Isolation and characterization of renal cancer stem cells from patient-derived xenografts

// Meriem Hasmim 1, 2, * , Stefania Bruno 3, * , Sandy Azzi 2 , Cindy Gallerne 2 , Julien Giron Michel 2 , Giulia Chiabotto 4 , Vincent Lecoz 2 , Cristina Romei 5 , Grazia Maria Spaggiari 5 , Annalisa Pezzolo 6 , Vito Pistoia 6 , Eric Angevin 1, 7 , Sophie Gad 1, 8 , Sophie Ferlicot 1, 9 , Yosra Messai 1 , Claudine Kieda 10 , Denis Clay 11 , Federica Sabatini 12 , Bernard Escudier 1, 7 , Giovanni Camussi 4 , Pierre Eid 2 , Bruno Azzarone 5 , Salem Chouaib 1 1 INSERM U 1186, Equipe labellisée Ligue Contre le Cancer, Gustave Roussy Campus, Villejuif, France 2 INSERM UMR 1014, Lavoisier Building, Paul Brousse Hospital, Villejuif, France 3 Department of Molecular Biotechnology and Healthy Science, Molecular Biotechnology Center, University of Torino, Turin, Italy 4 Department of Medical Science, University of Torino, Medical School, Torino, Italy 5 DIMES, UNIGE, Genova, Italy 6 Laboratory of Oncology Giannina Gaslini Institute, Genoa, Italy 7 Medical Oncology Department, Gustave Roussy Campus, Villejuif, France 8 Laboratoire de Génétique Oncologique EPHE, Ecole Pratique des Hautes Etudes, Paris, France 9 Université Paris-Sud, Assistance Publique-Hôpitaux de Paris, Service d'Anatomo-Pathologie, Hôpital Bicêtre, Le Kremlin-Bicêtre, France 10 Centre de Biophysique Moléculaire, CNRS UPR 4301, Orléans, France 11 INSERM UMR 972, Paul Brousse Hospital, Villejuif, France 12 Stem Cell and Cell Therapy Laboratory, Istituto G. Gaslini, Genoa, Italy * These authors have contributed equally to this work Correspondence to: Salem Chouaib, e-mail: salem.chouaib@gustaveroussy.fr Bruno Azzarone, e-mail: bazzarone@hotmail.com Keywords: clear cell renal cell carcinoma, cancer stem cells, patient-derived xenografts, CD133, EpCAM Received: July 24, 2015 Accepted: October 08, 2015 Published: November 02, 2015 ABSTRACT As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133 high /CD105-/Ecad low and PDX-2 cells were CD133 low /CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAM low and RCC-41-PDX-2/CD133 + /EpCAM bright ). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133 − over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133 + over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132 + and RCC-41-PDX-2/CD133 + displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132 + tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution. Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.