Jun Nakayama

Spatial exosome analysis using cellulose nanofiber sheets reveals the location heterogeneity of extracellular vesicles

AbstractExtracellular vesicles (EVs), including exosomes, are recognized as promising functional targets involved in disease mechanisms. However, the intravital heterogeneity of EVs remains unclear, and the general limitation for analyzing EVs is the need for a certain volume of biofluids. Here, we present cellulose nanofiber (CNF) sheets to resolve these issues. We show that CNF sheets capture and preserve EVs from ~10 μL of biofluid and enable the analysis of bioactive molecules inside EVs. By attaching CNF sheets to moistened organs, we collect EVs in trace amounts of ascites, which is sufficient to perform small RNA sequence analyses. In an ovarian cancer mouse model, we demonstrate that CNF sheets enable the detection of cancer-associated miRNAs from the very early phase when mice did not have apparent ascites, and that EVs from different locations have unique miRNA profiles. By performing CNF sheet analyses in patients, we identify further location-based differences in EV miRNA profiles, with profiles reflecting disease conditions. We conduct spatial exosome analyses using CNF sheets to reveal that ascites EVs from cancer patients exhibit location-dependent heterogeneity. This technique could provide insights into EV biology and suggests a clinical strategy contributing to cancer diagnosis, staging evaluation, and therapy planning.

Ring-Finger Protein 126 (RNF126) Promotes Anoikis Resistance and Peritoneal Colonization in Ovarian Cancer

Ovarian cancer (OC) represents the most lethal gynecologic malignancy because the majority of patients with OC are diagnosed at advanced stages with peritoneal colonization of OC cells owing to subtle and nonspecific nature of symptoms. Thus, peritoneal colonization-directed therapeutic approaches are urgently needed for patients with advanced OC. Here, we investigated whether Ring-finger protein 126 (RNF126), an E3 ubiquitin ligase that is aberrantly upregulated in epithelial OC tissues, contributes to the peritoneal colonization of OC. RNF126-depleted OC cells showed comparable proliferation under normal culture conditions but displayed decreased growth under floating (anchorage-independent) conditions in vitro. Further analyses showed that RNF126 promoted anoikis resistance in vitro and increased peritoneal colonization in immunodeficient mice in a RING domain-dependent manner. Mechanistically, RNF126 activated the transcriptional factor NF-κB in OC cells under floating conditions in a RING domain-dependent manner, and this NF-κB activation was essential for anchorage-independent growth and peritoneal colonization of OC cells. Thus, RNF126 is a possible target for the prevention and/or therapy of peritoneally colonized OC.

Single‐Nucleus RNA Sequencing and Spatial Transcriptomics for Squamous Cell Carcinoma Arising From Ovarian Mature Teratoma

ABSTRACTSquamous cell carcinoma arising from mature teratoma (SCC‐MT) is a rare ovarian malignancy. The detailed molecular pathology of SCC‐MT is not well understood. Moreover, the prognosis of the patients remains poor because no standard treatment has been established. In this study, we performed single‐nucleus RNA sequencing and spatial transcriptomics using clinical SCC‐MT samples to identify novel therapeutic candidates. snRNA‐seq revealed three epithelial cell clusters, of which one was significantly associated with epidermis and keratinocyte development. Moreover, spatial transcriptomics revealed that the epithelial‐mesenchymal transition was significantly inhibited, and the MYC and E2F targets were significantly activated in cancer spots on specimen sections. We focused on KLF5, which was one of the upregulated genes in cancer cells, and performed a functional analysis using NOSCC‐1, a cell line derived from an SCC‐MT. KLF5 downregulation significantly decreased cell proliferation and increased apoptosis. Furthermore, we previously identified miR‐145‐5p as a downregulated miRNA in SCC‐MT. We demonstrated that miR‐145‐5p overexpression attenuated cell proliferation and decreased KLF5 expression. In conclusion, through multi‐omics analyses, we identified unique gene expression profiles of SCC‐MT and determined a role for KLF5 in SCC‐MT development. Therefore, KLF5‐related factors may be novel therapeutic targets, and further studies are needed to improve the diagnosis and treatment of SCC‐MT.