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Terapie Cellulari e Geniche per Neoplasie Solide

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.

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Terapie Cellulari e Geniche per Neoplasie Solide

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  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) Kidney Intestine Brain Lung Skeletal Muscle Skin Heart Liver Bone Dominici M et al. 2001, 2004, 2009

  4. How do Mesenchymal StromalCellsInteract with Tumors? BreastCancer Specimen

  5. The Mesenchymal Tumor Stroma: Tumor-associated Fibroblasts Tumor Cells Lymphocytes e NK cells Tumor-associated fibroblasts (TAF) NormalTissue Orimo AP et al. 1999; Orimo, AP et al. 2005

  6. 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

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

  8. Can WeArtificiallySubstitute TAF With Infused MSC For A Therapeutic Benefit?

  9. MSC Tumor Vessel MSC asCytopharmacologicalVehicles • MSC tropismhasbeenreported in a variety of tumor types • MSC are easilyaccessible from marrow and othersources and readily propagate in culture • MSC can be geneticallymodified to express desired gene products (retro/lenti/AAV) • MSC retain a high metabolicactivity for sufficient generation of therapeutic agents Bussolari et al. 2011

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

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

  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 on “transformed” cells sparing normal tissues Wiley SR. et al. 1995; Walczak H. et al. 1999

  13. TRAIL Mechanismsof Action KufeDW, et al 2003 Johnstone RW, 2008

  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 (HalpernW. et al. 2004) • PROSTATE CANCER (YuR. 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. rhTRAIL in Clinical Trials Johnstone RW, 2008; Ashkenzazi A, 2009

  16. Limits of rhTRAIL into Clinics • Short half-life after single injection • TRAIL lasts for approx. 30 minutes intopatientserum • Need of multiple injections:  Increasedtoxicity (GE, Liver, Neurological)  Increasedcosts Ashkenazi A. et al. 2008

  17. GM-MSC TRAIL NK TUMOR CELL

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

  19. PBMC + IL-2/INFg CD8+ Cells as Source of TRAIL cDNA 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 IRES pMIGR1 GFP 6065 bp GFP Transduced AD-MSC Grisendi G et al, 2010

  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 §§§ *** *** §§ *** ** § *** * §§§ ** §§ * § §§§ §§§ §§ ** * § §§ § ** * IMR32 BT549 LS174T BxPc3 IMR32 BT549 LS174T BxPc3 24 h 48 h 24 h 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 CombinatoryApproaches by pharmaceuticals and Cyto-pharamceuticalsaiming to synergisticeffects?

  24. BT549 + BORTEZO 48 h Bortezomib Up-regulates TRAIL Receptors Expression On BT549 • BT549 don’t express TRAIL receptors rTRAIL • The treatment of BT549 with • Bortezomibincreases the • expressionof TRAIL-R2

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

  26. Tumor induction: HeLa AD-MSC TRAIL or AD-MSC GFP 55 5 25 60 10 15 20 30 35 40 45 0 50 In vivo studies DAYS 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 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. INTRA-VENOUS AD-MSC TRAIL DELIVERY EXERT AN ANTI-TUMOR ACTIVITY IN VIVO *P=0.01 Grisendi G et al, 2010

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

  30.  Stemcellscan delivercytotoxicmoleculeshaving sub-optimalpharmacocineticsifsystemicallydeliveredasrecombinantfactors Stemcellscan delivercytotoxicmoleculeshavingrelvant side effectsifsystemicallydeliveredasrecombinantfactors Adipose StemCellsrepresentideal source for the delivery StemCellsas “Cyto-pharmaceutics” to be used for drugcombinatoryapproaches and drugdiscovery

  31. Azienda Ospedaliera – Universitaria diModena 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 Pietro Loschi Marco Pignatti Giorgio De Santis Fabrizio Di Benedetto Uliano Morandi Fabio Catani Serv. Trasfusionale Edwin Horwitz Istvan Petak

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