AG Eiseler: Exosomes in Metastasis and Inflammation

Projects

Pancreas adenocarcinoma and small extracellular vesicles

Pancreatic ductal adenocarcinoma (PDAC) are characterized by a dismal prognosis due to late-stage diagnosis and early metastasis1. It is therefore vital to understand which factors determine PDAC metastasis patterns. In the last years it has become more and more evident that small extracellular vesicles (sEVs, exosomes) that are released by tumor cells in high amounts are vital regulators of tumor progression and metastasis2-4. sEVs have a size range of 30-150 nm and are derived of the endo-lysosomal system. sEV originate in multivesicular bodies (MVBs) as intraluminal vesicles. Once sEVs have been released by tumors cells, they can be distributed via the circulation. Recipient cells are educated by binding of sEVs to surface receptors or intracellular release of their bioactive cargos, such as lipids, proteins, miRNAs or mRNA thereby modifying the behavior of cells2,4.

a. Intercellular communication during PDAC invasion and metastasis

Recently it was shown that distinct integrin combinations on the surface of sEVs from cancer cells are able to direct organotropic metastasis5,6. This is achieved by preferred uptake of the tumor-derived sEVs by cells in future metastatic organs e.g. fibroblasts in the lung6. The sEVs carry bioactive molecules which change the behavior of recipient cells to support the establishment of pre-metastatic niches6. Our group has recently shown that Protein Kinase D1 (PKD1) is downregulated in 72% of PDAC by more the 75%7. Loss of PKD1 strongly enhanced sEV secretion and directed metastasis of PDACs to the lung in a Prkd1KOKC mouse model. We demonstrate that this was facilitated by upregulation of integrins a6b4 in cells and on sEVs. These integrins are packaged into Prkd1KO-derived sEVs in a CD82-dependent manner7, by facilitating their recycling from the cell surface into MVBs. We also demonstrate that strongly increased sEV secretion upon loss of PKD1 expression is achieved via the PKD1 substrate Cortactin. When Cortactin is not phosphorylated by PKD1 at S298, it is able to promote the formation of branched actin filaments at the plasma membrane which are required for the efficient fusion of MVBs and thus sEV release (Fig. 1). Once these sEVs have reached the lung, we show uptake into lung fibroblasts facilitates the formation of pre-metastatic niches via S100A proteins6,7. As part of project C1 GRK2254 “Heterogeneity and Evolution in Solid Tumors (HEIST)”, we are currently investigating how sEVs released by PDACs define pre-metastatic niches in the lung or liver microenvironment on a physiological and molecular level.  Besides, we are elucidating how relevant cargos are packaged into sEVs and if this process can be targeted to qualitatively alter sEV cargo content and distant metastasis.

b. sEV mediated immune evasion in PDAC

The PDAC microenvironment is highly immunosuppressive, blocking CD8+ T-cell tumor recognition and clearance8,9. sEVs are associated with different human diseases, including cancer, as levels of plasma sEVs were shown to be extensively enhanced in cancers patients. Tumor-derived sEVs were shown to carry immune-inhibitory ligands such as PD-L1, FasL and CTLA-4 that can directly interact with receptors on target cells, interfering with immune cell function10-14. We are currently analyzing how immune-modulatory ligands are packaged into sEVs and how these factors modulate T-cell immune evasion via CD8+ and CD4+ T-cells, contributing to the highly immunosuppressive PDAC tumor microenvironment. To this end, we are characterizing sEV miRNA cargos and protein targets for functions in apoptosis, proliferation, T-cell polarization and activation.  Experiments are performed in vitro and in PDAC mouse models.

