)
13. Nucleoli (Proteasome) (@Synta)
(Also: Hexosaminidase C)
Lipid droplets
(Sudan III)
Early endosomes Mitochondria
(APP-C99) (Mito-Tracker)
Late endosomes
(Also: APP) (NPC1) (Also: Ceramide)
PM
(CXCR4) (Also: NPC2, MLN64, Secretory granules
ML4, and activated CXCR4) ER (ML4)
(Also: CRACM1, S1P1) (Insulin)
Tracking
Nuclei
(NFkB Cellular Targets Golgi
(Also: Mutants
NPC1, ML4)
c-Rel)
(Also: FcER)
(Also: 16Q)
Chromosome
(pERK)
Nucleoli Actin filaments
(Proteasome-i) Microtubules
(Also: 16Q) (a-tubulin) Centrosome
(Tubulin-YFP)
(Also: Spag6)
3

4. Study modes of action of new drugs in
discovery and development
1. Apilimod (c-Rel translocation, ERK translocation, vacuolation)
(@Synta)
2. Elesclomol (Proteasome inhibition, and induction of ROS and OH
radical generation) (@Synta)
3. MTi (Existing or new replacement, fragmentation of MT and
multinucleation) (@Synta)
4. CRAC inhibitors (Effect on cardiomyocytes; Stim-1 translocation by
activation of ER calcium release) (@Synta)
5. Gamma-secretase inhibitors (Accumulation of substrates in ERC)
(@MGH)
6. Natural compounds (Inducing insulin production) (@Synta)
7. Synergistic anti-cancer effects (Elesclomol with paclitaxel,
ascorbate and other anticancer agents) (@Synta)
ROS inducer (Elesclomol)
IL12 inhibitor (Apilimod)
MT
inhibitors
Determine
MoA -secretase inhibitors
CA4P
CRAC HSP90i
inhibitors
1+1>2
Anti-cancer synergy
(Elesclomol+Taxol, or VitC)
4

5. Screening Focused Libraries and
Optimizing HTS Hits
• Apoptosis-rescuing, Screening Project (Leader, assay,
screening, MoA) (@Synta)
• New Microtubule Inhibitor, (VDA) Program, (MoA in Lead-Opt)
(@Synta)
• CRAC Inhibitor Backup Screening Program (Assay
development, Hit-ID) (@Synta)
• CXCR4 Inhibitor Screening Program (Leader, assay, MoA,
chemistry, collaboration) (@Synta)
• New ROS Inducer Screening Program (MoA in Lead-Opt)
(@Synta)
Apoptosis-rescuing
Screening Project
(Leader, assay,
screening, MoA) New Microtubule Inhibitor
(VDA) Program
(MoA in Lead-Opt)
Screening and Optimization
CXCR4 Inhibitor CRAC Inhibitor Backup
Screening Program Screening Program
(Leader, assay, MoA, (Assay development, Hit-ID)
chemistry, collaboration)
New ROS Inducer
Screening Program
(MoA in Lead-Opt)
5

6. Topics
• Revealing disease mechanisms for lysosomal
storage disorders
• Tracking cellular targets of g-secretase
inhibitor for Alzheimer’s disease
• Determining MoA of drug Elesclomol for
metastatic melanoma
• Screening and optimization for CXCR4
inhibitors
Water immersions from Zeiss in LCBB NIDDK
Topic 1
Discovery of novel
mechanisms of NPC1 and
ML4 diseases
Co-culture of rat neurons and astrocytes
6

7. NPC1 disease
Co-culture of rat neurons and astrocytes
Niemann-Pick Disease, Type C (NPC) and
the Late Endocytic Pathway
Niemann-Pick Disease,
type C
• Age: 0-60 Recycling
• Genes: NPC1 and NPC2
• Symptoms: dementia
• Cellular abnormality:
Accumulation of cholesterol
in lysosomes
• Diagnosis:
– Mutations in NPC1
or NPC2
– Cholesterol
esterification
– Cholesterol in
lysosomes (filipin
staining)
• Therapy: miglustat (on trials) Degradation
7

