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In vivo animal imaging
Need for animal models with adequate genetic/phenotypicbackground and closed to what observed in humans.
o to analyze the first steps of tumor development
o to evaluate the relationship between genetic alterationsand tumor progression
o to identify new therapeutic targets
o etc…
Animal models in oncology
Tools for evaluation of tumor burden in vivo :
o magnetic resonance imaging (MRI)
o positron emission tomography (PET)
o bioluminescence
o optical fluorescence imaging (proteins, probes, near infrared dyes)
Science 299: 1972, 2002
Animal models in oncology
PET for preclinical evaluation of tumor prolifration[(18)F]-FLT = 3'-deoxy-3'[(18)F]-fluorothymidine
Limitationsocannot discriminate moderately proliferative, thymidine salvage‐driven tumors from those of high proliferative index that rely primarily upon de novo thymidine synthesis.o reflects proliferative indices to variable and potentially unreliable extents (unlike proliferation markers like Ki67)
PLoS One. 2013;8(3):e58938.
Optical imaging modalities
Bioluminescence (BLi) :
Cells are transfected with a luciferase gene that emits photons in the presence of Luciferin, ATP and oxygen
Featureso Detects the presence of cells expressing a Luciferase geneo No need for imaging agents
LimitationsoRequires the use of transgenic cellsoNot compatible with clinical studiesoLimited to imaging growing cells
(luciérnaga)
In vivo imaging using transgenic tumoral cells/bacteria
Bone marrowPlasma cell
Memory B cell
MantleGerminalcenter
Marginalzone
Precursor B cell
Naive B cell
Peripheral B cell malignancies (mature)Precursor B cell malignancies
t(14; 18)BCL2
t(8; 14)cMYC t(3q)
BCL6
t(11; 18)API2‐MALT
?Follicularlymphoma
Burkittlymphoma
Diffuselarge B
cell lymphoma
MALTlymphoma
Chroniclymphocyticleukemia
splenic/marginal zone lymphoma
Leukemia/lymphoma
lymphoblastic/o B
Chroniclymphocyticleukemia
MantleCell
lymphoma
Lympho‐plasmacyticlymphoma
MultipleMyeloma
t(11;14)Cyclin D1
t(14; 18)BCL2
B cell lymphoid malignancies seem through the lymph node
Overall survival of patients with non‐Hodgkin lymphoma (Hospital Clinic, 2012)
Aggressive B‐cell lymphoma study group (IDIBAPS): main goals
Genetic, molecular and physiopathologicalstudy of various B‐NHL subtypes for thedesign of potent and selective therapies:
o Search for selective therapies against deregulatedsignaling pathways in preclinical models of MCL, FL,DLBCL and DH lymphoma
o Mechanism of resistance to conventional therapies(mutations, microenvironment, ..)
o HTS screening of new therapeutic compounds. Molecularbases underlying drug efficacy and selectivity
o In vivo validation in xenotransplant mouse models
Experimental design
Peripheral blood or tissue Purification
of lymphoid cells
B‐NHL cell lines (n=32)
Mouse xenotransplant(SCID/NSG)
Single agent or combined therapies
Cytotoxic assays, apoptosis measurement and molecular determination of anti‐tumoral signaling
n > 600fully
characterized and cryopreservedCLL/MCL/FL and DLBCL samples
24h
casp‐3/‐7 ROS+ G2/M blockadeΔΨm loss sub‐G1cell shrinkage
24h
48h
MCLprimaries(n=3)
24h
48h
24h
48h
Multiparametic detection of novel putative antitumoral agents by flow cytometry
MCLcell lines(n=5)
48h
24h
48h
24h
48h
24h
48h
24h
48h
24h
CT
Cpd 1 Cpd 2CT
Cpd 1 Cpd 2CT
Cpd 1 Cpd 2CT
Cpd 1 Cpd 2CT
Cpd 1 Cpd 2
CT
Cpd 1 Cpd 2
Sample processing and analysis
1. Production of GFP‐luciferasa Lentivirus2. Infection of B‐NHL cell lines (MOI determination, cell sorting
(GFP) and luciferase activity in vitro)3. Subcutaneous (s.c.) or intravenous (i.v.) injection in
immunocompromised mice (SCID or NSG) and luminescence detection
Hamamatsuimaging system
Introduction of LUC‐GFP genes in lymphoid cell lines using lentiviral particles
Envelopegenes
Capsidgenes
3‐step protocol for GFP‐LUC lentivirusproduction
2.5 1 0.5 0.1 nb of cells (x106)
REC‐1
Z‐138
JeKo‐1
Granta‐519
GFP/mCherry signal (CMF)
Luciferase activity in vitro
oExperiment: Z138‐Luc oSCID miceo107 cells/mouse (s.c.)o75mg/kg luciferine (i.p.)
