Metabolic Heterogeneity and Potential Immunotherapeutic Responses Revealed by Single-Cell Transcriptomics of Breast Cancer.

BACKGROUND: Breast cancer (BC) exhibits remarkable heterogeneity. However, the transcriptomic heterogeneity of BC at the single-cell level has not been fully elucidated. METHODS: We acquired BC samples from 14 patients. Single-cell RNA sequencing (scRNA-seq), bioinformatic analyses, along with immunohistochemistry (IHC) and immunofluorescence (IF) assays were carried out. RESULTS: According to the scRNA-seq results, 10 different cell types were identified. We found that Cancer-Associated Fibroblasts (CAFs) exhibited distinct biological functions and may promote resistance to therapy. Metabolic analysis of tumor cells revealed heterogeneity in glycolysis, gluconeogenesis, and fatty acid synthetase reprogramming, which led to chemotherapy resistance. Furthermore, patients with multiple metastases and progression were predicted to benefit from immunotherapy based on a heterogeneity analysis of T cells and tumor cells. CONCLUSIONS: Our findings provide a comprehensive understanding of the heterogeneity of BC, provide comprehensive insight into the correlation between cancer metabolism and chemotherapy resistance, and enable the prediction of immunotherapy responses based on T-cell heterogeneity.

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.

Altered regulation of LncRNA analysis of human alcoholic hepatitis with Mallory-Denk Bodies (MDBs) is revealed by RNA sequencing.

Mallory-Denk Bodies (MDBs) are prevalent in a variety of liver diseases including alcoholic hepatitis (AH) and are formed in mice livers by feeding DDC. Long noncoding RNAs (lncRNAs) are considered as emerging new gene regulators, which participates in many functional activities through diverse mechanisms. We previously reported the mechanisms involved in the formation of liver MDBs in mouse model and in AH livers where MDBs had formed. To investigate the regulation of mRNAs expression and the probable role of lncRNAs in AH livers with MDBs, RNA-Seq analyses was further conducted to determine the mRNA and lncRNA expression profiles of the AH livers compared with the normal livers. It showed that different lncRNAs have different information contribution degrees by principal component analysis, and the integrated analysis of lncRNA-mRNA co-expression networks were linked to endocytosis, cell cycle, p53 signaling pathways in the human. Based on the co-expression networks, we identify 36 mRNAs that could be as potential biomarkers of alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). To our knowledge, this is the first report on the regulatory network of lncRNAs associated with liver MDB formation in human, and these results might offer new insights into the molecular mechanisms of liver MDB formation and the progression of AH to HCC.

Single-Cell Transcriptomic Map of the Human and Mouse Bladders.

BACKGROUND: Having a comprehensive map of the cellular anatomy of the normal human bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer. METHODS: We used single-cell RNA sequencing (scRNA-seq) of 12,423 cells from healthy human bladder tissue samples taken from patients with bladder cancer and 12,884 cells from mouse bladders to classify bladder cell types and their underlying functions. RESULTS: We created a single-cell transcriptomic map of human and mouse bladders, including 16 clusters of human bladder cells and 15 clusters of mouse bladder cells. The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified. We also discovered two novel types of human bladder cells. One type is ADRA2A+ and HRH2+ interstitial cells which may be associated with nerve conduction and allergic reactions. The other type is TNNT1+ epithelial cells that may be involved with bladder emptying. We verify these TNNT1+ epithelial cells also occur in rat and mouse bladders. CONCLUSIONS: This transcriptomic map provides a resource for studying bladder cell types, specific cell markers, signaling receptors, and genes that will help us to learn more about the relationship between bladder cell types and diseases.

Cordycepin exhibits a suppressive effect on T cells through inhibiting TCR signaling cascade in CFA-induced inflammation mice model.

