Adjacent Normal Kidney Research Articles

14 Natural killer cells have impaired cytotoxicity in advanced renal cell carcinoma

Abstract Background Natural killer (NK) cells are key mediators of anti-tumor activity in multiple cancers, including clear cell renal cell carcinoma (ccRCC). However, the phenotype and function of NK cells across different disease stages of ccRCC is incompletely characterized. Methods Single-cell RNA (scRNA)-sequencing (10x Genomics) data from tumor and adjacent normal kidney from ccRCC was analyzed at multiple clinical stages (localized – stage I/II/III; and advanced – stage IV). Graph-based clustering and lineage markers were used to identify distinct NK cell populations. Differential gene expression analysis was performed to further investigate NK cell phenotype and to derive a gene expression signature (GES). Gene signatures from NK cell subclusters of interest were used to interrogate bulk transcriptomic datasets and expression with clinical outcomes. Tumor-infiltrating NK cell function (cytokine production and cytotoxicity) was assessed by isolation of live NK cells from ccRCC tissue, co-culture with K562 target cells, and measurement of cytokine (IFNgamma) and cytotoxicity (CD107a) markers by flow cytometry. Results Single-cell transcriptomic data were analyzed from 13 patients with ccRCC (tumor and normal kidney), resulting in 21,139 high-quality NK cells. Patients with advanced/metastatic RCC had a lower proportion of NK cells versus total immune cells in the tumor microenvironment compared to normal kidney (p=0.036) and localized ccRCC (p=0.088). Clustering analysis revealed 6 distinct NK cell subsets. The C0.Bright-like NK cell cluster was significantly enriched in advanced ccRCC compared to localized ccRCC (p=0.048) and normal kidney (p=0.0059), expressed markers of tissue residency (ZNF683, ITGA1, CD9, ITGAE), and had decreased expression of cytotoxicity genes (GZMB, PRF1). A GES signature composed of genes upregulated in the dysfunctional C0.Bright-like NK cell cluster was used to interrogate bulk RNA-sequencing data from The Cancer Genome Atlas clear cell cohort, providing validation in a large, independent cohort, of an increase in this dysfunctional NK subset in advanced ccRCC compared to localized ccRCC (p=0.00039) and normal tissue (p=5.3e-05). To functionally confirm the decreased cytotoxicity of this dysfunctional NK population, CD45+CD56+CD3- NK cells were isolated from advanced ccRCC, and co-cultured with K562 target cells. Consistent with the transcriptomic phenotype, tumor-resident CD49a+CD9+ NK cells had cytokine production (IFNgamma) but decreased markers of cytotoxicity (CD107a) compared to CD49a-CD9- NK cells. By contrast, CD49a+CD9+ NK cells isolated from peripheral blood or normal kidney tissue maintained cytotoxic activity. Conclusions Among patients with ccRCC, a single-cell transcriptomic analysis revealed heterogeneous NK cell populations. A dysfunctional tumor resident NK cell phenotype was enriched among patients with advanced disease in ccRCC, and functionally confirmed to have diminished cytotoxicity. This study therefore provides insight into a new axis of immune dysfunction – decreased NK-mediated cytotoxicity – in advanced ccRCC. An improved understanding of NK cell dysfunction within ccRCC may provide a foundation for therapeutic restoration of NK-mediated anti-tumor activity in ccRCC. DOD CDMRP Funding: yes

