Myofibroblastic cancer-associated fibroblast subtype heterogeneity in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a fibrotic stroma that has both tumor-promoting and tumor-restraining properties. Different types of cancer-associated fibroblasts (CAFs) have been described. Here, we investigated whether CAFs within the same subtype exhibit heterogeneous functions. METHODS: We evaluated the gene and protein expression differences in two myofibroblastic CAF (myCAF) lines using single-cell and bulk RNA-sequencing. We utilized proliferation and migration assays to determine the effect of different CAF lines on a tumor cell line. RESULTS: We found that myCAF lines express an activated stroma subtype gene signature, which is associated with a shorter survival in patients. Although both myCAF lines expressed α-smooth muscle actin (α-SMA), platelet-derived growth factor-α (PDGFR-α), fibroblast-activated protein (FAP), and vimentin, we observed heterogeneity between the two lines. Similarly, despite being consistent with myCAF gene expression overall, heterogeneity within specific genes was observed. We found that these differences extended to the functional level where the two myCAF lines had different effects on the same tumor cell line. The myCAF216 line, which had slightly increased inflammatory CAF-like gene expression and higher protein expression of α-SMA, PDGFR-α, and FAP was found to restrain migration of tumor cells. CONCLUSIONS: We found that two myCAF lines with globally similar expression characteristics had different effects on the same tumor cell line, one promoting and the other restraining migration. Our study highlights that there may be unappreciated heterogeneity within CAF subtypes. Further investigation and attention to specific genes or proteins that may drive this heterogeneity will be important.

Designing Hybrid Antibiotic Peptide Conjugates To Cross Bacterial Membranes.

We design hybrid antibiotic peptide conjugates that can permeate membranes. Integration of multiple components with different functions into a single molecule is often problematic, due to competing chemical requirements for different functions and to mutual interference. By examining the structure of antimicrobial peptides (AMPs), we show that it is possible to design and synthesize membrane active antibiotic peptide conjugates (MAAPCs) that synergistically combine multiple forms of antimicrobial activity, resulting in unusually strong activity against persistent bacterial strains.

Long-read sequencing to interrogate strain-level variation among adherent-invasive Escherichia coli isolated from human intestinal tissue.

Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohn's disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn's disease and non-Crohn's tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.