Objective: Cordycepin has been shown to exhibit multiple pharmacological activities, such as antitumor, antifungi, antivirus, and immune-regulation activities, and is involved in the regulation of T cells. However, cordycepin that affects T cell activity is still not clear, and the molecular mechanism of cordycepin in regulation of TCR signaling has not yet been elucidated. In this study, the potential effect of cordycepin on T cells was observed in CFA-induced inflammation mice model, and the function of cordycepin in regulating TCR signaling cascade was investigated.Methods: A CFA-induced inflammation mice model was established for observing the effect of cordycepin on the thymus and spleen swellings, and T cell infiltration in paw tissue was detected by immunohistochemistry. The protein expression or phosphorilation was detected by western blotting, and the NFAT1 nuclear translocation was determined by fluorescence imaging. The cell proliferation, apoptosis, and IL-2 production were analyzed by CCK-8 method, flow cytometry, and ELISA.Results: In the mice model, the thymus and spleen swellings were suppressed and the T cell infiltration in paw tissue was inhibited by cordycepin at a concentration of 10 mg/kg. Although the expressions of ZAP70 and PLCγ1 were not significantly changed in the human T cell line Jurkat with cordycepin pretreatment, the CD3-antibody-induced phosphorylations of ZAP70 and PLCγ1 were markedly blocked. The protein level of p85 decreased when Jurkat cells were pretreated with cordycepin, and cordycepin blocked TCR downstream molecule Erk phosphorylation and NFAT1 nuclear translocation. Further investigation revealed that cordycepin inhibited T cell proliferation, reduced IL-2 production, and induced T cell apoptosis. Conclusions: These findings suggest that cordycepin regulates TCR signaling to inhibit excessive T cell activation in inflammation. Thus, cordycepin may be a potential therapeutic application in inflammation-associated diseases.

Characterization of Amphioxus IFN Regulatory Factor Family Reveals an Archaic Signaling Framework for Innate Immune Response.

The IFN regulatory factor (IRF) family encodes transcription factors that play important roles in immune defense, stress response, reproduction, development, and carcinogenesis. Although the origin of the IRF family has been dated back to multicellular organisms, invertebrate IRFs differ from vertebrate IRFs in genomic structure and gene synteny, and little is known about their functions. Through comparison of multiple amphioxus genomes, in this study we suggested that amphioxus contains nine IRF members, whose orthologs are supposed to be shared among three amphioxus species. As the orthologs to the vertebrate IRF1 and IRF4 subgroups, Branchiostoma belcheri tsingtauense (bbt)IRF1 and bbtIRF8 bind the IFN-stimulated response element (ISRE) and were upregulated when amphioxus intestinal cells were stimulated with poly(I:C). As amphioxus-specific IRFs, both bbtIRF3 and bbtIRF7 bind ISRE. When activated, they can be phosphorylated by bbtTBK1 and then translocate into nucleus for target gene transcription. As transcriptional repressors, bbtIRF2 and bbtIRF4 can inhibit the transcriptional activities of bbtIRF1, 3, 7, and 8 by competing for the binding of ISRE. Interestingly, amphioxus IRF2, IRF8, and Rel were identified as target genes of bbtIRF1, bbtIRF7, and bbtIRF3, respectively, suggesting a dynamic feedback regulation among amphioxus IRF and NF-κB. Collectively, to our knowledge we present for the first time an archaic IRF signaling framework in a basal chordate, shedding new insights into the origin and evolution of vertebrate IFN-based antiviral networks.

Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling.

Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate.

Overexpression of microRNA-21 strengthens stem cell-like characteristics in a hepatocellular carcinoma cell line.

BACKGROUND: Liver cancer stem cells (LCSCs) have been shown to express higher levels of microRNA-21 (miR-21). Here, we examine the possible contributions of miR-21 to the phenotype of LCSCs in culture and in xenograft tumors in nude mice. METHODS: The hepatocellular carcinoma cell line MHCC-97H was stably transformed with a retroviral vector to establish cells overexpressing miR-21, while a cell line transformed with empty vector served as a negative control. RT-PCR and Western blotting were used to evaluate the effects of miR-21 overexpression on the expression of various LCSC markers, a Transwell assay was used to assess the effects on cell migration and invasion, and a spheroid formation assay was used to examine the effects on clonogenesis. The effects of miR-21 overexpression were also examined in tumors in nude mice. RESULTS: An MHCC-97H cell line was constructed that stably overexpresses miR-21 at 7.78 ± 1.51-fold higher levels than the negative control cell line. Expression of the LCSC markers CD13, Ep-CAM, CD90, and OCT4 was significantly higher in the miR-21-overexpressing cell line than in the negative control at both mRNA and protein levels. The overexpressing cell line formed larger, tighter, and more numerous spheroids. Overexpression of miR-21 was associated with greater cell migration and invasion. Tumors of overexpressing cells in nude mice had a significantly larger mean volume after 34 days of growth (773.62 ± 163.46 mm3) than tumors of negative control cells (502.79 ± 33.94 mm3, p = 0.048), as well as greater mean weight (0.422 ± 0.019 vs. 0.346 ± 0.006 g, p = 0.003). CONCLUSIONS: Overexpression of miR-21 strengthens the phenotype of LCSCs, facilitating invasion, migration, and tumorigenesis in hepatocellular carcinoma.

Genome-wide analyses of amphioxus microRNAs reveal an immune regulation via miR-92d targeting C3.