54 MicroRNA Signatures in Clear Cell Renal Cell Carcinoma: Exploring Potential Implications for Prognosis

Abstract Background Renal cell carcinoma (RCC), the most common malignancy of the renal epithelium, accounts for over 90% of kidney cancer cases, with clear cell RCC (ccRCC) representing about 75% of these diagnoses. MicroRNAs (miRNAs), short non-coding RNAs of 21 to 23 nucleotides, play significant roles in RNA silencing and post-transcriptional regulation of gene expression. They are also crucial in tumorigenesis. We aimed to identify differentially expressed miRNA between ccRCC tumor and normal-adjacent samples, aiming to find miRNA signatures for ccRCC diagnostics and prognosis. Methods We collected tumor and normal-adjacent kidney samples from the Dartmouth Renal Tumor Biobank spanning 1994 to 2009. Samples were mechanically and enzymatically disassociated and preserved at -80 Celsius until processing. The single-stranded sequencing data was processed using the smrnaseq (v2.2.4) Nextflow analysis pipeline to map miRNAs to the reference database. The preprocessed counts data was imported into R (v4.3.3). We employed strict quality control measures excluding samples with low counts with a mean log-transcripts per million of less than -2, a read length below 25% within the range of 20-25 nucleotides, a microRNA fraction less than 20%, non-primate reads exceeding 70%, or a mapping rate greater than 1.5 times the interquartile range. Differential expression analysis was conducted using the Bioconductor DESeq2 package (v1.42.1), comparing tumor and normal samples as well as across different survival time categories. miRNAs with a false discovery rate (FDR) <0.05 and log2 fold change >2 were considered differentially expressed (DE) after adjustment for batch effects, tumor grade, sex, and tumor stage. An overrepresentation enrichment analysis was performed for the DE microRNAs using the miRNA Enrichment Analysis and AnnotationTool (miEAA). Survival analysis, utilizing stepwise selection in both directions, was conducted using identified DE miRNAs while adjusting for the covariates above plus age at diagnosis. Mature and hairpin miRNAs were analyzed separately for DE and survival. Results Among 213 samples with mature microRNA data, 198 samples (excluding one control) passed the quality control. These comprised 153 tumor samples and 45 normal samples, originating from a cohort of 159 patients. Among these patients, 13.8% have survived longer than 5 years, while 51.6% survived between 1 to 5 years, and 34.6% less than 1 year. 35 mature microRNAs were found to be differentially expressed between tumor and normal samples. Three mature miRNAs were under-expressed among samples with longer survival compared to those with short to medium survival. The DE mature miRNAs were overrepresented in vascular diseases (FDR=4.44 E-4), carcinoma (FDR=1.87 E-3) and clear cell renal cell carcinoma (FDR=5.65 E-3). miR-187-3p, miR-224-5p, miR-155-5p, miR-514a-3p, miR-490-5p were significantly associated with survival after adjustment (p <0.05). Out of the 219 samples analyzed for hairpin microRNA, 200 samples (excluding one control) passed quality control (155 tumor samples and 45 normal samples from 160 patients). Among these patients, 13.7% survived longer than 5 years, 51.9% survived between 1 to 5 years, and 34.4% survived less than 1 year. Differential expression analysis identified 28 hairpin microRNAs showing significant differences between tumor and normal samples. Notably, the differentially expressed hairpin microRNAs were overrepresented in renal cell carcinoma (FDR=5.38e-4) and renal clear cell carcinoma (FDR=0.01), as well as pathways associated with transcription factor TGFB1 (FDR=7.8×e−3), while being underrepresented in pathways associated with transcription factor KDM5B (FDR=9.5×e−3). Furthermore, hsa-mir-21, hsa-mir-155, hsa-mir-1291, and hsa-mir-506 were found to be significantly associated with survival after adjustment (p < 0.05). Conclusions This exploratory analysis identified distinct miRNA differential expression between ccRCC tumors and normal tissue, suggesting a potential miRNA signature for ccRCC. Our findings will be further explored using miRNA clustering analysis, functional analysis, network analysis, and differential transcript usage analysis. DOD CDMRP Funding: yes

High-resolution and highly accelerated MRI T2 mapping as a tool to characterise renal tumour subtypes and grades

BackgroundClinical imaging tools to probe aggressiveness of renal masses are lacking, and T2-weighted imaging as an integral part of magnetic resonance imaging protocol only provides qualitative information. We developed high-resolution and accelerated T2 mapping methods based on echo merging and using k-t undersampling and reduced flip angles (TEMPURA) and tested their potential to quantify differences between renal tumour subtypes and grades.MethodsTwenty-four patients with treatment-naïve renal tumours were imaged: seven renal oncocytomas (RO); one eosinophilic/oncocytic renal cell carcinoma; two chromophobe RCCs (chRCC); three papillary RCCs (pRCC); and twelve clear cell RCCs (ccRCC). Median, kurtosis, and skewness of T2 were quantified in tumours and in the normal-adjacent kidney cortex and were compared across renal tumour subtypes and between ccRCC grades.ResultsHigh-resolution TEMPURA depicted the tumour structure at improved resolution compared to conventional T2-weighted imaging. The lowest median T2 values were present in pRCC (high-resolution, 51 ms; accelerated, 45 ms), which was significantly lower than RO (high-resolution; accelerated, p = 0.012) and ccRCC (high-resolution, p = 0.019; accelerated, p = 0.008). ROs showed the lowest kurtosis (high-resolution, 3.4; accelerated, 4.0), suggestive of low intratumoural heterogeneity. Lower T2 values were observed in higher compared to lower grade ccRCCs (grades 2, 3 and 4 on high-resolution, 209 ms, 151 ms, and 106 ms; on accelerated, 172 ms, 160 ms, and 102 ms, respectively), with accelerated TEMPURA showing statistical significance in comparison (p = 0.037).ConclusionsBoth high-resolution and accelerated TEMPURA showed marked potential to quantify differences across renal tumour subtypes and between ccRCC grades.Trial registrationClinicalTrials.gov, NCT03741426. Registered on 13 November 2018.Relevance statementThe newly developed T2 mapping methods have improved resolution, shorter acquisition times, and promising quantifiable readouts to characterise incidental renal masses.Graphical

Expression of hsa-miRNA-15b, -99b, -181a and Their Relationship to Angiogenesis in Renal Cell Carcinoma.