Nº Ratón
# 0
# 1
# 2
8 days 15 days 22 days
5 MIN 45s3 MIN
Luciferase activity in vivo
Day 1 (5’) Day 15 (5’) Day 21 (3’)
15
17
18
MouseFRONT FRONT BACKFRONT BACK
Day 28 (2’’) FRONT BACK
15
17
18
Mouse
Luciferase activity in vivo
oExperiment: RL‐LucoNSG miceo107 cells/mouse (i.v.)o75mg/kg luciferine (i.p.)
Enlargedovary
Enlargedovaries
Normal ovary
Enlargedovary
Normal ovary
Luciferase activity in vivo
Flow cytometry detection of CD45+/CD20+/CD10+ cells
1.Peripheral blood (PB)2. Bone marrow (BM)3. Spleen4. Brain5. Ovary
0
50000
100000
150000
200000
250000
300000
350000
400000
17 18
Total tum
or cells
Mouse number
Bone Marrow SPLEEN Ovary Brain
FL cell recount by flow cytometry
# 1
# 2
# 0
30 days
45s
Luciferase activity in vivo
0
500
1000
1500
2000
2500
3000
0 7 15 22 30
Tum
or v
olum
e (m
m3)
Z138Luc
oDaratumumab (human anti‐CD38) activity in subcutunaeous xenograft models
(SCID) with the FL cell line RL‐Luc
o Cell line shows strong CD38 expression and good Dara‐induced ADCC in vitro
o i.p. treatment after tumor growth. Treatment with a loading dose of 20mg/kg
to ensure target saturation, followed by weekly dosing of 10mg/kg .
Day 1 Day 20 Day 27 Day 34 Day 41 Day 42
Example#1: anti‐CD38 therapy in FL‐bearingmice
0
500
1000
1500
2000
2500
0 10 20 30 40 50
Volu
me
(mm
3 )
Days
Control IgG1-B12 Daratumumab
0,0
1,0
2,0
3,0
4,0
5,0
Control Daratumumab
Tumor weight (g)
CT dara
Example#1: anti‐CD38 therapy in FL‐bearingmice
96
74
73
8493
57
56
9
94
3
75
Day 8 (3’) Day 15 (5’) Day 22 (2’’) Day 29 (3’’) Day 36 (1’’)Control
Daratumumab
Mouse
Moreno et al, J Pathol , 2014
Levels of CXCR4 surface expression correlate with aggressiveness after injection into mice.
*
CXCR4 inhibition through Plerixafor (AMD3100) rescues mice from DLBCL agressiveness
Example#2: Role of surface CXCR4 in DLBCL
Bioluminescence (BLi): applications for drugdiscovery in lymphoma research
Limitations:oRequires the use of transgenic cellsoNot compatible with clinical studiesoLimited to imaging growing cells
Evolución de peso
15,0
16,0
17,0
18,0
19,0
20,0
21,0
22,0
23,0
24,0
0 5 10 15 20 25 30 35
Días
Peso
(gr)
SUDHL6 iv
Brain
Spleen
Ovary
*OCT +FFPE blocks
*
How to monitorize tumor burden while using hard‐to‐transfect malignant B cells?