Recently, amphioxus has served as a model for studying the origin and evolution of vertebrate immunity. However, little is known about how microRNAs (miRNAs) are involved in the immune defense in amphioxus. In this article, we present a systematic study of amphioxus miRNAs in the acute-phase response to bacterial infection; miR-92d was found to regulate the complement pathway in this basal chordate. We identified all 155 possible miRNAs present in the amphioxus Branchiostoma belcheri genome by bioinformatics analyses, including 57 newly identified miRNAs (called bbe-miRNAs), and characterized the miRNA expression pattern. Four miRNAs (bbe-miR-7, bbe-miR-4868a, bbe-miR-2065, and bbe-miR-34b) were upregulated and bbe-miR-92d was downregulated under the challenge of both Vibrio anguillarum and Staphylococcus aureus bacteria. We further predicted miRNA targets and identified mRNA targets of immune-related miRNA using the hybrid PCR method. We propose that miR-92d regulates the complement pathway through targeting C3 for controlling the acute immune response to bacterial infections. This study provides evidence for the complex immune regulation of miRNAs in the acute-phase response in basal chordates.

Rapid production of a H9 N2 influenza vaccine from MDCK cells for protecting chicken against influenza virus infection.

H9N2 subtype avian influenza viruses are widespread in domestic poultry, and vaccination remains the most effective way to protect the chicken population from avian influenza pandemics. Currently, egg-based H9N2 influenza vaccine production has several disadvantages and mammalian MDCK cells are being investigated as candidates for influenza vaccine production. However, little research has been conducted on low pathogenic avian influenza viruses (LPAIV) such as H9N2 replicating in mammalian cells using microcarrier beads in a bioreactor. In this study, we present a systematic analysis of a safe H9N2 influenza vaccine derived from MDCK cells for protecting chickens against influenza virus infection. In 2008, we isolated two novel H9N2 influenza viruses from chickens raised in southern China, and these H9N2 viruses were adapted to MDCK cells. The H9N2 virus was produced in MDCK cells in a scalable bioreactor, purified, inactivated, and investigated for use as a vaccine. The MDCK-derived H9N2 vaccine was able to induce high titers of neutralizing antibodies in chickens of different ages. Histopathological examination, direct immunofluorescence, HI assay, CD4(+)/CD8(+) ratio test, and cytokine evaluation indicated that the MDCK-derived H9N2 vaccine evoked a rapid and effective immune response to protect chickens from influenza infection. High titers of H9N2-specific antibodies were maintained in chickens for 5 months, and the MDCK-derived H9N2 vaccine had no effects on chicken growth. The use of MDCK cells in bioreactors for LPAIV vaccine production is an attractive option to prevent outbreaks of LPAIV in poultry.

Aldosterone's impact on kidney health: exploring the benefits of mineralocorticoid receptor antagonists for renal protection.

Aldosterone, a hormone synthesized by the adrenal cortex, plays a crucial role in regulating sodium and potassium levels in the kidneys through interaction with the mineralocorticoid receptor (MR) in the distal tubules and collecting ducts. While aldosterone aids in maintaining fluid balance by promoting sodium reabsorption and potassium secretion, elevated levels can lead to inflammation, oxidative stress, and organ damage. Experimental evidence highlights aldosterone's involvement in renal inflammation, collagen deposition, and fibrosis, often exacerbating the effects of therapies like angiotensin-converting enzyme inhibitors (ACEIs) by increasing proteinuria and vascular damage. Conversely, mineralocorticoid receptor antagonists (MRAs) show promise in mitigating these harmful effects. This review integrates current knowledge on aldosterone and MRAs, emphasizing their roles in renal health from both clinical and experimental perspectives. Additionally, the novel drug finerenone has shown favorable renal and cardiovascular outcomes in patients with diabetes and chronic kidney disease (CKD), warranting exploration of its potential use in other disease populations in future research.

Resveratrol inhibits NF-kB signaling through suppression of p65 and IkappaB kinase activities.

Resveratrol has been shown to possess multiple pharmacological activities including anti-tumor, anti-inflammation and immunomodulation, and participates in the regulation of the NF-kappaB signaling pathway. However, the mechanism of the NF-kappaB signaling pathway inhibited by resveratrol remains obscure. In this study, we first examined the effect of resveratrol on endogenous and TNF-alpha-induced NF-kappaB activation, and found that resveratrol suppressed NF-kappaB activation in a dose dependent manner. Resveratrol reduced the transcriptional activity of p65, but neither affected the DNA-binding activity of NF-kappaB nor blocked the nuclear translocation of p65. Moreover, resveratrol had no effect on the expression level of IkappaBalpha protein and inhibited IkappaBalpha degradation. Further investigation revealed that resveratrol blocked the ubiquitination of NEMO and inhibited IkappaB kinase(beta)-mediated NF-kappaB activation. These results demonstrated that resveratrol effectively suppressed NF-kappaB signaling through inhibiting the activities of NF-kappaB and IkappaB kinase. Therefore, resveratrol may provide a novel approach to treating inflammation-associated diseases and cancer.

