The SARS-CoV-2 Omicron variant is characterized by its high transmissibility, which has caused a worldwide epidemiological event. Yet, it turns ominous once the disease progression degenerates into severe pneumonia and sepsis, presenting a horrendous lethality. To elucidate the alveolar immune or inflammatory landscapes of Omicron critical-ill patients, we performed single-cell RNA-sequencing (scRNA-seq) of bronchoalveolar lavage fluid (BALF) from the patients with critical pneumonia caused by Omicron infection, and analyzed the correlation between the clinical severity scores and different immune cell subpopulations. In the BALF of Omicron critical patients, the alveolar violent myeloid inflammatory environment was determined. ISG15+ neutrophils and CXCL10+ macrophages, both expressed the interferon-stimulated genes (ISGs), were negatively correlated with clinical pulmonary infection score, while septic CST7+ neutrophils and inflammatory VCAN+ macrophages were positively correlated with sequential organ failure assessment. The percentages of ISG15+ neutrophils were associated with more protective alveolar epithelial cells, and may reshape CD4+ T cells to the exhaustive phenotype, thus preventing immune injuries. The CXCL10+ macrophages may promote plasmablast/plasma cell survival and activation as well as the production of specific antibodies. As compared to the previous BALF scRNA-seq data from SARS-CoV-2 wild-type/Alpha critical patients, the subsets of neutrophils and macrophages with pro-inflammatory and immunoregulatory features presented obvious distinctions, suggesting an immune disparity in Omicron variants. Overall, this study provides a BALF single-cell atlas of Omicron critical patients, and suggests that alveolar interferon-responsive neutrophils and macrophages may extricate SARS-CoV-2 Omicron critical patients from the nasty fate of sepsis.

BACKGROUND: Neutrophil extracellular traps (NETs) are related to the prognosis of cancer patients. Nevertheless, the potential prognostic values of NETs in skin cutaneous melanoma (SKCM) remains largely unknown.
METHODS: The NET-related gene signature was constructed by LASSO Cox regression analysis using the TCGA-SKCM cohort. The overall survival (OS) and immune status in SKCM patients between the high- and low-NET score (high-score, low-score) groups were explored. The scRNA-seq dataset GSE115978 was used to understand the role of NET score in SKCM at single cell resolution.
RESULTS: A five NET genes-based signature (TLR2, CLEC6A, PDE4B, SLC22A4 and CYP4F3) was constructed as the NET-related prognostic model for SKCM. The OS of SKCM patients with low-score was better than that in patients with high-score. Additionally, NET score was negatively associated with infiltration of some immune cells (e.g. type I Macrophages, CD8-T cells, CD4-T cells). Moreover, patients with high-score had low stromal, immune and ESTIMATE scores. Furthermore, drug sensitivity analysis results showed that Lapatinib, Trametinib and Erlotinib may have better therapeutic advantages in patients with high-score.
CONCLUSIONS: We established a NET-related five gene signature in SKCM and found that the NET-related signature may exhibit a good predictive ability for SKCM prognosis. The NET score may not only predict the survival outcome and drug sensitivity in SKCM, but also reflect the immune conditions of SKCM patients.

Despite substantial breakthroughs in the treatment of hepatocellular carcinoma (HCC) in recent years, many patients are diagnosed in the middle or late stages, denying them the option for surgical excision. Therefore, it is of great importance to find effective therapeutic targets of HCC. In this study, it is found that Gap junction protein beta-2 (GJB2) is highly enriched in malignant cells based on single-cell RNA sequencing and higher expression of GJB2 indicates a worse prognosis. The localization of GJB2 in HCC cancer cells is changed compared with normal liver tissue. In cancer cells, GJB2 tends to be located in the cytoplasm and nucleus, while in normal tissues, GJB2 is mainly located on the cell membrane. GJB2 is related to glycolysis, promoting NF-κB pathway via inducing the ubiquitination degradation of IκBa, and activating HIF-1α/GLUT-1/PD-L1 pathway. In addition, GJB2 knockdown reshapes tumor immune microenvironment and Salvianolic acid B inhibits the activity of GJB2. In conclusion, GJB2 promotes HCC progression by activating glycolysis through cytoplasmic translocation and generating a suppressive tumor microenvironment. Salvianolic acid B inhibits the expression of GJB2 and enhances the sensitivity of anti-PD1 therapy, which may provide insights into the development of novel combination therapeutic strategies for HCC.

