To live or to die, that's a problem of Fas signal: 成功大學醫學院 微生物暨免疫學研究所 17/11/2004 張金瑛 王琳雅 吳怡靜 黃建勳 鄭柏吟 楊倍昌 蘇重禎 張雅南 黃偉倫 黃俊淵 林裕萍

Apoptosis 秋天樹葉的凋零飄落 'Apoptosis' is of Greek origin, means "falling off or dropping off", in analogy to leaves falling from trees or petals from flowers. 成功大學醫學院旁

The term 'apoptosis' describes the molecular and morphological processes leading to controlled cellular self-destruction and was first introduced in a publication by Kerr, Wyllie and Currie. Br. J. Cancer, 1972, 26: 239

Two types of cell death Apoptosis Necrosis

Detection of apoptotic cells Hoechst stain DNA ladder Comet assay TUNEL stain; immunohistochmical (terminal transferase) FACS analysis (Annexin V, MC540, propidium iodine for subG0, TUNEL)

Apoptosis vs. necrosis

The Fas/Fas-ligand system Nagata, S. (Fas); Krammer, P. (Apo1) CD95/CD95-L Transducing death signal Involving in cytotoxicity, immune homeostasis, tumor formation, etc.

Shigekazu Nagata Lab member: total 21 Cell Death and Diff. Genes to Cell Apoptosis Int. Immunol. Immunity Exp. Cell Res. Biochem. Biophys. Acta IUBMB/Life Science Int. J. Cancer Cancer Cell Japan Academy Prize, Imperial Prize (Japan) Person of Cultural Merits (Japan)

Peter H. Krammer, 2002 Lab member: total 34

Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis Nature 407, 789 – 795(2000)

十大癌症在台灣 (2001): 1. 肺癌 2. 肝癌 3. 結腸直腸癌 4. 胃癌 5. 女性乳癌 6. 子宮頸癌 7. 口腔癌 8. 攝護腺癌 9. 非何杰金淋巴癌 10. 胰臟癌

Evidence for active host defense against cancer 80 years of immunotherapy. (Currie GA, 1972, Br. J. Cancer 26: 141) A critique of the evidence for active host defense against cancer, based on personal studies of 27 murine tumors of spontaneous origin. (Hewitt HB, et al. 1976, Br. J. Cancer 33:241)

A fight between immune cells and cancer

But, sometimes we lose

The great escape: immune evasion versus tumor progression

Resistance to killing Defective Fas pathway Resistance to Granzyme Fas-L and Neutrophil Cytotoxic T cells Innate immunity

In vivo consequences of Fas-L expression by tumors (using over-expression system) Enhanced rejection Delayed rejection Renca, MH134, L5178Y, B16- BL6, CT26* Note: *:syngenic, nude, SCID # : allogenic, lpr Ref: Lejeune FJ et al., 1998, Curr. Op. Immunol. CT26 # B16-F10

Immune privilege or inflammation? Joe O'Connell et al. Nature Medicine 7, (2001)

Fas-L positive tumors with heavily infiltrated lymphocytes Nasal pharyngeal carcinoma Glioblastoma Hematoxylin/eosin staining CD3 + cells Immune cells are not eliminated

Fas-L ribozyme /EGFP plasmid Hammerhead ribozyme. 54-mers. Inserted into the pEGFP-N1. Driven by CMV promoter. Br J Cancer, (2001) 85:

U-373MG cells transfected with Fas-L ribozyme /EGFP plasmid Light microscopy Fluorescent microscopy

Down-regulation of Fas-L by Fas-L ribozyme RT-PCR 1. U-373MG 2. EGFP-N1 3. EGFP/Fas-L ribozyme FACScan U-373MG

Animal model C57B/6 Melanoma cell line B6F10 Gene knockdown by FasL ribozyme Subcutis/lung

2002/4-2004/1 工作的結論 : FasL on tumor cells mediates inactivation of neutrophils. J Immunol (2003) 171: Crosstalk between tumor cells and neutrophils through the Fas (APO-1, CD95)/Fas-L system: human glioma cells enhance cell viability and stimulate cytokine production in neutrophils. J leukocyte Biol (2003)73: Induction of IL-10 in T cell lines upon contact with human glioma cells, that is mediated by Fas signaling through a PKA-independent pathway. J Immunol (2003) 171: Fas FasL T cells Tumor Tissue environment ? Cytokines ? IL-10, IL-6, IL-8 Neutrophils To help or not to help ? FasL-high FasL-low Subcutis vs lung 追求卓越計畫 : 基因調控與訊息傳遞

B16-F10 (melanoma) in B6 mice Control Fas-L Ribozyme

Vector controlsFas-L-ribozyme Depletion of CD4, CD8 T cells and PMNs affected subcutaneous tumor formation

Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behavior (B16F10 in C57BL/6) Br J Cancer, 87: 359 (2002)

Depletion of CD4, CD8 T cells and PMNs affected lung metastasis Vector controlsFas-L ribozyme What happened here?

