Compound Screening Using Cancer Stem Cells<br />RuggeroDeMaria<br />Giorgio Stassi<br />Milan Bhagat<br />
Significance of CancerStemCells<br /> Cancer stem cells are responsible for the development,  maintenance and spreading of...
Research Options<br />CharacterizationofCancerStemCells<br />Immunohistochemistry Screening: FormalinFixedCancerStemCellAr...
Drs. De Maria and Stassihavepioneered<br />cancerstemcellresearch in solid tumors<br />Identification and expansion of hum...
CancerStemCells (CSC)<br />CSCs are also known as Tumorogenic Cells, Tumor Intiating Cells etc.<br /> Minor subpopulation ...
Asymmetric<br />division<br />Symmetric<br />division<br />Tumorigenic<br />cancerstemcells<br />Non tumorigenic<br />diff...
Human CancerStemCells<br />Leukemia<br />Breast<br />Brain<br />Prostate<br />Colon<br />Lung<br />Liver<br />Melanoma<br ...
Cancerstemcellsare a potential targetforthedevelopment<br />of effectiveanti-cancertherapies<br />Therapieseffective on ca...
CancerStemCells<br />Cancer stem cells are isolated from solid tumors using specific<br />Culture conditions that allow CS...
CD133<br />Control<br />Colon cancer stem cell sphere, serial sections<br />
H&E<br />Tumor<br /> database<br />Available Resources<br /> Diagnosis and Tissue Banking<br />TumorDissociation<br />Stem...
CytoinclusionMicroarrays of CancerStemCells As a Tool forCancer Research<br />	Donor Samples   	Cancer Type<br />	12		Colo...
In vitro drug testing<br /> Drug screening is performed by an established<br />high-throughput system that allows monitori...
CSCsdisplaybroadresistance to anti-cancer drugs<br />CSCs derived from 4 colon cancer patients (horizontal strips) were tr...
Inhibition of IL-4 SensitizesColonCancerSpheres<br />to ChemotherapyandTrial-inducedCellDeath<br />Untreated<br />Oxalipl....
In Vivo Drug Testing<br />Cancer Stem Cells are able to generate Xenografts that<br />Histologically and Phenotypically Re...
EGFP<br />CMV<br />PGK<br />LUCIFERASE<br />LTR<br />LTR<br />presorting<br />postsorting<br />GFP+<br />Imaging of CSC-ba...
Colon Cancer Stem Cell-based Models<br />Orthotopic<br />Lung metastasis<br />Liver metastasis<br />Haematoxylin & Eosin s...
Breast Cancer Stem Cells Grow As Spheres Expressing ALDH<br />InfiltratingDuctalBreastCancer<br />CK14<br />PrimaryCancerC...
Colon Cancer Stem Cell-based Models<br />100<br />PBS<br />80<br />IL-4 DM<br />60<br />cell viability (%)<br />40<br />20...
Colon Cancer Stem Cell-based Models<br />Blocking IL-4 in combination with chemotherapy prevents tumor growth generated by...
Thanks!<br />Tristar Technology Group<br />9700 Great Seneca Highway<br />Rockville, MD 20850<br />U.S.A<br />www.tristarg...
