A study on the impact of marital status on the survival status of prostate cancer patients based on propensity score matching.

Marital status is an independent prognostic factor for survival in many types of cancers, but its prognostic impact on patients with prostate cancer (PCa) has not been established. The aim of this study was to explore the independent prognostic factors of PCa and to investigate the effect of marital status on survival outcomes in patients with different stratified by PCa. Using the surveillance, epidemiology, and end results (SEER) database, we collected data on 584,655 PCa patients diagnosed between 1975 and 2019. Marital status was classified as married, divorced, widowed, and single. We used the Kaplan-Meier analysis and single multivariate Cox proportional hazards regression analysis to determine the effect of marital status on overall survival (OS) and cancer-specific survival (CSS). In addition, we performed subgroup analyses for different ages, Gleason score and PSA values, and performed a 1:1 propensity score matching (PSM) to reduce the impact of confounding factors to obtain more accurate matching results. According to our findings, marital status was an independent prognostic factor for the survival of PCa patients and a better prognosis of married patients. Moreover, we also found that factors such as age, TNM stage, Gleason score, and PSA concentration were also considered as important predictors for the prognosis of PCa. The above findings can facilitate early detection and treatment of high-risk PCa patients, prolong their life and reduce family burden.

Single-cell sequencing reveals the heterogeneity of B cells and tertiary lymphoid structures in muscle-invasive bladder cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is a highly aggressive disease with a poor prognosis. B cells are crucial factors in tumor suppression, and tertiary lymphoid structures (TLSs) facilitate immune cell recruitment to the tumor microenvironment (TME). However, the function and mechanisms of tumor-infiltrating B cells and TLSs in MIBC need to be explored further. METHODS: We performed single-cell RNA sequencing analysis of 11,612 B cells and 55,392 T cells from 12 bladder cancer patients and found naïve B cells, proliferating B cells, plasma cells, interferon-stimulated B cells and germinal center-associated B cells, and described the phenotype, gene enrichment, cell-cell communication, biological processes. We utilized immunohistochemistry (IHC) and immunofluorescence (IF) to describe TLSs morphology in MIBC. RESULTS: The interferon-stimulated B-cell subtype (B-ISG15) and germinal center-associated B-cell subtypes (B-LMO2, B-STMN1) were significantly enriched in MIBC. TLSs in MIBC exhibited a distinct follicular structure characterized by a central region of B cells resembling a germinal center surrounded by T cells. CellChat analysis showed that CXCL13 + T cells play a pivotal role in recruiting CXCR5 + B cells. Cell migration experiments demonstrated the chemoattraction of CXCL13 toward CXCR5 + B cells. Importantly, the infiltration of the interferon-stimulated B-cell subtype and the presence of TLSs correlated with a more favorable prognosis in MIBC. CONCLUSIONS: The study revealed the heterogeneity of B-cell subtypes in MIBC and suggests a pivotal role of TLSs in MIBC outcomes. Our study provides novel insights that contribute to the precision treatment of MIBC.

Distinct regulatory machineries underlying divergent chromatin landscapes distinguish innate lymphoid cells from T helper cells.

Innate lymphoid cells (ILCs), as the innate counterpart of CD4+ T helper (Th) cells, play crucial roles in maintaining tissue homeostasis. While the ILC subsets and their corresponding Th subsets demonstrate significant similarities in core programming related to effector function and regulatory mechanisms, their principal distinctions, given their innate and adaptive lymphocyte nature, remain largely unknown. In this study, we have employed an integrative analysis of 294 bulk RNA-sequencing results across all ILC and Th subsets, using scRNA-seq algorithms. Consequently, we identify two genesets that predominantly differentiate ILCs from Th cells, as well as three genesets that distinguish various immune responses. Furthermore, through chromatin accessibility analysis, we find that the ILC geneset tends to rely on specific transcriptional regulation at promoter regions compared with the Th geneset. Additionally, we observe that ILCs and Th cells are under differential transcriptional regulation. For example, ILCs are under stronger regulation by multiple transcription factors, including RORα, GATA3, and NF-κB. Otherwise, Th cells are under stronger regulation by AP-1. Thus, our findings suggest that, despite the acknowledged similarities in effector functions between ILC subsets and corresponding Th subsets, the underlying regulatory machineries still exhibit substantial distinctions. These insights provide a comprehensive understanding of the unique roles played by each cell type during immune responses.