Urinary SPP1 has potential as a non-invasive diagnostic marker for focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a type of chronic glomerular nephropathy showing characteristic glomerular sclerosis, diagnosed by kidney biopsy. However, it is difficult and expensive to monitor disease progression with repeated renal biopsy in clinical practice, and thus here we explored the feasibility of urine biomarkers as non-invasive diagnostic tools. We downloaded scRNA-seq datasets of 20 urine cell samples and 3 kidney tissues and obtained two gene lists encoding extracellular proteins for bioinformatic analysis; in addition, we identified key EP-Genes by immunohistochemical staining and performed bulk RNA sequencing with 12 urine samples. We report that urine cells and kidney cells were correlated. A total of 64 EP-Genes were acquired by intersecting genes of distal tubular cluster with extracellular proteins. Function enrichment analysis showed that EP-Genes might be involved in the immune response and extracellular components. Six key EP-Genes were identified and correlated with renal function. IMC showed that key EP-Genes were located mainly in tubules. Cross verification and examination of a urine RNAseq dataset showed that SPP1 had diagnostic potential for FSGS. The presence of urine SPP1 was primarily associated with macrophage infiltration in kidney, and the pathogenesis of FSGS may be related to innate immunity. Urinary cells seemed to be strongly similar to kidney cells. In summary, SPP1 levels reflect renal function and may have potential as a biomarker for non-invasive diagnosis of FSGS.

Urinary PART1 and PLA2R1 Could Potentially Serve as Diagnostic Markers for Diabetic Kidney Disease Patients.

Background: Diabetic kidney disease (DKD) is a chronic renal disease which could eventually develop into renal failure. Though albuminuria and estimated glomerular filtration rate (eGFR) are helpful for the diagnosis of DKD, the lack of specific biomarkers reduces the efficiency of therapeutic interventions. Methods: Based on bulk-seq of 56 urine samples collected at different time points (including 11 acquired from DKD patients and 11 from healthy controls), in corporation of scRNA-seq data of urine samples and snRNA-seq data of renal punctures from DKD patients (retrieved from NCBI GEO Omnibus), urine-kidney specific genes were identified by Multiple Biological Information methods. Results: Forty urine-kidney specific genes/differentially expressed genes (DEGs) were identified to be highly related to kidney injury and proteinuria for the DKD patients. Most of these genes participate in regulating glucagon and apoptosis, among which, urinary PART1 (mainly derived from distal tubular cells) and PLA2R1 (podocyte cell surface marker) could be used together for the early diagnosis of DKD. Moreover, urinary PART1 was significantly associated with multiple clinical indicators, and remained stable over time in urine. Conclusion: Urinary PART1 and PLA2R1 could be shed lights on the discovery and development of non-invasive diagnostic method for DKD, especially in early stages.

Establishment of a risk score model for bladder urothelial carcinoma based on energy metabolism-related genes and their relationships with immune infiltration.

Bladder urothelial carcinoma (BLCA) is a common malignant tumor of the human urinary system, and a large proportion of BLCA patients have a poor prognosis. Therefore, there is an urgent need to find more efficient and sensitive biomarkers for the prognosis of BLCA patients in clinical practice. RNA sequencing (RNA-seq) data and clinical information were obtained from The Cancer Genome Atlas, and 584 energy metabolism-related genes (EMRGs) were obtained from the Reactome pathway database. Cox regression analysis and least absolute shrinkage and selection operator analysis were applied to assess prognostic genes and build a risk score model. The estimate and cibersort algorithms were used to explore the immune microenvironment, immune infiltration, and checkpoints in BLCA patients. Furthermore, we used the Human Protein Atlas database and our single-cell RNA-seq datasets of BLCA patients to verify the expression of 13 EMRGs at the protein and single-cell levels. We constructed a risk score model; the area under the curve of the model at 5 years was 0.792. The risk score was significantly correlated with the immune markers M0 macrophages, M2 macrophages, CD8 T cells, follicular helper T cells, regulatory T cells, and dendritic activating cells. Furthermore, eight immune checkpoint genes were significantly upregulated in the high-risk group. The risk score model can accurately predict the prognosis of BLCA patients and has clinical application value. In addition, according to the differences in immune infiltration and checkpoints, BLCA patients with the most significant benefit can be selected for immune checkpoint inhibitor therapy.