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TriStar CSC

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TriStar CSC

  1. 1. Compound Screening Using Cancer Stem Cells<br />RuggeroDeMaria<br />Giorgio Stassi<br />Milan Bhagat<br />
  2. 2. Significance of CancerStemCells<br /> Cancer stem cells are responsible for the development, maintenance and spreading of tumor cells<br /> Tumor relapse results from residual cancer stem cells that survive therapeutic treatment<br /> Cancer stem cells should represent the primary target for new therapeutic strategies aimed at tumor eradication<br /> Cancer stem cell analysis may provide considerable information for prognostic study and patient stratification<br />
  3. 3. Research Options<br />CharacterizationofCancerStemCells<br />Immunohistochemistry Screening: FormalinFixedCancerStemCellArray<br />mRNA and proteinanalysis: Western Blot, RTPCR, Flow Cytometry,<br />Immunoflourescence, ConfocalAnalysis<br />Functionalassays<br />In vitro drug screening <br />In vivo drugvalidation<br />
  4. 4. Drs. De Maria and Stassihavepioneered<br />cancerstemcellresearch in solid tumors<br />Identification and expansion of human <br />colon-cancer-intiating cells<br />Lucia Ricci-Vitiani1, Dario G. Lombardi2, Emanuela Pilozzi3, Mauro Biffoni1, Matilde Todaro4, Cesare Peschle1 <br />& Rugerro De Maria1,2<br />Nature. 2007; 445:111-5.<br /> Established several patient derived CSC lines<br /> Stem cell phenotype maintained over long periods in culture<br /> Technology that allows us to isolate and expand in vitro from several different tumors<br />Identification and expansion of the tumorigenic lung <br />cancer stem cell population<br />A Eramo1, F Lotti2, G Sette2, E Pilozzii3, M Biffoni4, A Di Virgilio4, C Conticello2, L Ruco3, Cesare Peschle1 <br />& Rugerro De Maria*,1<br />Cell Death and Differentation. 2008; 15: 504-514.<br />Colon Cancer Stem Cells Dictate Tumor growth and <br />Resist Cell Death by Production of Interleukin-4<br />Matilde Todaro1, Mileidys Perez Alea1, Anna B. Di Stefano1, Patrizia Cammereri3, Louis Vermeulen5, Flora Iovino1,<br />Claudio Tripodo4,, Antonio Russo2, Gaspare Gulotta3, Jan Paul Medema5,*, and Giorgio Stassi1,*<br />Cell Stem Cell 1, 389-402, October 2007<br />
  5. 5. CancerStemCells (CSC)<br />CSCs are also known as Tumorogenic Cells, Tumor Intiating Cells etc.<br /> Minor subpopulation of tumor cells<br /> Undifferentiated neoplastic cells with some characteristics that represent stem cells<br /> Are able to proliferate indefinitely & produce differentiated progeny<br /> Able to form tumor xenografts when injected into immuno-compromised mice<br /> Based on current reviews, likely responsible for metastatis formation<br />
  6. 6. Asymmetric<br />division<br />Symmetric<br />division<br />Tumorigenic<br />cancerstemcells<br />Non tumorigenic<br />differentiated cell<br />Non tumorigenic transient amplifying progenitors<br />Oldview<br />New view<br />Non<br />Metastatic<br />Potentially<br />Metastatic<br />Potentially<br />metastatic<br />Potentially<br />metastatic<br />Non<br />Metastatic<br />Potentially<br />Metastatic<br />
  7. 7. Human CancerStemCells<br />Leukemia<br />Breast<br />Brain<br />Prostate<br />Colon<br />Lung<br />Liver<br />Melanoma<br />Head and neck<br />Pancreas<br />Ovary<br />Kidney<br />Thyroid<br />Colon<br />Lung<br />Thyroid<br />
  8. 8. Cancerstemcellsare a potential targetforthedevelopment<br />of effectiveanti-cancertherapies<br />Therapieseffective on cancerstemcells<br />CSC<br />Tumordegeneration<br />Therapieseffective on differentiatedcancercells<br />CSC<br />CSC<br />Tumorrelapse<br />Tumorregression<br />
  9. 9. CancerStemCells<br />Cancer stem cells are isolated from solid tumors using specific<br />Culture conditions that allow CSC-containing “tumor spheres” to grow<br />Culture in serum free medium,<br />Containing EGF and FGF2<br />Sorting<br />Xenograft<br />