Physical trauma
a. Role of sEVs in the host inflammatory trauma response

Trauma is the leading cause of death under 44 years of age15,16. A traumatic event triggers the release of damage- and pathogen-associated molecular patterns (DAMPS, PAMPs) causing neutrophil-mediated inflammation and barrier disruption17-20. Severe injuries in combination with comorbidities can also cause a runaway systemic inflammatory response that can even manifests in multiple-organ-dysfunction (MODS)21. In particular thorax trauma (TxT) is associated with one quarter of all trauma-related deaths22. Thus, it is important to understand how such an inflammatory response to a physical trauma is coordinated and whether it can be mitigated. Small extracellular vesicles (sEVs, exosomes) were described to be important mediators of stress signalling and are involved in immune modulatory processes4,12. However, molecular mechanisms and functions of sEVs after a traumatic insult have not been elucidated in detail. As part of the CRC 1149 “Danger Response, Disturbance Factors and Regenerative Potential after Acute Trauma” we are currently investigating whether qualitative and qualitative changes in sEVs after a traumatic insult will be implicated in propagating local, distant and systemic inflammatory processes.

 

 

 

b. Regulation of neutrophil chemotaxis and endothelial barrier stability

Neutrophils are important mediators of the innate immune defense and of the host response to a physical trauma23. We have previously reported that inhibition of Protein Kinase D by the small molecule inhibitor CRT0066101 was able to inhibit transendothelial migration of neutrophils towards a chemotactic gradient. To this end, PKD phosphorylates the Cofilin-phosphatase Slingshot-2L (SSH-2L). SSH-2L in turn dynamically regulates Cofilin activity and actin polymerization in response to a neutrophil chemotactic stimulus, such as fMLP23. Impaired PKD activity also significantly reduced neutrophil deformability as determined by optical stretcher analysis. Indeed, we were able to inhibit transmigration (diapedesis) of neutrophil-like differentiated NB4 cells and primary PMNs23. Thus, our data suggest that inhibition of PKD may affect trauma outcome by directly acting on neutrophil transmigration. In addition, we have elucidated a molecular mechanism by which inhibition of PKD activity is implicated in stabilizing epithelial24 and potentially also endothelial barriers via its substrate Cortactin24. We are therefore currently investigating in a DFG funded project, whether inhibition of PKD will improve outcome after thorax trauma (TxT) and mitigate a neutrophil-driven inflammatory response in vivo.    

PKD family of serine/threonine kinases

PKD kinases are also known to drive vesicle fission from the trans-Golgi network25. We are investigating how specific PKD isoforms, in particular PKD2, regulate and execute different steps of vesicle budding and fission by a network of protein interactions involving the small GTPase Arf1 26. Also, regulation of Cortactin and actin polymerization during vesicle separation from Golgi membranes27 and fusion of MVBs with the plasma membrane is investigated7. In order to address these questions, we are studying compartment-specific protein dynamics, interactions and activation processes utilizing high-end confocal microscopy techniques, such as Fluorescence-Recovery-after-Photobleaching (FRAP) and Foerster Energy Transfer (FRET) complemented by biochemical assays.

Staff

Postdoc / Principal Investigator

  • Profilbild von Dr. rer.nat. Tim Eiseler

    Dr. rer.nat. Tim Eiseler

    Senior PostDoc, Group Leader "Exosomes in metastases and inflammation"

    Schwerpunkte

    Actin Dynamics, Secretion, Exosomes

Current group members

Funding

Deutsche Forschungsgemeinschaft (DFG)

Cooperation partners

  • Prof. Dr. Johan Van Lint, Katholieke Universiteit Leuven, Belgium
  • Prof. Dr. med. Alexander Kleger, Department for Internal Medicine I, University of Ulm
  • Prof. Dr. Franz Oswald, Department for Internal Medicine I, University of Ulm
  • Prof. Dr. Mechthild Haztfeld, Division of Pathobiochemistry, Martin-Luther University, Halle
  • Dr. Martin Müller, Center for Internal Medicine I, University of Ulm
  • Dr. Angelika Rück, Core Facility for Confocal and Multiphoton Microscopy
  • Dr. Stephan Paschke, Department of Visceral Surgery, University of Ulm
  • Prof. Dr. Ralf Kemkemer, Max Planck Institute for Intelligent Systems, Stuttgart/University Reutlingen
  • Dr. Julia von Blume, Max Planck Institute for Biochemistry, Munich