Single-cell transcriptome sequencing of B-cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy.

BACKGROUND: Breast cancer (BC) is a highly heterogeneous disease, and although immunotherapy has recently increased patient survival in a number of solid and hematologic malignancies, most BC subtypes respond poorly to immune checkpoint blockade therapy (ICB). B cells, particularly those that congregate in tertiary lymphoid structures (TLS), play a significant role in antitumour immunity. However, B-cell heterogeneity at single-cell resolution and its clinical significance with TLS in BC need to be explored further. METHODS: Primary tumour lesions and surrounding normal tissues were taken from 14 BC patients, totaling 124,587 cells, for single-cell transcriptome sequencing and bioinformatics analysis. RESULTS: Based on the usual markers, the single-cell transcriptome profiles were classified into various clusters. A thorough single-cell study was conducted with a focus on tumour-infiltrating B cells (TIL-B) and tumour-associated neutrophils (TAN). TIL-B was divided into five clusters, and unusual cell types, such as follicular B cells, which are strongly related to immunotherapy efficacy, were identified. In BC, TAN and TIL-B infiltration are positively correlated, and at the same time, compared with TLS-high, TAN and TIL-B in TLS-low group are significantly positively correlated. CONCLUSIONS: In conclusion, our study highlights the heterogeneity of B cells in BC, explains how B cells and TLS contribute significantly to antitumour immunity at both the single-cell and clinical level, and offers a straightforward marker for TLS called CD23. These results will offer more pertinent information on the applicability and effectiveness of tumour immunotherapy for BC.

Landscape of Peripheral Blood Mononuclear Cells and Soluble Factors in Severe COVID-19 Patients With Pulmonary Fibrosis Development.

Resulting from severe inflammation and cell destruction, COVID-19 patients could develop pulmonary fibrosis (PF), which remains in the convalescent stage. Nevertheless, how immune response participates in the pathogenesis of PF progression is not well defined. To investigate that question, 12 patients with severe COVID-19 were included in the study. Peripheral mononuclear cell (PBMC) samples were collected shortly after their admission and proceeded for single-cell RNA sequencing (scRNA-seq). After 14 days of discharge, the patients were revisited for chest CT scan. PF index (FI) was computed by AI-assisted CT images. Patients were categorized into FIhi and FIlo based on median of FI. By scRNA-seq analysis, our data demonstrated that frequency of CD4+ activated T cells and Treg cells were approximately 3-fold higher in FIhi patients compared with FIlo ones (p < 0.034 for all). By dissecting the differentially expressed genes, we found an overall downregulation of IFN-responsive genes (STAT1, IRF7, ISG15, ISG20, IFIs, and IFITMs) and S100s alarmins (S100A8, S100A9, S100A12, etc.) in all T-cell clusters, and cytotoxicity-related genes (GZMB, PRF1, and GNLY) in CTLs and γδ T cells in the FIhi cohort, compared with FIlo subjects. The GSEA analysis illustrated decreased expression of genes enriched in IFN signaling, innate immune response, adaptive immune response in T cells, NK cells, and monocytes in FIhi patients compared with FIlo ones. In conclusion, these data indicated that the attenuated IFN-responsive genes and their related signaling pathways could be critical for PF progression in COVID-19 patients.

Identification of Immune-Related Subtypes and Construction of a Novel Prognostic Model for Bladder Urothelial Cancer.

The purpose of this study was to explore the relationship between bladder urothelial cancer (BLCA) and immunity, to screen prognosis-related immune genes (PIGs), and to construct an immune-related prognosis model (IRPM). We processed the relevant data of The Cancer Genome Atlas (TCGA-BLCA) and GSE13507 using R software and Perl. We divided BLCA into high-immunity and low-immunity subtypes. There were significant differences in the two subtypes. In addition, we identified 13 PIGs of BLCA by jointly analyzing the gene expression data and survival information of GSE13507 and TCGA-BLCA, and constructed IRPM through nine of them. The low-risk group had better survival outcome than the high-risk group. We also constructed a nomogram based on clinicopathological information and risk scores of the patients. Moreover, the prognosis of BLCA patients was significantly impacted by the expression of almost every gene used to calculate the risk score. The result of real-time fluorescence quantitative polymerase chain reaction revealed that all the genes used to calculate the risk score were differentially expressed between BLCA and adjacent normal tissues, except PDGFRA. Our research provided potential targets for the treatment of BLCA and a reference for judging the prognosis of BLCA.

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.].

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.