1. 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

2. 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

3. Potentials of Mesenchymal Stem/Stromal Cells (MSC) Lung Skin Liver Heart Skeletal Muscle Brain Intestine Kidney Dominici M et al. 2001, 2004, 2009 Bone

4. How do Mesenchymal Stromal Cells Interact with Tumors? Breast Cancer Specimen

5. 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

6. 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

7. The Origin of TAF Grisendi G et al, 2011

8. Can We Artificially Substitute TAF With Infused MSC For A Therapeutic Benefit?

9. 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

10. Gene Therapy Ex Vivo Donor/ Recipient Cell Vector Manipulated Cells Expansion of cells

11. Going Back to Immunology: How do CTL/NK Cells Kill Tumors? & TRAIL TRAIL-R

12. 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 ontransformed cells sparing normal tissues Wiley SR. et al. 1995; Walczak H. et al. 1999

13. TRAIL Mechanisms of Action Johnstone RW, 2008 Kufe DW, et al 2003

14. 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)

15. Johnstone RW, 2008; Ashkenzazi A, 2009 rhTRAIL in Clinical Trials

16. 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

17. NK TUMOR CELL GM-MSC TRAIL

18. Adipose Derived MSC as Vehicles Collagenase Centrifugation Turk stain Culture Adipose Tissue Specimen TRASH Lipoaspiration Grisendi G et al, 2010

19. 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 IRES GFP pMIGR1 GFP 6065 bp TRAIL cDNA PBMC + IL-2/INFg

20. GENE-MODIFIED AD-MSC EXPRESS TRAIL BOTH AS MEMBRANE BOUND PROTEIN AND SOLUBLE LIGAND Grisendi G et al, 2010

21. AD-MSC ARMED WITH TRAIL DISPLAY AN ANTITUMOR ACTIVITY IN VITRO Grisendi G et al, 2010

22. TESTING OTHER TARGETS: Colon, Panceras, Breast and Neuroblastoma BT549 IMR32 24 h48 h 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 For BT549 and IMR32 P>0.05 Grisendi G et al, 2010

23. Can We Use Combinatory Approaches by pharmaceuticals and Cyto-pharamceuticals aiming to synergistic effects? +

24. 48 h Bortezomib Up-regulates TRAIL Receptors Expression On BT549 rTRAIL BT549 + BORTEZO BT549 dont express TRAIL receptors The treatment of BT549 with Bortezomib increases the expression of TRAIL-R2

25. Bortezomib Sensitizes Resistant BT549 To AD-MSC TRAIL * P=0.01

26. In vivo studies Tumor induction: HeLa 0 5 101520 25 30354045 50 55 60 AD-MSC TRAIL or AD-MSC GFP SUBCUTANEOUSLY INJECTION (S.C.): mice sub-cutaneously flank injected once with 2x10 5 HeLa, as soon as tumor burden appeared (15-20 days) mice were treated with multiple bi-weekly intratumor injections of 10 6 AD-MSC TRAIL or GFP INTRAVENOUS INJECTION (I.V.): mice sub-cutaneously flank injected once with 2x10 5 HeLa, as soon as tumor burden appeared (15-20 days) mice were treated with multiple bi-weekly tail intravenous injections of 10 6 AD- MSC TRAIL or GFP DAYS Grisendi G et al, 2010

27. SUB-CUTANOUS AD-MSC TRAIL DELIVERY EXERT AN ANTI-TUMOR ACTIVITY IN VIVO *P=0.006; **P=0.002 Grisendi G et al, 2010

28. *P=0.01 INTRA-VENOUS AD-MSC TRAIL DELIVERY EXERT AN ANTI-TUMOR ACTIVITY IN VIVO Grisendi G et al, 2010

29. AD-MSC TRAIL LOCALIZE INTO TUMOR Anti-GFP Ab – HRP - DAB Grisendi G et al, 2010

31. Giulia Grisendi Rita Bussolari Elena Veronesi Luigi Cafarelli Serena Piccinno Jorge Burns Naomi DSouza Alba Murgia Carlotta Spano Sara Caldrer Cristiano Rosafio Olivia Candini Gaetano Santo Valeria Rasini Paolo Paolucci Pierfranco Conte Pietro Loschi Marco Pignatti Giorgio De Santis Fabrizio Di Benedetto Uliano Morandi Fabio Catani Serv. Trasfusionale Edwin Horwitz Istvan Petak Azienda Ospedaliera – Universitaria di Modena