title: Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine.
abstract: Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients.
  
  
  
  ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling.
  
  
  
  The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures.
  
  
  
  The ability to establish primary cultures of ccRCC cells and matched normal kidney epithelial cells from almost every patient provides a resource for future development of novel therapies and personalized medicine for ccRCC patients.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Nazleen C
authors.initials: NC
authors.lastname: Lobo
authors.affiliation: School of Biomedical Sciences and Pharmacy, University of Newcastle, Hunter Medical Research Institute, Newcastle, NSW, Australia.
authors.forename: Craig
authors.initials: C
authors.lastname: Gedye
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Apostoli
authors.affiliation: Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, ON, Canada.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Brown
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Joshua
authors.initials: J
authors.lastname: Paterson
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Natalie
authors.initials: N
authors.lastname: Stickle
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Robinette
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Neil
authors.initials: N
authors.lastname: Fleshner
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Hamilton
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Girish
authors.initials: G
authors.lastname: Kulkarni
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Zlotta
authors.affiliation: Laboratory Medicine Program, Department of Pathology, University Health Network, Toronto, ON, Canada.
authors.forename: Andrew
authors.initials: A
authors.lastname: Evans
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Antonio
authors.initials: A
authors.lastname: Finelli
authors.affiliation: Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, ON, Canada.
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Michael A S
authors.initials: MA
authors.lastname: Jewett
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada. lailles@uhnresearch.ca.|Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada. lailles@uhnresearch.ca.
authors.forename: Laurie
authors.identifier: 0000-0001-7289-8882
authors.initials: L
authors.lastname: Ailles
mesh_terms: D000818:Animals; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D064593:Heterografts; D006801:Humans; D007150:Immunohistochemistry; D007680:Kidney Neoplasms / Q000235:genetics / Q000473:pathology; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D057285:Precision Medicine / Q000379:methods*; D061251:Primary Cell Culture / Q000379:methods*; D059467:Transcriptome
keywords: Clear cell renal cell carcinoma; In vitro models; Primary cell culture; Renal cancer; VHL