Circulating tumor cell clustering modulates RNA splicing and polyadenylation to facilitate metastasis.

Circulating tumor cell (CTC) clusters exhibit significantly higher metastatic potential compared to single CTCs. However, the underlying mechanism behind this phenomenon remains unclear, and the role of posttranscriptional RNA regulation in CTC clusters has not been explored. Here, we conducted a comparative analysis of alternative splicing (AS) and alternative polyadenylation (APA) profiles between single CTCs and CTC clusters. We identified 994 and 836 AS events in single CTCs and CTC clusters, respectively, with ∼20% of AS events showing differential regulation between the two cell types. A key event in this differential splicing was observed in SRSF6, which disrupted AS profiles and contributed to the increased malignancy of CTC clusters. Regarding APA, we found a global lengthening of 3' UTRs in CTC clusters compared to single CTCs. This alteration was primarily governed by 14 core APA factors, particularly PPP1CA. The modified APA profiles facilitated the cell cycle progression of CTC clusters and indicated their reduced susceptibility to oxidative stress. Further investigation revealed that the proportion of H2AFY mRNA with long 3' UTR instead of short 3' UTR was higher in CTC clusters than single CTCs. The AU-rich elements (AREs) within the long 3' UTR of H2AFY mRNA enhance mRNA stability and translation activity, resulting in promoting cell proliferation and invasion, which potentially facilitate the establishment and rapid formation of metastatic tumors mediated by CTC clusters. These findings provide new insights into the mechanisms driving CTC cluster metastasis.

Single-cell transcriptomics of peripheral blood reveals anti-tumor systemic immunity induced by oncolytic virotherapy.

Rationale: Oncolytic virus (OV) therapy as a cancer therapy that improves immune status makes it a favorable candidate for optimizing immunotherapy strategies. Existing studies have focused on characterizing the disturbance of the tumor microenvironment (TME) by OV therapy. However, the changes in systemic immunity induced by OV were largely ignored, which would prevent the further understanding and optimization of oncolytic viruses. Methods: The HSV-2-based oncolytic virus OH2 was used to treat tumor-bearing mouse models. The peripheral blood samples were then collected for single-cell RNA sequencing (scRNA-seq). The scRNA-seq data were analyzed using Cell Ranger, Seurat, and other bioinformatics tools. Key findings were further validated by ELISA, immunohistochemistry, flow cytometry, in vivo experiments, and clinical samples. Results: Our data showed that OH2 therapy effectively activated systemic immunity and induced a sustained anti-tumor immune response. One major impact of OH2 on systemic immunity was to boost Ccl5 production, which correlated with clinical response. Besides, the cytotoxic ability of peripheral cytotoxic Cd8+ T cells and mature NK cells was elevated by OH2. Further analysis revealed that the interaction of monocytes with T cells and NK cells was critical for systemic immune remodeling and activation. We also found that systemic immune responses induced by OH2 could effectively reshape the microenvironment of distant tumor lesions and inhibit their progression. Conclusions: This study is the first to comprehensively characterize the effects of OV therapy on systemic immunity, which not only sheds new light on the anti-tumor mechanisms of OH2, but also contributes to the establishment of companion diagnostics for OH2 treatment and the improvement of oncolytic therapy strategies.

SIRPα antibody combined with oncolytic virus OH2 protects against tumours by activating innate immunity and reprogramming the tumour immune microenvironment.

BACKGROUND: The combination of oncolytic viruses (OVs) with immune checkpoint blockades is a research hotspot and has shown good efficacy. Here, we present the first attempt to combine oncolytic herpes simplex virus 2 (OH2) with an anti-SIRPα antibody as an antitumour treatment. Our results provide unique insight into the combination of innate immunity with OV. METHODS: We verified the polarization and activation of OH2 in RAW264.7 cells in vitro. Subsequently, we evaluated the antitumour ability of OH2 and anti-SIRPα combined therapy in a tumour-bearing mouse model. RNA-seq and Single-cell RNA-seq were used to characterize the changes in the tumour microenvironment. RESULTS: The OH2 lysates effectively stimulated RAW264.7 cells to polarize towards the M1 but not the M2 phenotype and activated the function of the M1 phenotype in vitro. In the macrophage clearance experiment, OH2 therapy induced polarization of M1 macrophages and participated in the antitumour immune response in a tumour-bearing mouse model. Treatment with a combination of OH2 and anti-SIRPα effectively inhibited tumour growth and significantly prolonged the survival time of the mice, and this result was more obvious in the mouse model with a larger tumour volume at the beginning of the treatment. These results suggest that combination therapy can more profoundly reshape the TME and activate stronger innate and adaptive immune responses. CONCLUSIONS: Our data support the feasibility of oncolytic virus therapy in combination with anti-SIRPα antibodies and suggest a new strategy for oncolytic virus therapy.

Multi-omics analysis reveals RNA splicing alterations and their biological and clinical implications in lung adenocarcinoma.

Alternative RNA splicing is one of the most important mechanisms of posttranscriptional gene regulation, which contributes to protein diversity in eukaryotes. It is well known that RNA splicing dysregulation is a critical mechanism in tumor pathogenesis and the rationale for the promising splice-switching therapeutics for cancer treatment. Although we have a comprehensive understanding of DNA mutations, abnormal gene expression profiles, epigenomics, and proteomics in lung adenocarcinoma (LUAD), little is known about its aberrant alternative splicing profiles. Here, based on the multi-omics data generated from over 1000 samples, we systematically studied the RNA splicing alterations in LUAD and revealed their biological and clinical implications. We identified 3688 aberrant alternative splicing events (AASEs) in LUAD, most of which were alternative promoter and exon skip. The specific regulatory roles of RNA binding proteins, somatic mutations, and DNA methylations on AASEs were comprehensively interrogated. We dissected the functional implications of AASEs and concluded that AASEs mainly affected biological processes related to tumor proliferation and metastasis. We also found that one subtype of LUAD with a particular AASEs pattern was immunogenic and had a better prognosis and response rate to immunotherapy. These findings revealed novel events related to tumorigenesis and tumor immune microenvironment and laid the foundation for the development of splice-switching therapies for LUAD.

