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Summary: Von Hippel-Lindau (VHL) syndrome is a rare autosomal dominant disorder, where renal cell carcinoma (RCC) serves as a significant cause of mortality. We collected peripheral blood from 61 VHL-RCC patients and 31 healthy individuals, along with 19 paired RCC tumor and adjacent non-malignant samples. Using liquid chromatography-mass spectrometry, we identified 238 plasma and 241 tissue differentially abundant metabolites (DAMs), highlighting key pathways such as arginine and proline metabolism. The top 10 of the 23 DAMs, common to both plasma and tissue, were instrumental in constructing a high-performance diagnostic model. These DAMs demonstrated significant correlations with VHL gene mutation types. Cox regression analysis revealed that plasma levels of N2,N2-dimethylguanosine were associated with the timing of RCC onset in VHL patients, acting as an independent predictive factor. This study enhances diagnostic accuracy for this rare condition and opens new avenues for exploring metabolic mechanisms of the disease and potential therapeutic directions.

Summary: Background: Cholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. Methods: Enabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. Findings: Surprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIFα and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness in vitro and in vivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. Interpretation: Collectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. Funding: This work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No. 2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

Summary: The efficacy of chimeric antigen receptor (CAR) T cell immunotherapy is limited by insufficient infiltration and activation of T cells due to the immunosuppressive tumor microenvironment. Preclinical studies with optimized mouse CAR T cells in immunocompetent mouse cancer models will help define the mechanisms underlying immunotherapy resistance. Here, we present a protocol for preparing mouse T cells and generating CAR T cells. We then detail procedures for testing their therapeutic efficacy and tracking them in a syngeneic mouse glioma model.For complete details on the use and execution of this protocol, please refer to Zhang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract A lipidome comprises thousands of lipid species, many of which are isomers and isobars. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), although widely used for lipidomic profiling, faces challenges in differentiating lipid isomers. Herein, we address this issue by leveraging the orthogonal separation capabilities of hydrophilic interaction liquid chromatography (HILIC) and trapped ion mobility spectrometry (TIMS). We further integrate isomer-resolved MS/MS methods onto HILIC-TIMS, which enable pinpointing double bond locations in phospholipids and sn-positions in phosphatidylcholine. This system profiles phospholipids at multiple structural levels with short analysis time (

Abstract Background The management of advanced clear cell renal cell carcinoma (ccRCC) remains a major challenge in clinical practice, and the construction of more reliable prognostic prediction models and the further elucidation of key molecular mechanisms of tumor progression are topics in urgent need of in-depth investigation. Methods We used CIBERSORT to estimate the proportion of 22 tumor-infiltrating immune cell types in the TCGA-KIRC cohort. Weighted gene co-expression network analysis, least absolute shrinkage and selection operator regression analysis were used to build risk prediction models. Expression patterns and clinical significance of TRAF2 were determined through bioinformatics analysis, real-time qPCR, Western Blot, immunohistochemistry. GSEA analysis, transmission electron microscopy, 2D/3D colony formation assay, cell migration and invasion assay, and tube-formation assay were used to investigate the underlying function and mechanism of the TRAF2/M2 macrophage/autophagy axis. Results We constructed a novel prognostic prediction model based on M2 macrophage-related genes, which was identified as an accurate, independent and specific prognostic risk model for ccRCC patients. A reliable nomogram was constructed to predict 1-, 3-, and 5-year overall survival for patients with ccRCC. As one of the constituent genes of the risk model, TRAF2 was determined to be upregulated in ccRCC and associated with poor clinical prognosis. We found that TRAF2 promotes malignant progression of ccRCC by regulating macrophage polarization, migration and angiogenesis. Mechanistically, we found that TRAF2 promotes the polarization of M2 macrophages, and this chemotaxis is achieved in an autophagy-dependent pathway. Orthotopic tumor growth assay results revealed that TRAF2 plays a key role as a promotor of ccRCC growth and metastasis. Conclusions In conclusion, this risk model is highly predictive of prognostic in ccRCC patients, which is expected to promote improved treatment evaluation and comprehensive management of ccRCC. Moreover, our findings reveal that the TRAF2/M2 macrophage/autophagy axis plays a key regulatory role in the malignant progression of ccRCC, and suggest that TRAF2 is a potential novel therapeutic target for advanced ccRCC.

Abstract Recent evidences have suggested that Zinc finger protein 582 (ZNF582) plays different important roles in various tumors, but its clinical role, biological function and regulatory mechanism in clear cell renal cell carcinoma (ccRCC) are still vague. Through analyzing GEO and TCGA-KIRC data and validation with local samples, we identified the low expression pattern of ZNF582 in ccRCC. Decreased ZNF582 expression is correlated with higher tumor stage and grade, distant metastasis and poor prognosis. By analyzing the DNA methylation data of ccRCC in TCGA-KIRC and using Massarray DNA methylation and demethylation analysis, we confirmed the hypermethylation status of ZNF582 in ccRCC and its negative regulation on ZNF582 expression. Using cell phenotype experiments and orthotopic kidney tumor growth models, we determined the inhibitory effect of ZNF582 overexpression on ccRCC growth and metastasis in vivo and in vitro. Mechanistically, using TMT (Tandem mass tags) quantitative proteomics test, Co-IP (Co-immunoprecipitation) and Western Blot experiments, we clarified that ZNF582 binds to TJP2 and up-regulates TJP2 protein expression. Increased TJP2 protein combines with ERK2 to promote ERK2 protein expression and suppresses the phosphorylation of ERK2, thereby inhibiting the growth and metastasis of ccRCC. In general, our findings provide the first solid theoretical rationale for targeting ZNF582/TJP2/ERK2 axis to improve ccRCC treatment.

Abstract Background Clear cell renal cell carcinoma (ccRCC) is characterized by the accumulation of lipid-reactive oxygen species. Ferroptosis, due to the lipid peroxidation, has been reported to be strongly correlated with tumorigenesis and progression. However, the functions of the ferroptosis process in ccRCC remain unclear. Methods After sample cleaning, data integration, and batch effect removal, we used the Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases to screen out the expression and prognostic value of ferroptosis-related lncRNAs and then performed the molecular subtyping using the K-means method. Then, the functional pathway enrichment and immune microenvironment infiltration between the different clusters were carried out. The results showed a significant difference in immune cell infiltration between the two clusters and the associated marker responded to individualized differences in treatment. Then, least absolute shrinkage and selection operator (LASSO) Cox regression was used to establish a prognostic signature based on 5 lncRNAs. This signature could accurately predicted patient prognosis and served as an independent clinical risk factor. We then combined significant clinical parameters in multivariate Cox regression and the prognostic signature to construct a clinical predictive nomogram, which provides appropriate guidance for predicting the overall survival of ccRCC patients. Results The prognostic differentially expressed ferroptosis-related LncRNAs (DEFRlncRNAs) were found, and 5 lncRNAs were finally used to establish the prognostic signature in the TCGA cohort, with subsequently validation in the internal and external cohorts. Moreover, we conducted the molecular subtyping and divided the patients in the TCGA cohort into two clusters showing differences in Hallmark pathways, immune infiltration, immune target expression, and drug therapies. Differences between clusters contributed to individualizing treatment. Furthermore, a nomogram was established to better predict the clinical outcomes of the ccRCC patients. Conclusions Our study conducted molecular subtyping and established a novel predictive signature based on the ferroptosis-related lncRNAs, which contributed to the prognostic prediction and individualizing treatment of ccRCC patients.