MicroRNAs (miRNAs) play a regulatory role in various human cancers. The roles of hsa-miR-15a-5p, hsa-miR-99b-5p, and hsa-miR-181a-5p have not been fully explored in the angiogenesis of renal cell carcinoma (RCC). The present study aimed to evaluate the expression of these miRNAs in tumorous and adjacent healthy tissues of RCC. Paired tumorous and adjacent normal kidney tissues from 20 patients were studied. The expression levels of hsa-miR-15b-5p, hsa-miR-99b-5p, and hsa-miR-181a-5p were quantified by TaqMan miRNA Assays. Putative targets were analyzed by qRT-PCR. Significant downregulation of all three miRNAs investigated was observed in tumorous samples compared to adjacent normal kidney tissues. Spearman analysis showed a negative correlation between the expression levels of miRNAs and the pathological grades of the patients. Increased expression of vascular endothelial growth factor-A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α), a tissue inhibitor of metalloproteinases-1 (TIMP-1), was observed in tumorous samples compared to adjacent normal tissues. Depletion of tissue inhibitors of metalloproteinase-2 (TIMP-2) and metalloproteinase-2 (MMP-2) was detected compared to normal adjacent tissues. The examined miRNAs might function as contributing factors to renal carcinogenesis. However, more prospective studies are warranted to evaluate the potential role of miRNAs in RCC angiogenesis.

Metabolite profiling of human renal cell carcinoma reveals tissue-origin dominance in nutrient availability.

The tumor microenvironment is a determinant of cancer progression and therapeutic efficacy, with nutrient availability playing an important role. Although it is established that the local abundance of specific nutrients defines the metabolic parameters for tumor growth, the factors guiding nutrient availability in tumor compared to normal tissue and blood remain poorly understood. To define these factors in renal cell carcinoma (RCC), we performed quantitative metabolomic and comprehensive lipidomic analyses of tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples collected from patients. TIF nutrient composition closely resembles KIF, suggesting that tissue-specific factors unrelated to the presence of cancer exert a stronger influence on nutrient levels than tumor-driven alterations. Notably, select metabolite changes consistent with known features of RCC metabolism are found in RCC TIF, while glucose levels in TIF are not depleted to levels that are lower than those found in KIF. These findings inform tissue nutrient dynamics in RCC, highlighting a dominant role of non-cancer-driven tissue factors in shaping nutrient availability in these tumors.

Metabolite profiling of human renal cell carcinoma reveals tissue-origin dominance in nutrient availability

The tumor microenvironment is a determinant of cancer progression and therapeutic efficacy, with nutrient availability playing an important role. Although it is established that the local abundance of specific nutrients defines the metabolic parameters for tumor growth, the factors guiding nutrient availability in tumor compared to normal tissue and blood remain poorly understood. To define these factors in renal cell carcinoma (RCC), we performed quantitative metabolomic and comprehensive lipidomic analyses of tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples collected from patients. TIF nutrient composition closely resembles KIF, suggesting that tissue-specific factors unrelated to the presence of cancer exert a stronger influence on nutrient levels than tumor-driven alterations. Notably, select metabolite changes consistent with known features of RCC metabolism are found in RCC TIF, while glucose levels in TIF are not depleted to levels that are lower than those found in KIF. These findings inform tissue nutrient dynamics in RCC, highlighting a dominant role of non-cancer-driven tissue factors in shaping nutrient availability in these tumors.

Abstract 6231: Multi-omic characterisation of clear cell renal carcinoma reveals endogenous and exogenous processes related to its aetiology

Abstract Endogenous and exogenous exposures are associated with distinctive molecular marks in somatic tissues, including human tumours. Integrative multi-omic approaches could help improving the resolution to detect the molecular marks associated with disease aetiology. Here, we leveraged the overlapping molecular information of clear cell renal cell carcinomas (ccRCC) tumours from Mutographs cohort by integrating DNA methylation, transcriptome, and whole-genome sequencing-based somatic mutation data to explore and gain new insights into exposures linked to ccRCC aetiology. Our novel framework identified important sources of biological variance across ccRCC tumours that were summarized as molecular components. We inferred these molecular ccRCC components into independent datasets to further explore their relationship with molecular and epidemiological features of ccRCC, normal adjacent kidney tissues, and other cancer types. Our results revealed a major ccRCC molecular component correlating with cellular mitotic age-related features, particularly the mitotic epigenetic clock (age-adjusted epiTOC2), clock-like DNA mutational signatures (SBS1/ID1), and telomere attrition. This molecular component was overrepresented in ccRCC tumours in comparison with normal paired kidney tissues, associating with PBRM1 and SETD2 somatic cancer driver mutations, genome stability, tumor stage, grade and ccRCC patient survival, independently to chronological age. Pan-cancer analysis supported the mitotic-age effect, represented by this molecular component, in other cancer types. Another ccRCC component was associated with tobacco usage, the presence of tobacco related DNA mutational and methylation signatures, increased total mutation burden, and sex. This component was also related to the epigenetic regulation of xenobiotic metabolism-related genes (e.g., GSTP1), further suggesting a relationship with genotoxic compounds. We further identified molecular components related to the ccRCC tumour microenvironment and cell proliferation, including one component related to BAP1 cancer driver mutations, pro-inflammatory immune cells, and ccRCC patient survival. In conclusion, our study reports a novel framework to molecular characterize ccRCC tumours using an integrative multi-omic approach, providing additional insights into the molecular footprints of endogenous and exogenous exposures in ccRCC tumours, including molecular components with prognostic value for patients. Citation Format: Ricardo Cortez Cardoso Penha, Alexandra Sexton Oates, Sergey Senkin, Han La Park, Nicolas Alcala, Matthieu Foll, Karl Smith-Byrne, Paul Brennan, James Mckay. Multi-omic characterisation of clear cell renal carcinoma reveals endogenous and exogenous processes related to its aetiology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6231.

