Terapie Cellulari e Geniche per Neoplasie Solide

Laboratory of Cell Biology and Advanced Cancer Therapy

Department of Oncology & HematologyHospital-University of Modena and Reggio

Emilia, Italy

Gaiato, July, 16th 2011

Obiettivo Laboratorio Terapie Oncologiche Avanzate

Sintetizzare innovative cito-terapie anti-cancro focalizzandosi su:

A) Cellule Staminali Adulte Cellule Staminali Ematopoietiche CD34+

Cellule Staminali Mesenchimali (MSC)

B) Effettori citotossici CD8+

NK

Potentials of Mesenchymal Stem/Stromal Cells (MSC)

Lung

Skin

LiverHeart

SkeletalMuscle

BrainIntestine

Kidney

Dominici M et al. 2001, 2004, 2009 Bone

How do Mesenchymal Stromal Cells Interact with Tumors?

Breast Cancer Specimen

Tumor Cells

Tumor-associated fibroblasts (TAF)

Normal Tissue

Lymphocytes e NK cells

Orimo AP et al. 1999; Orimo, AP et al. 2005

The Mesenchymal Tumor Stroma: Tumor-associated Fibroblasts

Secreting Growth Factors And Cytokines Which Promote Proliferation And Survival

Contributing To The Generation Of New Blood Vessels

Driving The Recruitment Of Inflammatory Cells

The Role of TAF

The Origin of TAF

Grisendi G et al, 2011

Can We Artificially Substitute TAF With Infused MSC For A

Therapeutic Benefit?

MSC as Cytopharmacological Vehicles

MSC tropism has been reported in a variety of tumor types

MSC are easily accessible from marrow and other sources and readily propagate in culture

MSC can be genetically modified to express desired gene products (retro/lenti/AAV)

MSC retain a high metabolic activity for sufficient generation of therapeutic agents

Bussolari et al. 2011

Tumor

MSC

Vessel

Gene Therapy

Ex Vivo

Donor/Recipient

CellVector

Manipulated Cells

Expansionof cells

Going Back to Immunology: How do CTL/NK Cells Kill

Tumors?

& TRAIL

TRAIL-R

What is TRAIL?

TNF-related apoptosis inducing ligand (TRAIL) belongs to the death ligand family (w/ FasL, TNF, RANKL)

TRAIL is naturally produced by NK and CD8+ T cells

TRAIL acts controlling “cellular disfunctions” Autoimmunity (i.e Diabetis) Viral Infection (i.e HIV)

CancerTRAIL acts on specific receptors specifically located on “transformed” cells sparing normal tissues

Wiley SR. et al. 1995; Walczak H. et al. 1999

TRAIL Mechanisms of Action

Johnstone RW, 2008

Kufe DW, et al 2003

TRAIL Receptors and Tumors

COLON CANCER(Jalving M. et al. 2006) LUNG CANCER (Jin H. et al. 2004) OVARIAN CANCER (Pukac L. et al. 2003) BREAST CANCER (Rahman M. et al. 2009) CERVICAL CARCINOMAS (Sheridan JP. et al. 1997) GLIOBLASTOMA (Pollack IF. et al. 2001) PANCREATIC CANCER (Halpern W. et al. 2004) PROSTATE CANCER (Yu R. et al. 2000) TYROID CANCER (Mitsiades N. et al. 2000) SARCOMAS (Petak I. et al. 2001) LYMPHOMAS (Daniel D. et al. 2007) MULTIPLE MYELOMA (Mitsiades CS. et al. 2001)

Johnstone RW, 2008; Ashkenzazi A, 2009

rhTRAIL in Clinical Trials

Limits of rhTRAIL into Clinics

Short half-life after single injection

TRAIL lasts for approx. 30 minutes into patient serum

Need of multiple injections:

Increased toxicity (GE, Liver, Neurological)