SUDHL-6 (DLBCL cell line), 8x106 cells by i.v.
H&E CD20Brain Ovary
H&E CD20
Recasens et al, unpublished data
Limitations:ovisible fluorophores do not offer optimal performance for all applicationsocells, animal tissue, plasticware, blotting membranes, and chemical compound libraries all possess intrinsic autofluorescence that can interfere with detection
Solution:oin the near‐infrared (NIR) spectral region (700‐900 nm), autofluorescent background is dramatically reduced.
Use of GFP‐expressing cells or fluoresent probes
Hemoglobin (Hb) and other tissue components strongly absorb visible light.In the NIR region, where IRDye agents are detected, tissue absorbance is
dramatically reduced. Above 820 nm, light absorbance by water increases and can affect performance.
Animal tissue absorbs visible light
IRDye® infrared dyes: first commercialized in 1993 by LI‐COR Biosciences:oreduced autofluorescentbackgroundohigh sensitivityoenhanced signal‐to‐noise ratiosowide dynamic rangeofastoeasy to conjugate to various moleculesonative cell lines may be usedopotential for clinical translation
Applications: Western Blotting, Multiplex Phosphorylation Analysis, ProteinMicroarrays, In‐Cell Western™ Immunofluorescent Assay, Electrophoretic Mobility Shift Assays (EMSA), IRDye FRET Assays for Protease Activity, Mycroscopy, In vivo Optical Imaging
Near‐infrared (NIR) fluorescence detection system using NIR dyes
IRDye fluorophore and characteristics
Pearl ImpulseFieldBrite™ Xi CCD-based optical system
oAffordable benchtop systemo Best NIR detection: FieldBrite Xio Simplicity – No adjustments.o Dynamic Range – No saturation, long term studieso Speed – Fluorescent images in 30 secondso Sensitivity – NIR allows detection of deeper targetso Reliability – The system simply WORKS!o Designed to work with IRDye 800CW
Odyssey® FcSystemFieldBrite™ XT optical system
oSystematic approachoExceptional reagentsoHighly specificoin vivo and in vitrooExcellent binding activityoEfficient clearanceoResearch versatility
NIR imaging systems
Acquisition and analysis: Image Studio v5.0
NIR optical imaging: non‐invasive study of molecular targets inside the body of the living animal to:ofollow the progression of diseaseoevaluate the effects of drug candidates on the target pathologyoanalyze the pharmacokinetic behavior of drug candidatesoto develop biomarkers indicative of disease and treatment outcomes.
IRDyes: workflow
IRDyes: administration site and clearence
i.p. i.v.
15min post‐injection
Comparison of target‐to‐background ratio (TBR) with IRDye® 800CW and Cy5.5 fluorescent conjugates.