Altered NCR3 Splice Variants May Result in Deficient NK Cell Function in Renal Cell Carcinoma Patients.

The natural killer (NK) cell function of patients with malignant tumours may be suppressed by deficiency, and the poor prognosis of renal cell carcinoma (RCC) patients may be due to escape from NK cell cytotoxicity, especially with respect to natural cytotoxicity receptors (NCRs) on the NK cell surface. However, the specific mechanism remains unclear. Therefore, in this study, we sought to explore the role of NCR, especially NCR3 splice variants, in the process of NK cell deficiency in RCC patients. We used flow cytometry to analyse the phenotype of NK cells from the peripheral blood and kidney tumour tissue of RCC patients. The NKp30-mediated NK cell killing function was measured by antibody-dependent cell-mediated cytotoxicity (ADCC) in NK and RCC cell coincubation. We extracted RNA from the peripheral blood mononuclear cells (PBMCs) of RCC patients and renal carcinoma tissue and carried out real-time quantitative PCR to detect the mRNA levels of NKp30a, NKp30b and NKp30c. mRNA expression levels of cytokines (IL-6, IL-8, IL-10, IL-18 and TGF-β) based on RNA extracted from renal carcinoma tissue and adjacent normal kidney tissues were also measured by real-time quantitative PCR. Regarding the phenotype of NK cells in RCC patients, the proportion of NK cells in tumour tissue was significantly reduced, with changes in the NK cell proportion being most obvious in NKp30+ NK cells. Furthermore, the results of the ADCC function assay showed limited NKp30+ NK cell-mediated cytotoxicity in RCC patients. Through real-time quantitative PCR, we found lower expression of NKp30a and NKp30b, the immunostimulatory splice variants of NCR3 encoding NKp30, in RCC patients. Moreover, expression of activating cytokines (IL-6 and IL-8) in renal cancer tissue was decreased, though inhibitory cytokine (TGF-β) expression remained unchanged, which may result in an immunosuppressive cytokine microenvironment. Decreased expression of immunostimulatory NCR3 splice variants and the inhibitory cytokine microenvironment in RCC patients may contribute to deficient NK cell cytotoxicity and renal carcinoma cell immune escape from NK cell killing, which may provide a theoretical basis for finding new immunotherapeutic targets for RCC.

KL is a favorable prognostic factor related immune for clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is a prevalent cancer in adult urology, often leading to metastasis and poor prognosis. Recently, advances in tumor immunology and aging research have opened up new possibilities for the treatment of kidney cancer. Therefore, the identification of potential targets and prognostic biomarkers for immunotherapy has become increasingly urgent.MethodsUsing GSE168845 data, we identified immune-aging-associated differentially expressed genes (IAR-DEGs) by intersecting differentially expressed immune-related genes and aging-related genes. The prognostic value of IAR-DEGs was determined via univariate and multivariate Cox regression analysis, revealing KL as an independent prognostic factor for ccRCC. We also investigated the correlation between KL and various immune-related factors, including immune cell infiltration, immune score, immune checkpoint, MSI, and TIED score. To confirm the expression of KL in ccRCC, we conducted qRT-PCR assays on both ccRCC cell lines and clinical tissue samples, and compared KL expression levels between normal kidney cell line (HK-2) and ACHN, a ccRCC cell line. Finally, we assessed KL protein expression levels in tissues using immunohistochemistry (IHC).ResultsIn this study, we utilized Venn diagram analysis to identify 17 co-expressed immune-aging related DEGs from GSE168845, import database, and MSigDB database. GO and KEGG analysis revealed that the functions of the 17 IAR-DEGs were primarily related to “aging”. Univariate and multivariate Cox analysis validated these 17 genes, and KL was determined to be an independent prognostic factor for ccRCC. The downregulation of KL was observed in ccRCC tissues and was negatively associated with T stage, M stage, pathological stage, and histologic grade (p < 0.05). This downregulation indicated disease deterioration and a shortened overall survival period. Our calibration curves and nomogram demonstrated the excellent predictive potential of KL. GSEA analysis showed that KL gene mediated immune and aging-related pathways, and was significantly correlated with immune infiltration and MS and TIED score. More research has revealed a significant reduction in KL mRNA expression levels in human renal cancer cells, particularly in ccRCC tissues compared to adjacent normal kidney tissues. Moreover, immunohistochemistry data have demonstrated a marked decrease in KL protein expression levels in ccRCC cells when compared to adjacent normal tissues.ConclusionsKL was a potential aging-related target for immunotherapy and valid prognostic biomarker for ccRCC patients.

