Mutation of the conserved late element in geminivirus CP promoters abolishes Arabidopsis TCP24 transcription factor binding and decreases H3K27me3 levels on viral chromatin.

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation.

A Network-Based Approach for Improving Annotation of Transcription Factor Functions and Binding Sites in Arabidopsis thaliana.

Transcription factors are an integral component of the cellular machinery responsible for regulating many biological processes, and they recognize distinct DNA sequence patterns as well as internal/external signals to mediate target gene expression. The functional roles of an individual transcription factor can be traced back to the functions of its target genes. While such functional associations can be inferred through the use of binding evidence from high-throughput sequencing technologies available today, including chromatin immunoprecipitation sequencing, such experiments can be resource-consuming. On the other hand, exploratory analysis driven by computational techniques can alleviate this burden by narrowing the search scope, but the results are often deemed low-quality or non-specific by biologists. In this paper, we introduce a data-driven, statistics-based strategy to predict novel functional associations for transcription factors in the model plant Arabidopsis thaliana. To achieve this, we leverage one of the largest available gene expression compendia to build a genome-wide transcriptional regulatory network and infer regulatory relationships among transcription factors and their targets. We then use this network to build a pool of likely downstream targets for each transcription factor and query each target pool for functionally enriched gene ontology terms. The results exhibited sufficient statistical significance to annotate most of the transcription factors in Arabidopsis with highly specific biological processes. We also perform DNA binding motif discovery for transcription factors based on their target pool. We show that the predicted functions and motifs strongly agree with curated databases constructed from experimental evidence. In addition, statistical analysis of the network revealed interesting patterns and connections between network topology and system-level transcriptional regulation properties. We believe that the methods demonstrated in this work can be extended to other species to improve the annotation of transcription factors and understand transcriptional regulation on a system level.

A Robust Personalized Classification Method for Breast Cancer Metastasis Prediction.

Accurate prediction of breast cancer metastasis in the early stages of cancer diagnosis is crucial to reduce cancer-related deaths. With the availability of gene expression datasets, many machine-learning models have been proposed to predict breast cancer metastasis using thousands of genes simultaneously. However, the prediction accuracy of the models using gene expression often suffers from the diverse molecular characteristics across different datasets. Additionally, breast cancer is known to have many subtypes, which hinders the performance of the models aimed at all subtypes. To overcome the heterogeneous nature of breast cancer, we propose a method to obtain personalized classifiers that are trained on subsets of patients selected using the similarities between training and testing patients. Results on multiple independent datasets showed that our proposed approach significantly improved prediction accuracy compared to the models trained on the complete training dataset and models trained on specific cancer subtypes. Our results also showed that personalized classifiers trained on positively and negatively correlated patients outperformed classifiers trained only on positively correlated patients, highlighting the importance of selecting proper patient subsets for constructing personalized classifiers. Additionally, our proposed approach obtained more robust features than the other models and identified different features for different patients, making it a promising tool for designing personalized medicine for cancer patients.

Loss of ATRX promotes aggressive features of osteosarcoma with increased NF-κB signaling and integrin binding.

Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here, we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvß3 expression, and ETS family transcription factor binding. Here, we characterize these changes in vitro, in vivo, and in a data set of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor-suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X α thalassemia intellectual disability syndrome.

2-Hydroxyglutarate destabilizes chromatin regulatory landscape and lineage fidelity to promote cellular heterogeneity.

The epigenome delineates lineage-specific transcriptional programs and restricts cell plasticity to prevent non-physiological cell fate transitions. Although cell diversification fosters tumor evolution and therapy resistance, upstream mechanisms that regulate the stability and plasticity of the cancer epigenome remain elusive. Here we show that 2-hydroxyglutarate (2HG) not only suppresses DNA repair but also mediates the high-plasticity chromatin landscape. A combination of single-cell epigenomics and multi-omics approaches demonstrates that 2HG disarranges otherwise well-preserved stable nucleosome positioning and promotes cell-to-cell variability. 2HG induces loss of motif accessibility to the luminal-defining transcriptional factors FOXA1, FOXP1, and GATA3 and a shift from luminal to basal-like gene expression. Breast tumors with high 2HG exhibit enhanced heterogeneity with undifferentiated epigenomic signatures linked to adverse prognosis. Further, ascorbate-2-phosphate (A2P) eradicates heterogeneity and impairs growth of high 2HG-producing breast cancer cells. These findings suggest 2HG as a key determinant of cancer plasticity and provide a rational strategy to counteract tumor cell evolution.

