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Marcin Cebula

02.11.2009

Hallmarks of cancer

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

METASTASIS

Loss of E-cadherinMMP

MMP

HOW CANCER CELLS AFFECT THE INFLAMMATORY MICROENVIROMENTHOW 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?

Incubation of bone marrow–derived macrophages with SF conditioned media from different cancer cell lines

Free of IL-6 & TNF

LLC – Lewis lung carcinomaLCM – Lewis lung carcinoma conditioned medium

mR

NA

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 shownIL-6 is not important for LLC metastasisTNF- is responsible for metastasis

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

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

In vivo role of TLR2 after inoculation LLC in WT and Tlr2-/-

mice on cytokine expression

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

TLR2-/- mice demonstrate higher survival and less metastasis in comparison to WT

Contribution of TLR2 signaling to survivalafter inoculation LLC in WT and Tlr2-/- mice

TLR2 is acting on BMD cellsWT 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

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

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

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

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

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

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

Summary

New point for anti-metastatic intervention???

Versican V1 Versican binds to TLR2 on

Macrophages

TNF-

Metastases development

Thank you for your attention