Abstract B019: Clinical and molecular characterization of chromophobe renal cell carcinoma: A focus on immunotherapy based regimens and the tumor immune microenvironment

Abstract Background: Chromophobe renal cell carcinoma (ChRCC) represents 5% of all kidney cancers. In contrast to clear cell RCC (ccRCC), the immune landscape of ChRCC and its response to immunotherapy remain poorly characterized. We sought to evaluate the clinical outcomes of patients with ChRCC treated with immuno-oncology (IO)-based regimens, and assess the immune cell composition, phenotypic state, and T cell specificity in the tumor microenvironment of ChRCC. Methods: Using real-world data from the International Metastatic RCC Database Consortium, we analyzed the survival outcomes and objective responses of patients with advanced ChRCC to currently adopted IO-based regimens (i.e. dual IO therapy or IO + vascular endothelial growth factor targeted therapy [VEGF-TT]) in the first-line setting, as compared to patients with ccRCC. Single-cell RNA sequencing (scRNA-seq) and single-cell T-cell receptor sequencing (scTCR-seq) were performed on ChRCC and related oncocytic neoplasms (i.e. renal oncocytoma [RO] and low-grade oncocytic tumor [LOT]) samples with matched normal kidney specimens. The infiltration of CD45+ immune cells in renal oncocytic tumors and ccRCC samples was quantified using immunohistochemistry (IHC). Results: Compared to patients with ccRCC (n=856) treated with first-line IO-based regimens, patients with ChRCC (n=31) had a lower overall survival (median: 24.7 vs. 50.5 months, p&amp;lt;0.001) and lower time to treatment failure (median: 4.5 vs. 11.0 months, p&amp;lt;0.001). Similarly, patients with ChRCC had a significantly lower overall response rate than those with ccRCC (12.0 vs. 47.1%, respectively; p&amp;lt;0.001). When evaluating immune cell infiltration, renal oncocytic tumors (ChRCC, RO, and LOT) exhibited a low density of CD45+ cells (mean: 739 ± 114 cells/mm2; n=5) compared to ccRCC (mean: 3,420 ± 1,979 cells/mm2; n=5) (p&amp;lt;0.05). Single-cell analysis was performed on 46,817 cells from 5 tumors (ChRCC: n=3, RO: n=1 and LOT: n=1) and 4 samples from adjacent normal kidney. Across all tumors, CD8+ T cell clusters displayed a lower expression of immune checkpoints (i.e. PDCD1, CTLA4, LAG3, HAVCR2, and TIGIT) as compared to CD8+ T-cells from ccRCC. This was further validated in the analysis of bulk RNA-seq data from the TCGA, with a significantly lower expression of all immune checkpoints in ChRCC compared to both ccRCC (p&amp;lt;0.01) and papillary RCC (pRCC; p&amp;lt;0.01). Analysis of the T cell receptor repertoire (scTCR-seq) of ChRCC, RO and LOT samples did not show any pattern of clonal expansion, and a higher proportion of T cells in ChRCC were inferred to have a viral specificity, compared to ccRCC (0.79 vs. 0.1%, respectively). Conclusions: Patients with metastatic ChRCC appear to display poor clinical outcomes when treated with IO-based regimens, compared to ccRCC. Renal oncocytic tumors, including ChRCC, exhibit a low infiltration of immune cells, and a non-exhausted immune phenotype. Citation Format: Michel Alchoueiry, Chris Labaki, Long Zhang, Yue Hou, Kevin Bi, Charbel Hobeika, J. Connor Wells, Kosuke Takemura, Ziad Bakouny, Sabrina Camp, Carmen Priolo, Damir Khabibullin, Nicholas Schindler, Renee Maria Saliby, Eddy Saad, Samer Salem, Melissa Daou, Rana McKay, Sumanta Pal, Daniel Heng, Eliezer Van Allen, Sachet Shukla, Toni Choueiri, David Braun, Elizabeth Henske. Clinical and molecular characterization of chromophobe renal cell carcinoma: A focus on immunotherapy based regimens and the tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B019.

