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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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