Cabotegravir is an integrase strand transfer inhibitor (INSTI) for HIV treatment and prevention. Cabotegravir-based long-acting pre-exposure prophylaxis (PrEP) presents an emerging paradigm for infectious disease control. In this scheme, a combination of a high efficacy and low solubility of anti-infection drugs permits the establishment of a pharmaceutical firewall in HIV-vulnerable groups over a long period. Although the structure-activity-relationship (SAR) of cabotegravir as an INSTI is known, the structural determinants of its low solubility have not been identified. In this work, we have integrated multiple experimental and computational methods, namely X-ray diffraction, solid-state NMR (SSNMR) spectroscopy, solution NMR spectroscopy, automated fragmentation (AF)-QM/MM and density functional theory (DFT) calculations, to address this question. The molecular organization of cabotegravir in crystal lattice has been determined. The combination of very-fast magic-angle-sample-spinning (VF MAS) SSNMR and solution NMR, as supported by AF-QM/MM and DFT calculations, permits the identification of structural factors that contribute to the low aqueous solubility of cabotegravir. Our study reveals the multitasking nature of pharmacophores in cabotegravir, which controls the drug solubility and, meanwhile, the biological activity. By unraveling these function-defining molecular features, our work could inspire further development of long-acting HIV PrEP drugs.

Background: Dysregulation of long noncoding RNAs (lncRNAs) has been reported to be associated with multiple tumors where they act as tumor suppressors or accelerators. The lncRNA CYTOR was identified as an oncogene involved in many cancers, such as gastric cancer, colorectal cancer, hepatocellular carcinoma, and renal cell carcinoma. However, the role of CYTOR in bladder cancer (BCa) has rarely been reported. Methods: Using cancer datasets from The Cancer Genome Atlas (TCGA) program, we analyzed the association between CYTOR expression and prognostic value, oncogenic pathways, antitumor immunity and immunotherapy response in BCa. The influence of CYTOR on the immune infiltration pattern in the urothelial carcinoma microenvironment was further verified in our dataset. Single-cell analysis revealed the role of CYTOR in the tumor microenvironment (TME) of BCa. Finally, we evaluated the expression of CYTOR in BCa in the Peking University First Hospital (PKU–BCa) dataset and its correlation with the malignant phenotype of BCa in vitro and in vivo. Results: The results indicated that CYTOR was highly expressed in multiple cancer samples, including BCa, and increased CYTOR expression contributed to poor overall survival (OS). Additionally, elevated CYTOR expression was significantly correlated with clinicopathological features of BCa, such as female sex, advanced TNM stage, high histological grade and non-papillary subtype. Functional characterization revealed that CYTOR may be involved in immune-related pathways and the epithelial mesenchymal transformation (EMT) process. Moreover, CYTOR had a significant association with infiltrating immune cells, including M2 macrophages and regulatory T cells (Tregs). CYTOR facilitates the crosstalk between cancer-associated fibroblasts (CAFs) and macrophages, and mediates M2 polarization of macrophages. Correlation analysis revealed a positive correlation between CYTOR expression and programmed cell death-1 (PD-1)/programmed death ligand 1 (PD-L1)/expression and other targets for specific immunotherapy in BCa, which are recognized to predict the efficacy of immunotherapy. Conclusions: These results suggest that CYTOR serves as a potential biomarker for predicting survival outcome, TME cell infiltration characteristics and immunotherapy response in BCa.

Due to fewer adverse events, faster onset of action, and longer durable responses compared to chemotherapy, immunotherapy has been widely used to treat advanced solid tumors. Moreover, immunotherapy can improve the autoimmune status, thus allowing patients to benefit from the treatment in the long term. The immune microenvironment status is closely associated with the response to chemotherapies. Here, we analyzed the characteristics of the immune microenvironment in pheochromocytoma and paraganglioma (PPGL). Immunohistochemistry showed that PD-L1 is sparely expressed in PPGL with low positive rates and low expression levels, an expression pattern, that is, not correlated with tumor malignancy. Moreover, the level of intratumoral CD4+ and CD8+ lymphocyte infiltration in PPGL is low, suggesting that the immune microenvironment in PPGL may be in “immune desertification” or “immune rejection” states in which CD4+ and CD8+ lymphocyte infiltration is prevented, rendering immunotherapy less effective. In sum, our results indicate that PPGL is a microsatellite-stable tumor with low tumor mutational burden (TMB) levels, weak neoantigen production, and poor tumor antigenicity, hinting at a poor response of PPGL to chemotherapies.

BackgroundIn bladder and breast cancer, the claudin-low subtype is widely identified, revealing a distinct tumor microenvironment (TME) and immunological feature. Although we have previously identified individual claudin members as prognostic biomarkers in clear cell renal cell carcinoma (ccRCC), the existence of an intrinsic claudin-low subtype and its interplay with TME and clinical outcomes remains unclear.MethodsTranscriptomic and clinical data from The Cancer Genome Atlas (TCGA)- kidney clear cell carcinoma (KIRC) cohort and E-MTAB-1980 were derived as the training and validation cohorts, respectively. In addition, GSE40435, GSE53757, International Cancer Genome Consortium (ICGC) datasets, and RNA-sequencing data from local ccRCC patients were utilized as validation cohorts for claudin clustering based on silhouette scores. Using weighted correlation network analysis (WGCNA) and multiple machine learning algorithms, including least absolute shrinkage and selection operator (LASSO), CoxBoost, and random forest, we constructed a claudin-TME related (CTR) risk signature. Furthermore, the CTR associated genomic characteristics, immunity, and treatment sensitivity were evaluated.ResultsA claudin-low phenotype was identified and associated with an inferior survival and distinct TME and cancer immunity characteristics. Based on its interaction with TME, a risk signature was developed with robust prognostic prediction accuracy. Moreover, we found its association with a claudin-low, stem-like phenotype and advanced clinicopathological features. Intriguingly, it was also effective in kidney chromophobe and renal papillary cell carcinoma. The high CTR group exhibited genomic characteristics similar to those of claudin-low phenotype, including increased chromosomal instability (such as deletions at 9p) and risk genomic alterations (especially BAP1 and SETD2). In addition, a higher abundance of CD8 T cells and overexpression of immune checkpoints, such as LAG3, CTLA4 and PDCD1, were identified in the high CTR group. Notably, ccRCC patients with high CTR were potentially more sensitive to immune checkpoint inhibitors; their counterparts could have more clinical benefits when treated with antiangiogenic drugs, mTOR, or HIF inhibitors.ConclusionWe comprehensively evaluated the expression features of claudin genes and identified a claudin-low phenotype in ccRCC. In addition, its related signature could robustly predict the prognosis and provide guide for personalizing management strategies.