Construction and Evaluation of Robust Interpretation Models for Breast Cancer Metastasis Prediction.

Interpretability of machine learning (ML) models represents the extent to which a model's decision-making process can be understood by model developers and/or end users. Transcriptomics-based cancer prognosis models, for example, while achieving good accuracy, are usually hard to interpret, due to the high-dimensional feature space and the complexity of models. As interpretability is critical for the transparency and fairness of ML models, several algorithms have been proposed to improve the interpretability of arbitrary classifiers. However, evaluation of these algorithms often requires substantial domain knowledge. Here, we propose a breast cancer metastasis prediction model using a very small number of biologically interpretable features, and a simple yet novel model interpretation approach that can provide personalized interpretations. In addition, we contributed, to the best of our knowledge, the first method to quantitatively compare different interpretation algorithms. Experimental results show that our model not only achieved competitive prediction accuracy, but also higher inter-classifier interpretation consistency than state-of-the-art interpretation methods. Importantly, our interpretation results can improve the generalizability of the prediction models. Overall, this work provides several novel ideas to construct and evaluate interpretable ML models that can be valuable to both the cancer machine learning community and related application domains.

Robust edge-based biomarker discovery improves prediction of breast cancer metastasis.

BACKGROUND: The abundance of molecular profiling of breast cancer tissues entailed active research on molecular marker-based early diagnosis of metastasis. Recently there is a surging interest in combining gene expression with gene networks such as protein-protein interaction (PPI) network, gene co-expression (CE) network and pathway information to identify robust and accurate biomarkers for metastasis prediction, reflecting the common belief that cancer is a systems biology disease. However, controversy exists in the literature regarding whether network markers are indeed better features than genes alone for predicting as well as understanding metastasis. We believe much of the existing results may have been biased by the overly complicated prediction algorithms, unfair evaluation, and lack of rigorous statistics. In this study, we propose a simple approach to use network edges as features, based on two types of networks respectively, and compared their prediction power using three classification algorithms and rigorous statistical procedure on one of the largest datasets available. To detect biomarkers that are significant for the prediction and to compare the robustness of different feature types, we propose an unbiased and novel procedure to measure feature importance that eliminates the potential bias from factors such as different sample size, number of features, as well as class distribution. RESULTS: Experimental results reveal that edge-based feature types consistently outperformed gene-based feature type in random forest and logistic regression models under all performance evaluation metrics, while the prediction accuracy of edge-based support vector machine (SVM) model was poorer, due to the larger number of edge features compared to gene features and the lack of feature selection in SVM model. Experimental results also show that edge features are much more robust than gene features and the top biomarkers from edge feature types are statistically more significantly enriched in the biological processes that are well known to be related to breast cancer metastasis. CONCLUSIONS: Overall, this study validates the utility of edge features as biomarkers but also highlights the importance of carefully designed experimental procedures in order to achieve statistically reliable comparison results.

Gene selection for optimal prediction of cell position in tissues from single-cell transcriptomics data.

Single-cell RNA-sequencing (scRNAseq) technologies are rapidly evolving. Although very informative, in standard scRNAseq experiments, the spatial organization of the cells in the tissue of origin is lost. Conversely, spatial RNA-seq technologies designed to maintain cell localization have limited throughput and gene coverage. Mapping scRNAseq to genes with spatial information increases coverage while providing spatial location. However, methods to perform such mapping have not yet been benchmarked. To fill this gap, we organized the DREAM Single-Cell Transcriptomics challenge focused on the spatial reconstruction of cells from the Drosophila embryo from scRNAseq data, leveraging as silver standard, genes with in situ hybridization data from the Berkeley Drosophila Transcription Network Project reference atlas. The 34 participating teams used diverse algorithms for gene selection and location prediction, while being able to correctly localize clusters of cells. Selection of predictor genes was essential for this task. Predictor genes showed a relatively high expression entropy, high spatial clustering and included prominent developmental genes such as gap and pair-rule genes and tissue markers. Application of the top 10 methods to a zebra fish embryo dataset yielded similar performance and statistical properties of the selected genes than in the Drosophila data. This suggests that methods developed in this challenge are able to extract generalizable properties of genes that are useful to accurately reconstruct the spatial arrangement of cells in tissues.

Manipulation of the Plant Host by the Geminivirus AC2/C2 Protein, a Central Player in the Infection Cycle.

