title: TGFBR2-dependent alterations of exosomal cargo and functions in DNA mismatch repair-deficient HCT116 colorectal cancer cells.
abstract: Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells.
  
  
  
  Exosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA.
  
  
  
  Frameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2-6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell.
  
  
  
  Our results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors. Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells.
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Department of Cancer Early Detection, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.
authors.forename: Fabia
authors.initials: F
authors.lastname: Fricke
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Department of Cancer Early Detection, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Present address: Tissue Genesis, Suite 1000, Tissue Genesis Tower, 810 Richards Street, Honolulu, HI, 96813, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Department of Cancer Early Detection, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.
authors.forename: Malwina
authors.initials: M
authors.lastname: Michalak
authors.affiliation: Functional Proteome Analysis and Core Facility Protein Analysis (B100), German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Warnken
authors.affiliation: Department of General Pathology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Hausser
authors.affiliation: Department of Medical Oncology, National Center for Tumor diseases (NCT), Tissue Imaging and Analysis Center, Bioquant, University Hospital Heidelberg, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
authors.forename: Meggy
authors.initials: M
authors.lastname: Suarez-Carmona
authors.affiliation: Department of Medical Oncology, National Center for Tumor diseases (NCT), Tissue Imaging and Analysis Center, Bioquant, University Hospital Heidelberg, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
authors.forename: Niels
authors.initials: N
authors.lastname: Halama
authors.affiliation: Functional Proteome Analysis and Core Facility Protein Analysis (B100), German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Schnölzer
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Department of Cancer Early Detection, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Kopitz
authors.affiliation: Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany. johannes.gebert@med.uni-heidelberg.de.|Department of Cancer Early Detection, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 224, 69120, Heidelberg, Germany. johannes.gebert@med.uni-heidelberg.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Gebert
mesh_terms: D018925:Chemokines / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000378:metabolism*; D053843:DNA Mismatch Repair; D004797:Enzyme-Linked Immunosorbent Assay; D055354:Exosomes / Q000378:metabolism* / Q000648:ultrastructure; D016368:Frameshift Mutation / Q000235:genetics; D045325:HCT116 Cells; D056945:Hep G2 Cells; D006801:Humans; D053842:Microsatellite Instability; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D018925:Chemokines; D010982:Platelet-Derived Growth Factor; D020543:Proteome; D018125:Receptors, Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II
keywords: Colorectal cancer; DNA mismatch repair deficiency; Exosomes; Intercellular communication; Microsatellite instability; Proteomics; Transforming Growth Factor Beta Receptor Type 2