8. Confocal / digital scopes, cell warmers
Zeiss 410 Lab-Tek covers Controllers
CoolSnapHQ
Lens warmer
Sandwich and
circulator
Cellular phenotype of NPC:
Cholesterol accumulation in lysosomes
Normal NPC CT60 Cells
Human Fibroblasts Patient Fibroblasts (NPC1 Null CHO)
Cholesterol
Filipin Staining
Filipin
8

9. Cholesterol-laden lysosomes are multilamellar
compartments
EM structure of a lysosome
Anti-LAMP / Filipin
Freeze Fracture
A human NPC fibroblast Freeze Fracture (Filipin)
From Nancy and Joan
NPC1 Protein
and the SSD
Millard, et al. JBC 2005
Davis, et al. JBC 2000
P691S
NPC1 GFP
9

10. NPC1-GFP, but not the P691S mutant, is functional in
clearing cholesterol accumulation.
NPC1-GFP Anti-NPC1 Cholesterol
SSD-NPC1-GFP (P691S) Anti-NPC1 Cholesterol
CT60 mutant cells
Cessation of mutant NPC1-GFP
trafficking in NPC cells
10

11. NPC1-GFP regenerates LET trafficking in
NPC cells
NPC1-GFP adenovirus is fully functional.
NPC1-GFP Cholesterol (Filipin)
Without
NPC1-GFP
With NPC1-GFP
Adenovirus
CT60 cells
11

12. Adenovirus-NPC1-GFP infected rat cortical neuron
(axon-like neurite)
Adenovirus-NPC1-GFP infected rat astrocytes
12

13. A Networking Trouble?
- A novel NPC cell defect
Normal cell fusion
NP-C cell fusion
Can NPC cells exchange lysosomal content ?
If not, what inhibits the dynamic networking?
Rhodamine-dextran loads
NPC1-GFP-containing late endosomes and
large lysosomes
NPC1-GFP RD SSD-NPC1-GFP RD
13

14. Procedure for examining lysosomal content exchange
• Normal cells
Incubate with fluorescent dextran
for 24 hrs and wash for 2 hrs • NP-C cells
• LDL
Trypsinize and mix cells
• LPDS
LPDS:
Plate mixed cells Lipoprotein-deficient serum
(bovine)
Fuse with Polyethylene Glycol (PEG)
Fix at various time points
(5 min to 48 hrs)
Complete exchange of contents in fused WT CHO
Normal cell fusion
Living Cells, 2 hrs
after fusion
Red Channel Green Channel
14

15. NPC1 transports and clears cholesterol out of
lysosomes; by doing this, NPC1 acts to:
• Maintain the dynamic networking
and communication among
lysososmes;
• Properly sort lysosomal glycolipids
and direct lipid trafficking to the
proper places;
• Keep efficient long distance
transportation along microtubules;
• Maintain CNS function (PNAS February
17, 2009 vol. 106 no. 7 2377–2382 Benny Liua, …
and John M. Dietschy.)
LET and microtubules
Disruption of microtubules prohibits content exchange
of late endocytic compartments in fused cells
Nocodazole treated
WT CHO cells, 2 hrs after fusion
15

16. Content exchange among lysosomes is blocked in
fused CT60 cells
2 h after fusion
Cholesterol-depletion partially regenerate
tubular trafficking
LDL
(cholesterol-
enriched)
LPDS
(cholesterol-
depleted)
16

17. Lysosomal content exchange is recovered in fused
CT60 cells depleted of cholesterol
2 h after fusion
Content exchange among lysosomes is regulated
by endosomal cholesterol content
LDL LPDS
-
17

18. Glycolipids traffics in NPC1 compartment
NPC1 Glycolipids mAb
GM2
Lac-Cer
GD3
CTH
fibroblasts
NPC1, NPC2, Endocytosis and Drug Discovery
18

