Cluster of Excellence –
University of Freiburg

Elucidating functional networks of shed and secreted proteins in complex tumor microenvironments

Prof. Dr. Thomas Reinheckel, Prof. Dr. Christoph Peters, PD Dr. Oliver Schilling (Institute of Molecular Medicine and Cell Research, University of Freiburg)

 

Proteomic analysis of tumor/stroma interactions is currently mostly relying on co-culture of various cell types in monolayer cultures or 3D spheroids. However, such cell cultures may not represent the genuine network of secreted proteins in the microenvironment of solid cancer, such as breast-, colon-, or pancreatic carcinomas. Therefore, we recently devised a method for isolation of interstitial fluid from cancers that is suitable for isotope-labeling with subsequent MS-based quantification and identification of secreted and shed proteins (Gomez-Auli et al. Biochimie 2015. E-pub ahead of print). We will further develop and to apply this new option for in vivo secretome analysis to various mouse models of human cancers, congenic murine and xenografted human tumors, as well as patient specimens.

Work flow of interstitial fluid proteomics. The interstitial fluid of isolated tumors/organs is obtained by low-speed centrifugation using an in-house constructed collector. After depletion of highly abundant proteins, for better proteome coverage, proteins are precipitated and digested. This is followed by isotopic labeling, which allows for multiplex comparisons, pre-fractionated and subjected to LC-MS/MS using a state of the art Mass Spectrometer (Thermo Q Exactive Plus), followed by data analysis.