IL-21R-STAT3 signalling initiates a differentiation program in uterine tissue-resident NK cells to support pregnancy.

Tissue-resident Natural Killer (trNK) cells are crucial components of local immunity that activate rapidly upon infection. However, under steady state conditions, their responses are tightly controlled to prevent unwanted tissue damage. The mechanisms governing their differentiation and activation are not fully understood. Here, we characterise uterine trNK cells longitudinally during pregnancy by single cell RNA sequencing and find that the combined expression pattern of 4-1BB and CD55 defines their three distinct stages of differentiation in mice. Mechanistically, an IL-21R-STAT3 axis is essential for initiating the trNK cell differentiation. The fully differentiated trNK cells demonstrate enhanced functionality, which is necessary for remodelling spiral arteries in the decidua. We identify an apoptotic program that is specific to the terminal differentiation stage, which may preclude tissue damage by these highly activated trNK cells. In summary, uterine trNK cells become intensely active and effective during pregnancy, but tightly controlled via a differentiation program that also limits potential harm, suggesting an intricate mechanism for harnessing trNK cells in maintaining pregnancy.

Proline uptake promotes activation of lymphoid tissue inducer cells to maintain gut homeostasis.

Metabolic regulation is integral to the proper functioning of innate lymphoid cells, yet the underlying mechanisms remain elusive. Here, we show that disruption of exogenous proline uptake, either through dietary restriction or by deficiency of the proline transporter Slc6a7, in lymphoid tissue inducer (LTi) cells, impairs LTi activation and aggravates dextran sodium sulfate-induced colitis in mice. With an integrative transcriptomic and metabolomic analysis, we profile the metabolic characteristics of various innate lymphoid cell subsets and reveal a notable enrichment of proline metabolism in LTi cells. Mechanistically, defective proline uptake diminishes the generation of reactive oxygen species, previously known to facilitate LTi activation. Additionally, LTi cells deficient in Slc6a7 display downregulation of Cebpb and Kdm6b, resulting in compromised transcriptional and epigenetic regulation of interleukin-22. Furthermore, our study uncovers the therapeutic potential of proline supplementation in alleviating colitis. Therefore, these findings shed light on the role of proline in facilitating LTi activation and ultimately contributing to gut homeostasis.

Genetic distinction between functional tissue-resident and conventional natural killer cells.

Tissue-residential natural killer (trNK) cells act as pioneering responders during infectious challenges. However, their discrimination with conventional NK (cNK) cells is still an issue. Through an integrative transcriptome comparison of the two NK subgroups from different tissues, we have defined two genesets capable of efficiently distinguishing them. Based on the two genesets, a fundamental difference between the activation of trNK and cNK is identified and further confirmed. Mechanistically, we have discovered a particular role of chromatin landscape in regulating the trNK activation. In addition, IL-21R and IL-18R are respectively highly expressed by trNK and cNK, indicating a role of cytokine milieu in determining their differential activation. Indeed, IL-21 is particularly critical in accessorily promoting trNK activation using a bunch of bifunctional transcription factors. Together, this study sheds light on the bona fide difference between trNK and cNK, which will further expand our knowledge about their distinct functionalities during immune responses.

A spatiotemporal steroidogenic regulatory network in human fetal adrenal glands and gonads.

Human fetal adrenal glands produce substantial amounts of dehydroepiandrosterone (DHEA), which is one of the most important precursors of sex hormones. However, the underlying biological mechanism remains largely unknown. Herein, we sequenced human fetal adrenal glands and gonads from 7 to 14 gestational weeks (GW) via 10× Genomics single-cell transcriptome techniques, reconstructed their location information by spatial transcriptomics. Relative to gonads, adrenal glands begin to synthesize steroids early. The coordination among steroidogenic cells and multiple non-steroidogenic cells promotes adrenal cortex construction and steroid synthesis. Notably, during the window of sexual differentiation (8-12 GW), key enzyme gene expression shifts to accelerate DHEA synthesis in males and cortisol synthesis in females. Our research highlights the robustness of the action of fetal adrenal glands on gonads to modify the process of sexual differentiation.