19. Cholesterol depletion study on mice
Survival Liver Neurode-
function generations
Controls
(Saline, Allo., No improv. No improv. No improv.
Ezetimibe)
CYCLO at Improv. Improv. Improv.
day 7.
beta-cyclodextrin
(‘CYCLO’) CYCLO at No improv. Improv. No improv.
day 49.
PNAS February 17, 2009 vol. 106 no. 7 2377–2382 Benny Liua, … and John M. Dietschy
Cholesterol depletion improved NPC1 disease
mouse model (npc1-/-)
PNAS February 17, 2009 vol. 106 no. 7 2377–2382
Benny Liua, … and John M. Dietschy
19

20. NPC1 disease - a trafficking disorder!
• NP-C disease has a defective late endosomal tubular
trafficking function due to cholesterol accumulation.
• Reducing cholesterol improves NPC1 in vitro as well
as in vivo (Dr. John Dietschy, PNAS 2009 106:2377).
cholesterol
NPC1 transports and clears cholesterol out of
lysosomes; by doing this, NPC1 acts to:
• Maintain the dynamic networking
and communication among
lysososmes;
• Properly sort lysosomal glycolipids
and direct lipid trafficking to the
proper places;
• Keep efficient long distance
transportation along microtubules;
• Maintain CNS function (PNAS February
17, 2009 vol. 106 no. 7 2377–2382 Benny Liua, …
and John M. Dietschy.)
LET and microtubules
20

21. Mucolipidosis IV disease
Mucolipin-1 is a channel protein
cytoplasm
HN2 COOH
F408del
membrane
D362Y
V446L
4(32aa del)
lumen
TRP cation channel domain
21

22. Mucolipin-1 Resides in the Late
Endocytic Compartment
Mucolipin-1 Rhodamine-dextran GFP-Mucolipin-1
Mucolipin-1-GFP
GFP-
Mucolipin1
Mucolipin1-
GFP
GFP
CHO Cells
Ion flow direction is depended upon
mucolipin-1 topology on lysosomal membrane.
Ion flow
Or
Ion flow
Late endosome / Lysosome Late endosome / Lysosome
22

23. Topological Analysis on Mucolipin-1 Indicates Ion Flow
from the Late Endocytic Compartment to the Cytoplasm
Ion flow
GFP-mucolipin-1 NPC2-RFP
Late endosome / Lysosome
Mucolipin-1-GFP NPC2-RFP
NPC2-GFP as a tool…
No leupeptin Leupeptin 12h
NPC2-RFP
Niemann-Pick Disease
type C2 protein
RFP is protease
resistant!
23

24. Mucolipin-1-GFP is delivered to lysosomes via a biosynthetic pathway
that bypasses the plasma membrane, which is different from LAMPII
LAMP2-GFP Mucolipin-1-GFP
Transfection 24 hr
Transfection 72 hr
Association of late endosomal mucolipin-1-GFP with the ER
GFP-Mucolipin-1 24hr
Rhodamine-dextran
24

25. F408del-GFP mutant is delivered to the late endocytic compartment
in MLIV fibroblasts
F408del-GFP V446L-GFP D362Y-GFP
F408del Rho-dextran
Calcium Orange Labeling:
Calcium accumulation in the late endocytic compartment in MLIV fibroblasts
Normal Niemann-Pick C
KpnI cut uncut (CWN) (GM3123)
KpnI cut 2527
2629 2527 2629
HeteroHomoNormal
MLIV MLIV
(GM2527) (GM2629)
25

26. Calcium Orange is
trapped in the lumen of
F408del-GFP defined
late endosomes and
lysosomes
Expression of mucolipin-1 prevents calcium accumulation in the
late endocytic compartment in MLIV fibroblasts
GFP-Mucolipin-1 Calcium Orange F408del-GFP Calcium Orange
Mucolipin-1-GFP Calcium Orange
Note: Loaded dye first then transfect:
Calcium Orange Mucolipin-1-GFP
26

