title: Impact of cathepsin B on the interstitial fluid proteome of murine breast cancers.
abstract: Carcinomas establish a molecular cross talk between malignant tumor cells and the activated non-malignant cells of the tumor stroma. This cell-cell communication in tumor-stroma interaction includes soluble, secreted proteins that act in a paracrine or autocrine manner. Proteases are crucial factors in tumor-stroma interaction by degrading or truncating secreted bioactive proteins. The cysteine protease cathepsin B is frequently overexpressed in several cancer types, including breast cancer. Its abundance often correlates with poor prognosis. In the murine polyoma virus middle T oncogene (PyMT) breast cancer model, cathepsin B is equally pro-tumorigenic. In this study, we investigate how cathepsin B shapes the secreted proteome of PyMT breast cancers. We employed a novel strategy to harvest tumor interstitial fluid (IF) in combination with chemical stable isotope tagging for quantitative proteomic comparison of IF stemming from PyMT tumors from wild-type mice, mice lacking cathepsin B, and mice over-expressing human cathepsin B. In three biological replicates, we achieve good proteome coverage (∼1700 proteins), with a large content (>70%) of secreted proteins. This characterizes IF as a robust source for the investigation of cancer secretomes. We also identified a large number of shed ectodomains, thus highlighting the importance of tumor-contextual cell surface proteolysis. Furthermore, IF contained >190 proteases and protease inhibitors, which span the entire range of absolute protein abundances; an observation testifying for an important role of proteolysis in tumor-stroma interaction. The cathepsin B genotype consistently affected proteins including alpha-1B-glycoprotein and major urinary proteins 11 and 8 (MUP8). Our study establishes tumor IF as a rich source for the investigation of secreted proteins in tumor biology and sheds light on complex proteolytic networks in the breast cancer secretome.
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, D-79104 Freiburg, Germany; Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Gomez-Auli
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Larissa Elisabeth
authors.initials: LE
authors.lastname: Hillebrand
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Martin Lothar
authors.initials: ML
authors.lastname: Biniossek
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, D-79104 Freiburg, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Peters
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, D-79104 Freiburg, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Reinheckel
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, D-79104 Freiburg, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany. Electronic address: oliver.schilling@mol-med.uni-freiburg.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D015153:Blotting, Western; D002401:Cathepsin B / Q000378:metabolism*; D002853:Chromatography, Liquid; D045604:Extracellular Fluid / Q000378:metabolism*; D008325:Mammary Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism*; D018345:Mice, Knockout; D008822:Mice, Transgenic; D010447:Peptide Hydrolases / Q000378:metabolism; D011120:Polyomavirus / Q000235:genetics / Q000276:immunology; D011480:Protease Inhibitors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D000952:Antigens, Polyomavirus Transforming; D011480:Protease Inhibitors; D011506:Proteins; D020543:Proteome; C035737:major urinary proteins; D010447:Peptide Hydrolases; D002401:Cathepsin B
keywords: Cathepsins; Ectodomain shedding; Interstitial fluid; Proteases; Proteomics; Tumor microenvironment