Selected publications

  1. Armacki M@, Polaschek S @, Waldenmaier M , Morawe M, Ruhland C, Schmid R, Lechel A, Tharehalli U , Christoph S, Bektas Y, Li H, Kraus JM, Kestler HA, Kruger S, Ormanns S, Walther P, Eiseler T$ and Seufferlein T1$. Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice. Gastroenterology in press. doi: 10.1053/j.gastro.2020.05.052 (@ equal contribution, $ shared senior author).
  2. Weeber F, Becher A, Seibold T, Seufferlein T, Eiseler T. Concerted regulation of actin polymerization during constitutive secretion by cortactin and PKD2. J Cell Sci. 2019 Dec 13;132(24):jcs232355. doi: 10.1242/jcs.232355. PMID: 31727638.
  3. Lackner I, Weber B, Baur M, Haffner-Luntzer M, Eiseler T, Fois G, Gebhard F, Relja B, Marzi I, Pfeifer R, Halvachizadeh S, Lipiski M, Cesarovic N, Pape HC, Kalbitz M; TREAT Research Group. Midkine Is Elevated After Multiple Trauma and Acts Directly on Human Cardiomyocytes by Altering Their Functionality and Metabolism. Front Immunol. 2019 Aug 21;10:1920. doi: 10.3389/fimmu.2019.01920. PMID: 31552013; PMCID: PMC6736577.
  4. Wille C*, Eiseler T*, Langenberger ST, Richter J, Mizuno K, Radermacher P, Knippschild U, Huber-Lang M, Seufferlein T, Paschke S. PKD regulates actin polymerization, neutrophil deformability, and transendothelial migration in response to fMLP and trauma. J Leukoc Biol. 2018 Sep;104(3):615-630. doi: 10.1002/JLB.4A0617-251RR. Epub 2018 Apr 14. PMID: 29656400 (equal contgribution).
  5. Armacki M, Trugenberger AK, Ellwanger AK, Eiseler T, Schwerdt C, Bettac L, Langgartner D, Azoitei N, Halbgebauer R, Groß R, Barth T, Lechel A, Walter BM, Kraus JM, Wiegreffe C, Grimm J, Scheffold A, Schneider MR, Peuker K, Zeißig S, Britsch S, Rose-John S, Vettorazzi S, Wolf E, Tannapfel A, Steinestel K, Reber SO, Walther P, Kestler HA, Radermacher P, Barth TF, Huber-Lang M, Kleger A, Seufferlein T. Thirty-eight-negative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure. J Clin Invest. 2018 Nov 1;128(11):5056-5072. doi: 10.1172/JCI97912. Epub 2018 Oct 15. PMID: 30320600; PMCID: PMC6205400.
  6. Becher A*, Eiseler T*, Porzner M, Walther P, Keil R, Bobrovich S, Hatzfeld M, Seufferlein T. The armadillo protein p0071 controls KIF3 motor transport. J Cell Sci. 2017 Oct 1;130(19):3374-3387. doi: 10.1242/jcs.200170. Epub 2017 Aug 14. PMID: 28808088 (equal contribution).
  7. Denk S, Taylor RP, Wiegner R, Cook EM, Lindorfer MA, Pfeiffer K, Paschke S, Eiseler T, Weiss M, Barth E, Lambris JD, Kalbitz M, Martin T, Barth H, Messerer DAC, Gebhard F, Huber-Lang MS. Complement C5a-Induced Changes in Neutrophil Morphology During Inflammation. Scand J Immunol. 2017 Sep;86(3):143-155. doi: 10.1111/sji.12580. PMID: 28671713; PMCID: PMC5773343.
  8. Durand N, Bastea LI, Döppler H, Eiseler T, Storz P. Src-mediated tyrosine phosphorylation of Protein Kinase D2 at focal adhesions regulates cell adhesion. Sci Rep. 2017 Aug 25;7(1):9524. doi: 10.1038/s41598-017-10210-7. PMID: 28842658; PMCID: PMC5573332.