Geminiviruses are a significant group of emergent plant DNA viruses causing devastating diseases in food crops worldwide, including the Southern United States, Central America and the Caribbean. Crop failure due to geminivirus-related disease can be as high as 100%. Improved global transportation has enhanced the spread of geminiviruses and their vectors, supporting the emergence of new, more virulent recombinant strains. With limited coding capacity, geminiviruses encode multifunctional proteins, including the AC2/C2 gene that plays a central role in the viral replication-cycle through suppression of host defenses and transcriptional regulation of the late viral genes. The AC2/C2 proteins encoded by mono- and bipartite geminiviruses and the curtovirus C2 can be considered virulence factors, and are known to interact with both basal and inducible systems. This review highlights the role of AC2/C2 in affecting the jasmonic acid and salicylic acid (JA and SA) pathways, the ubiquitin/proteasome system (UPS), and RNA silencing pathways. In addition to suppressing host defenses, AC2/C2 play a critical role in regulating expression of the coat protein during the viral life cycle. It is important that the timing of CP expression is regulated to ensure that ssDNA is converted to dsDNA early during an infection and is sequestered late in the infection. How AC2 interacts with host transcription factors to regulate CP expression is discussed along with how computational approaches can help identify critical host networks targeted by geminivirus AC2 proteins. Thus, the role of AC2/C2 in the viral life-cycle is to prevent the host from mounting an efficient defense response to geminivirus infection and to ensure maximal amplification and encapsidation of the viral genome.

Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance.

Acquired therapy resistance is a major problem for anticancer treatment, yet the underlying molecular mechanisms remain unclear. Using an established breast cancer cellular model, we show that endocrine resistance is associated with enhanced phenotypic plasticity, indicated by a general downregulation of luminal/epithelial differentiation markers and upregulation of basal/mesenchymal invasive markers. Consistently, similar gene expression changes are found in clinical breast tumours and patient-derived xenograft samples that are resistant to endocrine therapies. Mechanistically, the differential interactions between oestrogen receptor α and other oncogenic transcription factors, exemplified by GATA3 and AP1, drive global enhancer gain/loss reprogramming, profoundly altering breast cancer transcriptional programs. Our functional studies in multiple culture and xenograft models reveal a coordinated role of GATA3 and AP1 in re-organizing enhancer landscapes and regulating cancer phenotypes. Collectively, our study suggests that differential high-order assemblies of transcription factors on enhancers trigger genome-wide enhancer reprogramming, resulting in transcriptional transitions that promote tumour phenotypic plasticity and therapy resistance.

Network-Based Single-Cell RNA-Seq Data Imputation Enhances Cell Type Identification.

Single-cell RNA sequencing is a powerful technology for obtaining transcriptomes at single-cell resolutions. However, it suffers from dropout events (i.e., excess zero counts) since only a small fraction of transcripts get sequenced in each cell during the sequencing process. This inherent sparsity of expression profiles hinders further characterizations at cell/gene-level such as cell type identification and downstream analysis. To alleviate this dropout issue we introduce a network-based method, netImpute, by leveraging the hidden information in gene co-expression networks to recover real signals. netImpute employs Random Walk with Restart (RWR) to adjust the gene expression level in a given cell by borrowing information from its neighbors in a gene co-expression network. Performance evaluation and comparison with existing tools on simulated data and seven real datasets show that netImpute substantially enhances clustering accuracy and data visualization clarity, thanks to its effective treatment of dropouts. While the idea of netImpute is general and can be applied with other types of networks such as cell co-expression network or protein-protein interaction (PPI) network, evaluation results show that gene co-expression network is consistently more beneficial, presumably because PPI network usually lacks cell type context, while cell co-expression network can cause information loss for rare cell types. Evaluation results on several biological datasets show that netImpute can more effectively recover missing transcripts in scRNA-seq data and enhance the identification and visualization of heterogeneous cell types than existing methods.

Comparative evaluation of network features for the prediction of breast cancer metastasis.