Degenerative spinal stenosis is a chronic disease that affects the spinal ligaments and associated bones, resulting in back pain and disorders of the limbs among the elderly population. There are few preventive strategies for such ligament degeneration. We here aimed to establish a comprehensive transcriptomic atlas of ligament tissues to identify high-priority targets for pharmaceutical treatment of ligament degeneration. Here, single-cell RNA sequencing was performed on six degenerative ligaments and three traumatic ligaments to understand tissue heterogeneity. After stringent quality control, high-quality data were obtained from 32,014 cells. Distinct cell clusters comprising stromal and immune cells were identified in ligament tissues. Among them, we noted that collagen degradation associated with CTHRC1+ fibroblast-like cells and calcification linked to CRTAC1+ chondrocyte-like cells were key features of ligament degeneration. SCENIC analysis and further experiments identified ATF3 as a key transcription factor regulating the pathogenesis of CRTAC1+ chondrocyte-like cells. Typically, immune cells infiltrate localized organs, causing tissue damage. In our study, myeloid cells were found to be inflammatory-activated, and SPP1+ macrophages were notably enriched in degenerative ligaments. Further exploration via CellChat analysis demonstrated a robust interaction between SPP1+ macrophages and CRTAC1+ chondrocyte-like cells. Activated by SPP1, ATF3 propels the CRTAC1/MGP/CLU axis, fostering ligament calcification. Our unique resource provides novel insights into possible mechanisms underlying ligament degeneration, the target cell types, and molecules that are expected to mitigate degenerative spinal ligament. We also highlight the role of immune regulation in ligament degeneration and calcification, enhancing our understanding of this disease.

Accumulating evidence suggests that berberine (BBR) exhibits anti-cancer effects in hepatocellular carcinoma (HCC). However, the mechanisms by which BBR regulates the immunological microenvironment in HCC has not been fully elucidated. In this study, a mouse model of orthotopic HCC is established and treated with varying doses of BBR. BBR showed effectiveness in reducing tumor burden in mice with HCC. Cytometry by time-of-flight depicted the alterations in the tumor immune landscape following BBR treatment, revealing the enhancement in the T lymphocytes effector function. In particular, BBR decreased the proportion of TCRbhiPD-1hiCD69+CD27+ effector CD8+ T lymphocytes and increased the proportion of Ly6ChiTCRb+CD69+CD27+CD62L+ central memory CD8+ T lymphocytes. Single-cell RNA sequencing further elucidates the effects of BBR on transcriptional profiles of liver immune cells and confirms the phenotypical heterogeneity of T lymphocytes in HCC immune microenvironment. Additionally, it is found that BBR potentially regulated the antitumor immunity in HCC by modulating the receptor-ligand interaction among immune cells mediated by cytokines. In summary, the findings improve the understanding of BBR\'s impact on protecting against HCC, emphasizing BBR\'s role in regulating intrahepatic T cell heterogeneity. BBR has the potential to be a promising therapeutic strategy to hinder the advancement of HCC.

OBJECTIVE: The main focus of this study is to explore the molecular mechanism of IRF7 regulation on RPS18 transcription in M1-type macrophages in pancreatic adenocarcinoma (PAAD) tissue, as well as the transfer of RPS18 by IRF7 via exosomes to PAAD cells and the regulation of ILF3 expression.
METHODS: By utilising single-cell RNA sequencing (scRNA-seq) data and spatial transcriptomics (ST) data from the Gene Expression Omnibus database, we identified distinct cell types with significant expression differences in PAAD tissue. Among these cell types, we identified those closely associated with lipid metabolism. The differentially expressed genes within these cell types were analysed, and target genes relevant to prognosis were identified. Flow cytometry was employed to assess the expression levels of target genes in M1 and M2 macrophages. Cell lines with target gene knockout were constructed using CRISPR/Cas9 editing technology, and cell lines with target gene knockdown and overexpression were established using lentiviral vectors. Additionally, a co-culture model of exosomes derived from M1 macrophages with PAAD cells was developed. The impact of M1 macrophage-derived exosomes on the lipid metabolism of PAAD cells in the model was evaluated through metabolomics analysis. The effects of M1 macrophage-derived exosomes on the viability, proliferation, division, migration and apoptosis of PAAD cells were assessed using MTT assay, flow cytometry, EdU assay, wound healing assay, Transwell assay and TUNEL staining. Furthermore, a mouse PAAD orthotopic implantation model was established, and bioluminescence imaging was utilised to assess the influence of M1 macrophage-derived exosomes on the intratumoural formation capacity of PAAD cells, as well as measuring tumour weight and volume. The expression of proliferation-associated proteins in tumour tissues was examined using immunohistochemistry.
RESULTS: Through combined analysis of scRNA-seq and ST technologies, we discovered a close association between M1 macrophages in PAAD samples and lipid metabolism signals, as well as a negative correlation between M1 macrophages and cancer cells. The construction of a prognostic risk score model identified RPS18 and IRF7 as two prognostically relevant genes in M1 macrophages, exhibiting negative and positive correlations, respectively. Mechanistically, it was found that IRF7 in M1 macrophages can inhibit the transcription of RPS18, reducing the transfer of RPS18 to PAAD cells via exosomes, consequently affecting the expression of ILF3 in PAAD cells. IRF7/RPS18 in M1 macrophages can also suppress lipid metabolism, cell viability, proliferation, migration, invasion and intratumoural formation capacity of PAAD cells, while promoting cell apoptosis.
CONCLUSIONS: Overexpression of IRF7 in M1 macrophages may inhibit RPS18 transcription, reduce the transfer of RPS18 from M1 macrophage-derived exosomes to PAAD cells, thereby suppressing ILF3 expression in PAAD cells, inhibiting the lipid metabolism pathway, and curtailing the viability, proliferation, migration, invasion of PAAD cells, as well as enhancing cell apoptosis, ultimately inhibiting tumour formation in PAAD cells in vivo. Targeting IRF7/RPS18 in M1 macrophages could represent a promising immunotherapeutic approach for PAAD in the future.