  10. 10. CD133<br />Control<br />Colon cancer stem cell sphere, serial sections<br />
  11. 11. H&E<br />Tumor<br /> database<br />Available Resources<br /> Diagnosis and Tissue Banking<br />TumorDissociation<br />StemCell Culture<br />And Expansion<br />CancerStemCell<br />Banking<br />
  12. 12. CytoinclusionMicroarrays of CancerStemCells As a Tool forCancer Research<br /> Donor Samples Cancer Type<br /> 12 Colon<br /> 12 Lung<br /> 6 Breast<br /> 5 Ovarian<br /> 9 Melanoma<br /> 6 Glioblastoma<br /> 6 Thyroid<br />Colon cancer stem cells<br />
  13. 13. In vitro drug testing<br /> Drug screening is performed by an established<br />high-throughput system that allows monitoring the viability of CSCs using a luminescence-based assay that reliably measures cellular ATP levels. <br />Incubation<br />StemCell Culture<br />And Expansion<br />Single CellSuspensionDistributed in 96-well Plates<br /> Liquid handling station interconnected to a multimode plate reader <br />Readout<br />Resistant<br />Sensitive<br />
  14. 14. CSCsdisplaybroadresistance to anti-cancer drugs<br />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:<br />Anti-Cancer Activity <br />Results are rainbow coded, with more active compounds in the violetarea<br />(red: 0%, dark violet: 40%)<br />
  15. 15. Inhibition of IL-4 SensitizesColonCancerSpheres<br />to ChemotherapyandTrial-inducedCellDeath<br />Untreated<br />Oxalipl.<br />TRAIL<br />5-FU<br />Ox+5-FU<br />IgG<br />anti-IL-4<br />
  16. 16. In Vivo Drug Testing<br />Cancer Stem Cells are able to generate Xenografts that<br />Histologically and Phenotypically Reproduce the Patient Tumor<br />Colon Cancer<br />NSCLC<br />PatientTumor<br />CSC Xenograft<br />PatientTumor<br />CSC Xenograft<br />H&E<br />H&E<br />CDX2<br />HMW-CK<br />
  17. 17. EGFP<br />CMV<br />PGK<br />LUCIFERASE<br />LTR<br />LTR<br />presorting<br />postsorting<br />GFP+<br />Imaging of CSC-based tumors<br />The CSC samples are genetically labelled with markers that allowed the <br />non-invasive monitoring of tumor growth and response to treatment<br />108<br />106<br />Luciferaseactivity<br />104<br />102<br />100<br />Control<br />Tw-GFP-Luc<br />
  18. 18. Colon Cancer Stem Cell-based Models<br />Orthotopic<br />Lung metastasis<br />Liver metastasis<br />Haematoxylin & Eosin staining of an orthotopic tumor, lung and liver metastasis generated from colon cancer stem cells <br />
  19. 19. Breast Cancer Stem Cells Grow As Spheres Expressing ALDH<br />InfiltratingDuctalBreastCancer<br />CK14<br />PrimaryCancerCells<br />Serum-freecultures<br />with EGF + FGF<br />CK14<br />ALDH<br />2 days<br />7days<br />Breast cancer<br />spheres<br />CK14<br />Differentiation in presence of FBS<br />
  20. 20. Colon Cancer Stem Cell-based Models<br />100<br />PBS<br />80<br />IL-4 DM<br />60<br />cell viability (%)<br />40<br />20<br />0<br />Untreated<br />Doxo<br />w/o<br />Doxo<br />IL4DM +Doxo<br />IL4DM <br />Orange acridine/ethidium bromide staining of breast cancer spheres treated with Doxorubicin and IL-4 DM (IL-4 blocker)<br />
  21. 21. Colon Cancer Stem Cell-based Models<br />Blocking IL-4 in combination with chemotherapy prevents tumor growth generated by breast CSC<br />Doxo<br />IL-4DM<br />+Doxo<br />Control<br />IL-4DM<br />2.5<br />PBS<br />2<br />Tunel<br />1.5<br />Doxo<br />tumor size (cm3)<br />1<br />IL-4 DM <br />0.5<br />IL-4 DM+Doxo<br />Azan Mallory<br />0<br />0<br />7<br />14<br />21<br />28<br />35<br />days<br />
  22. 22. Thanks!<br />Tristar Technology Group<br />9700 Great Seneca Highway<br />Rockville, MD 20850<br />U.S.A<br />www.tristargroup.us<br />Rockville, MD<br />Headquarters<br />Hamburg, Germany<br />Main Repository<br />Array Manufacturing<br />Contract Research<br />Catania, Italy<br />Tissue Repository<br />

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