1. Dies ist nur ein Beispielbild! Marcin Cebula 02.11.2009

2. Hallmarks of cancer

3. METASTASIS Final stage in tumor progression → responsible for 90 % death associated with solid tumors Process of metastasis is, complex but can be simplified to certain steps: Local invasion Intravasation Survival in circulation Extravasation Colonisation Processes depends on genetic, epigenetic alterations, and tumor microenvironment

4. METASTASIS Loss of E-cadherin MMP

5. HOW CANCER CELLS AFFECT THE INFLAMMATORY MICROENVIROMENT HOW THIS INFLUENCES ON METASTASIS Progression and metastasis – correlated with presence of infitratates of myeloid and lymphoid cells Do the cancer cells secrete factors that directly activate myeloid cells to produce tumor–promoting cytokines?

6. Incubation of bone marrow–derived macrophages with SF conditioned media from different cancer cell lines Free of IL-6 & TNF  LLC – Lewis lung carcinoma LCM – Lewis lung carcinoma conditioned medium mRNA

7. Metastatogenic function of LLC induced cytokines (IL-6, TNF-  ) LLC cells injected to IL-6 -/- or TNF-  -/- knockout mice and WT IL-6 -/- little differences to WT were shown IL-6 is not important for LLC metastasis TNF-  is responsible for metastasis

8. Which TLR are involved in sensing LCM components Bone marrow M  from mice deficient in TLR2, TLR3, TLR4, TLR5 or their adaptors (Myd88 and TRIF) were incubated with LCM. IL-6 production was examined as BMDM activation marker. Signal from LCM is recognized by TLR2 / Myd88

9. What is the co-receptor involved in TLR2 signaling TLR2 uses TLR6 or CD14 as co-receptors Bacterial lipoprotein analog,to rule out bacterial lipoprotein contamination

10. In vivo role of TLR2 after inoculation LLC in WT and Tlr2 -/- mice on cytokine expression

11. Contribution of TLR2 signaling to LLC metastatogenesis WT but not Tlr -/- lungs showed clusters with dsRED-LLC with adjacent myeloid cells CD11b/ are myeloid cells markers

12. TLR2 -/- mice demonstrate higher survival and less metastasis in comparison to WT Contribution of TLR2 signaling to survival after inoculation LLC in WT and Tlr2 -/- mice

13. TLR2 is acting on BMD cells WT mice reconstituded with WT BM (WT/WT) or Tlr2 -/- BM (WT/ Tlr2 -/- ) (WT/Tlr2 -/- ) – improved survival Additionally to LLC inoculation in WT/WT or WT/Tlr2 -/-, serum free medium SFM or LLC conditioned medium was injected i.p Clear suggestion that LCM contains TLR2 activating factors

14. Identification of factors in LCM in terms of their ability to induce IL-6 LCM was seperated on mono-Q anion echange column Fractions inducing IL-6 were colected and separated on Superdex 200 sizing column Fractions inducing IL-6 eluted in high molecular fractions and contained several polypeptides > 200 kDa Fractions were pooled deglycosylated and subjected to MS Identified: Peptides from extracellular matrix proteins: -versican V1 -laminin-  1 -thrombospondin 2 -procollagen type III-  1

15. Which of identified peptides are factors inducing inflammatory cytokines BMDM incubated with LCM and respective antibodies to identified peptides in LCM -procollagen type III-  1 -versican V1 -laminin-  1

16. Role of indetified peptides in metastatogenesis shRNA silencing Silenced LLC cell lines Silenced LLC cell lines injected i.v to mice Not silenced versican Silenced LLC cell lines injected s.c to mice

17. What is versican V1 ??? Versican is able to regulate many cellular procesess including: -adhesion -proliferation -apoptosis -invasion via negatively-charged side chains Versican expression is elevated in cancer cells: -brain tumors, melanomas, osteosarcomas, lymphomas, breast, prostate, colon, lung, pancreatic, oral, and ovarian cancers -elevated levels of versican are associated with cancer relapse and poor prognosis

18. Confirmation of proinflamatory and prometastatic function of versican Low metastatic variant of LLC – P29-LLC P29-LLC conditioned medium do not induce IL-6 in BMDM P29-LLC conditioned medium contain little versican Ectopic expression of human versican increased

19. How versican activates macrophages His-tagged versican V1 – purified on Ni-chelate column Immunoprecipitation with versican specific antibody Lysats from Raw264.7 macrophages incubated or not with LCM

20. Summary Versican secretion is by LLC cells is necessary for metastatic spread to lung, liver and adrenal gland Process depends on TLR2-mediated myeloid cell activation and TNF- , (TNF-  can suppress apoptosis of cancer cells, stimulate their proliferation through NF-  B activation, it increases vascular permeability enhancing intra and extravasations of cancer cells) Response to versican requires co-receptor TLR6 Interaction of TLR2 and versican can be direct or mediated by versican ligand – hyaluronian which fragments can activate macropgagesTLR2 as well

21. New point for anti-metastatic intervention??? Versican V1 Versican binds to TLR2 on Macrophages TNF-  Metastases development

22. Thank you for your attention