Corrigendum: Single-cell transcriptomics reveals the complexity of the tumor microenvironment of treatment-naive osteosarcoma.

[This corrects the article DOI: 10.3389/fonc.2021.709210.].

Proximal and Distal Regions of Pathogenic Th17 Related Chromatin Loci Are Sequentially Accessible During Pathogenicity of Th17.

Pathogenic Th17, featured by their production of pro-inflammatory cytokines, are considered as a key player in most autoimmune diseases. The transcriptome of them is obviously distinct from that of conventional regulatory Th17. However, chromatin accessibility of the two Th17 groups have not been comprehensively compared yet. Here, we found that their chromatin-accessible regions(ChARs) significantly correlated with the expression of related genes, indicating that they might engage in the regulation of these genes. Indeed, pathogenic Th17 specific ChARs (patho-ChARs) exhibited a significant distribution preference in TSS-proximal region. We further filtered the patho-ChARs based on their conservation among mammalians or their concordance with the expression of their related genes. In either situation, the filtered patho-ChARs also showed a preference for TSS-proximal region. Enrichment of expression concordant patho-ChARs related genes suggested that they might involve in the pathogenicity of Th17. Thus, we also examined all ChARs of patho-ChARs related genes, and defined an opening ChAR set according to their changes in the Th17 to Th1 conversion. Interestingly, these opening ChARs displayed a sequential accessibility change from TSS-proximal region to TSS-distal region. Meanwhile, a group of patho-TFs (transcription factors) were identified based on the appearance of their binding motifs in the opening ChARs. Consistently, some of them also displayed a similar preference for binding the TSS-proximal region. Single-cell transcriptome analysis further confirmed that these patho-TFs were involved in the generation of pathogenic Th17. Therefore, our results shed light on a new regulatory mechanism underlying the generation of pathogenic Th17, which is worth to be considered for autoimmune disease therapy.

Single-cell transcriptomics reveals cell type diversity of human prostate.

Extensive studies have been performed to describe the phenotypic changes occurring during malignant transformation of the prostate. However, the cell types and associated changes that contribute to the development of prostate diseases and cancer remain elusive, largely due to the heterogeneous composition of prostatic tissues. Here, we conduct a comprehensive evaluation of four human prostate tissues by single-cell RNA sequencing (scRNA-seq) to analyze their cellular compositions. We identify 18 clusters of cell types, each with distinct gene expression profiles and unique features; of these, one cluster of epithelial cells (Ep) is found to be associated with immune function. In addition, we characterize a special cluster of fibroblasts and aberrant signaling changes associated with prostate cancer (PCa). Moreover, we provide insights into the epithelial changes that occur during the cellular senescence and aging. These results expand our understanding of the unique functional associations between the diverse prostatic cell types and the contributions of specific cell clusters to the malignant transformation of prostate tissues and PCa development.

Single-Cell Transcriptomics Reveals the Complexity of the Tumor Microenvironment of Treatment-Naive Osteosarcoma.

Osteosarcoma (OS), which occurs most commonly in adolescents, is associated with a high degree of malignancy and poor prognosis. In order to develop an accurate treatment for OS, a deeper understanding of its complex tumor microenvironment (TME) is required. In the present study, tissues were isolated from six patients with OS, and then subjected to single-cell RNA sequencing (scRNA-seq) using a 10× Genomics platform. Multiplex immunofluorescence staining was subsequently used to validate the subsets identified by scRNA-seq. ScRNA-seq of six patients with OS was performed prior to neoadjuvant chemotherapy, and data were obtained on 29,278 cells. A total of nine major cell types were identified, and the single-cell transcriptional map of OS was subsequently revealed. Identified osteoblastic OS cells were divided into five subsets, and the subsets of those osteoblastic OS cells with significant prognostic correlation were determined using a deconvolution algorithm. Thereby, different transcription patterns in the cellular subtypes of osteoblastic OS cells were reported, and key transcription factors associated with survival prognosis were identified. Furthermore, the regulation of osteolysis by osteoblastic OS cells via receptor activator of nuclear factor kappa-B ligand was revealed. Furthermore, the role of osteoblastic OS cells in regulating angiogenesis through vascular endothelial growth factor-A was revealed. C3_TXNIP+ macrophages and C5_IFIT1+ macrophages were found to regulate regulatory T cells and participate in CD8+ T cell exhaustion, illustrating the possibility of immunotherapy that could target CD8+ T cells and macrophages. Our findings here show that the role of C1_osteoblastic OS cells in OS is to promote osteolysis and angiogenesis, and this is associated with survival prognosis. In addition, T cell depletion is an important feature of OS. More importantly, the present study provided a valuable resource for the in-depth study of the heterogeneity of the OS TME.