Increased costs

Ashkenazi A. et al. 2008

NK

TUMOR CELL

GM-MSC TRAIL

Adipose Derived MSC as Vehicles

Collagenase CentrifugationTurk stain

CultureAdipose Tissue

Specimen

TRASH

Lipoaspiration

Grisendi G et al, 2010

CD8+ Cells as Source of TRAIL cDNA

Full-length human TRAIL gene was amplified from cDNA obtained from stimulated CD8+ cells

A bicistronic murine stem cell virus–derived viral vector (pMIGR1) encoding for green fluorescent protein (GFP) was generated including the full-length human TRAIL cDNA

Grisendi G et al, 2010

Transduced AD-MSC

IRE

S

GFP

pMIGR1 GFP 6065 bp

TRAIL cDNA

PBMC + IL-2/INFg

GENE-MODIFIED AD-MSC EXPRESS TRAIL BOTH AS MEMBRANE BOUND

PROTEIN AND SOLUBLE LIGAND

Grisendi G et al, 2010

AD-MSC ARMED WITH TRAIL DISPLAY AN ANTITUMOR ACTIVITY

IN VITRO

Grisendi G et al, 2010

TESTING OTHER TARGETS:

Colon, Panceras, Breast and Neuroblastoma

BT549 BT549IMR32 IMR32

24 h 48 h

BxPc3 LS174T

24 h

BxPc3 LS174T

48 h

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* **

**

§§

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§§

§§

§§

§§

§

§

§§§

§§§

For BxPc3:*P<0.005;**P<0.005;***P=0.02. For LS174T: §P<0.001; §§P<0.001; §§§P<0.01

For BT549 and IMR32 P>0.05

Grisendi G et al, 2010

Can We Use Combinatory Approaches by pharmaceuticals and Cyto-pharamceuticals

aiming to synergistic effects?

+

48 h

Bortezomib Up-regulates TRAIL

Receptors Expression On BT549

rTRAIL

BT549 + BORTEZO

BT549 don’t express TRAIL receptors

The treatment of BT549 with

Bortezomib increases the

expression of TRAIL-R2

Bortezomib Sensitizes Resistant

BT549 To AD-MSC TRAIL

*

P=0.01

In vivo studiesTumor induction:

HeLa

0 5 10 15 20 25 30 35 40 45 50 55 60

AD-MSC TRAIL or AD-MSC GFP

SUBCUTANEOUSLY INJECTION (S.C.): mice sub-cutaneously flank injected once with 2x105 HeLa, as soon as tumor burden appeared (15-20 days) mice were treated with multiple bi-weekly intratumor injections of 106 AD-MSC TRAIL or GFP

INTRAVENOUS INJECTION (I.V.): mice sub-cutaneously flank injected once with 2x105 HeLa, as soon as tumor burden appeared (15-20 days) mice were treated with multiple bi-weekly tail intravenous injections of 106 AD-MSC TRAIL or GFP

DAYS

Grisendi G et al, 2010

SUB-CUTANOUS AD-MSC TRAIL DELIVERY

EXERT AN ANTI-TUMOR ACTIVITY IN VIVO

*P=0.006; **P=0.002

Grisendi G et al, 2010

*P=0.01

INTRA-VENOUS AD-MSC TRAIL DELIVERY

EXERT AN ANTI-TUMOR ACTIVITY IN VIVO

Grisendi G et al, 2010

AD-MSC TRAIL LOCALIZE INTO TUMOR

Anti-GFP Ab – HRP - DAB

Grisendi G et al, 2010

Giulia Grisendi Rita BussolariElena VeronesiLuigi Cafarelli Serena PiccinnoJorge BurnsNaomi D’SouzaAlba MurgiaCarlotta SpanoSara CaldrerCristiano RosafioOlivia CandiniGaetano SantoValeria Rasini

Paolo Paolucci

Pierfranco Conte

Pietro LoschiMarco PignattiGiorgio De SantisFabrizio Di BenedettoUliano MorandiFabio CataniServ. Trasfusionale

Edwin Horwitz

Istvan Petak

Azienda Ospedaliera – Universitaria di Modena