1. Compound Screening Using Cancer Stem Cells RuggeroDeMaria Giorgio Stassi Milan Bhagat

2. Significance of CancerStemCells Cancer stem cells are responsible for the development, maintenance and spreading of tumor cells Tumor relapse results from residual cancer stem cells that survive therapeutic treatment Cancer stem cells should represent the primary target for new therapeutic strategies aimed at tumor eradication Cancer stem cell analysis may provide considerable information for prognostic study and patient stratification

3. Research Options CharacterizationofCancerStemCells Immunohistochemistry Screening: FormalinFixedCancerStemCellArray mRNA and proteinanalysis: Western Blot, RTPCR, Flow Cytometry, Immunoflourescence, ConfocalAnalysis Functionalassays In vitro drug screening In vivo drugvalidation

4. Drs. De Maria and Stassihavepioneered cancerstemcellresearch in solid tumors Identification and expansion of human colon-cancer-intiating cells Lucia Ricci-Vitiani1, Dario G. Lombardi2, Emanuela Pilozzi3, Mauro Biffoni1, Matilde Todaro4, Cesare Peschle1 & Rugerro De Maria1,2 Nature. 2007; 445:111-5. Established several patient derived CSC lines Stem cell phenotype maintained over long periods in culture Technology that allows us to isolate and expand in vitro from several different tumors Identification and expansion of the tumorigenic lung cancer stem cell population A Eramo1, F Lotti2, G Sette2, E Pilozzii3, M Biffoni4, A Di Virgilio4, C Conticello2, L Ruco3, Cesare Peschle1 & Rugerro De Maria*,1 Cell Death and Differentation. 2008; 15: 504-514. Colon Cancer Stem Cells Dictate Tumor growth and Resist Cell Death by Production of Interleukin-4 Matilde Todaro1, Mileidys Perez Alea1, Anna B. Di Stefano1, Patrizia Cammereri3, Louis Vermeulen5, Flora Iovino1, Claudio Tripodo4,, Antonio Russo2, Gaspare Gulotta3, Jan Paul Medema5,*, and Giorgio Stassi1,* Cell Stem Cell 1, 389-402, October 2007

5. CancerStemCells (CSC) CSCs are also known as Tumorogenic Cells, Tumor Intiating Cells etc. Minor subpopulation of tumor cells Undifferentiated neoplastic cells with some characteristics that represent stem cells Are able to proliferate indefinitely & produce differentiated progeny Able to form tumor xenografts when injected into immuno-compromised mice Based on current reviews, likely responsible for metastatis formation

6. Asymmetric division Symmetric division Tumorigenic cancerstemcells Non tumorigenic differentiated cell Non tumorigenic transient amplifying progenitors Oldview New view Non Metastatic Potentially Metastatic Potentially metastatic Potentially metastatic Non Metastatic Potentially Metastatic

7. Human CancerStemCells Leukemia Breast Brain Prostate Colon Lung Liver Melanoma Head and neck Pancreas Ovary Kidney Thyroid Colon Lung Thyroid

8. Cancerstemcellsare a potential targetforthedevelopment of effectiveanti-cancertherapies Therapieseffective on cancerstemcells CSC Tumordegeneration Therapieseffective on differentiatedcancercells CSC CSC Tumorrelapse Tumorregression

9. CancerStemCells Cancer stem cells are isolated from solid tumors using specific Culture conditions that allow CSC-containing “tumor spheres” to grow Culture in serum free medium, Containing EGF and FGF2 Sorting Xenograft

10. CD133 Control Colon cancer stem cell sphere, serial sections

11. H&E Tumor database Available Resources Diagnosis and Tissue Banking TumorDissociation StemCell Culture And Expansion CancerStemCell Banking

12. CytoinclusionMicroarrays of CancerStemCells As a Tool forCancer Research Donor Samples Cancer Type 12 Colon 12 Lung 6 Breast 5 Ovarian 9 Melanoma 6 Glioblastoma 6 Thyroid Colon cancer stem cells

13. In vitro drug testing Drug screening is performed by an established high-throughput system that allows monitoring the viability of CSCs using a luminescence-based assay that reliably measures cellular ATP levels. Incubation StemCell Culture And Expansion Single CellSuspensionDistributed in 96-well Plates Liquid handling station interconnected to a multimode plate reader Readout Resistant Sensitive

14. CSCsdisplaybroadresistance to anti-cancer drugs CSCs derived from 4 colon cancer patients (horizontal strips) were treated with 80 anti-cancer drugs (vertical bars) for 72 and viability was measured by cell titer glo: Anti-Cancer Activity Results are rainbow coded, with more active compounds in the violetarea (red: 0%, dark violet: 40%)

15. Inhibition of IL-4 SensitizesColonCancerSpheres to ChemotherapyandTrial-inducedCellDeath Untreated Oxalipl. TRAIL 5-FU Ox+5-FU IgG anti-IL-4

16. In Vivo Drug Testing Cancer Stem Cells are able to generate Xenografts that Histologically and Phenotypically Reproduce the Patient Tumor Colon Cancer NSCLC PatientTumor CSC Xenograft PatientTumor CSC Xenograft H&E H&E CDX2 HMW-CK

17. EGFP CMV PGK LUCIFERASE LTR LTR presorting postsorting GFP+ Imaging of CSC-based tumors The CSC samples are genetically labelled with markers that allowed the non-invasive monitoring of tumor growth and response to treatment 108 106 Luciferaseactivity 104 102 100 Control Tw-GFP-Luc

18. Colon Cancer Stem Cell-based Models Orthotopic Lung metastasis Liver metastasis Haematoxylin & Eosin staining of an orthotopic tumor, lung and liver metastasis generated from colon cancer stem cells

19. Breast Cancer Stem Cells Grow As Spheres Expressing ALDH InfiltratingDuctalBreastCancer CK14 PrimaryCancerCells Serum-freecultures with EGF + FGF CK14 ALDH 2 days 7days Breast cancer spheres CK14 Differentiation in presence of FBS

20. Colon Cancer Stem Cell-based Models 100 PBS 80 IL-4 DM 60 cell viability (%) 40 20 0 Untreated Doxo w/o Doxo IL4DM +Doxo IL4DM Orange acridine/ethidium bromide staining of breast cancer spheres treated with Doxorubicin and IL-4 DM (IL-4 blocker)

21. Colon Cancer Stem Cell-based Models Blocking IL-4 in combination with chemotherapy prevents tumor growth generated by breast CSC Doxo IL-4DM +Doxo Control IL-4DM 2.5 PBS 2 Tunel 1.5 Doxo tumor size (cm3) 1 IL-4 DM 0.5 IL-4 DM+Doxo Azan Mallory 0 0 7 14 21 28 35 days

22. Thanks! Tristar Technology Group 9700 Great Seneca Highway Rockville, MD 20850 U.S.A www.tristargroup.us Rockville, MD Headquarters Hamburg, Germany Main Repository Array Manufacturing Contract Research Catania, Italy Tissue Repository