Glioblastomas are generally resistant to treatment with immune checkpoint inhibitors. Here the authors show that IL6 blockade, in combination with a CD40 agonist, overcomes macrophage-mediated immunosuppression and sensitizes glioblastoma to immune checkpoint blockade in preclinical models.

Background: Early detection and precise prognostic evaluation of clear cell renal cell carcinoma (ccRCC) are crucial for patient life expectancy. Ion channel-related genes (ICRGs) are of great diagnostic and prognostic value as components that maintain the normal structure of the kidney. Therefore, we systematically explored the diagnostic, prognostic, and therapeutic value of ICRGs in ccRCC using the multi-database.Methods: RNA transcriptome profiles and clinical data of ccRCC patients were extracted and integrated from public databases including The Cancer Genome Atlas, ICGC, GEO, and E-MTAB databases. Ion channel-related genes were obtained from the literature collection. The diagnostic signature was performed using the LASSO and SVM-REF analyses. Meanwhile, the prognostic signature was conducted using the LASSO analyses. Molecular subtyping was performed using the ConsensusClusterPlus and the corresponding therapeutic targets were evaluated using the pRRophetic package. In addition, a prognostic nomogram was constructed based on the results of cox regression analyses.Results: We successfully constructed diagnostic signatures for five ICRGs and prognostic signatures for 10 ICRGs with AUC values greater than 0.7, showing good predictive performance. Based on the median risk score, we found that high-risk patients had a significantly worse prognosis. We also divided ccRCC patients into two clusters according to prognostic ICRGs, and there was a significant survival outcome between the two clusters and different sensitivity to diverse clinical therapeutic strategies. Meanwhile, we constructed a nomogram based on clinical molecules and signatures, and its predictive efficacy was better than the signature or the present tumor-node-metastasis staging system.Conclusion: In this study, we established useful signatures for early detection, prognosis evaluation, and individualized treatment for ccRCC. Moreover, KCNJ16 deserves to be explored comprehensively in the future.

Abstract Background Emerging evidence confirms that lncRNAs (long non-coding RNAs) are potential biomarkers that play vital roles in tumors. ZNF582-AS1 is a novel lncRNA that serves as a potential prognostic marker of cancers. However, the specific clinical significance and molecular mechanism of ZNF582-AS1 in ccRCC (clear cell renal cell carcinoma) are unclear. Methods Expression level and clinical significance of ZNF582-AS1 were determined by TCGA-KIRC data and qRT-PCR results of 62 ccRCCs. DNA methylation status of ZNF582-AS1 promoter was examined by MSP, MassARRAY methylation and demethylation analysis. Gain-of-function experiments were conducted to investigate the biological roles of ZNF582-AS1 in the phenotype of ccRCC. The subcellular localization of ZNF582-AS1 was detected by RNA FISH. iTRAQ, RNA pull-down and RIP-qRT-PCR were used to identify the downstream targets of ZNF582-AS1. rRNA MeRIP-seq and MeRIP-qRT-PCR were utilized to examine the N(6)-methyladenosine modification status. Western blot and immunohistochemistry assays were used to determine the protein expression level. Results ZNF582-AS1 was downregulated in ccRCC, and decreased ZNF582-AS1 expression was significantly correlated with advanced tumor stage, higher pathological stage, distant metastasis and poor prognosis. Decreased ZNF582-AS1 expression was caused by DNA methylation at the CpG islands within its promoter. ZNF582-AS1 overexpression inhibited cell proliferative, migratory and invasive ability, and increased cell apoptotic rate in vitro and in vivo. Mechanistically, we found that ZNF582-AS1 overexpression suppressed the N(6)-methyladenosine modification of MT-RNR1 by reducing rRNA adenine N(6)-methyltransferase A8K0B9 protein level, resulting in the decrease of MT-RNR1 expression, followed by the inhibition of MT-CO2 protein expression. Furthermore, MT-RNR1 overexpression reversed the decreased MT-CO2 expression and phenotype inhibition of ccRCC induced by increased ZNF582-AS1 expression. Conclusions This study demonstrates for the first time that ZNF582-AS1 functions as a tumor suppressor gene in ccRCC and ZNF582-AS1 may serve as a potential biomarker and therapeutic target of ccRCC.

Abstract TP53 is a classic tumor suppressor, but its role in kidney cancer remains unclear. In our study, we tried to explain the role of p53 in kidney cancer through the p53-related enhancer RNA pathway. Functional experiments were used to explore whether P53-bound enhancer regions 2 (p53BER2) has a role in the cell cycle and senescence response of TP53-wild type (WT) renal cancer cells in vitro or vivo. RNA-sequencing was used to identify the potential target of p53BER2. The results showed that the expression level of P53BER2 was downregulated in renal cancer tissues and cell lines, further dual-luciferase experiments and APR-256-reactivated experiments showed p53BER2 expresses in a p53-dependent way. Moreover, knockdown p53BER2 could reverse nutlin-3-induced cytotoxic effect in TP53-WT cell lines. Further exploration showed the downregulation of p53BER2 could reverse nutlin-3-induced G1-arrest and senescence in TP53-WT cell lines. What is more, the knockdown of p53BER2 showed resistance to nutlin-3 treatment in vivo. Additionally, we found BRCA2 could be regulated by p53BER2 in vitro and vivo; further experiment showed p53BER2 could induce cell-cycle arrest and DNA repair by mediating BRCA2. In summary, the p53-associated enhancer RNA-p53BER2 mediates the cell cycle and senescence of p53 in TP53-WT renal cancer cells.

Abstract Prostate cancer (PCa) is a complex disease. An ongoing accumulation of mutations results in increased genetic diversity, with the tumor acquiring distinct subclones. However, non‐genetic intra‐tumoral heterogeneity, the cellular differentiation state and the interplay between subclonal evolution and transcriptional heterogeneity are poorly understood. Here, the authors perform single‐cell RNA sequencing from 14 untreated PCa patients. They create an extensive cell atlas of the PCa patients and mapped developmental states onto tumor subclonal evolution. They identify distinct subclones across PCa patients and then stratify tumor cells into four transcriptional subtypes, EMT‐like (subtype 0), luminal A‐like (subtype 1), luminal B/C‐like (subtype 2), and basal‐like (subtype 3). These subtypes are hierarchically organized into stem cell‐like and differentiated status. Strikingly, multiple subclones within a single primary tumor present with distinct combinations of preferential subtypes. In addition, subclones show different communication strengths with other cell types within the tumor ecosystem, which may modulate the distinct transcriptional subtypes of the subclones. Notably, by integrating TCGA data, they discover that both tumor cell transcriptional heterogeneity and cellular ecosystem diversity correlate with features of a poor prognosis. Collectively, their study provides the analysis of subclonal and transcriptional heterogeneity and its implication for patient prognosis.

