An exosome‑based liquid biopsy signature for pre‑operative identification of lymph node metastasis in patients with pathological high‑risk T1 colorectal cancer

Background: According to current guidelines, more than 70% of patients with invasive submucosal colorectal cancer (T1 CRC) undergo a radical operation with lymph node dissection, even though only ~ 10% have lymph node metastasis (LNM). Hence, there is imperative to develop biomarkers that can help robustly identify LNM-positive patients to prevent such overtreatments. Given the emerging interest in exosomal cargo as a source for biomarker development in cancer, we examined the potential of exosomal miRNAs as LNM prediction biomarkers in T1 CRC. Methods: We analyzed 200 patients with high-risk T1 CRC from two independent cohorts, including a training (n = 58) and a validation cohort (n = 142). Cell-free and exosomal RNAs from pre-operative serum were extracted, followed by quantitative reverse-transcription polymerase chain reactions for a panel of miRNAs. Results: A panel of four miRNAs (miR-181b, miR-193b, miR-195, and miR-411) exhibited robust ability for detecting LNM in the exosomal vs. cell-free component. We subsequently established a cell-free and exosomal combination signature, successfully validated in two independent clinical cohorts (AUC, 0.84; 95% CI 0.70–0.98). Finally, we developed a risk-stratification model by including key pathological features, which reduced the false positive rates for LNM by 76% without missing any true LNM-positive patients. Conclusions: Our novel exosomal miRNA-based liquid biopsy signature robustly identifies T1 CRC patients at risk of LNM in a preoperative setting. This could be clinically transformative in reducing the significant overtreatment burden of this malignancy

Transcription factor TFEB cell-autonomously modulates susceptibility to intestinal epithelial cell injury in vivo

Understanding the transcription factors that modulate epithelial resistance to injury is necessary for understanding intestinal homeostasis and injury repair processes. Recently, transcription factor EB (TFEB) was implicated in expression of autophagy and host defense genes in nematodes and mammalian cells. However, the in vivo roles of TFEB in the mammalian intestinal epithelium were not known. Here, we used mice with a conditional deletion of Tfeb in the intestinal epithelium (Tfeb ΔIEC) to examine its importance in defense against injury. Unperturbed Tfeb ΔIEC mice exhibited grossly normal intestinal epithelia, except for a defect in Paneth cell granules. Tfeb ΔIEC mice exhibited lower levels of lipoprotein ApoA1 expression, which is downregulated in Crohn’s disease patients and causally linked to colitis susceptibility. Upon environmental epithelial injury using dextran sodium sulfate (DSS), Tfeb ΔIEC mice exhibited exaggerated colitis. Thus, our study reveals that TFEB is critical for resistance to intestinal epithelial cell injury, potentially mediated by APOA1

Distinct expression patterns of Notch ligands, Dll1 and Dll4, in normal and inflamed mice intestine

Reports have suggested that the two Notch ligands, Dll1 and Dll4, are indispensable to maintain the homeostasis of the intestinal epithelium. However, within the intestinal epithelium, the precise distribution of the cells that express those ligands at the protein level remains largely unknown. Here, we show a series of immunohistochemical analysis through which we successfully identified mice intestinal epithelial cells (IECs) that endogenously express Dll1 or Dll4. Results showed that Dll1-positive (Dll1+ve) IECs reside exclusively within the crypt, whereas Dll4-positive (Dll4+ve) IECs can locate both in the crypt and in the villus of the small intestine. Also in the colon, Dll1+ve IECs resided at the lower part of the crypt, whereas Dll4+ve IECs resided at both upper and lower part of the crypt, including the surface epithelium. Both Dll1+ve and Dll4+ve IECs were ATOH1-positive, but Hes1-negative cells, and located adjacent to Hes1-positive cells within the crypts. A sub-population of both Dll1+ve and Dll4+ve IECs appeared to co-express Muc2, but rarely co-expressed other secretory lineage markers. However, as compared to Dll1+ve IECs, Dll4+ve IECs included larger number of Muc2-postive IECs, suggesting that Dll4 is more preferentially expressed by goblet cells. Also, we identified that Dll4 is expressed in the Paneth cells of the small intestine, whereas Dll1 and Dll4 is expressed in the c-kit-positive IECs of the colon, indicating that Dll1+ve and Dll4+ve IECs may contribute to constitute the intestinal stem cell niche. Compared to the normal colon, analysis of DSS-colitis showed that number of Dll1+ve IECs significantly decrease in the elongated crypts of the inflamed colonic mucosa. In sharp contrast, number of Dll4+ve IECs showed a significant increase in those crypts, which was accompanied by the increase in number of Hes1-positive IECs. Those Dll4+ve IECs were mostly found adjacent to the Hes1-positive IECs, suggesting that Dll4 may act as a major Notch ligand in the crypts of the inflamed colonic mucosa. Our results illustrate distinct expression patterns of Dll1 and Dll4 within the intestinal epithelium, and suggest that these two ligands may have different roles in normal and inflamed mucosa

Efficacy and safety of repeated endoscopic radial incision and cutting procedure for benign esophageal stricture

Background: Radial incision and cutting (RIC) is indicated for refractory benign esophageal strictures after curative treatment for esophageal cancer and has shown favorable short-term outcomes. However, re-stricture after RIC may occur in the long term, and RIC is performed repeatedly in such cases, but the efficacy and safety of repeated RIC are unclear. Therefore, we aimed to demonstrate the efficacy and safety of the repeated RIC for refractory benign esophageal strictures after surgical and non-surgical treatment. Methods: Between April 2008 and September 2019, we enrolled patients who were treated with the first RIC for benign esophageal strictures. The RIC was indicated for the refractory stricture and repeatedly performed for re-refractory esophageal stricture after RIC. We retrospectively evaluated the 6-month refractory stricture-free rate, and adverse events in the first RIC and repeated RICs. Results: Forty-six patients (39 men, 7 women; median age, 71 years, range 49–85) were included. RIC was performed once in 24 patients (non-repeated RIC group) and two or more times in 22 patients (repeated RIC group). In all patients, the 6-month refractory stricture-free rate after the first RIC were 42.3%. In the repeated RIC group, the 6-month refractory stricture-free rate after the first and repeated RICs were 18.2% vs 18.2%, respectively. No adverse events were noted. Conclusions: The repeated RIC could not be a curative treatment, but can be effective in the short-term and safe, even for patients with refractory benign esophageal stricture after the first RIC