27. Calcium starvation damages MLIV fibroblasts
Ca2+ Ca2+ Ca2+ Ca2+
200mg/L Free 200mg/L Free
A B G H
MLIV
Normal
fibroblasts1
human
(GM2527)
fibroblasts
1
C D I J
Normal MLIV
human fibroblasts2
fibroblasts (GM2629)
2
E F
NPC1
fibroblasts
Calcium depletion induces death of MLIV fibroblasts
Ca2+ 200mg/L Ca2+ Free
live dead live dead
Normal
human
fibroblasts 1
Normal
human
fibroblasts 2
Live
NPC1
fibroblasts
Dead
MLIV
fibroblasts1
(GM2527)
MLIV
fibroblasts2
(GM2629)
27

28. Mucolipidosis IV has defective protein
trafficking and endocytic calcium accumulation
• ML4 disease has defective mucolipin protein
trafficking and some mutants are stuck in the ER.
• ML4 disease presents intracellular calcium
homeostasis defect.
• Modulating calcium homeostasis may improve
ML4 patients.
The Whole Story in One Big Movie
28

29. Topic 2
Drug modes of action study
with in vitro imaging
- Elesclomol is a special
proteasome inhibitor
Microtubules in CHO cells expressing tubulin-YFP
Elesclomol
Elesclomol was known as:
• HSP70 inducer
• Cytoskeleton disorganizer
• Paclitaxel enhancer (in vivo)
Taxol treated MCF7 cells expressing tubulin-YFP
29

30. Elesclomol induces accumulation of
Tubulin-YFP at the centrosome in CHO cells
DMSO Elesclomol
CHO cells (12hr, 500nM)
Elesclomol and known proteasome inhibitors
cause similar phenotypes in CHO cells
5hr 24hr
DMSO Elesclomol Elesclomol ALLN
MG132 Lactacystin
MG132 Lactacystin
30

31. IXMicro solution is now involved in
centrosome !
HCS analysis for the centrosomal
1. effect
Multiple known proteasome inhibitors
2. 80 compounds from MicroSource (‘Cancer Plate’)
31

32. Quantitative analysis of centrosomal proteasome inhibition by
Elesclomol and other proteasome inhibitors
“Plate 3, 24-26hr treatment”
Z factor = 0.50-0.68
Centrosomal Average Intensity
Elesclomol vs. derivatives and proteasome inhibitors
32

33. Quantitative analysis of centrosomal proteasome inhibition
by 80 control compounds (Cancer Plate)
“Plate 4, 24-26hr treatment”
Centrosomal Average Intensity
Elesclomol vs. 80 anti-cancer drugs
33

