Terapie Cellulari e Geniche
per Neoplasie Solide
Laboratory of Cell Biology and
Advanced Cancer Therapy
Department of Oncology & Hematology
Hospital-University of Modena and Reggio Emilia, Italy
Gaiato, July, 16th 2011
Obiettivo Laboratorio Terapie
Oncologiche Avanzate
Sintetizzare innovative cito-terapie anticancro 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)
Kidney
Intestine
Brain
Lung
Skeletal
Muscle
Skin
Heart
Liver
Dominici M et al. 2001, 2004, 2009
Bone
How do Mesenchymal
Stromal Cells Interact with Tumors?
Breast Cancer Specimen
The Mesenchymal Tumor Stroma:
Tumor-associated Fibroblasts
Tumor Cells
Lymphocytes e NK cells
Tumor-associated fibroblasts (TAF)
Normal Tissue
Orimo AP et al. 1999; Orimo, AP et al. 2005
The Role of TAF
 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 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
Tumor
Vessel
 MSC retain a high metabolic activity for
sufficient generation of therapeutic agents
Bussolari et al. 2011
Gene Therapy
Ex Vivo
Vector
Cell
Expansion
of cells
Donor/
Recipient
Manipulated Cells
Going Back to Immunology:
How do CTL/NK Cells Kill Tumors?
TRAIL-R
& TRAIL
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)
 Cancer
TRAIL 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
Kufe DW, et al 2003
Johnstone RW, 2008
TRAIL Receptors and Tumors
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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)
rhTRAIL in Clinical Trials
Johnstone RW, 2008; Ashkenzazi A, 2009
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
GM-MSC
NK TRAIL
TUMOR CELL
Adipose Derived MSC as Vehicles
TRASH
Culture
Adipose Tissue
Specimen
Lipoaspiration
Grisendi G et al, 2010
Collagenase
Centrifugation
Turk stain
CD8+ Cells as Source of TRAIL cDNA
pMIGR1 GFP
6065 bp
IRES
TRAIL cDNA
PBMC + IL-2/INFg
Transduced AD-MSC
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
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
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BxPc3
LS174T
24 h
BxPc3
LS174T
48 h
For BxPc3:*P<0.005;**P<0.005;***P=0.02.
For LS174T: §P<0.001; §§P<0.001; §§§P<0.01
Grisendi G et al, 2010
BT549
IMR32
24 h
BT549
IMR32
48 h
For BT549 and IMR32 P>0.05
+
Can We Use Combinatory Approaches by
pharmaceuticals and Cyto-pharamceuticals
aiming to synergistic effects?
Bortezomib Up-regulates TRAIL
Receptors Expression On BT549
 BT549 don’t express TRAIL receptors
48 h
rTRAIL
BT549 + BORTEZO
 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 studies
Tumor induction:
AD-MSC TRAIL or AD-MSC GFP
HeLa
DAYS
0
5
10 15 20 25 30 35 40 45 50 55 60
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 ADMSC TRAIL or GFP
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
INTRA-VENOUS AD-MSC TRAIL DELIVERY
EXERT AN ANTI-TUMOR ACTIVITY IN VIVO
*P=0.01
Grisendi G et al, 2010
AD-MSC TRAIL LOCALIZE INTO TUMOR
Anti-GFP Ab – HRP - DAB
Grisendi G et al, 2010
Giulia Grisendi
Rita Bussolari
Elena Veronesi
Luigi Cafarelli
Serena Piccinno
Jorge Burns
Naomi D’Souza
Alba Murgia
Carlotta Spano
Sara Caldrer
Cristiano Rosafio
Olivia Candini
Gaetano Santo
Valeria Rasini
Paolo Paolucci
Pierfranco Conte
Azienda Ospedaliera – Universitaria di Modena
Pietro Loschi
Marco Pignatti
Giorgio De Santis
Fabrizio Di Benedetto
Uliano Morandi
Fabio Catani
Serv. Trasfusionale
Edwin Horwitz
Istvan Petak