BACKGROUND: Discovering a highly accurate and robust gene signature for the prediction of breast cancer metastasis from gene expression profiling of primary tumors is one of the most challenging tasks to reduce the number of deaths in women. Due to the limited success of gene-based features in achieving satisfactory prediction accuracy, many methodologies have been proposed in recent years to develop network-based features by integrating network information with gene expression. However, evaluation results are inconsistent to confirm the effectiveness of network-based features, because of many confounding factors involved in classification model learning process, such as data normalization, dimension reduction, and feature selection. An unbiased comparative evaluation is essential for uncovering the strength of network-based features. METHODS: In this study, we compared several types of network-based features obtained using different mathematical operators (Mean, Maximum, Minimum, Median, Variance) on geneset (i.e., a gene and its' neighbors in the network) in protein-protein interaction network and gene co-expression network for their ability in predicting breast cancer metastasis using gene expression data from more than 10 patient cohorts. RESULTS: While network-based features are usually statistically more significant than gene-based feature, a consistent improvement of prediction performance using network-based features requires a substantial number of patients in the dataset. In contrary to many previous reports, no evidence was found to support the robustness of network-based features and we argue some of the robustness may be due to the inherent bias associated with node degree in the network. In addition, different types of network features seem to cover different pathways and are complementary to each other. Consequently, an ensemble classifier combining different network features was proposed and was found to significantly outperform classifiers based on gene-based feature or any single type of network-based features. CONCLUSIONS: Network-based features and their combination show promise for improving the prediction of breast cancer metastasis but may require a large amount of training data. Robustness claim of network-based features needs to be re-examined with network node degree and other confounding factors in consideration.

Spatial mapping of single cells in the Drosophila embryo from transcriptomic data based on topological consistency.

The advancement in single-cell RNA sequencing technologies allow us to obtain transcriptome at single cell resolution. However, the original spatial context of cells, a crucial knowledge for understanding cellular and tissue-level functions, is often lost during sequencing. To address this issue, the DREAM Single Cell Transcriptomics Challenge launched a community-wide effort to seek computational solutions for spatial mapping of single cells in tissues using single-cell RNAseq (scRNA-seq) data and a reference atlas obtained from in situ hybridization data. As a top-performing team in this competition, we approach this problem in three steps. The first step involves identifying a set of most informative genes based on the consistency between gene expression similarity and cell proximity. For this step, we propose two different approaches, i.e., an unsupervised approach that does not utilize the gold standard location of the cells provided by the challenge organizers, and a supervised approach that relies on the gold standard locations. In the second step, a Particle Swarm Optimization algorithm is used to optimize the weights of different genes in order to maximize matches between the predicted locations and the gold standard locations. Finally, the information embedded in the cell topology is used to improve the predicted cell-location scores by weighted averaging of scores from neighboring locations. Evaluation results based on DREAM scores show that our method accurately predicts the location of single cells, and the predictions lead to successful recovery of the spatial expression patterns for most of landmark genes. In addition, investigating the selected genes demonstrates that most predictive genes are cluster specific, and stable across our supervised and unsupervised gene selection frameworks. Overall, the promising results obtained by our methods in DREAM challenge demonstrated that topological consistency is a useful concept in identifying marker genes and constructing predictive models for spatial mapping of single cells.

Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer.

Advanced prostate cancer is a very heterogeneous disease reflecting in diverse regulations of oncogenic signaling pathways. Aberrant spatial dynamics of epidermal growth factor receptor (EGFR) promote their dimerization and clustering, leading to constitutive activation in oncogenesis. The EphB2 and Src signaling pathways are associated with the reorganization of the cytoskeleton leading to malignancy, but their roles in regulating EGFR dynamics and activation are scarcely reported. Using single-particle tracking techniques, we found that highly phosphorylated EGFR in the advanced prostate cancer cell line, PC3, was associated with higher EGFR diffusivity, as compared with LNCaP and less aggressive DU145. The increased EGFR activation and biophysical dynamics were consistent with high proliferation, migration, and invasion. After performing single-cell RNA-seq on prostate cancer cell lines and circulating tumor cells from patients, we identified that upregulated gene expression in the EphB2 and Src pathways are associated with advanced malignancy. After dasatinib treatment or siRNA knockdowns of EphB2 or Src, the PC3 cells exhibited significantly lower EGFR dynamics, cell motility, and invasion. Partial inhibitory effects were also found in DU145 cells. The upregulation of parts of the EphB2 and Src pathways also predicts poor prognosis in the prostate cancer patient cohort of The Cancer Genome Atlas. Our results provide evidence that overexpression of the EphB2 and Src signaling pathways regulate EGFR dynamics and cellular aggressiveness in some advanced prostate cancer cells.

The International Conference on Intelligent Biology and Medicine (ICIBM) 2018: genomics with bigger data and wider applications.

