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Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma.

ABSTRACT: Rare cancer stem cells (CSC) are proposed to be responsible for tumour propagation and re-initiation and are functionally defined by identifying tumour-initiating cells (TICs) using the xenotransplantation limiting dilution assay (LDA). While TICs in clear cell renal cell carcinoma (ccRCC) appeared rare in NOD/SCID/IL2R?(-/-) (NSG) mice, xenografts formed more efficiently from small tumour fragments, indicating the LDA underestimated ccRCC TIC frequency. Mechanistic interrogation of the LDA identified multiple steps that influence ccRCC TIC quantitation. For example, tissue disaggregation destroys most ccRCC cells, common assays significantly overestimate tumour cell viability, and microenvironmental supplementation with human extracellular factors or pharmacological inhibition of anoikis increase clonogenicity and tumourigenicity of ccRCC cell lines and primary tumour cells. Identification of these previously uncharacterized concerns that cumulatively lead to substantial underestimation of TICs in ccRCC provides a framework for development of more accurate TIC assays in the future, both for this disease and for other cancers.

SUBMITTER: Gedye C

PROVIDER: S-EPMC4848484 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma.

Gedye Craig C Sirskyj Danylo D Lobo Nazleen C NC Meens Jalna J Hyatt Elzbieta E Robinette Michael M Fleshner Neil N Hamilton Robert J RJ Kulkarni Girish G Zlotta Alexandre A Evans Andrew A Finelli Antonio A Jewett Michael A S MA Ailles Laurie E LE

Scientific reports 20160428

Rare cancer stem cells (CSC) are proposed to be responsible for tumour propagation and re-initiation and are functionally defined by identifying tumour-initiating cells (TICs) using the xenotransplantation limiting dilution assay (LDA). While TICs in clear cell renal cell carcinoma (ccRCC) appeared rare in NOD/SCID/IL2Rγ(-/-) (NSG) mice, xenografts formed more efficiently from small tumour fragments, indicating the LDA underestimated ccRCC TIC frequency. Mechanistic interrogation of the LDA iden ...[more]

PMID: 27121191

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Project description:BackgroundManagement of clear cell renal cell carcinoma (ccRCC) has changed rapidly in recent years with the advent of immune checkpoint inhibitors (ICIs). However, only a limited number of patients can sustainably respond to immune checkpoint inhibitors and many patients develop resistance to therapy, creating an additional need for therapeutic strategies to improve the efficacy of systemic therapies.MethodsBinding probability and target genes prediction using online databases, invasion, migration, and apoptosis assays as well as the inhibition of cancer stem cells (CSCs) markers in ccRCC cell lines were used to select the most promising phytochemicals (PTCs). Mixed lymphocyte tumor cell culture (MLTC) system and flow cytometry were performed to confirm the potential combination strategy. The potential immunotherapeutic targets and novel CSC markers were identified via the NanoString analysis. The mRNA and protein expression, immune signatures as well as survival characteristics of the marker in ccRCC were analyzed via bioinformation analysis.ResultsShikonin was selected as the most promising beneficial combination partner among 11 PTCs for ipilimumab for the treatment of ccRCC patients due to its strong inhibitory effect on CSCs, the significant reduction of FoxP3+ Treg cells in peripheral blood mononuclear cells (PBMCs) of patients and activation of the endogenous effector CD3+CD8+ and CD3+CD4+ T cells in response to the recognition of tumor specific antigens. Based on NanoString analysis VCAM1, CXCL1 and IL8 were explored as potential immunotherapeutic targets and novel CSC markers in ccRCC. The expression of VCAM1 was higher in the tumor tissue both at mRNA and protein levels in ccRCC compared with normal tissue, and was significantly positively correlated with immune signatures and survival characteristics in ccRCC patients.ConclusionWe propose that a combination of shikonin and ipilimumab could be a promising treatment strategy and VCAM1 a novel immunotherapeutic target for the treatment of ccRCC.

