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

BackgroundPatients 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.MethodsccRCC 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2539-z) contains supplementary material, which is available to authorized users.

Clonal heterogeneity influences drug responsiveness in renal cancer assessed by ex vivo drug testing of multiple patient‐derived cancer cells

“…Functional studies based on model systems to recapitulate genetic and phenotypic characteristics of the patient profile such as patient‐derived xenograft (PDX)/tissue grafts (TG), organoids and patient‐derived cultures that have recently been established from RCC . TG models were generally well‐characterized based on histology, gene expression and genomics, and are known to retain regional ITH . TG models only allow for small scale drug testing studies, however, may recapitulate the drug responsiveness in patients .…”

The potential of organoids in urological cancer research

“…Another issue with these organoids is the absence of nerves, blood vessels, and immune cells in the system. For example, a big difference in transcriptional profiles between VHL -mutant cells and their matched primary tumour specimens was observed by Lobo and colleagues 55 . However, further analysis indicated that the differentially expressed genes in the tumour component were mainly involved in immune functions, meaning that these genes were lost in VHL -mutant cultures owing to the absence of the immune cells.…”

“…Prostate organoids can be derived from nonmalignant human and mouse prostate stem cells and mimic the morphology of a nonmalignant prostate. The use of patient-derived organoids to study cancers is expanding, with studies in bladder 50 , prostate 53 , kidney 54,55 , pancreatic 44 , and colorectal cancers 56 already published (FIG. 1b).…”

“…The gene expression pattern of these cells was maintained for up to 21 days in 3D cultures, but was lost in cells in 2D culture. Moreover, Lobo and colleagues 55 established patient-matched malignant and nonmalignant primary cell cultures from sample from patients with ccRCC with VHL mutations. Surprisingly, the unselected tumour cell lines derived from primary ccRCC samples failed to show the VHL mutations that matching their parental tumour samples, which means that these cell lines are not cancer cells.…”

“…For example, both the basal-cell-derived and luminal-cell-derived prostate organoids transduced with MYC–myrAKT1 did not display any vague glandular structures or lumen, but some MYC–myrAKT1-transduced luminal organoids showed adenosquamous differentiation 93 . The systems developed for patient-derived bladder cell culture system and kidney organoids 55,80 , could be used for studying prostate cancer oncogenic events, regeneration, and transformation to investigate the cell of origin.…”