Radiation causes a collapse of bone marrow cells and elimination of microvasculature. To understand how bone marrow recovers after radiation, we focused on mesenchymal lineage cells that provide a supportive microenvironment for hematopoiesis and angiogenesis in bone. We recently discovered a nonproliferative subpopulation of marrow adipogenic lineage precursors (MALPs) that express adipogenic markers with no lipid accumulation. Single-cell transcriptomic analysis revealed that MALPs acquire proliferation and myofibroblast features shortly after radiation. Using an adipocyte-specific Adipoq-Cre, we validated that MALPs rapidly and transiently expanded at day 3 after radiation, coinciding with marrow vessel dilation and diminished marrow cellularity. Concurrently, MALPs lost most of their cell processes, became more elongated, and highly expressed myofibroblast-related genes. Radiation activated mTOR signaling in MALPs that is essential for their myofibroblast conversion and subsequent bone marrow recovery at day 14. Ablation of MALPs blocked the recovery of bone marrow vasculature and cellularity, including hematopoietic stem and progenitors. Moreover, VEGFa deficiency in MALPs delayed bone marrow recovery after radiation. Taken together, our research demonstrates a critical role of MALPs in mediating bone marrow repair after radiation injury and sheds light on a cellular target for treating marrow suppression after radiotherapy.

ObjectiveWhole-exon sequencing (WES) is a commercially available tool for hereditary disease testing. However, little is known about hereditary upper-tract urothelial carcinoma (UTUC) in the Chinese population. This study aims to investigate the prevalence of Lynch syndrome (LS) in UTUC patients with high-risk features and identify the germline mutations of genetic predisposition gene mutations in those patients.MethodsIn total, 354 consecutive UTUC patients undergoing surgery were universally recruited, of whom 108 patients under 60 years old or with a personal/family history of cancer underwent universal immunohistochemistry staining to detect the expression of mismatch repair (MMR) proteins (MLH1, MSH2, MSH6 and PMS2). Patients with deficient or weak MMR protein staining or meeting the Amsterdam II criterion were defined as suspected LS patients, who further experienced microsatellite instability (MSI) (BAT25, BAT26, BAT40, D2S123, D5S346, D17S250) detection and performed WES analysis to explore germline pathogenic/likely pathogenic (P/LP) alterations.ResultsOf 108 patients, 90 (83.3%) cases were included due to younger than 60 years, and 18 cases due to personal/family history. IHC staining identified 21 patients with deficient MMR protein staining and 15 cases with weak MMR protein staining. Three cases met the Amsterdam II criterion but with proficient MMR protein staining. Finally, WES analysis was performed in 38 suspected LS patients and P/LP germline mutations were identified in 22 individuals. Genetic testing confirmed 5 LS cases, including 3 cases with novel mutations. MSI-harboring tumor was discovered in 4 LS cases, one of whom had weak MMR protein staining. Germline P/LP variants in DNA damage repair genes were found in 11 cases. In addition, we found that 11 patients had high- or moderate- penetrance P/LP mutations other than MMR genes. The common P/LP variants in high- or moderate-penetrance genes were 4 in ATM, 3 in MSH6 and KIT, and 2 in APC, NF1 and DICER.ConclusionsWe identified approximately 11% of UTUC cases as suspected LS and at least 1.4% patients with confirmed LS-associated UTUC. In addition, broader germline genetic testing could be considered to screen for cancer severity in hereditary UTUC patients.

Renal cell carcinoma (RCC) is one of the most common urological cancers. RCC has a poor prognosis and is considered a metabolic disease. It has been reported that many metabolic pathways are associated with the development of RCC. However, the prognostic value of metabolism-related genes in RCC is unclear. We herein aimed to establish a scoring system based on the gene expression profile of metabolic genes to evaluate the response to immunotherapy and predict the prognosis of RCC. In this study, we collected multicentre RCC data and performed integrated analysis to characterize the role of tumour metabolism in RCC and explore the relationship between metabolism and prognosis and immune therapy. Based on transcriptomic data, metabolism-related genes were used for nonnegative matrix factorization clustering. We obtained three subclasses of RCC (M1, M2, and M3), and they are associated with different prognoses and immune infiltrate levels. Then, based on the pathway activity of 113 metabolism-related gene signatures, we classified patients into three distinct metabolism-related signatures. Finally, we provide a metabolism-related pathway score (MRPScore) that is significantly associated with RCC prognosis and the response to immunotherapy. Taken together, in this study, we established an RCC classification system based on metabolic gene expression profiles that could further the understanding of the diversity of RCC. We also present the MRPScore, which may be used as an indicator to predict the response to clinical immune therapy.

Abstract Background Accumulating evidence indicates that long non-coding RNAs (lncRNAs) are potential biomarkers and key regulators of tumour development and progression. SOX2 overlapping transcript (SOX2OT) is a novel lncRNA that acts as a potential biomarker and is involved in the development of cancer and cancer stem cells. However, the clinical significance and molecular mechanism of SOX2OT in bladder cancer are still unknown. Methods The expression level of SOX2OT was determined by RT-qPCR in a total of 106 patients with urothelial bladder cancer and in different bladder cancer cell (BCC) lines. Bladder cancer stem cells (BCSCs) were isolated from BCCs using flow cytometry based on the stem cell markers CD44 and ALDH1. Loss-of-function experiments were performed to investigate the biological roles of SOX2OT in the stemness phenotype of BCSCs. Comprehensive transcriptional analysis, RNA FISH, dual-luciferase reporter assays and western blots were performed to explore the molecular mechanisms underlying the functions of SOX2OT. Results SOX2OT was highly expressed in bladder cancer, and increased SOX2OT expression was positively correlated with a high histological grade, advanced TNM stage and poor prognosis. Further experiments demonstrated that knockdown of SOX2OT inhibited the stemness phenotype of BCSCs. Moreover, inhibition of SOX2OT delayed xenograft tumour growth and decreased metastases in vivo. Mechanistically, we found that SOX2OT was mainly distributed in the cytoplasm and positively regulated SOX2 expression by sponging miR-200c. Furthermore, SOX2 overexpression reversed the SOX2OT silencing-induced inhibition of the BCSC stemness phenotype. Conclusion This study is the first to demonstrate that SOX2OT plays an important regulatory role in BCSCs and that SOX2OT may serve as a potential diagnostic biomarker and therapeutic target in bladder cancer.