Telomerase positive CTCs with PSMA high expression associated with prostate cancer metastasis.

Background: Whether circulating tumor cells (CTCs) with prostate-specific membrane antigen (PSMA) high expression was related to the metastatic progress in prostate cancer (PCa) remains explored. This study aimed to provide evidence to elucidate this relationship via the telomerase reverse transcriptase (TERT)-based CTC detection method. Methods: A total of 71 patients were enrolled and divided into the local PCa group (n=44) and metastatic PCa group (n=27). TERT-based CTC detection (TBCD) was used to detect CTCs. CTCs single-cell sequencing data were analyzed using gene ontology (GO) functional classification and enrichment. Results: The mean 'TERT+ CTCs' number was 6.11±9.63 in the metastatic group and 4.09±3.41 in the local group. GO enrichment analysis for 77 prostate CTCs single-cell sequencing confirmed that proliferation-related terms were enriched in the PSMA-high expression group, and 27 metastasis-related gene panels also had high expression in this group. Then, PSMA antibody was applied to mark the 'TERT+ CTCs'. The proportion of patients with 'TERT+ PSMA+ CTCs' was positively associated with the Gleason score. Furthermore, the proportion of 'TERT+ PSMA+ CTCs' patients was 48.15% in the metastatic group, significantly higher than 22.72% in the local group. Conclusions: This study suggested that TERT positive CTCs with high PSMA expression were associated with the PCa metastatic progress.

Establishment of gastric signet ring cell carcinoma organoid for the therapeutic drug testing.

Signet ring cell carcinoma (SRCC) has specific oncogenesis and phenotypic and treatment resistance heterogeneity. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. Tumor organoids have recently emerged as an ideal model for drug testing and screening. Here, we report gastric organoids established from tumor tissues comprising four SRCCs and eight non-SRCCs. Tumor organoids demonstrated different growth characteristics and morphologies. Changes in the original tumor genome were maintained during long-term culture from whole-exome sequencing (WES) analysis. Immunohistochemistry and H&E staining showed that the tissue characteristics of the primary tumor could be recapitulated. In addition, organoid lines successfully formed tumors in immunodeficient mice and maintained tumorigenic character. Different responses to 5-fluorouracil, oxaliplatin, docetaxel and irinotecan treatment were observed in SRCC and non-SRCC organoids. These results demonstrate that gastric organoid drug models, including SRCC, were highly similar to the original tumors in phenotypic and genotypic profiling and could be as living biomarkers for drug response testing.

Systematic Profiling of mRNA Splicing Reveals the Prognostic Predictor and Potential Therapeutic Target for Glioblastoma Multiforme.

Despite many changes in alternative splicing events (ASEs) are frequently involved in various cancers, prognosis-related ASEs and drug treatment targets in glioblastoma multiforme (GBM) have not been well explored. ASEs participate in many biological behaviors in the initiation and progression of tumors, the aberrant ASE has been considered another hallmark of cancer, and the systematic study of alternative splicing may provide potential biomarkers for malignancies. In this study, we carried out a systematic analysis to characterize the ASE signatures in GBM cohort. Through comparing GBM tissues and nontumor tissues, a total of 48,191 differently expressed ASEs from 10,727 genes were obtained, and these aberrant ASEs play an important role in the oncogenic process. Then, we identified 514 ASEs independently associated with patient survival in GBM by univariate and multivariate Cox regression, including exon skip in CD3D, alternate acceptor site in POLD2, and exon skip in DCN. Those prognostic models built on ASEs of each splice type can accurately predict the outcome of GBM patients, and values for the area under curve were 0.97 in the predictive model based on alternate acceptor site. In addition, the splicing-regulatory network revealed an interesting correlation between survival-associated splicing factors and prognostic ASE corresponding genes. Moreover, these three hub splicing factors in splicing regulation network are the potential targets of some drugs. In conclusion, a systematic analysis of ASE signatures in GBM could serve as an indicator for identifying novel prognostic biomarkers and guiding clinical treatment.

The landscape and biological relevance of aberrant alternative splicing events in esophageal squamous cell carcinoma.

Aberrant alternative splicing events (AASEs) are key biological processes for tumorigenesis and the rationale for designing splice-switching oligonucleotides (SSOs). However, the landscape of AASEs in esophageal squamous cell carcinoma (ESCC) remains unclear, which undermines the development of SSOs for ESCC. Here, we profiled AASEs based on 125 pairs of RNA-seq libraries. We identified 14,710 AASEs in ESCC, most of which (92.67%) affected coding genes. The first exon of transcripts was frequently changed in ESCC. We constructed a regulatory network where 74 RNA-binding proteins regulated 2142 AASEs. This network was enriched in apoptotic pathways and various adhesion/junction-related processes. Somatic mutations in ESCC regulating ASEs were mainly through trans-regulatory mode and were enriched in intron regions. Isoform switches of apoptotic genes and binding genes both tended to induce "noncoding transcripts" and "domain loss," disrupting the apoptotic and Hippo signaling pathways. All ESCC samples were grouped into three clusters with different AASEs patterns and the second cluster was identified as "cold tumor," with a low abundance of immune cells, activated immune pathways, and immunomodulators. Our work comprehensively profiled the landscape of AASEs in ESCC, revealed novel AASEs related to tumorigenesis and immune microenvironment, and suggested promising directions for designing SSOs for ESCC.