Probes for INS

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

Deciphering intra- and inter-tumoural heterogeneity is essential for understanding the biology of gastric cancer (GC) and its metastasis and identifying effective therapeutic targets. However, the characteristics of different organ-tropism metastases of GC are largely unknown.Ten fresh human tissue samples from six patients, including primary tumour and adjacent non-tumoural samples and six metastases from different organs or tissues (liver, peritoneum, ovary, lymph node) were evaluated using single-cell RNA sequencing. Validation experiments were performed using histological assays and bulk transcriptomic datasets.Malignant epithelial subclusters associated with invasion features, intraperitoneal metastasis propensity, epithelial-mesenchymal transition-induced tumour stem cell phenotypes, or dormancy-like characteristics were discovered. High expression of the first three subcluster-associated genes displayed worse overall survival than those with low expression in a GC cohort containing 407 samples. Immune and stromal cells exhibited cellular heterogeneity and created a pro-tumoural and immunosuppressive microenvironment. Furthermore, a 20-gene signature of lymph node-derived exhausted CD8+ T cells was acquired to forecast lymph node metastasis and validated in GC cohorts. Additionally, although anti-NKG2A (KLRC1) antibody have not been used to treat GC patients even in clinical trials, we uncovered not only malignant tumour cells but one endothelial subcluster, mucosal-associated invariant T cells, T cell-like B cells, plasmacytoid dendritic cells, macrophages, monocytes, and neutrophils may contribute to HLA-E-KLRC1/KLRC2 interaction with cytotoxic/exhausted CD8+ T cells and/or natural killer (NK) cells, suggesting novel clinical therapeutic opportunities in GC. Additionally, our findings suggested that PD-1 expression in CD8+ T cells might predict clinical responses to PD-1 blockade therapy in GC.This study provided insights into heterogeneous microenvironment of GC primary tumours and organ-specific metastases and provide support for precise diagnosis and treatment.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.