Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity.

Fibroblasts display extensive transcriptional heterogeneity, yet functional annotation and characterization of their heterocellular relationships remains incomplete. Using mass cytometry, we chart the stromal composition of 18 murine tissues and 5 spontaneous tumor models, with an emphasis on mesenchymal phenotypes. This analysis reveals extensive stromal heterogeneity across tissues and tumors, and identifies coordinated relationships between mesenchymal and immune cell subsets in pancreatic ductal adenocarcinoma. Expression of CD105 demarks two stable and functionally distinct pancreatic fibroblast lineages, which are also identified in murine and human healthy tissues and tumors. Whereas CD105-positive pancreatic fibroblasts are permissive for tumor growth in vivo, CD105-negative fibroblasts are highly tumor suppressive. This restrictive effect is entirely dependent on functional adaptive immunity. Collectively, these results reveal two functionally distinct pancreatic fibroblast lineages and highlight the importance of mesenchymal and immune cell interactions in restricting tumor growth.

A novel method for the production of fully modified K-Ras 4B.

Post-translational modifications in proteins play a major functional role. Post-translational modifications affect the way proteins interact with each other, bind nucleotides, and localize in cellular compartments. Given the importance of post-translational modifications in protein biology, development of methods to produce post-translationally modified proteins for biochemical and biophysical studies is timely and significant. At the same time, obtaining post-translationally modified proteins in bacterial expression systems is often problematic. Here, we describe a novel recombinant approach to prepare human K-Ras 4B, a protein that is post-translationally farnesylated, proteolytically cleaved, and methylated in its C-terminus. K-Ras 4B is a member of the Ras subfamily of small GTPases and is of interest because it is frequently mutated in human cancer. The method relies on separate production of two structural domains-the N-terminal catalytic domain and the C-terminal peptide chemically modified with S-farnesyl-L-cysteine methyl ester. After the two domains are prepared, they are ligated together using the transpeptidase enzyme, sortase. Our procedure starts with the use of the plasmid of K-Ras 4B catalytic domain containing the sortase recognition sequence. After this, we describe the bacterial expression and purification steps used to purify K-Ras 4B and the preparation of the conjugated C-terminal peptide. The procedure ends with the sortase-mediated ligation technique. The produced post-translationally modified K-Ras 4B is active in a number of assays, including a GTP hydrolysis assay, Raf-1 binding assay, and surface plasmon resonance-based phospholipid binding assay.