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