Shaping T cell functions by tumor extracellular matrix.

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 NCKU

2. Fight cancer, But, sometimes we lose NCKU

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

4. This is not a single case. 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) 115:67–72Immune 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) NCKU

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. 179:4589-4597. • Hor WS, et al. (2003) J Leukocyte Biol. 73: 363-368. • Can those cells modulate consequent immune reaction? • IL-10 & TGF-b; 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. NCKU

8. 黃俊淵 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. NCKU

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. NCKU

10. Rational: • 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. NCKU

11. Transmigration of T cells through the tumor monolayers. NCKU

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

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

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. NCKU

15. Tenascin-C is a disulfide-bonded hexamer composed of subunits with molecular weights in the range of 120–300 kD Tenascin-C can interact with perlecan and fibronectin, and cell surface receptors including integrins a2b1, avb3 and a9b1, and annexin I. Nature Reviews Cancer 5, 436-446 (2005) NCKU

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

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

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

19. Tumor contact activates ERK activity in Jurkat cells, which is required for the transmigration of Jurkat cells. ERK and actin colocalize at the front of lamellipodia of Jurkat cells. Further study on migration machinery (focal adhesion complex) is on going. NCKU

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

21. Conclusion: Tenascin-C inhibits the polarization and transmigrationof T cells, that consequently prevents a direct contact of glioma tumor and T cells. Are those T cells alive ? 蘇重禎 NCKU

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

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

24. FasLribozyme/EGFP plasmid U373(R) U118(R) U118(v) U373(v) Fas-ligand -tubulin Br J Cancer 85 (2002):1185-1192 NCKU BC Yang

25. A B C D E No death occurs in Jurkat cells during coculture with gliomas. PI-staining A: Jurkat cells alone B: CH-11 (1ng/ml) C: with U-118MG(V) D: with U118MG (R) E: ZB4 (100ng/ml)/CH-11 NCKU

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

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

28. Direct cell contact is required. NCKU

29. Integrin signal is required Coculture with U118 NCKU

30. How Fas signal-mediated death is suppressed in T cells? 25 M LY294002 for 1 h NCKU

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

32. 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. NCKU

33. Stepwise hijacking of infiltrating T cells by tumor: Paralyzing → Keeping alive → modifying (IL-10/TGF) • Why are T cells trapped in peripheral area of tumor mass?Tenascin-C of glioma. • Are those T cells alive? ECM signal suppressesapoptosis! (IL-6/IL-8 for neutrophils) NCKU

34. Jurkat Molt-4 1 2 3 1 2 3 IL-10 b-actin Glioma-associated IL-10 induction in T cell lines NCKU

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

36. Jurkat U-373MG U-118MG - V R V R IL-10 b-actin Molt-4U-373MG U-118MG - V R V R IL-10 b-actin Fas/Fas-L signal-associated IL-10 induction NCKU

37. Thank you for listening NCKU