Abstract B003: Increasing branched-chain amino acid metabolism reduces clear cell renal cell carcinoma growth

Abstract North America has the highest incidence of renal cancer in the world with the most common subtype being clear cell renal cell carcinoma (ccRCC). Metabolic dysfunction is a hallmark of ccRCC based on molecular analysis and its clear cell histologic appearance due to significant cytoplasmic accumulation of lipid droplets and glycogen. Using publicly available RNA-Seq and proteomic datasets, we identified reduced expression of branched-chain amino acid (BCAA) enzymes BCAA transaminase 2 (BCAT2) and the branched-chain ketoacid dehydrogenase complex (BCKDH) in ccRCC compared to healthy kidney tissue. We validated these results by confirming that BCAT2 and the BCKDH complex are decreased at the mRNA and protein level in ccRCC tumors and cell lines compared to pair-matched normal adjacent kidney tissue. The reduced expression of the BCKDH complex was reinforced at the genome level by copy number loss of BCKDH subunits DBT and BCKDHB, underscoring the selection for suppression of this pathway. Metabolomics analysis then showed that the abundance of BCAAs were decreased in ccRCC tumors. Additionally, public datasets revealed that ccRCC patients presenting low mRNA expression of any of the four BCKDH subunits DBT, BCKDHB, BCKDHA, and DLD have a worse overall survival compared to patients with higher expression. To test if reactivating the BCAA pathway would impact ccRCC growth, I inhibited BCKDK, a kinase that inactivates the BCKDH complex, pharmacologically with BT2 and genetically with shRNA. Multiple ccRCC cell lines demonstrated reduced proliferation due to apoptosis after being treated with shRNA targeting BCKDK. Cell viability could be rescued by the antioxidant N-acetyl cysteine, suggesting loss of BCKDK increased the production of reactive oxygen species (ROS). Flow cytometry showed that knocking down BCKDK increases cellular ROS. Our data reveal that BCAA catabolism is decreased in ccRCC and reactivating the pathway reduces ccRCC growth in vitro by inducing apoptosis through increased production of ROS. This suggests that BCKDK could be a therapeutic target in ccRCC. Citation Format: Nathan J. Coffey, Romain Riscal, Nicolas Skuli, Nicholas P. Lesner, Michael Noji, Christine Jiang, Laura C. Kim, Xu Han, Megan C. Blair, Brian Keith, Zoltan Arany, M. Celeste Simon. Increasing branched-chain amino acid metabolism reduces clear cell renal cell carcinoma growth [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B003.

Identification of ATP6V0A4 as a potential biomarker in renal cell carcinoma using integrated bioinformatics analysis.

Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of renal cancer, and is associated with a high mortality rate, which is related to high rates of tumor recurrence and metastasis. The aim of the present study was to identify reliable molecular biomarkers with high specificity and sensitivity for ccRCC. A total of eight ccRCC-related expression profiles were downloaded from Gene Expression Omnibus for integrated bioinformatics analysis to screen for significantly differentially expressed genes (DEGs). Reverse transcription-quantitative (RT-q)PCR, western blotting and immunohistochemistry staining assays were performed to evaluate the expression levels of candidate biomarkers in ccRCC tissues and cell lines. In total, 255 ccRCC specimens and 165 adjacent normal kidney specimens were analyzed, and 344 significant DEGs, consisting of 115 upregulated DEGs and 229 downregulated DEGs, were identified. The results of Gene Ontology analysis suggested a significant enrichment of DEGs in 'organic anion transport' and 'small molecule catabolic process' in biological processes, in 'apical plasma membrane' and 'apical part of the cell' in cell components, and in 'anion transmembrane transporter activity' and 'active transmembrane transporter activity' in molecular functions. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the DEGs were significantly enriched in the 'phagosome', the 'PPAR signaling pathway', 'complement and coagulation cascades', the 'HIF-1 signaling pathway' and 'carbon metabolism'. Next, 7 hub genes (SUCNR1, CXCR4, VCAN, CASR, ATP6V0A4, VEGFA and SERPINE1) were identified and validated using The Cancer Genome Atlas database. Survival analysis showed that low expression of ATP6V0A4 was associated with a poor prognosis in patients with ccRCC. Additionally, received operating characteristic curves indicated that ATP6V0A4 could distinguish ccRCC samples from normal kidney samples. Furthermore, RT-qPCR, western blotting and immunohistochemistry staining results showed that ATP6V0A4 was significantly downregulated in ccRCC tissues and cell lines. In conclusion, ATP6V0A4 may be involved in tumor progression and regarded as a potential therapeutic target for the recurrence and metastasis of ccRCC.

Speckle-type POZ protein could play a potential inhibitory role in human renal cell carcinoma