Single-cell transcriptomes reveal characteristic features of cell types within the human adrenal microenvironment.

Various cells within the adrenal microenvironment are important in maintaining the body homeostasis. However, our understanding of adrenal disease pathogenesis is limited by an incomplete molecular characterization of the cell types responsible for the organ's multiple homeostatic functions. We report a cellular landscape of the human adrenal gland using single-cell RNA sequencing. We reveal characteristic features of cell types within the human adrenal microenvironment and found immune activation of nonimmune cells in the adrenal endothelial cells. We also reveal that abundant immune cells occupied a lot of space in adrenal gland. Additionally, Sex-related diversity in the adrenocortical cells and different gene expression profiles between the left and right adrenal gland are also observed at single-cell resolution. Together, at single-cell resolution, the transcriptomic map presents a comprehensive view of the human adrenal gland, which serves as a fundamental baseline description of this organ and paves a way for the further studies of adrenal diseases.

Molecular mechanisms, immune cell infiltration, and potential drugs for prostate cancer.

BACKGROUND: The molecular mechanisms involved in the prostate cancer and their relationship with immune cell infiltration are not fully understood. The prostate cancer patients undergoing standard androgen deprivation therapy eventually develop castration resistant prostate cancer (CRPC) for which there is no effective treatment currently available, and the hub genes involved in this process remain unclear. OBJECTIVE: To study prostate cancer systematically and comprehensively. METHODS: Differentially expressed genes (DEGs) of prostate cancer were screened in The Cancer Genome Atlas (TCGA) database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Connectivity Map (Cmap) software was applied to discover potential treatment drugs. A protein-protein interaction (PPI) analysis was performed to obtained the hub genes, and the relationship between hub genes and immune cell infiltration was investigated. Next, RNAseq data of hormone-sensitive prostate cancer samples and CRPC samples obtained from TCGA database was further analyzed to identify DEGs. Finally, a PPI analysis was performed to obtain the hub genes. RESULTS: A total of 319 DEGs were identified between prostate cancer samples and normal adjacent samples from TCGA database using comparative analysis. The KEGG pathway analysis showed significant correlations with drug metabolism, metabolism of xenobiotics by cytochrome P450, and chemical carcinogenesis. AMACR, FOLH1 and NPY, three hub genes, were found to be upregulated. FOLH1 was positively correlated with CD8+ T cell infiltration. FOLH1, AMACR, and NPY were negatively correlated with CD4+ T cell infiltration. A total of 426 DEGs were identified from RNAseq data of hormone-sensitive prostate cancer samples and CRPC samples using further comparative analysis. KEGG pathway enrichment analysis showed significant correlations with arachidonic acid metabolism, PPAR signaling pathway, AMPK signaling pathway, and metabolic pathways. The top 10 hub genes in PPI network were screened out, including PPARG, SREBF1, SCD, HMGCR, FASN, PTGS2, HMGCS2, SREBF2, FDFT1, and INSIG1. Among them, SCD and FASN are expected to be the potential therapeutic targets for CRPC. CONCLUSIONS: AMACR, FOLH1 and NPY may be effective therapeutic targets and specific diagnostic markers for prostate cancer. AMACR, FOLH1, and NPY are also closely associated with immune cell infiltration in prostate cancer. Moreover, aminoglutethimide and resveratrol were found to be the promising drugs for treating prostate cancer. The progression of hormone-sensitive prostate cancer to CRPC may be related to arachidonic acid metabolism, PPAR signaling pathway, AMPK signaling pathway, and other metabolic pathways. SCD and FASN are expected to be the potential therapeutic targets for CRPC.

