Dir ComIGR,MMB,05 _ 2008 Identification of cancer stem cell properties in the human cell line IGR-CAP1, established from a localized epithelial prostate cancer Anne Chauchereau , Catherine Gaudin, Nader Al Nakouzi, Daniel Compagno, Paule Opolon, Elisabeth Connault, Nathalie Auger, Patrick Gonin, Sylvestre Le Moulec, Christophe Massard, Karim Fizazi, Institut Gustave Roussy, Villejuif, France Introduction Conclusion ORTHOTOPIC XENOGRAFTS IN NUDE MICE Pan CK KI67 HES PAP PSMA IGR-CaP1 parental cell line,106 cells injected in the prostate EXPRESSION OF BASAL CYTOSKELETAL MARKERS CK19 The absence of Androgen receptor (AR) and PSA expression and the cytokeratin pattern of the IGR-CaP1 cell line suggests that these cells correspond to prostate epithelial basal cells. After clonally selection, 9 clones were derived from the IGR-CaP1 cell line, expanded and characterized : - Genotyping of DNA Short Tandem Repeat (STR) shows that each clone differ at 1 to 6 loci compared to the parental cells - The derived clone did not expressed AR nor PSA - The derived clone expressed adhesion markers EpCAM and α2β1 integrin - The derived clones expressed the cytokeratin 19 (CK19) - All the derived clones expressed basal cytokeratin markers Cytosketal Markers IGR-CaP1 cell line Clone 3A11 Clone 3C11 CK5.8 +++ (100%) +++ (100%) +++ (100%) CK8 - (0%) - (0%) + (17%) CK14 +++ (100%) +++ (100%) +++ (97%) IGR- CaP1 CD44 CD133 CXCR4 Clone 3A11 Clone 3C11 Antibodies: CD44-FITC (Clone G44-26, BD Biosciences), CD133-APC (AC133, Miltenyi Biotec) et CXCR4- APC (CD184 clone , BD Pharmingen). For three-color staining analysis, CD184-PE (BD Pharmingen) was used. Samples were analyzed with FACSCalibur cytometer (Becton Dickinson). SUBCUTANEOUS XENOGRAFTS IN NUDE MICE IGR-CaP1 parental cell line,10x 106 cells injected subcutaneously without matrigel PAP PSMA HES KI67 P504S CK14 EXPRESSION OF CANCER STEM CELL MARKERS METASTASIS DISSEMINATION FROM THE TUMOR ON ECTOPIC OR ORTHOTOPIC XENOGRAFTS Metastases observation 3 months subcutaneously post-injection of IGR-CaP1 cells, 7 weeks intraprostaticpost- injection of IGR-CaP1 cells Liver Lung Lung In vivo studies on nude mice with IGR-CaP1 cells showed that : The intra-prostatic tumor was largely undifferentiated The tumors were highly aggressive The presence of many ascini showed a glandular differentiation Prostate specific markers PSAP and PSMA were expressed in xenografts Our in vivo model based on IGR-CaP1 cell line reconstituted the human prostate adenocarcinoma We observed metastases, mainly on liver, around kidney and spleen We detected small metastases or micro metastases on lung Our in vivo model based on IGR-CaP1 cell line reconstituted the natural history of undifferentiated prostate tumors observed with a high Gleason score HIGH EXPRESSION OF CANCER STEM CELL MARKERS IN TWO DERIVED CLONES CD44+/ CXCR4+ CD44+/ CXCR4+/ CD133+ Clone 3A11 Clone 3C11 -Only 2/9 clones show high expression of CSC markers -Triple positive cells were cell-sorted, cloned and cultured. -CSC marker expression of subsequent cells are ongoing BONE METASTASIS FROM ECTOPIC XENOGRAFTS WERE DETECTED 10x 106 cells of 3C11 clone derived from the IGR-CaP1 cell line were injected subcutaneously in Nude mice. Six months post- injection, the mice were analyzed with CT-scan to detect bone metastasis. 100% of the injected mice (n=3) showed bone defects by CT- scan CT-scan data highly suggest bone metastasis formation that should further be analyzed by HIC MIP image (Maximum Intensity Projection) We have established a new human prostate cancer cell line, IGR-CaP1, from a localized epithelial prostate cancer. Summary of the molecular characterization of IGR-CaP1 cell line: Epithelial morphology Spontaneously immortalized High telomerase activity High rate of in vitro proliferation Tetraploid cells Prostatic markers: Absence of AR and PSA expression (at mRNA and protein levels) Mesenchymal markers: Absence of expression of STRO-1, CD73, and CD105 markers Neuroendocrine markers: Absence of Chromogranine A expression Adhesion molecule markers: High expression of vimentin, low expression of E-Cadherin Preliminary data showed that the IGR-CaP1 cell line and derived clones were more resistant to docetaxel chemotherapy. 0.0 0.3 0.6 0.9 1.2 1.5 1.8 0 25 50 75 100 125 IGR-CaP1 3A11 Log ([Docetaxel] (nM)) % survival WST1 Survival assay after 3 days of treatment with increasing concentrations of docetaxel ACKNOWLEDGMENTS We thank Dr. Alain Le Pape (Plateforme CDTA CNRS, Orléans) for his help on CT-Scan acquisitions. We also thank SANOFI-AVENTIS for kindly gift of Docetaxel. This study was supported in part by the Institut Gustave Roussy Foundation, by the Association pour la Recherche sur le Cancer (ARC), by the Ligue Contre le Cancer and by the INCA OBJECTIVE To establish a prostate cancer model recapitulating the natural history of human prostate cancer To establish a model for studying the early process of oncogenesis To use this model to study the response to conventional therapies To use this model to develop new therapeutic approaches • We obtained a new Prostate Cancer Cell Line, IGR-CaP1, established from a localized tumor • IGR-CaP1 was spontaneously immortalized • These cell line showed Prostate Epithelial Basal Cell phenotype • IGR-CaP1 cells and derived clones are highly tumorigenic in Nude mice and likely produce bone metastasis • IGR-CaP1 cell line and derived-clones showed some of the characteristic identified for Cancer Stem Cells. • These new models may help developing new therapeutic approaches targeting prostate cancer stem cells. Perspectives and Ongoing Studies We are studying in more details the molecular characterization of these clones : -We study the capacity of these clones to differentiate into the different prostatic epithelial cells. -We try to establish mice models to follow the tumor growth initiated by these cells. -We study the response of these cells to different therapies: Hormone treatment, chemotherapy and radiation. RESPONSE TO CHEMOTHERAPY IGR-CaP1-DERIVED CLONES Corresponding author: anne.chauchereau@igr.fr # 2035 CK18 CK18 LNCaP IGR-CaP1 Cell lines can provide powerful models to study several aspects of tumorigenesis and progression of prostatic carcinoma. But most of the cellular models have been obtained from metastasis and do not permit the study the molecular pathways implicated in early events of oncogenesis It’s thus important to develop new prostate cancer models that accurately reflect the progression of the human disease. Considerable efforts are currently being directed toward the identification of markers associated with the initiation and progression of prostate cancer, potentially identifying cancer stem cells (CSC). We report the identification and characterization of a new cell line that show cancer stem cell characteristics. There is now increasing evidence that CSC are resistant to anti-cancer drugs and irradiation. Thus, the CSC might be a crucial target for the therapy. Cancer cell lines containing such CSC could be an attractive source of cells for CSC research and might help to develop new therapeutic approaches.