1. IDENTIFICATION AND CHARACTERIZATION OF Cancer STEM CELLS IN CLEAR CELL
Dipartimento dell’Emergenza e dei Trapianti di Organi
Sezione di Urologia, Andrologia e Trapianto di Rene
Università di Bari
IDENTIFICATION AND CHARACTERIZATION OF
Cancer STEM CELLS IN CLEAR CELL
RENAL CELL CARCINOMA
Lucarelli G, Galleggiante V, Rutigliano M, Ditonno P, Bettocchi C,
Vavallo A, Selvaggi FP,, Battaglia M.

2. What is a cancer stem cell (CSC)?
The CSC paradigm refers to the ability of a subpopulation of cancer cells to initiate tumorigenesis by undergoing self-renewal and differentiation, like normal stem cells, whereas the remaining majority of the cells are more “differentiated” and lack these properties.
Science June 2009

3. AIMS OF THE STUDY
Investigate if tumor stem cells manifest the characteristic properties typical of CSCs and study the difference in comparison to the healthy counterpart (ARPCs) .
Study the gene expression profile of Renal Cancer Stem Cells CD (RCSCs)
Find a marker of membrane that was able to discriminate the population of Adult Renal Progenitor Cells (ARPCs) from the RCSCs.

4. Clinical and patological charecteristics of patients

5. CS CD133+
CD 24+
TUB
CS CD133-
HEALTHY tissue
CS CD133+
CD 24+
GLOM
CS CD133-
NEPHRECTOMIES
CS CD133+
CD 24+
TUMORAL tissue
RCSC
CS CD133-

6. Renal Cancer Stem Cell (RCSC), Adult/Progenitor Renal Cell (ARPC)
ARPC TUB
ARPC GLOM
From the same patient

7. Cells CD133+
92%
Cells CD24+
99%
characterization
CD133+
CD24+
85%

8. Mesenchymal Phenotyping
MSC
ARPC TUB
RCSC TUM
CD 73+
CD 73+
CD 73+
CD 90+
CD 90-
CD 90-
CD 105+
CD 105-
CD 105-
CD 45-
CD 14-
CD 20-
CD 34-
CD 45-
CD 14-
CD 20-
CD 34-
CD 45-
CD 14-
CD 20-
CD 34-

9. Characterization of Renal Cancer Stem Cell:
PROTEOME PROFILER
RCSC
ARPC GLOM
ARPC TUB
*p ≤ 0,05
**p ≤ 0,01

10. Self renewal after 10/15 days
CLONING
LIMITING DILUTION:
Self renewal after 10/15 days

11. Differentiation RCSC 133+/24+ ENDOTHELIAL DIFFERENTIATION
CK-19
RCSC 133+/24+
ENDOTHELIAL
DIFFERENTIATION
ZO-1 C-
DIFF
RCSC 133+/24+
OSTEOGENIC
DIFFERENTIATION
RCSC 133+/24+
ADIPOGENIC
DIFFERENTIATION

12. Tumorigenity
Soft Agar Assay in vitro
Caki-2
RCSC
ARPC

13. WHOLE-GENOME GENE EXPRESSION APPROACH
6 tubular ARPCs lines
6 glomerular ARPCs lines
6 RCSC lines
3 RPTEC (control -)
3 MSC (control +)
RNA
RNA hybridized on ILLUMINA Human HT-12 v4 Expression Beadchips
Analysis of data with several softwares
Statistical Analysis
Validation Real time

14. 149 modulated genes (FDR 0.05)

15. Fold change > 1.5 72 geni Principal Component Analysis RCSC
ARPC GLOM
ARPC TUB

16. Biological Functions in which selected genes are involved

17. Validation of microarray analysis** * * **
*p<0.05
**p<0.01

18. CTR2 EXPRESSION 59.3 % 54.6% RCSC CD 133 CTR2 0.21 % tARPC CD 133 CTR2

19. CD 133 *
*p<0.01
CTR2
MERGE
RCSC
tARPC
immunofluorescence

20. CONCLUSIONS
The isolated stem cells, from tumor tissues, express markers CD133
Almost all CD 133+ cells expressed the CD24 renal marker
The isolated cells express a high stemness potential (stem cell profiler)
CD133+/24+ cells from tumoral tissues are tumorigenic in vitro
Specific RCSC gene expression profile has been identified for the first time
CTR2 is localized on plasma membrane allowing the separation of RCSCs from ARPCs