Abstract Upper tract urothelial carcinomas (UTUCs) are rare entities that are usually diagnosed at advanced stages. Research on UTUC pathobiology and clinical management has been hampered by the lack of models accurately reflecting disease nature and diversity. In this study, a modified organoid culture system is used to generate a library of 25 patient‐derived UTUC organoid lines retaining the histological architectures, marker gene expressions, genomic landscapes, and gene expression profiles of their parental tumors. The study demonstrates that the responses of UTUC organoids to anticancer drugs can be identified and the model supports the exploration of novel treatment strategies. This work proposes a modified protocol for generating patient‐derived UTUC organoid lines that may help elucidate UTUC pathophysiology and assess the responses of these diseases to various drug therapies in personalized medicine.

Background. FXYD2, a gene coding for the γ subunit of Na+/K+-ATPase, was demonstrated to involve in carcinogenesis recently. However, the specific role of FXYD2 in clear cell renal cell carcinoma (ccRCC) remains unknown. The current study was conducted to investigate the expression, biological function, and potentially immune-related mechanisms of FXYD2 in ccRCC. Materials and methods. The data from TCGA-KIRC, ICGC, GEO, Oncomine, ArrayExpress, TIMER, HPA datasets, and our clinical samples were used to determine and validate the expression level, prognostic roles, and potentially immune-related mechanisms in ccRCC. Cell function assays were performed to investigate the biological role of FXYD2 in vitro. Results. FXYD2 was identified to be downregulated in ccRCC tissue compared to normal tissue, which was confirmed by our RT-PCR, WB, and IHC analyses. Kaplan-Meier survival analysis and Cox regression analysis suggested that downregulated FXYD2 could independently predict poor survival of ccRCC patients. Through the ESTIMATE algorithm, ssGSEA algorithm, CIBERSORT algorithm, TIMER database, and our laboratory experiment, FXYD2 was found to correlate with the immune landscape, especially regulatory T cells (Treg), in ccRCC. Gain-of-function experiment revealed that FXYD2 could restrain cell proliferation, migration, and invasion in vitro. Functional enrichment analysis illustrated that TGF-β-SMAD2/3, Notch, and PI3K-Akt-mTOR signaling pathways may be potential signaling pathways of FXYD2 in ccRCC. Conclusions. Downregulation of FXYD2 is associated with ccRCC tumorigenesis, poor prognosis, and increased Treg infiltration in ccRCC, which may be related to TGF-β-SMAD2/3, Notch, and PI3K-Akt-mTOR signaling pathways. This will probably provide a novel prognostic marker and potential therapeutic target for ccRCC.

Tumor staging of upper tract urothelial carcinomas (UTUCs) is relatively difficult to assert accurately before surgery. Here, we used copy number (CN) signatures as a tool to explore their clinical significance of molecular stratification in UTUC. CN signatures were extracted by non-negative matrix factorization from the whole-genome sequencing (WGS) data of 90 Chinese UTUC primary tumor samples. A validation UTUC cohort (n = 56) and a cohort from urinary cell-free DNA (cfDNA) of urothelial cancer patients (n = 94) and matched primary tumors were also examined. Survival analyses were measured using the Kaplan–Meier, and Cox regression was used for multivariate analysis. Here, we identified six CN signatures (Sig1–6). Patients with a high contribution of Sig6 (Sig6high) were associated with higher microsatellite instability level and papillary architecture and had a favorable outcome. Patients with a low weighted genome integrity index were associated with positive lymph node and showed the worst outcome. Sig6high was identified to be an independently prognostic factor. The predictive significance of CN signature was identified by a validation UTUC cohort. CN signatures retained great concordance between primary tumor and urinary cfDNA. In conclusion, our results reveal that CN signature assessment for risk stratification is feasible and provides a basis for clinical studies that evaluate therapeutic interventions and prognosis.

Background: Metastatic bladder cancer (BLCA) is a lethal disease with an unmet need for study. Transgelin (TAGLN) is an actin-binding protein that affects the dynamics of the actin cytoskeleton indicating its robust potential as a metastasis initiator. Here, we sought to explore the expression pattern of TAGLN and elucidate its specific functioning and mechanisms in BLCA. Methods: A comprehensive assessment of TAGLN expression in BLCA was performed in three cohorts with a total of 847 patients. The potential effects of TAGLN on BLCA were further determined using clinical genomic analyses that guided the subsequent functional and mechanistic studies. In vitro migration, invasion assays and in vivo metastatic mouse model were performed to explore the biological functions of TAGLN in BLCA cells. Immunofluorescence, western blot and correlation analysis were used to investigate the molecular mechanisms of TAGLN. Findings: TAGLN was highly expressed in BLCA and correlated with advanced prognostic features. TAGLN promoted cell colony formation and cell migration and invasion both in vitro and in vivo by inducing invadopodia formation and epithelial-mesenchymal transition, during which a significant correlation between TAGLN and Slug was observed. The progression-dependent correlation between TGF-β and TAGLN was analysed at both the cellular and tissue levels, while TGF-β-mediated migration was abolished by the suppression of TAGLN. Interpretation: Overall, TAGLN is a promising novel prognosis biomarker of BLCA, and its metastatic mechanisms indicate that TAGLN may represent a novel target agent that can be utilized for the clinical management of invasive and metastatic BLCA. Fund: This work was supported by the National Natural Science Foundation of China [81772703, 81672546, 81602253]; the Natural Science Foundation of Beijing [71772219, 7152146]. and Innovative Fund for Doctoral Students of Peking University Health Science Center (BUM2018BSS002). Funders had no role in the design of the study, data collection, data analysis, interpretation, or the writing of this report. Keywords: Bladder cancer, Transgelin, Metastasis, EMT, Invadopodia, TGF-β

Purpose: Gleason score (GS) system is one of the most widely used histological grading methods for prostate cancer (PCa) all over the world. GS can be obtained by adding the primary Gleason pattern (GP) and secondary GP. Different proportions of GP 4 and GP 5 in prostate specimens can both lead to GS 9. In this study, we explored whether GP 5 + 4 or GP 4 + 5 was associated with different prognoses among patients with GS 9 PCa.Materials and methods: A retrospective population-based study was conducted on 10,124 subjects diagnosed with GS 9 PCa between 2004 and 2009 from the Surveillance, Epidemiology, and End Results program. A 1:1 propensity-score matching (PSM) was performed to balance the baseline characteristics between the GP 4 + 5 and 5 + 4 groups and to compare the prognoses between the two groups. Cox regression analysis and Fine-Gray competing risk regression models were adopted to screen the covariates significantly associated with all-cause mortality (ACM) and cancer-specific mortality (CAM).Results: GP 5 + 4 was associated with higher risks of ACM and CSM before or after PSM than GP 4 + 5. In the original cohort, there were eight independent predictors for ACM, which were age at diagnosis, race, AJCC NM stage, PSA levels, treatments, GP, and marital status, confirmed by the Cox analysis; and nine independent predictors for CSM, which were age at diagnosis, race, AJCC TNM stage, PSA levels, treatments, GP, and marital status, confirmed by the competing-risk model.Conclusion: GP 5 + 4 was associated with a poorer overall survival and cancer-specific survival compared with GP 4 + 5.