  9. Hohwieler M, Illing A, Hermann PC, Mayer T, Stockmann M, Perkhofer L, Eiseler T, Antony JS, Müller M, Renz S, Kuo CC, Lin Q, Sendler M, Breunig M, Kleiderman SM, Lechel A, Zenker M, Leichsenring M, Rosendahl J, Zenke M, Sainz B Jr, Mayerle J, Costa IG, Seufferlein T, Kormann M, Wagner M, Liebau S, Kleger A. Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling. Gut. 2017 Mar;66(3):473-486. doi: 10.1136/gutjnl-2016-312423. Epub 2016 Sep 15. PMID: 27633923; PMCID: PMC5534761.
  10. Perkhofer L, Walter K, Costa IG, Carrasco MC, Eiseler T, Hafner S, Genze F, Zenke M, Bergmann W, Illing A, Hohwieler M, Köhntop R, Lin Q, Holzmann KH, Seufferlein T, Wagner M, Liebau S, Hermann PC, Kleger A, Müller M. Tbx3 fosters pancreatic cancer growth by increased angiogenesis and activin/nodal-dependent induction of stemness. Stem Cell Res. 2016 Sep;17(2):367-378. doi: 10.1016/j.scr.2016.08.007. Epub 2016 Aug 10. PMID: 27632063.
  11. Eiseler T*, Wille C*, Koehler C, Illing A, Seufferlein T. Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion. J Biol Chem. 2016 Jan 1;291(1):462-77. doi: 10.1074/jbc.M115.673582. Epub 2015 Oct 27. PMID: 26507660; PMCID: PMC4697185 (equal contribution).
  12. Sroka R, Van Lint J, Katz SF, Schneider MR, Kleger A, Paschke S, Seufferlein T, Eiseler T. Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation. J Cell Sci. 2016 Jun 15;129(12):2416-29. doi: 10.1242/jcs.184721. Epub 2016 May 13. PMID: 27179075.
  13. Russell R, Ilg M, Lin Q, Wu G, Lechel A, Bergmann W, Eiseler T, Linta L, Kumar P P, Klingenstein M, Adachi K, Hohwieler M, Sakk O, Raab S, Moon A, Zenke M, Seufferlein T, Schöler HR, Illing A, Liebau S, Kleger A. A Dynamic Role of TBX3 in the Pluripotency Circuitry. Stem Cell Reports. 2015 Dec 8;5(6):1155-1170. doi: 10.1016/j.stemcr.2015.11.003. PMID: 26651606; PMCID: PMC4682344.
  14. Qin R, Schmid H, Münzberg C, Maass U, Krndija D, Adler G, Seufferlein T, Liedert A, Ignatius A, Oswald F, Eiseler T, von Wichert G. Phosphorylation and turnover of paxillin in focal contacts is controlled by force and defines the dynamic state of the adhesion site. Cytoskeleton (Hoboken). 2015 Feb;72(2):101-12. doi: 10.1002/cm.21209. Epub 2015 Feb 7. PMID: 25620625.
  15. Wille C, Seufferlein T, Eiseler T. Protein Kinase D family kinases: roads start to segregate. Bioarchitecture. 2014;4(3):111-5. doi: 10.4161/bioa.29273. Epub 2014 May 21. PMID: 24847910; PMCID: PMC4201600.
  16. Wille C, Köhler C, Armacki M, Jamali A, Gössele U, Pfizenmaier K, Seufferlein T*, Eiseler T*. Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases. Mol Biol Cell. 2014 Feb;25(3):324-36. doi: 10.1091/mbc.E13-06-0334. Epub 2013 Dec 11. PMID: 24336522; PMCID: PMC3907273 (shared senior author).
  17. Müller M, Schröer J, Azoitei N, Eiseler T, Bergmann W, Köhntop R, Lin Q, Costa IG, Zenke M, Genze F, Weidgang C, Seufferlein T, Liebau S, Kleger A. A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells. Sci Rep. 2015 Jul 7;5:11742. doi: 10.1038/srep11742. PMID: 26148697; PMCID: PMC4493579.
  18. Eiseler T*, Köhler C*, Nimmagadda SC, Jamali A, Funk N, Joodi G, Storz P, Seufferlein T. Protein kinase D1 mediates anchorage-dependent and -independent growth of tumor cells via the zinc finger transcription factor Snail1. J Biol Chem. 2012 Sep 21;287(39):32367-80. doi: 10.1074/jbc.M112.370999. Epub 2012 Jul 12. PMID: 22791710; PMCID: PMC3463335 (equal contribution).