BackgroundSpeckle-type POZ protein(SPOP), a substrate adaptor of Cul3 ubiquitin ligase, plays crucial roles in solid neoplasms by promoting the ubiquitination and degradation of substrates. Limited studies have shown that SPOP is overexpressed in human renal cell carcinoma (RCC) tissue. However, the exact role of SPOP in RCC remains unclear and needs to be further elucidated. The present study showed that SPOP was expressed at different levels in different RCC cell lines. The purpose of this study was to explore the roles of SPOP in the biological features of RCC cells and the expression levels of SPOP in human tissue microarray (TMA) and kidney tissues.MethodsHere, SPOP was overexpressed by lentiviral vector transfection in ACHN and Caki-1 cells, and SPOP was knocked down in Caki-2 cells with similar transfection methods. The transfection efficiency was evaluated by quantitative PCR and western blotting analyses. The role of SPOP in the proliferation, migration, invasion and apoptosis of cell lines was determined by the MTT, wound-healing, transwell and flow cytometry assays. Moreover, the cells were treated with different drug concentrations in proliferation and apoptosis assays to investigate the effect of sunitinib and IFN-α2b on the proliferation and apoptosis of SPOP-overexpressing cells and SPOP-knockdown RCC cells. Finally, immunohistochemical staining of SPOP was performed in kidney tissues and TMAs, which included RCC tissues and corresponding adjacent normal tissues.ResultsOverexpression of SPOP inhibited cell proliferation, migration and invasion and increased cell apoptosis. Interestingly, sunitinib and IFN-α2b at several concentrations increased the proliferation inhibitory rate and total apoptosis rate of cells overexpressing SPOP. The findings of the present study showed that the SPOP protein was significantly expressed at low levels in most clear cell RCC (ccRCC) tissues and at relatively high levels in the majority of adjacent normal tissues and kidney tissues. Kaplan–Meier survival analysis showed that there was no statistically significant difference in cumulative survival based on the data of different SPOP expression levels in TMA and patients.ConclusionsIn contrast to previous studies, our findings demonstrated that overexpression of SPOP might suppress the progression of RCC cells, which was supported by cell experiments and immunohistochemical staining. SPOP could be a potential tumour inhibitor in RCC.

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ Tcell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

A comprehensive investigation discovered the novel methyltransferase METTL24 as one presumably prognostic gene for kidney renal clear cell carcinoma potentially modulating tumor immune microenvironment.

BackgroundRecently, an increasing number of studies have uncovered the aberrant expression of methyltransferase-like family (METTL) plays an important role in tumorigenesis, such as METTL3 (an m6A writer). In our recent work, we discovered METTL24 expression was highly associated with the hazard ratio (HR) of kidney renal clear cell carcinoma (KIRC) compared to other tumors, implying a special function of METTL24 in KIRC carcinogenesis. Until now, the functions and mechanisms of METTL24 in KIRC have remained mostly unknown.MethodsThe mRNA expression of METTL24 in KIRC was analyzed using the TIMER 2.0, GEPIA, and UALCAN databases. The immunohistochemical assay was performed to validate METTL24 expression in our self-built Chinese cohort (n tumor = 88, n normal = 85). The gene set enrichment analysis (GSEA) was used to investigate the biological processes in which METTL24 might be engaged. The Spearman analysis was used to evaluate the expression correlations between METTL24 and a range of immunological variables, and the effects of METTL24 on the infiltration levels of multiple immune cells were explored using TCGA data. The upstream transcription factors of METTL24 were screened through a multi-omics analysis.ResultsMETTL24 expression in KIRC tissues was significantly decreased compared to normal adjacent kidney tissues, which was associated with the lower survival rate of KIRC patients. METTL24 potentially participated in the immune-relevant biological processes such as cytokine binding, NF-kappa B binding, MHC protein complex, and interleukin-12 action. Besides, METTL24 expression was linked to a number of immune checkpoints, cytokines, chemokines, and chemokine receptors, and also correlated with the infiltration levels of 10 types of immune cells in KIRC. Meanwhile, METTL24 expression differently affected the overall survival rates (OS) of KIRC patients with high or low levels of immune infiltration. Finally, CTCF and EP300 were discovered to be the probable transcription factors of METTL24 in KIRC.ConclusionThis study revealed that METTL24 might serve as a prognostic marker in KIRC and as one immune-relevant target for clinical treatment.

A transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer in adults. When ccRCC is localized to the kidney, surgical resection or ablation of the tumor is often curative. However, in the metastatic setting, ccRCC remains a highly lethal disease. Here we use fresh patient samples that include treatment-naive primary tumor tissue, matched adjacent normal kidney tissue, as well as tumor samples collected from patients with bone metastases. Single-cell transcriptomic analysis of tumor cells from the primary tumors reveals a distinct transcriptional signature that is predictive of metastatic potential and patient survival. Analysis of supporting stromal cells within the tumor environment demonstrates vascular remodeling within the endothelial cells. An in silico cell-to-cell interaction analysis highlights the CXCL9/CXCL10-CXCR3 axis and the CD70-CD27 axis as potential therapeutic targets. Our findings provide biological insights into the interplay between tumor cells and the ccRCC microenvironment.

Integrated TCR repertoire analysis and single-cell transcriptomic profiling of tumor-infiltrating T cells in renal cell carcinoma identifies shared and tumor-restricted expanded clones with unique phenotypes.