PurposeVon Hippel-Lindau (VHL) disease is a hereditary kidney cancer syndrome, with which patients are more likely to get affected by renal cell carcinoma (RCC), pancreatic cyst or tumor (PCT), central nervous system hemangioblastoma (CHB), retinal angiomas (RA), and pheochromocytoma (PHEO). Mutations of VHL gene located in 3p25 may impair the function of the VHL protein and lead to the disease. It’s unclear why obvious phenotype varieties exist among VHL patients. Here we aimed to ascertain whether the mutation types and locations affect the phenotype.MethodsWe enrolled 577 Chinese VHL patients from 211 families and divided them into three groups and six subgroups according to their mutation types and locations. Cox survival analysis and Kaplan-Meier analysis were used to compare intergroup age-related tumor risks.ResultsPatients with nonsense or frameshift mutations that were located before residues 117 of VHL protein (NoF1 subgroup) hold lower age-related risks of VHL associated tumors (HR = 0.638, 95%CI 0.461–0.883, p = 0.007), CHB (HR = 0.596, 95%CI 0.409–0.868, p = 0.007) or PCT (HR = 0.595, 95%CI 0.368–0.961, p = 0.034) than patients whose mutations were located after residues 117 (NoF2 subgroup). Patients in NoF1 subgroup still had lower age-related risks of CHB (HR = 0.652, 95%CI 0.476–0.893, p = 0.008) and PCT (HR = 0.605, 95%CI 0.398–0.918, p = 0.018) compared with those in combined NoF2 subgroup and other truncating mutation patients. NoF1 subgroup correspondingly had a longer estimated median lifespan (64 vs. 55 year, p = 0.037) than NoF2 subgroup. Among patients with missense mutations of VHL, only a small minority (23 of 286 missense mutations carriers) carried mutations involving neither HIF-α binding region nor elongin C binding region, who were grouped in MO subgroup. MO subgroup seemed to have a higher age-related risk of PHEO. In the whole cohort (n = 577), PHEO was an independent protective factor for CHB (p = 0.001) and survival (p = 0.005). RA and CHB failed to predict the age-related risk of each other.ConclusionThe mutation types and locations of VHL gene are associated with phenotypes. Genetic counselors could predict phenotypes more accurately based on more detailed genotype-phenotype correlations. Further genotype-phenotype studies should focus on the prediction of tumor recurrence, progression, and metastasis. The deep molecular mechanism of genotype-phenotype correlation is worth further exploring.

RNA-binding proteins (RBPs) are a kind of gene regulatory factor that presents a significant biological effect in the initiation and development of various tumors, including bladder cancer (BLCA). However, the RBP-based prognosis signature for BLCA has not been investigated. In this study, we attempted to develop an RBP-based classifier to predict overall survival (OS) for BLCA based on transcriptome analysis. We extracted data of BLCA patients from The Cancer Genome Atlas database (TCGA) and UCSC Xena. Finally, a total of 398 cases without missing clinical data were enrolled and six RBPs (FLNA, HSPG2, AHNAK, FASTKD3, POU5F1, and PCSK9) associated with OS of BLCA were identified through univariate and multivariate Cox regression analysis. Online analyses and immunohistochemistry validated the prognostic value and expression of six RBPs. Risk scores were calculated to divide patients into high-risk and low-risk level, and patients in the high-risk group tended to have a poor prognosis. In addition, the receiver operating characteristic (ROC) curve analysis was performed to assess the prognostic value of RBPs, and the area under the curve (AUC) values were 0.711 and 0.706, respectively, in the training set and validating set. The findings were further validated in an external validation set. Subsequently, the 6-RBP-based signature and pathological stage were used to construct the nomogram to predict the 3- and 5-years OS of BLCA patients. Also, this 6-RBP-based signature was highly related to recurrence-free survival of BLCA. Weighted co-expression network analysis (WGCNA) combined with functional enrichment analysis contributed to study the potential pathways of six RBPs, including keratinocyte differentiation, RHO GTPases activate PNKs, epithelial tube morphogenesis, establishment or maintenance of cell polarity, and so on. In summary, the 6-RBP-based signature holds the potentiality to serve as a novel prognostic predictor of OS for BLCA.

Bone marrow mesenchymal lineage cells are a heterogeneous cell population involved in bone homeostasis and diseases such as osteoporosis. While it is long postulated that they originate from mesenchymal stem cells, the true identity of progenitors and their in vivo bifurcated differentiation routes into osteoblasts and adipocytes remain poorly understood. Here, by employing large scale single cell transcriptome analysis, we computationally defined mesenchymal progenitors at different stages and delineated their bi-lineage differentiation paths in young, adult and aging mice. One identified subpopulation is a unique cell type that expresses adipocyte markers but contains no lipid droplets. As non-proliferative precursors for adipocytes, they exist abundantly as pericytes and stromal cells that form a ubiquitous 3D network inside the marrow cavity. Functionally they play critical roles in maintaining marrow vasculature and suppressing bone formation. Therefore, we name them marrow adipogenic lineage precursors (MALPs) and conclude that they are a newly identified component of marrow adipose tissue.