The Endogenous Alterations of the Gut Microbiota and Feces Metabolites Alleviate Oxidative Damage in the Brain of LanCL1 Knockout Mice.

Altered composition of the gut microbiota has been observed in many neurodegenerative diseases. LanCL1 has been proven to protect neurons and reduce oxidative stress. The present study was designed to investigate alterations of the gut microbiota in LanCL1 knockout mice and to study the interactions between gut bacteria and the brain. Wild-type and LanCL1 knockout mice on a normal chow diet were evaluated at 4 and 8-9 weeks of age. 16s rRNA sequence and untargeted metabolomics analyses were performed to investigate changes in the gut microbiota and feces metabolites. Real-time polymerase chain reaction analysis, AB-PAS staining, and a TUNEL assay were performed to detect alterations in the gut and brain of knockout mice. The serum cytokines of 9-week-old knockout mice, which were detected by a multiplex cytokine assay, were significantly increased. In the central nervous system, there was no increase of antioxidant defense genes even though there was only low activity of glutathione S-transferase in the brain of 8-week-old knockout mice. Interestingly, the gut tight junctions, zonula occludens-1 and occludin, also displayed a downregulated expression level in 8-week-old knockout mice. On the contrary, the production of mucus increased in 8-week-old knockout mice. Moreover, the compositions of the gut microbiota and feces metabolites markedly changed in 8-week-old knockout mice but not in 4-week-old mice. Linear discriminant analysis and t-tests identified Akkermansia as a specific abundant bacteria in knockout mice. Quite a few feces metabolites that have protective effects on the brain were reduced in 8-week-old knockout mice. However, N-acetylsphingosine was the most significant downregulated feces metabolite, which may cause the postponement of neuronal apoptosis. To further investigate the effect of the gut microbiota, antibiotics treatment was given to both types of mice from 5 to 11 weeks of age. After treatment, a significant increase of oxidative damage in the brain of knockout mice was observed, which may have been alleviated by the gut microbiota before. In conclusion, alterations of the gut microbiota and feces metabolites alleviated oxidative damage to the brain of LanCL1 knockout mice, revealing that an endogenous feedback loop mechanism of the microbiota-gut-brain axis maintains systemic homeostasis.

rs2274911 polymorphism in GPRC6A associated with serum E2 and PSA in a Southern Chinese male population.

BACKGROUND: rs2274911 (Pro91Ser, G > A) is a missense mutation located on the second exon of the GPRC6A gene. Increasing evidence revealed a significant association between the A allele of rs2274911 and male diseases, such as oligospermia, cryptorchidism, and prostate tumor. However, the function of rs2274911 in healthy males is unclear. SUBJECTS AND METHODS: A total of 1742 healthy men were selected from the Fangchenggang Area Male Health and Examination Survey (FAMHES). The association between rs2274911 and phenotype was evaluated. The cell characteristics of rs2274911 mutation (mu), wild-type GPRC6A (WT), and RFP control in human embryonic kidney (293T) and human prostate cancer (PC3) cells were analyzed. RNA sequencing was performed on PC3 cells. RESULTS: E2 and PSA serum levels increased with the accumulation of the A allele (E2: G vs. A, -0.029 [-0.050, -0.008], P < 0.01, P trend = 0.027; PSA: G vs. A, -0.040 [-0.079, 0.000], P < 0.05, P trend = 0.048). rs2274911 enhanced the proliferation and invasion ability of PC3 or 293T cells and activated the ERK pathway. The genes were identified as rs2274911 mu-affected genes through RNA sequential analysis of rs2274911 mu, GPRC6A WT, and RFP control of PC3 cells. Most of these genes were related to cancer development processes, cAMP, and the ERK cell signaling pathway. CONCLUSION: This project represents that rs2274911 is associated with E2 and PSA serum levels in Southern Chinese men. Rs2274991 mutation promotes 293T and PC3 cell proliferation in vitro. These results suggest that rs2274911 is a functional variant of GPRC6A.