BACKGROUND: Glioma is the most frequent and lethal form of primary brain tumor. The molecular mechanism of oncogenesis and progression of glioma still remains unclear, rendering the therapeutic effect of conventional radiotherapy, chemotherapy, and surgical resection insufficient. In this study, we sought to explore the function of HEC1 (highly expressed in cancer 1) in glioma; a component of the NDC80 complex in glioma is crucial in the regulation of kinetochore.
METHODS: Bulk RNA and scRNA-seq analyses were used to infer HEC1 function, and in vitro experiments validated its function.
RESULTS: HEC1 overexpression was observed in glioma and was indicative of poor prognosis and malignant clinical features, which was confirmed in human glioma tissues. High HEC1 expression was correlated with more active cell cycle, DNA-associated activities, and the formation of immunosuppressive tumor microenvironment, including interaction with immune cells, and correlated strongly with infiltrating immune cells and enhanced expression of immune checkpoints. In vitro experiments and RNA-seq further confirmed the role of HEC1 in promoting cell proliferation, and the expression of DNA replication and repair pathways in glioma. Coculture assay confirmed that HEC1 promotes microglial migration and the transformation of M1 phenotype macrophage to M2 phenotype.
CONCLUSIONS: Altogether, these findings demonstrate that HEC1 may be a potential prognostic marker and an immunotherapeutic target in glioma.

Keratins are typical intermediate filament proteins of the epithelium that exhibit highly specific expression patterns related to the epithelial type and stage of cellular differentiation. They are important for cytoplasmic stability and epithelial integrity and are involved in various intracellular signaling pathways. Several keratins are associated with enamel formation. However, information on their expression patterns during tooth development remains lacking. In this study, we analyzed the spatiotemporal expression of keratin family members during tooth development using single-cell RNA-sequencing (scRNA-seq) and microarray analysis. scRNA-seq datasets from postnatal Day 1 mouse molars revealed that several keratins are highly expressed in the dental epithelium, indicating the involvement of keratin family members in cellular functions. Among various keratins, keratin 5 (Krt5), keratin 14 (Krt14), and keratin 17 (Krt17) are highly expressed in the tooth germ; KRT17 is specifically expressed in the stratum intermedium (SI) and stellate reticulum (SR). Depletion of Krt17 did not affect cell proliferation in the dental epithelial cell line SF2 but suppressed their differentiation ability. These results suggest that Krt17 is essential for SI cell differentiation. Furthermore, scRNA-seq results indicated that Krt5, Krt14, and Krt17 exhibited distinct expression patterns in ameloblast, SI, and SR cells. Our findings contribute to the elucidation of novel mechanisms underlying tooth development.

As 3D culture technology advances, new avenues have opened for the development of physiological human cancer models. These preclinical models provide efficient ways to translate basic cancer research into clinical tumor therapies. Recently, cancer organoids have emerged as a model to dissect the more complex tumor microenvironment. Incorporating cancer organoids into preclinical programs have the potential to increase the success rate of oncology drug development and recapitulate the most efficacious treatment regimens for cancer patients. In this review, four main types of cancer organoids are introduced, their applications, advantages, limitations, and prospects are discussed, as well as the recent application of single-cell RNA-sequencing (scRNA-seq) in exploring cancer organoids to advance this field.

Aggrephagy, a type of autophagy, degrades the aggregation of misfolded protein in cells. However, the role of aggrephagy in multiple myeloma (MM) has not been fully demonstrated. In this study, we first investigated the correlation between aggrephagy signaling, MM immune microenvironment composition and disease prognosis. Single-cell RNA-seq data, including the expression profiles of 12,187 single cells from seven MM bone marrow (BM) and seven healthy BM samples, were analyzed by non-negative matrix factorization for 44 aggrephagy-related genes. Bulk RNA-seq cohorts from the Gene Expression Omnibus database were used to evaluate the prognostic value of aggrephagy-related immune cell subtypes and predict immune checkpoint blockade immunotherapeutic response in MM. Compared with healthy BM, MM BM exhibited different patterns of aggrephagy-related gene expression. In MM BM, macrophages, CD8+ T cells, B cells and natural killer cells could be grouped into four to nine aggrephagy-related subclusters. The signature of aggrephagy signaling molecule expression in the immune cells correlates with the patient\'s prognosis. Our investigation provides a novel view of aggrephagy signaling in MM tumor microenvironment cells, which might be a prognostic indicator and potential target for MM treatment.