Abstract Background Metastasis is the primary cause of death in renal cell carcinoma (RCC). Loss of cell-to-cell adhesion, including tight junctions (TJs) is the initial step in the process of metastasis. Claudin-7 (CLDN7) is a major component of TJs. However, the clinical significance and its regulation of kidney tumorigenesis remain poorly understood. Methods A total of 120 fresh clear cell RCC (ccRCC) specimens and 144 primary RCC and adjacent nonmalignant renal paraffin specimens were obtained from Department of Urology, Peking University First Hospital. Expression of CLDN7 in ccRCC tissues and cell lines were determined using bioinformatic data mining, quantitative real-time PCR (qRT-PCR), Western blotting and immunostaining. The clinical significance of CLDN7 expression and promoter DNA methylation status was analyzed in ccRCC patients from Peking University First Hospital and The Cancer Genome Atlas. Additionally, the methylation specific-PCR, bisulfite genomic sequencing and demethylation analysis of CLDN7 were performed. Biological functions of CLDN7 were investigated by examining cell proliferation using MTS assays and EdU incorporation assays, cell migration by in vitro wound healing assays and transwell migration assays, cell invasion by transwell invasion assays, and cell apoptosis by flow cytometry. Mouse model experiments were performed to confirm the effects of CLDN7 on tumor growth and metastasis in vivo. The molecular mechanism of CLDN7 function was investigated using gene-set enrichment analysis (GSEA) and high-throughput cDNA sequencing (RNA-Seq) and confirmed by qRT-PCR, Western blot and immunostaining in vitro and in vivo. Results Our findings revealed that CLDN7 is frequently downregulated via hypermethylation of its promoter in ccRCC. CLDN7 can help predict aggressive tumor status and poor prognosis in ccRCC patients. Interestingly, hypermethylation of the CLDN7 promoter was related to advanced ccRCC status and poor prognosis. Moreover, overexpression of CLDN7 induced cell apoptosis, suppressed proliferation, migration and invasion abilities of ccRCC cells both in vitro and in vivo. Additionally, GSEA and RNA-Seq results showed that CLDN7 had negative effects in cancer-associated signaling pathways and (epithelial-mesenchymal transition) EMT-related pathways. These results were validated by qRT-PCR, Western blot and immunostaining. Conclusions We have demonstrated a previously undescribed role of CLDN7 as a ccRCC suppressor and suggest that loss of CLDN7 potentiates EMT and tumor progression. CLDN7 may serve as a functional tumor suppressor in tumor progression and a potential biomarker and target in patients with ccRCC.

Background/Aims: Chronic Lymphocytic leukemia (CLL) is characterized by accumulation of cells in the G0/G1 phase of the cell cycle and resistance to apoptosis due to gene mutation or abnormal gene expression. In our previous study, constitutively photomorphogenic 1 (COP1) was shown to be upregulated in Binet C-phase CLL patients. Based on the negative regulation of COP1 in the repair of DNA damage, we further studied the function of COP1 in CLL cell apoptosis induced by fludarabine in vitro and in vivo. Methods: We analyzed the sensitivity of primary CLL cells to the fludarabine by CCK-8, and detected the expression of p53 in cells after drug treatment by western blot. Next, we constructed COP1 overexrpessing CLL cell line HG3, and analyzed the effect of COP1 overexpression on the HG3 cell’s apoptosis, and HG3 transplant mice survival with drug treatment. Results: Here, we found that primary CLL cells with high expression of COP1 showed low sensitivity to the drug and presented delayed enrichment of p53 protein than cells with low COP1 expressed. COP1 overexpression reduced HG3 cell sensitivity to the fludarabine treatment and inhibited cell apoptosis, and also retarded itself via autoubiquitination. The further study showed that COP1 promoted ubiquitin-dependent p53 degradation, which further disrupts the formation of the p53-Brn-3a complex and activation of Bcl-2 transcription. Moreover, mice engrafted with cells overexpressing COP1 showed a shortened survival, increased tumor cells burden in spleen and bone marrow (BM), and reduced tumor cell apoptosis even when fludarabine combined cyclophosphamide (F+C) therapy was administered. Conclusion: This study demonstrates that COP1 contributes to drug resistance of CLL cells to the fludarabine treatment in vitro and in vivo.

Abstract Background Accumulating evidences have indicated that long non-coding RNAs (lncRNAs) are potential biomarkers that play key roles in tumor development and progression. Differentiation antagonizing non-protein noding RNA (DANCR) is a novel lncRNA that acts as a potential biomarker and is involved in the development of cancers. However, the clinical significance and molecular mechanism of DANCR in bladder cancer is still unknown. Methods The relative expression level of DANCR was determined by Real-Time qPCR in a total of 106 patients with urothelial bladder cancer and in different bladder cancer cell lines. Loss-of-function experiments were performed to investigate the biological roles of DANCR on bladder cancer cell proliferation, migration, invasion and tumorigenicity. Comprehensive transcriptional analysis, RNA-FISH, dual-luciferase reporter assay and western blot were performed to explore the molecular mechanisms underlying the functions of DANCR. Results In this study, we found that DANCR was significantly up-regulated in bladder cancer. Moreover, increased DANCR expression was positively correlated with higher histological grade and advanced TNM stage. Further experiments demonstrated that knockdown of DANCR inhibited malignant phenotypes and epithelial-mesenchymal transition (EMT) of bladder cancer cells. Mechanistically, we found that DANCR was distributed mostly in the cytoplasm and DANCR functioned as a miRNA sponge to positively regulate the expression of musashi RNA binding protein 2 (MSI2) through sponging miR-149 and subsequently promoted malignant phenotypes of bladder cancer cells, thus playing an oncogenic role in bladder cancer pathogenesis. Conclusion This study is the first to demonstrate that DANCR plays a critical regulatory role in bladder cancer cell and DANCR may serve as a potential diagnostic biomarker and therapeutic target of bladder cancer.

Resistance to anti-angiogenic therapies often occurs in patients. Here, the authors demonstrate the role of PDGF signaling in GBM resistance to anti-VEGF treatment via a mechanism that involves endothelial-mesenchymal transformation and transcriptional regulation of VEGFR-2.

Background: MicroRNAs(miRNAs) are involved in the formation, maintenance, and metastasis of urologic cancer. Here, we aim to gather and evaluate all of the evidence regarding the potential role of miRNAs as novel predictors of urologic cancer survival. Methods: A systematic review was performed to identify and score all of the published studies that evaluated the prognostic effects of miRNAs in kidney (KCa), bladder (BCa) or prostate cancer (PCa). Where appropriate, the summary effects of miRNAs on urologic cancer were meta-analysed. The reliability of those results was then further validated by an integrated analysis of the TCGA cohort and miRNA panel. Results: Of 151 datasets, 80 miRNAs were enrolled in this systematic review. A meta-analysis of the prognostic qualities of each miRNA identified an objective association between miRNA and prognosis. miR-21 was identified as an unfavourable miRNA with the overall survival (HR:2.699, 1.76–4.14, P

Abstract Background 5-Hydroxymethylcytosine (5hmC) is converted from 5-methylcytosine (5mC) by a group of enzymes termed ten-eleven translocation (TET) family dioxygenases. The loss of 5hmC has been identified as a hallmark of most types of cancer and is related to tumorigenesis and progression. However, the role of 5hmC in bladder cancer is seldom investigated. Vitamin C was recently reported to induce the generation of 5hmC by acting as a cofactor for TET dioxygenases. In this study, we explored the role of 5hmC in bladder cancer and the therapeutic efficacy of vitamin C in increasing the 5hmC pattern. Results 5hmC was decreased in bladder cancer samples and was related to patient overall survival. Genome-wide mapping of 5hmC in tumor tissues and vitamin C-treated bladder cancer cells revealed that 5hmC loss was enriched in cancer-related genes and that vitamin C treatment increased 5hmC levels correspondingly. Vitamin C treatment shifted the transcriptome and inhibited the malignant phenotypes associated with bladder cancer cells in both in vitro cell lines and in vivo xenografts. Conclusions This study provided mechanistic insights regarding the 5hmC loss in bladder cancer and a rationale for exploring the therapeutic use of vitamin C as a potential epigenetic treatment for bladder cancer.