The sixth International Conference on Intelligent Biology and Medicine (ICIBM) took place in Los Angeles, California, USA on June 10-12, 2018. This conference featured eleven regular scientific sessions, four tutorials, one poster session, four keynote talks, and four eminent scholar talks. The scientific program covered a wide range of topics from bench to bedside, including 3D Genome Organization, reconstruction of large scale evolution of genomes and gene functions, artificial intelligence in biological and biomedical fields, and precision medicine. Both method development and application in genomic research continued to be a main component in the conference, including studies on genetic variants, regulation of transcription, genetic-epigenetic interaction at both single cell and tissue level and artificial intelligence. Here, we write a summary of the conference and also briefly introduce the four high quality papers selected to be published in BMC Genomics that cover novel methodology development or innovative data analysis.

A novel algorithm for network-based prediction of cancer recurrence.

To develop accurate prognostic models is one of the biggest challenges in "omics"-based cancer research. Here, we propose a novel computational method for identifying dysregulated gene subnetworks as biomarkers to predict cancer recurrence. Applying our method to the DNA methylome of endometrial cancer patients, we identified a subnetwork consisting of differentially methylated (DM) genes, and non-differentially methylated genes, termed Epigenetic Connectors (EC), that are topologically important for connecting the DM genes in a protein-protein interaction network. The ECs are statistically significantly enriched in well-known tumorgenesis and metastasis pathways, and include known epigenetic regulators. Importantly, combining the DMs and ECs as features using a novel random walk procedure, we constructed a support vector machine classifier that significantly improved the prediction accuracy of cancer recurrence and outperformed several alternative methods, demonstrating the effectiveness of our network-based approach.

Adipokines Deregulate Cellular Communication via Epigenetic Repression of Gap Junction Loci in Obese Endometrial Cancer.

Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro coculture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort (n = 141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell-cell interactions in vitro. Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. SIGNIFICANCE: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer.

The International Conference on Intelligent Biology and Medicine (ICIBM) 2018: bioinformatics towards translational applications.

The 2018 International Conference on Intelligent Biology and Medicine (ICIBM 2018) was held on June 10-12, 2018, in Los Angeles, California, USA. The conference consisted of a total of eleven scientific sessions, four tutorials, one poster session, four keynote talks and four eminent scholar talks, which covered a wild range of aspects of bioinformatics, medical informatics, systems biology and intelligent computing. Here, we summarize nine research articles selected for publishing in BMC Bioinformatics.

The International Conference on Intelligent Biology and Medicine (ICIBM) 2018: systems biology on diverse data types.

Between June 10-12, 2018, the International Conference on Intelligent Biology and Medicine (ICIBM 2018) was held in Los Angeles, California, USA. The conference included 11 scientific sessions, four tutorials, one poster session, four keynote talks and four eminent scholar talks that covered a wide range of topics in 3D genome structure analysis and visualization, next generation sequencing analysis, computational drug discovery, medical informatics, cancer genomics and systems biology. Systems biology has been a main theme in ICIBM 2018, with exciting advances presented in many areas of systems biology, covering various different data types such as gene regulation, circular RNAs expression, single-cell RNA-Seq, inter-chromosomal interactions, metabolomics, proteomics and phosphoproteomics. Here, we describe ten high quality papers to be published in BMC Systems Biology.

Intratumoral CD56bright natural killer cells are associated with improved survival in bladder cancer.

BACKGROUND: Natural killer (NK) cells are effective at killing tumors in a non-MHC restricted manner and are emerging targets for cancer therapy but their importance in bladder cancer (BC) is poorly defined. NK cells are commonly subdivided into populations based on relative surface expression of CD56. Two major subsets are CD56bright and CD56dim NK cells. METHODS: The prevalence of intratumoral lymphocytes was examined via flow cytometric analysis of bladder tissue from a local cohort of patients with non-invasive and invasive BC (n=28). The association of NK cell subsets with cancer-specific survival (CSS) and overall survival (OS) was examined in 50 patients with BC using Cox regression. Fluorescence-activated cell sorting (FACS) of intratumoral lymphocytes isolated CD56 NK cell subsets were used for examination of function, including cytokine production and in vitro cytotoxicity. RESULTS: NK cells predominated among bladder intratumoral lymphocytes. Intratumoral CD56bright NK cells showed increased cytokine production and cytotoxicity compared to their CD56dim counterparts and were associated with improved CSS and OS independent of pathologic tumor stage. On the other hand, CD56dim NK cells were not associated with improved outcomes but were associated with higher pathologic stage. CONCLUSIONS: NK cells are frequent among intratumoral lymphocytes in BC. Bladder intratumoral CD56bright NK cells are functional and prognostically relevant whereas CD56dim NK cells are dysfunctional and prevalent in higher stage tumors. Thus, CD56bright NK cells are promising targets in BC.