  19. Pusapati GV*, Eiseler T*, Rykx A, Vandoninck S, Derua R, Waelkens E, Van Lint J, von Wichert G, Seufferlein T. Protein kinase D regulates RhoA activity via rhotekin phosphorylation. J Biol Chem. 2012 Mar 16;287(12):9473-83. doi: 10.1074/jbc.M112.339564. Epub 2012 Jan 6. PMID: 22228765; PMCID: PMC3308793. (equal contribution)
  20. Döppler H, Bastea LI, Eiseler T, Storz P. Neuregulin mediates F-actin-driven cell migration through inhibition of protein kinase D1 via Rac1 protein. J Biol Chem. 2013 Jan 4;288(1):455-65. doi: 10.1074/jbc.M112.397448. Epub 2012 Nov 12. PMID: 23148218; PMCID: PMC3537043.
  21. Busch T, Armacki M, Eiseler T, Joodi G, Temme C, Jansen J, von Wichert G, Omary MB, Spatz J, Seufferlein T. Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells. J Cell Sci. 2012 May 1;125(Pt 9):2148-59. doi: 10.1242/jcs.080127. Epub 2012 Feb 17. PMID: 22344252; PMCID: PMC3367938.
  22. Eiseler T, Hausser A, De Kimpe L, Van Lint J, Pfizenmaier K. Protein kinase D controls actin polymerization and cell motility through phosphorylation of cortactin. J Biol Chem. 2010 Jun 11;285(24):18672-83. doi: 10.1074/jbc.M109.093880. Epub 2010 Apr 2. PMID: 20363754; PMCID: PMC2881792.
  23. Ziegler S, Eiseler T, Scholz RP, Beck A, Link G, Hausser A. A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration. Mol Biol Cell. 2011 Mar 1;22(5):570-80. doi: 10.1091/mbc.E10-05-0427. Epub 2011 Jan 5. PMID: 21209314; PMCID: PMC3046055.
  24. Cowell CF, Yan IK, Eiseler T, Leightner AC, Döppler H, Storz P. Loss of cell-cell contacts induces NF-kappaB via RhoA-mediated activation of protein kinase D1. J Cell Biochem. 2009 Mar 1;106(4):714-28. doi: 10.1002/jcb.22067. PMID: 19173301; PMCID: PMC4556359.
  25. Eiseler T, Döppler H, Yan IK, Kitatani K, Mizuno K, Storz P. Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot. Nat Cell Biol. 2009 May;11(5):545-56. doi: 10.1038/ncb1861. Epub 2009 Mar 29. PMID: 19329994; PMCID: PMC2761768.
  26. Eiseler T, Döppler H, Yan IK, Goodison S, Storz P. Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion. Breast Cancer Res. 2009;11(1):R13. doi: 10.1186/bcr2232. Epub 2009 Feb 25. PMID: 19243594; PMCID: PMC2687718.
  27. Eiseler T, Schmid MA, Topbas F, Pfizenmaier K, Hausser A. PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration. FEBS Lett. 2007 Sep 4;581(22):4279-87. doi: 10.1016/j.febslet.2007.07.079. Epub 2007 Aug 10. PMID: 17707375.
  28. Brändlin I, Hübner S, Eiseler T, Martinez-Moya M, Horschinek A, Hausser A, Link G, Rupp S, Storz P, Pfizenmaier K, Johannes FJ. Protein kinase C (PKC)eta- mediated PKC mu activation modulates ERK and JNK signal pathways. J Biol Chem. 2002 Feb 22;277(8):6490-6. doi: 10.1074/jbc.M106083200. Epub 2001 Dec 6. PMID: 11741879.
  29. Brändlin I, Eiseler T, Salowsky R, Johannes FJ. Protein kinase C(mu) regulation of the JNK pathway is triggered via phosphoinositide-dependent kinase 1 and protein kinase C(epsilon). J Biol Chem. 2002 Nov 22;277(47):45451-7. doi: 10.1074/jbc.M205299200. Epub 2002 Sep 9. PMID: 12223477.