1 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

2 Fight cancer, But, sometimes we lose

3 NPC (CD3 stain) Why T cells infiltrate and accumulate here? Nature Med. 1999, 5:874

4 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.

5 Leukocytes migration through endothelia cells

6 Molecules involve in cell-cell interaction

7 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:  Hor WS, et al. (2003) J Leukocyte Biol. 73: Can those cells modulate consequent immune reaction?  IL-10 & TGF-  ; Yang BC, et al. (2003) J Immunol. 171: Do they affect tumor growth or metastasis?  (depletion study) Chen YL, et al (2002) Br J Cancer 87:  Chen YL, et al. (2003) J Immunol. 171: etc.

8 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

9 Consist of different combinations of fibrous collagen proteins, hyaluronic acid, fibronectin, etc. Bind to cell-surface receptor: integrin, laminin receptors, etc. Are required for cell proliferation, differentiation, morphogenesis, etc.

10 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:

11 Transmigration of T cells through the tumor monolayers.

12 Extracellular matrix recaptures the effect of tumor monolayers. It is integrin-dependant. ECM was extracted by 0.5% Triton X-100.

13 Jurkat cells spread poorly on glioma ECM. (F-actin accumulated at front of lamellipodia)

14 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.

15 Tenascin-C can interact with perlecan and fibronectin, and cell surface receptors including integrins  2  1,  v  3 and  9  1, and annexin I. Nature Reviews Cancer 5, (2005) Tenascin-C is a disulfide-bonded hexamer composed of subunits with molecular weights in the range of 120–300 kD

16 Tenascin-C gene of glioma cells is knocked down by shRNA method. Tenascin-C-low transfectant cells allow better transmigration of Jurkat cells.

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

18 Tumor contact activates ERK activities in Jurkat cells, which are required for the transmigration of Jurkat cells. Question: How does tenascin-C work?

19 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.

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

21 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

22 No death occurs in Jurkat cells during coculture with gliomas.

23 Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis Nature 407, 789 5(2000)

24 FasL ribozyme /EGFP plasmid Br J Cancer 85 (2002): BC Yang  -tubulin Fas-ligand U373(v) U373(R) U118(v) U118(R)

25 No death occurs in Jurkat cells during coculture with gliomas. AB CDE 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

26 Coculture with tumor cells inhibits the Fas-mediated apoptosis.

27 Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis Apoptosis program is inhibited.

28 Direct cell contact is required.

29 Integrin signal is required Coculture with U118

30 How Fas signal-mediated death is suppressed in T cells? 25 M LY for 1 h

31 Fas-mediated death is suppressed by inhibitor of PI3K but not by inhibitors of MAPK, NF  B, PKA, HSP70.

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

33 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)

34 Jurkat Molt IL-10  -actin Glioma-associated IL-10 induction in T cell lines

35 U373(V/R) or U118(V/R) Jurkat cells IL-10  -actin Direct cell-to-cell contact is required 1. Jurkat alone 2. Jurkat separate chamber 3. Jurkat in upper chamber /coculture in low chamber 4. Jurkat in coculture U118(V)

36 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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