Adapted from Adams, KE et al. J Biomed Optics 12: 024017 (2007)
IRDyes: improved target‐to‐background ratio
Increased glucose metabolism imaged with IRDye® 800CW 2‐DG. Subcutaneous A431 tumor was detected. Image acquired with Pearl® Impulse
Uptake of IRDye® 800CW 2‐DG probe by hypoxic tissue surrounding the necrotic center of a tumor. A431 tumor was excised, fixed, and embedded. Tissue sections were then imaged at 21 μmresolution with Odyssey® Classic Imager. Green indicates probe fluorescence (800 nm) and red indicates tissue autofluorescence
Example#1: use of IRDye 2‐DG optical probe forthe monitoring of tumor outgrowth and validationof novel therapeutic agents in B‐cell lymphoma
Proteasome Inh
CD20
AbαCD40
CD40L
CD40
CD40L
CD40
TRAIL‐R
TRAIL
AbαDR4 AbαDR5
CXCR4
SDFα1
PI3K
Akt
mTOR
NF‐ΚB
BCL‐2 Inh
IAPsAntagonists
IL6TNFα
IAPsIAPs
PI3K/Akt/mTORAxis Inh
NF‐ΚB Inh
Mitochondria
stromal cellsnurse‐like cellsfolicullar dentritic cellsmacrophages
Nucleus
IL‐4
Nucleus
B cell neoplasm
BAFF
APRIL
TACI
BCMA
HSP‐90
HSP90 Inhibitors
SrcK
CD38
CD31
BCR
BAFF‐R
PK Inh
AbαCD20
AbαCD38
T Lymph
VEGF
VEGF‐R
ImiDS
IL10ImiDS
DNMT/HDAC Inhibitors
NucleosideAnalogs
Saborit-Villarroya et al, Curr Drug Targets. 2010
⇑ NoxaATF3 ATF4
H2ABortezomib
Bax
⇓ ΔΨm
SmacCyt C
APOPTOSIS
Bak
⇑ Mcl-1
⇑ Undegraded proteins
ER stressROS generation
Unfolded ProteinResponse (UPR)
UPRAdaptive response
SURVIVAL Perez‐Galan et al, Blood 2006Perez‐Galan et al, Blood 2007Wang Q et al, PNAS 2009Roue et al, Blood 2011Weniger et al, CCR 2011
BH3 MIMETICS
GX15‐070 MCL (Bortezomib) phase I/II Protocol ID GEM012
Bortezomib in B‐NHL: proposed mechanism of action
MYELOMA IRF4
NES=1.87; FDR=8x10-3
ACSS2ADAM9AMPD1APOBEC3BAPOBEC3DAPOBEC3HATF3ATF4AXIN1BCL2CAV1CCDC134CCL3CCNCCCPG1CD38CDK6CDKN1BCENPFCMTM8CYP51A1DDIT3DEPDC1DNAJB9EIF2C2ELL2FBXO16FHL1FKBP11GAAGFPT1GLDCGNG7GPT2GTF2IRD1HSPB1HVCN1IRF4ITGB7ITPKAKLF2KRASLDLRLDLRAP1MALAT1MAN2A1MICBMNAT1MTHFD1LMYBNFIL3NUP205PABPC4PDK1PRDM1RAB33ASKP2SLC3A2SORT1SPATS2
242-4
CCL3CD38CD59CYP51A1FKBP11GFI1IER3INSRPRDM1RGS1RRAGDSDC1SELPLGSSR3TNFAIP3WNT5B
PLASMA CELLVS B-CELL
NES=2.21 FDR=0
262-6
BLIMP TARGETS
232-3
AICDAALOX5ANP32AARPC2ATP2A3BCKDHABCL6BLNKBTKCD180CD19CD22CD24CD27CD37CD52CD53CD79ACD86CDKN2CCHKACORO1ACSKCYFIP2FCRLAGCET2GNB2IFI16IL16INPP5DIRF8LYNMAP2K1MYBL2MYO1EPAG1PHKG2POU2AF1POU2F2RASSF7RPS6KA1SH3KBP1SP110SP140SPIBSREBF2SYKTLR10TRAF5UCP2VNN2VPREB3WIPF1
NES=1.86; FDR=3x10-3
BLOOD PAN-B CELL
NES=-1.93; FDR=1x10-3
242-4
AIM2BANK1BLKCACNA1ACCR6CD19CD22CD24CD37CD40CD72CD79BCNTNAP2FADS3FCRL1FCRL2FCRLAIFT57LARGEMETTL8MTSS1OSBPL10P2RY10PAWRPCDH9QRSL1SWAP70SYT17ZCCHC7
Moros A et al. Leukemia. 2014 Oct;28(10):2049‐59.
c‐myc
β‐actin
IRF4
42
cereblon
Lenalidomide: a post‐translational inhibitor of IRF4
43Adapted from Filippakopoulos P, Knapp S. Nat Rev Drug Discov. 2014 May;13(5):337-56.
