Single-Exosome Profiling Identifies ITGB3+ and ITGAM+ Exosome Subpopulations as Promising Early Diagnostic Biomarkers and Therapeutic Targets for Colorectal Cancer.

Tumor metastasis is a hallmark of colorectal cancer (CRC), in which exosome plays a crucial role with its function in intercellular communication. Plasma exosomes were collected from healthy control (HC) donors, localized primary CRC and liver-metastatic CRC patients. We performed proximity barcoding assay (PBA) for single-exosome analysis, which enabled us to identify the alteration in exosome subpopulations associated with CRC progression. By in vitro and in vivo experiments, the biological impact of these subpopulations on cancer proliferation, migration, invasion, and metastasis was investigated. The potential application of exosomes as diagnostic biomarkers was evaluated in 2 independent validation cohorts by PBA. Twelve distinct exosome subpopulations were determined. We found 2 distinctly abundant subpopulations: one ITGB3-positive and the other ITGAM-positive. The ITGB3-positive cluster is rich in liver-metastatic CRC, compared to both HC group and primary CRC group. On the contrary, ITGAM-positive exosomes show a large-scale increase in plasma of HC group, compared to both primary CRC and metastatic CRC groups. Notably, both discovery cohort and validation cohort verified ITGB3+ exosomes as potential diagnostic biomarker. ITGB3+ exosomes promote proliferation, migration, and invasion capability of CRC. In contrast, ITGAM+ exosomes suppress CRC development. Moreover, we also provide evidence that one of the sources of ITGAM+ exosomes is macrophage. ITGB3+ exosomes and ITGAM+ exosomes are proven 2 potential diagnostic, prognostic, and therapeutic biomarkers for management of CRC.

m6A Regulator-Based Exosomal Gene Methylation Modification Patterns Identify Distinct Microenvironment Characterization and Predict Immunotherapeutic Responses in Colon Cancer.

Recent studies have highlighted the biological significance of exosomes and m6A modifications in immunity. Nonetheless, it remains unclear whether the m6A modification gene in exosomes of body fluid has potential roles in the tumor microenvironment (TME). Herein, we identified three different m6A-related exosomal gene modification patterns based on 59 m6A-related exosomal genes, which instructed distinguishing characteristics of TME in colon cancer (CC). We demonstrated that these patterns could predict the stage of tumor inflammation, subtypes, genetic variation, and patient prognosis. Furthermore, we developed a scoring mode-m6A-related exosomal gene score (MREGS)-by detecting the level of m6A modification in exosomes to classify immune phenotypes. Low MREGS, characterized by prominent survival and immune activation, was linked to a better response to anti-PDL1 immunotherapy. In contrast, the higher MREGS group displayed remarkable stromal activation, high activity of innate immunocytes, and a lower survival rate. Hence, this work provides a novel approach for evaluating TME cell infiltration in colon cancer and guiding more effective immunotherapy strategies.

Inhibition of GABAA receptors in intestinal stem cells prevents chemoradiotherapy-induced intestinal toxicity.

Lethal intestinal tissue toxicity is a common side effect and a dose-limiting factor in chemoradiotherapy. Chemoradiotherapy can trigger DNA damage and induce P53-dependent apoptosis in LGR5+ intestinal stem cells (ISCs). Gamma-aminobutyric acid (GABA) and its A receptors (GABAAR) are present in the gastrointestinal tract. However, the functioning of the GABAergic system in ISCs is poorly defined. We found that GABAAR α1 (GABRA1) levels increased in the murine intestine after chemoradiotherapy. GABRA1 depletion in LGR5+ ISCs protected the intestine from chemoradiotherapy-induced P53-dependent apoptosis and prolonged animal survival. The administration of bicuculline, a GABAAR antagonist, prevented chemoradiotherapy-induced ISC loss and intestinal damage without reducing the chemoradiosensitivity of tumors. Mechanistically, it was associated with the reduction of reactive oxygen species-induced DNA damage via the L-type voltage-dependent Ca2+ channels. Notably, flumazenil, a GABAAR antagonist approved by the U.S. Food and Drug Administration, rescued human colonic organoids from chemoradiotherapy-induced toxicity. Therefore, flumazenil may be a promising drug for reducing the gastrointestinal side effects of chemoradiotherapy.

Resolving the difference between left-sided and right-sided colorectal cancer by single-cell sequencing.

Colorectal cancers (CRCs) exhibit differences in incidence, pathogenesis, molecular pathways, and outcome depending on the location of the tumor. The transcriptomes of 27,927 single human CRC cells from 3 left-sided and 3 right-sided CRC patients were profiled by single-cell RNA-Seq (scRNA-Seq). Right-sided CRC harbors a significant proportion of exhausted CD8+ T cells of a highly migratory nature. One cluster of cells from left-sided CRC exhibiting states preceding exhaustion and a high ratio of preexhausted/exhausted T cells were favorable prognostic markers. Notably, we identified a potentially novel RBP4+NTS+ subpopulation of cancer cells that exclusively expands in left-sided CRC. Tregs from left-sided CRC showed higher levels of immunotherapy-related genes than those from right-sided CRC, indicating that left-sided CRC may have increased responsiveness to immunotherapy. Antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) induced by M2-like macrophages were more pronounced in left-sided CRC and correlated with a good prognosis in CRC.

M6A methylation of DEGS2, a key ceramide-synthesizing enzyme, is involved in colorectal cancer progression through ceramide synthesis.

N6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulator in cancer. However, the understanding of m6A modification on lipid metabolism regulation in colorectal cancer (CRC) is very limited. Here, we observed that human CRCs exhibited increased m6A mRNA methylation mediated by dysregulation of m6A erasers and readers. By performing methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and transcriptomic sequencing (RNA-seq), we identified DEGS2 as a downstream target of m6A dysregulation. Overexpression or knockdown of DEGS2 confirmed the role of DEGS2 in proliferation, invasion and metastasis of CRC both in vitro and in vivo. Mechanistic studies identified the specific m6A modification site within DEGS2 mRNA, and mutation of this target site was found to drastically enhance the proliferative and invasive ability of CRC cells in vitro and promote tumorigenicity in vivo. Lipidome analysis showed that lipid metabolism was dysregulated in CRC. Moreover, ceramide synthesis was suppressed due to DEGS2 upregulation mediated by m6A modification in CRC tissues. Our findings highlight that the function of DEGS2 m6A methylation in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.