Soft tissue sarcomas (STS) are a group of rare and heterogeneous cancers. While large-scale genomic and epigenomic profiling of STS have been undertaken, proteomic analysis has thus far been limited. Here we utilise sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) for proteomic profiling of formalin fixed paraffin embedded (FFPE) specimens from a cohort of STS patients (n=36) across four histological subtypes (leiomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma). We quantified 2951 proteins across all cases and show that there is a significant enrichment of gene sets associated with smooth muscle contraction in leiomyosarcoma, RNA splicing regulation in synovial sarcoma and leukocyte activation in undifferentiated pleomorphic sarcoma. We further identified a subgroup of STS cases (independent of histological subtype) that have a distinct expression profile in a panel of 133 proteins, with worse survival outcomes when compared to the rest of the cohort. Our study highlights the value of comprehensive proteomic characterisation as a means to identify histotype-specific STS profiles that describe key biological pathways of clinical and therapeutic relevance; as well as for discovering new prognostic biomarkers in this group of rare and difficult-to-treat diseases.

Summary: Pseudomonas aeruginosa produces extracellular DNA which functions as a cell‐to‐cell interconnecting matrix component in biofilms. Comparison of extracellular DNA and chromosomal DNA by the use of polymerase chain reaction and Southern analysis suggested that the extracellular DNA is similar to whole‐genome DNA. Evidence that the extracellular DNA in P. aeruginosa biofilms and cultures is generated via lysis of a subpopulation of the bacteria was obtained through experiments where extracellular β‐galactosidase released from lacZ‐containing P. aeruginosa strains was assessed. Experiments with the wild type and lasIrhlI, pqsA, pqsL and fliMpilA mutants indicated that the extracellular DNA is generated via a mechanism which is dependent on acyl homoserine lactone and Pseudomonas quinolone signalling, as well as on flagella and type IV pili. Microscopic investigation of flow chamber‐grown wild‐type P. aeruginosa biofilms stained with different DNA stains suggested that the extracellular DNA is located primarily in the stalks of mushroom‐shaped multicellular structures, with a high concentration especially in the outer part of the stalks forming a border between the stalk‐forming bacteria and the cap‐forming bacteria. Biofilms formed by lasIrhlI, pqsA and fliMpilA mutants contained less extracellular DNA than biofilms formed by the wild type, and the mutant biofilms were more susceptible to treatment with sodium dodecyl sulphate than the wild‐type biofilm.

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self‐produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.

The ESKAPE pathogen Pseudomonas aeruginosa is recognized as an important cause of chronic infections due to its ability to form biofilms where the bacteria are present in aggregates encased in a self-produced extracellular matrix and are difficult or impossible to eradicate with antibiotic treatment. P. aeruginosa is causing chronic infections in the lungs of patients with cystic fibrosis and chronic obstructive lung disease, as well as chronic urinary tract infections in patients with permanent bladder catheter, and ventilator-associated pneumonia in intubated patients, and is also an important pathogen in chronic wounds. Antibiotic treatment cannot eradicate these biofilm infections due to their intrinsic antibiotic tolerance and the development of mutational antibiotic resistance. The tolerance of biofilms to antibiotics is multifactorial involving physical, physiological and genetic determinants, whereas the antibiotic resistance of bacteria in biofilms is caused by mutations and driven by the repeated exposure of the bacteria to high levels of antibiotics. In this review both the antimicrobial tolerance and the development of resistance to antibiotics in P. aeruginosa biofilms is discussed. Possible therapeutic approaches based on the understanding of the mechanisms involved in the tolerance and resistances of biofilms to antibiotics are also addressed.