34. CHEMISTRY
O
H
N
ID MOLENAME
80 cpds for centrosome assay Chemistry 34
NH3
1501189 DEOXYURIDIN
O O
Pt
NH3
HO O
Chemistry 1 100537 SOLASODINE O Cl
O
HN Cl Chemistry 35 1502106 CARBOPLATIN
O-
O
O O
O
N+ OH N
O
NH3
O OH OH OH Cl
O CHLORAMPHEN O
OH
Pt
O
Chemistry 13 1500174 OL
HO
O
TAMOXIFEN NH3
O
Chemistry 24 1500557 CITRATE Cl
Cl N
Chemistry 2 200013 ROTENONE
O O Chemistry 36 1502107 CISPLATIN
HO P OH HO P OH S
O O HN OH
O
OH
OH
H O
N
HCl
HN HN
N
O N
OH O OH O N
CHLOROQUIN H2N N N HO O
O
OH Chemistry 14 1500179 DIPHOSPHAT
AKLAVINE Chemistry 25 1500573 THIOGUANINE N+ N
N-
Chemistry 3 200022 HYDROCHLORIDE Cl
Chemistry 37 1502109 ZIDOVUDINE [AZ
Cl -
H2O O N
NH2
O O
O
O P N N
NH Cl H2N N+ NH2 N O
OH
O CYCLOPHOSPH
O
Chemistry 15 1500213 MIDE HYDRAT ACRIFLAVINIUM O
O OH
Chemistry 26 1500618 HYDROCHLORIDE
OH
Chemistry 4 201138 DEGUELIN(-) O HN
O
N
S O Chemistry 38 1502111 5-AZACYTIDIN
O OH HO OH H N
2
N N
O
O O
OH
O OH
H O
O OH O O
O OH O NH
DIETHYLCARBA S H 2N
HO Chemistry 16 1500242 AZINE CITRAT
HO OH
PENTAMIDINE OH NH
Chemistry 27 1500641 ISETHIONATE
O
Chemistry 5 201664 CELASTROL O
HCl
O
N Chemistry 39 1502112 CYCLOHEXIMID
O O
H Cl
H
N O
O O O
N-
HO +
O N
O
OH O O OH
Chemistry 17 1500272 EMETINE MYCOPHENOLIC
OH O NH2
Chemistry 28 1500674 ACID
Chemistry 6 300038 JUGLONE H
N O H2N N N Chemistry 40 1502113 AZASERINE
OH H
N N
O S O O N O
O
-
O
NH H
+ F NH2 N
N OH F O
O O O
HO O
O
Chemistry 18 1500305 FLUOROURAC Chemistry 29 1500679 AMINOPTERIN H2N OH p-
SANGUINARINE FLUOROPHENY
Chemistry 7 310035 SULFATE O
OH O
Chemistry 41 1502114 LANINE
O
N
HN
HN
H
O O
O
O N N
+ HN NH 1,2-
O N
O
O
O H2N O O
N
HCl DIMETHYLHYDR
O
O Cl -
O NH2
NH
N
BERBERINE ZINE
H
O O Chemistry 19 1500344 HYDROXYURE
O
N N
Chemistry 30 1500811 CHLORIDE Chemistry 42 1502116 HYDROCHLORID
O
O
Cl
Chemistry 8 330001 DACTINOMYCIN OH O OH O
HO
O O
S N
OH
O O Cl HO
METHYLMETHANE MECHLORETHA O PHENETHYL
Chemistry 9 330003 SULFONATE Chemistry 20 1500375 NE Chemistry 43 1502209 CAFFEATE (CAP
Chemistry 31 1500898 EMODIN
O
N
N S N
HCl
O - N
N+ H N
N
N N
S
O HN N N OH
O
NH 2H C l
O
N O N
HN H
N N OH
N OH O
N MERCAPTOPUR O
Chemistry 21 1500387 E PUROMYCIN Chemistry 44 1502232 CAMPTOTHECI
Chemistry 10 1500133 AZATHIOPRINE
Chemistry 32 1501105 HYDROCHLORIDE
HCl
O
N
O O N
O
S O HCl
O O
O O
O S NH HO
O O
O OH
HO OH
HO OH OH
O Cl N
HO O
H
OH
QUINACRINE O OH
Chemistry 11 1500152 BUSULFAN Chemistry 22 1500522 HYDROCHLORI Chemistry 45 1502244 AMYGDALIN
Chemistry 33 1501136 NERIIFOLIN
O OH O
O
O
OH O OH
O F
O
NH
H NH Cl HO OH
HO H
HO OH
O Cl O N O
HO NH N
OH HO O
N O
+
CHLORAMPHENIC O O
- N
O O
OL OH
Chemistry 23 1500543 STREPTOZOS 5-FLUORO-5'- Chemistry 46 1502245 ELLAGIC ACID
Chemistry 12 1500173 HEMISUCCINATE
Chemistry 34 1501189 DEOXYURIDINE
The proteasome effect might be a consequence of
ROS induction…
Multi-
Tubulin-YFP ubiquitinated In Vitro
Proteosensor
Hsp70 accumulation Protein Proteasome
accumulation in
induction accumulation inhibitory
in cells cells
activity
in cells
Proteasome
+ + + + +
inhibitors
Elesclomol + + + + -
34

35. Topic 3
Tracking cellular targets of
-secretase inhibitors for
Alzheimer’s disease
-secretase inhibition and
APP accumulation in the ERC
35