The Third face of Fas signal Not only “not quite dead enough”! Stout Rex, 1944

No death occurred in Jurkat cells during coculture with gliomas PI-staining 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

Fas Fas-L Immune cells Tumor Tissue environment ? Cytokines ? (IL-10, TGF-  ) What will happen when Fas-stimulated immune cells resist to die?

Fas Engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1 , and the production of interferon-  in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses. J Exp Med 192: Rescigno M., et al. 2000

Hohlbaum AM, et al. 2001, Fas ligand engagement of resident peritoneal macrophages in vivo induces apoptosis and the production of neutrophil chemotactic factors. J Immunol 167:

Coculture with gliom cells induced the expression of IL-10 in T cell lines 1. T cells alone 2. In coculture with U-373MG 3. In coculture with U-118MG

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)

The IL-10 induction is associated with tumor Fas-L 2: U-373MG(V); 3: U373MG(R); 4: U-118MG(V); 5: U118MG(R)

IL-10  -actin %SubG 0 10% 50% 65% 73% IL-10 expression upon Fas-activation in Jurkat cells by CH : 0, 0.1, 0.5, 1 ng/ml of CH-11 for 24 h

Interrupting Fas/Fas-L interaction suppressed the glioma-induced IL-10 expression. 1-3: Jurkat alone 4-6: Jurkat/U-118MG 2,4: ZB-4:100ng/ml 3,5: ZB-4:200ng/ml

Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis Nature 407, 789 – 795(2000)

The IL-10 induction in Jurkat cells required caspase activities

Fas/FasL interaction may create an immunosuppresive environment Induction of IL-10 in T cell lines upon contact with human glioma cells, that is mediated by Fas signaling through a PKA-independent pathway. J Immunol (2003) 171:

J. Immunol. 171: Integrin ?

Cells can be kept in suspension by rotating the culture chamber to enhance transfection efficiency Microcarriers are used as substrates for adherent cells which can grow as a 3-D tissue mass Long-term high-performance can be achieved Cell spheroids

在動物體內, 細胞從來不 會這樣 攤平 生長. In culture dish Fas-L R Control U-118MG in nude mice FasL associated TIL in particular sites Invasion of tumor cells

Structure of tumor spheroid Yuhas JM, et al. (1977) Cancer Res 37: Culture on 0.5% Nobel Agar

The morphology of spheroids U118R U118V 1 w 2 w 3 w 4 w 100  m

The surface morphology of spheroids. (4 weeks; by a scanning electronic microscope) U118V U118R

Morphology of cells on the surface are different from that of cells in the center of spheroids.  Differentiate potential 1 w 2 w 4 w 6 w U118R U118V The structure of spheroids

Jurkat cells destroy the FasL high -spheroids. U118V ~250  m U118R ~260  m U-118MG (4 weeks) + Jurkat cells (1:5); 3 days coculture 5x10 5 cells Note: Jurkat cells do not kill tumor cells in dish culture condition HL-60 (Yes), BJAB (No)

HL-60 BJAB U118R U118V Human acute myeloid leukemia cell lineHuman B cell line Only U-118V spheroids but not U-118R spheroids were destroyed by the presence of or HL-60 cells.

Jurkat cells do not kill tumor cells in 2-D culture (U-118MG; PI+ stain) U-118MG + JurkatU-118MG + Jurkat + Neutrophils PI-positive U-118MG Why FasL + tumor spheroids are destroyed?

2-D versus 3-D growth On plate Spheroid Modification Reduced enhanced references points IEF SDS

FAK (125) C-src (60) Talin (235) Pyk2 (116) R V R V R V Spheroids Vinculin (116) ? Proteins associated with extracellular matrix

Fas FasL T cells Tumor Tissue environment ? (ECM) Cytokines ? IL-10, IL-6, IL-8 Neutrophils To live or to die, that's always dependent Constitute specific tumor environment