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Shaping T cell functions by tumor extracellular matrix.

Bei-Chang Yang (楊倍昌 )

• Institute of Basic Medical Sciences (基醫所 ), • Department of Microbiology and Immunology (微免所 ), College of

Medicine, National Cheng Kung University, Tainan 70428, Taiwan.

鄭宇容，林育萍，黃俊淵，蘇重禎

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Fight cancer, But, sometimes we lose

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NPC (CD3 stain)

Why T cells infiltrate and accumulate here?

Nature Med. 1999, 5:874

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Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer. Peter J.K. Kuppen et al, Histochem Cell Biol (2001) 115:67–72

Immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor.

Brest cancer (invasive)

This is not a single case.

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Leukocytes migration through endothelia cells

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Molecules involve in cell-cell interaction

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Questions:

Why are T cells trapped in peripheral area of tumor mass?

Are those T cells alive ? Su CC, et al (2007) J Immunol. 179:4589-4597. Hor WS, et al. (2003) J Leukocyte Biol. 73: 363-368.

Can those cells modulate consequent immune reaction?

IL-10 & TGF-; Yang BC, et al. (2003) J Immunol. 171:3947-3954.

Do they affect tumor growth or metastasis? (depletion study) Chen YL, et al (2002) Br J Cancer 87:359-365. Chen YL, et al. (2003) J Immunol. 171:1183-1192.

etc.

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Q: Why do immune cells appear only in peripheral area

of tumor nest?

Possibilities:

Receptor-mediated

Secreted proteins

Extracellular matrix

A: Tumor extracellular matrix may play a role.

黃俊淵

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Consist of different combinations of fibrous collagen protein

s, hyaluronic acid, fibronectin, etc.

Bind to cell-surface receptor: integrin, laminin receptors, etc.

Are required for cell proliferation, differentiation, morphogen

esis, etc.

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Why extracellular matrix? Tumor cells secrete distinctive ECM that form a basal

membrane surrounding the tumor mass. During the course of tumor infiltration, immune cells

have to migrate out of blood vessels and travel some

distance to get into the tumor site. There, they

encounter a distinct environment composed of various

ECM components, which are very different from those

of the bloodstream.

Rational:

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Transmigration of T cells through the tumor monolayers.

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Extracellular matrix recaptures the effect of tumor monolayers.

It is integrin-dependant.

ECM was extracted by 0.5% Triton X-100.

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Jurkat cells spread poorly on glioma ECM.

(F-actin accumulated at front of lamellipodia)

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The transcripts of collagen IV (COL IV), fibronectin (FN), laminin-g1 (LAM) and tenascin-C (TN-C) were detected by RT-PCR.

Glioma cells express high amount of tenascin-C.

Immunohistochemical stain for tenascin-C on a glioma tumor sample.

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Tenascin-C can interact with perlecan and fibronectin, and cell surface receptors including integrins 21, v3 and 91, and annexin I.

Nature Reviews Cancer 5, 436-446 (2005)

Tenascin-C is a disulfide-bonded hexamer composed of subunits with molecular weights in the range of 120–300 kD

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Tenascin-C gene of glioma cells is knocked down by shRNA method.

Tenascin-C-low transfectant cells allow better transmigration of Jurkat cells.

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A better spreading of Jurkat cells on monolayer and ECM of U-118MG(TNCshRNA) cells.

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Tumor contact activates ERK activities in Jurkat cells, which are required for the transmigration of Jurkat cells.

Question: How does tenascin-C work?

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ERK and actin colocalize at the front of lamellipodia of Jurkat cells.Further study on migration machinery (focal adhesion complex) is on going.

Tumor contact activates ERK activity in Jurkat cells, which is required for the transmigration of Jurkat cells.

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CD3+T cells are trapped in tenascin-C-rich area.

Tenascin-C CD3+ cells

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Conclusion:

Tenascin-C inhibits the polarization and transmigration of T cells, that consequently prevents a direct contact of glioma tumor and T cells.

Are those T cells alive ?

蘇重禎

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No death occurs in Jurkat cells during coculture with gliomas.

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Caspase 3 Apaf-1Caspase 9Death substrates

Apoptosis

Nature 407, 789 5(2000)

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FasLribozyme/EGFP plasmid

Br J Cancer 85 (2002):1185-1192

BC Yang

-tubulin

Fas-ligand

U37

3(v)

U37

3(R

)

U11

8(v)

U11

8(R

)

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No death occurs in Jurkat cells during coculture with gliomas.

A B

C D E

A: Jurkat cells alone

B: CH-11 (1ng/ml)

C: with U-118MG(V)

D: with U118MG (R)

E: ZB4 (100ng/ml)/CH-11

PI-staining

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Coculture with tumor cells inhibits the Fas-mediated apoptosis.

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Caspase 3 Apaf-1Caspase 9

Death substrates Apoptosis

Apoptosis program is inhibited.

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Direct cell contact is required.

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Integrin signal is required

Coculture with U118

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How Fas signal-mediated death is suppressed in T cells?

25 M LY294002 for 1 h

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Fas-mediated death is suppressed by inhibitor of PI3K but not by inhibitors of MAPK, NFB, PKA, HSP70.

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Su CC, et al. 2007 Phosphatidylinositol 3-Kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells. J Immunol 179:4589-4597.

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Why are T cells trapped in peripheral area of tumor mass? Tenascin-C of glioma.

Are those T cells alive? ECM signal suppresses apoptosis!

(IL-6/IL-8 for neutrophils)

Stepwise hijacking of infiltrating T cells by tumor:Paralyzing → Keeping alive → modifying (IL-10/TGF)

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Jurkat Molt-4 1 2 3 1 2 3 IL-10

-actin

Glioma-associated IL-10 induction in T cell lines

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U373(V/R) or U118(V/R)

Jurkat cells 1 2 3 4

IL-10

-actin

Direct cell-to-cell contact is required

1. Jurkat alone2. Jurkat separate chamber3. Jurkat in upper chamber /coculture in low chamber4. Jurkat in coculture

U118(V)

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Fas/Fas-L signal-associated IL-10 induction

Jurkat U-373MG U-118MG - V R V R IL-10 -actin

Molt-4 U-373MG U-118MG - V R V R IL-10

-actin

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