36. Early Endosomal Recycling Pathway
Recycling
APP Processing and Alzheimer’s Disease
36

37. C99-GFP accumulates intracellularly in stable clone
when  -secretase is inhibited (24h treatment)
DMSO DAPT
C99-GFP, accumulates following treatment with
PS/-secretase inhibitors
DMSO DAPT L-685,458
APP-C99-GFP
actin
37

38. Accumulation of APP-C99-GFP occurs first on the
cell surface and then in the recycling compartment
DAPT treatment
1 hr 3.5 hr 10.5 hr
Bottom
Morphological changes of APP-C99-GFP -containing
compartments during Nocodazole or BFA treatment
(Clone-27C)
DAPT only DAPT + Nocodazole DAPT + BFA
38

39. AD mutants APP-CTF-GFP accumulate intracellularly
when treated with DAPT
EGg V717F V717I
D
M
S
O
D
A
P
T
APP-C99-GFP accumulates both in endosomes and on the plasma
membrane following treatment with the PS/-secretase inhibitor DAPT
Top 2 3
4 5 Bottom
39

40. Accumulated C99-GFP is in the early endosomes
when -secretase is inhibited
C99-GFP Rho-Tfn
C99-GFP Rho-dextran
APP-C99-GFP accumulates in ERC + PM following treatment with
DAPT and L-685,458 in cells stably transfected with APP-C99-GFP
APP-C99-GFP
Transferrin
DMSO
C99-GFP
actin
DAPT
L-685,
458
40

41. APP-CTF-YFP accumulates in the recycling compartment following
treatment with two different PS/-secretase inhibitors in cells
transiently transfected with full length APP-YFP
DMSO DAPT L-685,458
APP
Plasma Membrane
APP(full-length)
BACE1
APP
A
Anti-GFP
APP-
CTF
blotting APP-CTF
* (free YFP)
Secretase
Complex
(PS1, PEN2,
actin
Nicastrin, APH1) APP-YFP + Transferrin
Development of gamma-secretase inhibitors
 Eli Lilly with LY450139 in Phase II
 Merck with MK0752 in Phase II
 Eisai with E2012 in Phase I
 Elan in preclinical
41

42. Summary
 GFP- and digital imaging-based live cell
analysis is an ideal technology to identify
defects of intracellular trafficking of
proteins.
 GFP- and microscopy-based cell biology
technologies can be applied to protease
inhibitor assays (lysosomal protease and
early endocytic g-secretase) in live cells.
Conclusions
 Inhibition of gamma-secretase causes
APP-CTF accumulates in the early
endocytic pathway.
 This cellular phenotype (APP-C-GFP
accumulation) is applicable to HCS for
secretase inhibitor drug discovery.
42

43. Topic 4
Screening and
optimization for CXCR4
inhibitors
Dual color masking of Chem-1-CXCR4 cells for ‘happy feet’ formation
CXCR4 and SDF1 mediate cancer metastasis
CXCR4
SDF1a (CXCL12)
Note:
CXCL12 = SDF1
Luker and Luker
(2005) Cancer
Letters 238:30-41
43

44. Key assays in SDF1 signaling pathways
3
Assays:
1. Calcium Mobilization
2. Cell Migration
1
4 3. Receptor Binding
4. cAMP production
5. Endocytosis
2
5
The platform
High content analysis
Cell-based assays
High throughput screen
Medicinal chemistry
Outsource
collaboration
Computational
Chemistry
Academic collaboration
44

45. Transflour assay Cyto-Nuc Translocation
HCA, HCS
Assays
Transwell migration
HCA - HCS
Pseudopodia formation Endothelial tube formation
The BioImaging Network:
JPEG, BMP, Excel
JPEG, BMP, Excel
Regular Microscopy Data
IXMicro HCS Data in in Local Drive
Local Drive (Biol)
JPEG, AVI
(Biol)
Videomicroscopy
Data in Local Drive
(Biol)
BioImaging
AcuityExpress Network Drive
(Biol and IT) (Biol)
JPEG, AVI, Excel
JPEG, BMP, Excel IncuCyte Data
In Local Drive
(Biol)
Tape Recording (IT)
45