Abstract Background To evaluate the prognostic significance of the novel index combining preoperative hemoglobin and albumin levels and lymphocyte and platelet counts (HALP) in renal cell carcinoma (RCC) patients. Methods We enrolled 1360 patients who underwent nephrectomy in our institution from 2001 to 2010. The cutoff values for HALP, neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio were defined by using X-tile software. Survival was analyzed by the Kaplan–Meier method, with differences analyzed by the log-rank test. Multivariate Cox proportional-hazards model was used to evaluate the prognostic significance of HALP for RCC. Results Low HALP was significantly associated with worse clinicopathologic features. Kaplan-Meier and log-rank tests revealed that HALP was strongly correlated with cancer specific survival (P

Macrophages in the tumour microenvironment (TME) acquire tumour-promoting functions upon M2 polarization. Here the authors show, in a mouse model of glioblastoma, that endothelial cells in the TME induce macrophage M2 polarization via IL-6 and that depletion of endothelial IL-6 improves survival.

Background: Von Hippel-Lindau (VHL) disease is an autosomal-dominant hereditary cancer syndrome. Currently, studies on tyrosine kinase inhibitor (TKI) therapy for VHL disease are scarce. In this study, we retrospectively evaluated the efficacy and safety of four TKIs in patients with VHL disease.Methods: Patients diagnosed with VHL disease who were receiving TKIs were recruited. Patients were treated with sunitinib (n = 12), sorafenib (n = 11), axitinib (n = 6), or pazopanib (n = 3). The therapeutic response was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1.Results: From July 2009 to September 2018, 32 patients with VHL disease were eligible and included in this study. The median duration of TKI therapy was 22 months (IQR 8.5–44.75), and the median follow-up period was 31.5 months (IQR 13.5–63.5). According to the RECIST, 9 (28%) of 32 patients showed a partial response, 15 (47%) achieved stable disease, and eight exhibited continued disease progression. A partial response was observed in 11 (31%) of 36 renal cell carcinomas, 4 (27%) of 15 pancreatic lesions, and 1 (20%) of five central nervous system (CNS) hemangioblastomas. The average tumor size decreased significantly for renal cell carcinomas (P = 0.0001), renal cysts (P = 0.027), and pancreatic lesions (P = 0.003) after TKI therapy. Common side effects included hand–foot skin reactions, diarrhea, alopecia, thrombocytopenia, and fatigue.Conclusions: Partial alleviation of VHL disease-related tumors can be achieved by TKI therapies in some patients, providing an alternative treatment strategy, and the side effects of TKIs are acceptable. Larger prospective studies are warranted to further evaluate the efficacy and safety of TKIs in patients with VHL disease.

The key prognostic factor at the time of diagnosis of upper tract urothelial carcinomas (UTUC) is whether the tumor is in the muscle-invasive or non-muscle invasive stage. It is critical to identify novel molecular biomarkers for early detection and target therapy. Plasma proteins secreted by tumor tissues have excellent potential as biomarkers for UTUC. In this study, we conducted a systematic study to identify plasma markers for UTUC based on RNA-seq data from five UTUC tissues and paired adjacent noncancerous mucosa. Through bioinformatics analysis, we found secreted phosphoprotein 1 (SPP1) was the most significant gene that coding secretory protein. Then, qRT-PCR and enzyme-linked immunosorbent assay were performed to evaluate the expression and clinical significance of SPP1 in UTUC. Results found that SPP1 mRNA was upregulated in UTUC cells and tissues, and high SPP1 mRNA expression level was closely related to advanced stage and high grade. Moreover, it is suggested that plasma SPP1 may be a potential biomarker to help identify early-stage UTUC patients and predict invasive and high-grade UTUC. In conclusion, plasma SPP1 is a novel biomarker for UTUC.

In sustainability research, archetype analysis reveals patterns of factors and processes that repeatedly shape social-ecological systems. These patterns help improve our understanding of global concerns, including vulnerability, land management, food security, and governance. During the last decade, the portfolio of methods used to investigate archetypes has been growing rapidly. However, these methods differ widely in their epistemological and normative underpinnings, data requirements, and suitability to address particular research purposes. Therefore, guidance is needed for systematically choosing methods in archetype analysis. We synthesize strengths and weaknesses of key methods used to identify archetypes. Demonstrating that there is no "one-size-fits-all" approach, we discuss advantages and shortcomings of a range of methods for archetype analysis in sustainability research along gradients that capture the treatment of causality, normativity, spatial variations, and temporal dynamics. Based on this discussion, we highlight seven analytical frontiers that bear particular potential for tackling methodological limitations. As a milestone in archetype analysis, our synthesis supports researchers in reflecting on methodological implications, including opportunities and limitations related to causality, normativity, space, and time considerations in view of specific purposes and research questions. This enables innovative research designs in future archetype analysis, thereby contributing to the advancement of sustainability research and decision-making.

Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.

The aim of this study was to determine the effect of human adipose derived stem cells (ADSCs) on the viability and apoptosis of human bladder cancer cells. EJ and T24 cells were cocultured with ADSCs or cultured with conditioned medium of ADSCs (ADSC-CM), respectively. The cell counting and colony formation assay showed ADSCs inhibited the proliferation of EJ and T24 cells. Cell viability assessment revealed that the secretions of ADSCs, in the form of conditioned medium, were able to decrease cancer cell viability. Wound-healing assay suggested ADSC-CM suppressed migration of T24 and EJ cells. Moreover, the results of the flow cytometry indicated that ADSC-CM was capable of inducing apoptosis of T24 cells and inducing S phase cell cycle arrest. Western blot revealed ADSC-CM increased the expression of cleaved caspase-3 and cleaved PARP, indicating that ADSC-CM induced apoptosis in a caspase-dependent way. PTEN/PI3K/Akt pathway and Bcl-2 family proteins were involved in the mechanism of this reaction. Our study indicated that ADSCs may provide a promising and practicable manner for bladder tumor therapy.

In the title compound, C38H32O4P2·2H2O, the dihedral angle between the methoxyphenol rings is 84.11 (7)°. O—H...O hydrogen bonds connect the water molecules of crystallization with the main molecule.

The title compound, C20H18O6, was prepared from resveratrol {systematic name: 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol}, which can be isolated from grapes, through triacetylation with using acetic anhydride in pyridine. The two benzene rings are approximately coplanar, making a dihedral angle of 6.64 (14)°, and the three acetoxy group are located on the same side of the plane. The skeleton of the compound resembles a table with three legs. In the crystal, molecules are linked via C—H...O interactions, forming inversion dimers. These dimers are further linked via C—H...O interactions, forming a three-dimensional structure.