Clear cell renal cell carcinoma (ccRCC) has a poor prognosis despite novel biological targeted therapies. Tumor aggressiveness and poor survival may correlate with tumor grade at diagnosis and with complex metabolic alterations, also involving glucose and lipid metabolism. However, currently no grade-specific metabolic therapy addresses these alterations. Here we used primary cell cultures from ccRCC of low- and high-grade to investigate the effect on energy state and reduced pyridine nucleotide level, and on viability and proliferation, of specific inhibition of glycolysis with 2-deoxy-D-glucose (2DG), or fatty acid oxidation with Etomoxir. Our primary cultures retained the tissue grade-dependent modulation of lipid and glycogen storage and aerobic glycolysis (Warburg effect). 2DG affected lactate production, energy state and reduced pyridine nucleotide level in high-grade ccRCC cultures, but the energy state only in low-grade. Rather, Etomoxir affected energy state in high-grade and reduced pyridine nucleotide level in low-grade cultures. Energy state and reduced pyridine nucleotide level were evaluated by ATP and reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) dye quantification, respectively. 2DG treatment impaired cell proliferation and viability of low-grade ccRCC and normal cortex cultures, whereas Etomoxir showed a cytostatic and cytotoxic effect only in high-grade ccRCC cultures. Our data indicate that in ccRCC the Warburg effect is a grade-dependent feature, and fatty acid oxidation can be activated for different grade-dependent metabolic needs. A possible grade-dependent metabolic therapeutic approach in ccRCC is also highlighted.
Renal-cell carcinomas (RCC) arise from the renal epithelium, account for about 85% of renal cancers, and are characterized by different subtypes having different incidences. The clear-cell (RCCcc) and papillary (RCCpap) subtypes of sporadic RCC account for about 75% and 12% of cases, respectively, and have distinct genetic abnormalities.
The existence and identification of adult renal stem cells is a controversial issue. In this study, renal stem cells were identified from cultures of clonal human nephrospheres. The cultured nephrospheres exhibited the activation of stem cell pathways and contained cells at different levels of maturation. In each nephrosphere the presence of 1.12-1.25 cells mirroring stem cell properties was calculated. The nephrosphere cells were able to generate three-dimensional tubular structures in 3D cultures and in vivo. In clonal human nephrospheres a PKH(high) phenotype was isolated using PKH26 epifluorescence, which can identify quiescent cells within the nephrospheres. The PKH(high) cells, capable of self-renewal and of generating a differentiated epithelial, endothelial and podocytic progeny, can also survive in vivo maintaining the undifferentiated status. The PKH(high) status, together with a CD133(+)/CD24(-) phenotype, identified a homogeneous cell population displaying in vitro self-renewal and multipotency capacity. The resident adult renal stem cell population isolated from nephrospheres can be used for the study of mechanisms that regulate self-renewal and differentiation in adult renal tissue as well as in renal pathological conditions.
Human clear cell renal cell carcinoma (ccRCC) is therapy resistant; therefore, it is worthwhile studying in depth the molecular aspects of its progression. In ccRCC the biallelic inactivation of the VHL gene leads to stabilization of hypoxia-inducible factors (HIFs). Among the targets of HIF-1α transcriptional activity is the LOX gene, which codes for the inactive proenzyme (Pro-Lox) from which, after extracellular secretion and proteolysis, derives the active enzyme (Lox) and the propeptide (Lox-PP). By increasing stiffness of extracellular matrix by collagen crosslinking, Lox promotes tumor progression and metastasis. Lox and Lox-PP can reenter the cells where Lox promotes cell proliferation and invasion, whereas Lox-PP acts as tumor suppressor because of its Ras recision and apoptotic activity. Few data are available concerning LOX in ccRCC. Using an in vitro model of ccRCC primary cell cultures, we performed, for the first time in ccRCC, a detailed study of endogenous LOX and also investigated their transcriptomic profile. We found that endogenous LOX is overexpressed in ccRCC, is involved in a positive-regulative loop with HIF-1α, and has a major action on ccRCC progression through cellular adhesion, migration, and collagen matrix stiffness increment; however, the oncosuppressive action of Lox-PP was not found to prevail. These findings may suggest translational approaches for new therapeutic strategies in ccRCC.
BackgroundClear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues.MethodsWe established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation).ResultsA very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed.ConclusionsccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches aimed to study genes or pathways involved in ccRCC etiopathogenesis and to identify novel clinical markers or therapeutic targets. Moreover, SNP array technology proved to be a powerful tool to better define the cell composition and homogeneity of RCC primary cultures.
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