Objective responses of metastatic renal cell carcinoma (RCC) associated with systemic immunotherapies suggest the potential for T-cell-mediated tumor clearance. Recent analyses associate clonally expanded T cells present in the tumor at diagnosis with responses to immune checkpoint inhibitors (ICIs). To identify and further characterize tumor-associated, clonally expanded T cells, we characterized the density, spatial distribution, T-cell receptor (TCR) repertoire, and transcriptome of tumor-infiltrating T cells from 14 renal tumors at the time of resection and compared them with T cells in peripheral blood and normal adjacent kidney. Multiplex immunohistochemistry revealed that T-cell density was higher in clear cell RCC (ccRCC) than in other renal tumor histologies with spatially nonuniform T-cell hotspots and exclusion zones. TCR repertoire analysis also revealed increased clonal expansion in ccRCC tumors compared with non-clear cell histologies or normal tissues. Expanded T-cell clones were most frequently CD8+ with some detectable in peripheral blood or normal kidney and others found exclusively within the tumor. Divergent expression profiles for chemokine receptors and ligands and the Ki67 proliferation marker distinguished tumor-restricted T-cell clones from those also present in blood suggesting a distinct phenotype for subsets of clonally expanded T cells that also differed for upregulated markers of T-cell activation and exhaustion. Thus, our single-cell level stratification of clonally expanded tumor infiltrating T-cell subpopulations provides a framework for further analysis. Future studies will address the spatial orientation of these clonal subsets within tumors and their association with treatment outcomes for ICIs or other therapeutic modalities.

Abstract 982: A new transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma

Abstract Introduction: Renal cell carcinoma, particularly the clear cell histological subtype (ccRCC), is the most common kidney cancer in adults, accounting for 75-85% of all cases. When it is localized to the kidney, it is often cured with surgical resection or thermal ablation. One quarter of the patients have evidence of metastatic disease at the time of diagnosis, and in this setting ccRCC still is a highly lethal disease with few effective treatment options. In this study we used single-cell RNA-sequencing to characterize tumor cell clonality, metastatic signatures and the tumor microenvironment in ccRCC at the single-cell level. Methodological approach: Here we took advantage of fresh patient samples that included treatment-naive primary tumor tissue, matched adjacent normal kidney tissue, as well as tumor samples collected from patients with bone metastases. We used single cell RNA sequencing (10X Genomics, Chromium 3’ v2 kit, 10x Genomics), that was analyzed with PAGODA and CONOS. Summary of new unpublished data: Our analysis revealed a distinct transcriptional signature that predicts metastatic potential and patient survival was disclosed through the single-cell analysis. In-depth exploration of the supporting stromal cells displayed a phenotype of vascular remodeling within the endothelial cells. The fibroblasts showed a proliferative signature that was associated with poor survival. An in silico cell-to-cell interaction analysis highlighted the CXCL9/CXCL10-CXCR3 axis and the CD70-CD27 axis as potential therapeutic targets. Conclusions: Our discoveries in ccRCC provide complementary biological insights, specifically in the interplay between tumor cells and their microenvironment in ccRCC. We demonstrate important biological insights into ccRCC biology including: (1) identification of a distinct metastatic signature that predicts survival outcome, (2) discovery of a proximal tubule cell population that may represent the ccRCC cell of origin, and (3) a detailed exploration in the immunosuppressive environment and the stromal alterations in treatment-naïve patients. In addition, we stress potential therapeutic targets for further evaluation in preclinical models. Citation Format: Adele Mirna Alchahin, Shenglin Mei, Ioanna Tsea, Taghreed Hirz, Youmna Kfoury, Douglas Dahl, Chin-Lee Wu, Alexander O. Subtelny, Shulin WU, David T. Scadden, John H. Shin, Philip J. Saylor, David B. Sykes, Peter V. Kharchenko, Ninib Baryawno. A new transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 982.

Single-cell transcriptomics reveals a low CD8+ T cell infiltrating state mediated by fibroblasts in recurrent renal cell carcinoma

PurposeRecurrent renal cell carcinoma(reRCC) is associated with poor prognosis and the underlying mechanism is not yet clear. A comprehensive understanding of tumor microenvironment (TME) of reRCC may aid in designing...

LDLR promotes growth and invasion in renal cell carcinoma and activates the EGFR pathway.

Previous studies identified an association of low-density lipoprotein (LDL) levels and LDL receptor (LDLR) with renal cell carcinoma (RCC) development. This study investigated the expression and roles of LDLR in RCC. LDLR expression was examined in clear cell RCC (ccRCC) and adjacent normal kidney tissues, and its clinicopathological significance was analyzed. The role of LDLR in RCC cell proliferation, cell cycle, and invasion were assessed in RCC cells with LDLR stable knockdown. LDLR expression was higher in ccRCC tissues than in normal kidney tissues and increased with RCC progression. LDLR knockdown in RCC cells inhibited cell growth, migration and invasion, and induced G1/S cell cycle arrest. We identified an interaction between LDLR and EGFR, and EGFR signaling protein expression was reduced after LDLR knockdown. Our findings reveal that LDLR plays an important role in RCC carcinogenesis, suggesting that LDL and LDLR might be potential targets for therapeutic intervention in RCC.