Project description:To retrospectively assess whether magnetic resonance (MR) imaging with opposed-phase and in-phase gradient-echo (GRE) sequences and MR feature analysis can differentiate angiomyolipomas (AMLs) that contain minimal fat from clear cell renal cell carcinomas (RCCs), with particular emphasis on small (<3-cm) masses.Institutional review board approval and a waiver of informed consent were obtained for this HIPAA-compliant study. MR images from 108 pathologically proved renal masses (88 clear cell RCCs and 20 minimal fat AMLs from 64 men and 44 women) at two academic institutions were evaluated. The signal intensity (SI) of each renal mass and spleen on opposed-phase and in-phase GRE images was used to calculate an SI index and tumor-to-spleen SI ratio. Two radiologists who were blinded to the pathologic results independently assessed the subjective presence of intravoxel fat (ie, decreased SI on opposed-phase images compared with that on in-phase images), SI on T1-weighted and T2-weighted images, cystic degeneration, necrosis, hemorrhage, retroperitoneal collaterals, and renal vein thrombosis. Results were analyzed by using the Wilcoxon rank sum test, two-tailed Fisher exact test, and multivariate logistic regression analysis for all renal masses and for small masses. A P value of less than .05 was considered to indicate a statistically significant difference.There were no differences between minimal fat AMLs and clear cell RCCs for the SI index (8.05%±14.46 vs 14.99%±19.9; P=.146) or tumor-to-spleen ratio (-8.96%±16.6 and -15.8%±22.4; P=.227) when all masses or small masses were analyzed. Diagnostic accuracy (area under receiver operating characteristic curve) for the SI index and tumor-to-spleen ratio was 0.59. Intratumoral necrosis and larger size were predictive of clear cell RCC (P<.001) for all lesions, whereas low SI (relative to renal parenchyma SI) on T2-weighted images, smaller size, and female sex correlated with minimal fat AML (P<.001) for all lesions.The diagnostic accuracy of opposed-phase and in-phase GRE MR imaging for the differentiation of minimal fat AML and clear cell RCC is poor. In this cohort, low SI on T2-weighted images relative to renal parenchyma and small size suggested minimal fat AML, whereas intratumoral necrosis and large size argued against this diagnosis.

Project description:BackgroundClear cell renal cell carcinoma (ccRCC) is common in urinary system tumors. Cuproptosis is a non-apoptotic cell death pathway. Copper binds to fatty acylated mitochondrial proteins and activates various forms of cell death. LncRNA LINC02154 is significantly highly expressed in cells and tissues of many types of tumors, and the risk signature of LINC02154 in some tumors has been validated for effectiveness.MethodsWe constructed a risk prognostic signature by obtaining differentially expressed long noncoding RNAs (lncRNAs) associated with ccRCC outcomes and cuproptosis from The Cancer Genome Atlas (TCGA). We used TCGA to construct training and testing sets to analyze the risk signature and the impact of LINC02154, and we performed relevant survival analyses. Tumor mutational burdens were analyzed in different LINC02154 expression groups and risk score groups. We next analyzed the immune microenvironment of LINC20154. We performed LINC20154-related drug sensitivity analyses. We also investigated the cellular function of LINC02154 in the ACHN cell line and performed CCK-8 assay, EdU, wound-healing assay, and Transwell assay. The essential genes FDX1 and DLST of cuproptosis were detected by western blot.ResultsWe demonstrated that LINC02154's impact on outcomes was statistically significant. We also demonstrated the association of different ages, genders, stages, and grades with LINC02154 and risk models. The results showed a significant difference in tumor mutation burden between the groups, which was closely related to clinical prognosis. We found differences in immune cells among groups with different levels of LINC02154 expression and significant differences in immune function, immunotherapeutic positive markers, and critical steps of the immune cycle. The sensitivity analysis showed that differential expression of LINC02154 discriminated between sensitivity to axitinib, doxorubicin, gemcitabine, pazopanib, sorafenib, sunitinib, and temsirolimus. This difference was also present in the high-risk group and low-risk group. We demonstrated that the proliferation and migration of t ACHN cells in the LINC02154 knockdown group were inhibited. The western blot results showed that the knockdown of LINC02154 significantly affected the expression of FDX1 and DLST, critical genes of cuproptosis.ConclusionFinally, we demonstrated that LINC02154 and our constructed risk signature could predict outcomes and have potential clinical value.

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Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma.

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Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma.

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