MYC
BCL2
NF-κBtargetgenes
CDK6
Aurora
kinase
CPI203 (BETi): interferes with BRD4 and MYC transcription
CPI203
Moros A et al. Leukemia. 2014 Oct;28(10):2049‐59.
0
10
20
30
40
50
60
70
80
90
100
CPI203 Lenalidomide Combo
Cyt
osta
tic e
ffect
(% o
f con
trol)
CI = 0.29
p = 0.009p < 0.001
MYC/β-actin ratio 0.56 0.36 0.52 0.19 0.12 0.11
MYC
β-actin
IRF4
Apoptosis (%) 16.7 15.3 16.5 17.2 73.1 79.8
+CPI203
[lenalidomide] 0 1 5 0 1 5(μM)
IRF4/β-actin ratio 1.30 0.71 0.94 0.81 0.26 0.21
Synergistic activity of lenalidomide and CPI203 inbz‐resistant MCL (in vitro)
Vehicle CPI203 lenalidomide
IRF4
H&E
combo
c-Myc
p-histone H3
act. caspase-3
01000200030004000500060007000
13 18 23 28 33
Tum
or v
olum
e (m
m3)
±SE
M
Days post-inoculation
VehicleLenalidomide 50 mg/KgCPI-203 2.5 mg/kgcombo ****
*
vehicle lenalidomide CPI203 combo
Moros A et al. Leukemia. 2014 Oct;28(10):2049‐59.
Synergistic activity of lenalidomide and CPI203 in mouse model of bz‐resistant MCL
ABT‐199 and CPI203 combo in MYC+/BCL2+ double hit lymphoma
Juan Garcia, Anna Esteve, David Gonzalez, 2015
control ABT 10 nM ABT 50 nM
n= 4 DH lymphoma cell lines
Sin CPI 48hCPI 0.1 48hCPI 0.5 48h
Rel
ativ
e nu
mbe
r of c
ells
ControlCPI203 100nMCPI203 500nM
0
10
20
30
40
AnnexinV+ cells casp‐3/‐7+ cells
% of cells
controlCPI203ABT199combo
c-Myc
β-actin
Bcl-2
ct[CPI203]
Mcl-1
0,00
0,20
0,40
0,60
0,80
1,00
1,20
Rel
ativ
enu
mbe
rof c
ells
ControlSiRNA 0,5SiRNA 5
ct
[ABT-199]
Scramble siRNAMCL-1 siRNA (0.5uM)MCL-1 siRNA (5uM)
Synergistic activity of ABT‐199 and CPI203 in vivo
Juan Garcia, Anna Esteve, David Gonzalez, 2015
0
0,2
0,4
0,6
0,8
1
1,2
Control CPI‐203 Lena/Dexa Combo
Prolife
ración
relativ
a a control
***
*
Prolife
ración
relativ
a a control
*****
*
Cell lines(n=7)ANOVA test: p=0,0003
MM primary cultures (n=9)
ANOVA test: p<0,0001
CPI203 and lenalidomide/dexamethasone combo in multiple myeloma
Tania Diaz, 2015
0
20
40
60
80
100
120
Control CPI‐203 Lena/Dexa Combo
RFU
Control ComboLena/Dexa CPI203
Tania Diaz, 2015
Synergistic activity of CPI203 and Len/Dex therapy MM xenostransplant
Celgene CorporationAntonia Lopez-Girona
AcknowledgementsHospital Clínic i Provincial de Barcelona,
Hematopathology UnitAntonio Martinez
Elias CampoDolors Colomer
Hematology DepartmentArmando Lopez-Guillermo
Financial support
Constellation Pharmaceuticals
Emmanuel NormantPeter Sandy
IDIBAPSAggressive B-cell lymphoma
study groupAlexandra MorosDavid Gonzalez
Anna EsteveIvan Dlouhy
Clara RecasensVanina Rodríguez
Patricia Pérez-GalánGaël Roué
Hemato-Oncology DptTania Diaz
Carlos Fernandez
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