在生物體中，惡性細胞所形成的腫瘤組織，是以複雜而立體的網絡結構組成，而在養分供應的機制、訊息傳遞的機制、細胞之間的接觸、以及細胞與胞外基質的交互作用等反應中，皆與正常的組織不同。在體外培養的模式中，部分腫瘤細胞在鋪有膠體的培養基上，可互相聚集，形成多細胞腫瘤球體 (multicellular tumor spheroid，MTS)。在腫瘤生物學以及器官分化學的研究方面，不同於平面培養模式，多細胞腫瘤球體培養系統提供了新的研究方向。本實驗中，利用多細胞腫瘤球體培養系統，研究Fas配體 (FasL) 對免疫監控能力及細胞移動能力的影響。Fas，又稱為CD95或APO-1，是細胞膜上的穿膜型蛋白，屬於腫瘤壞死因子接受器家族的一員。Fas配體，又稱為FasL或CD95L，是Fas的專一性配體，屬於腫瘤壞死因子家族的一員，是細胞膜上的穿膜型蛋白。Fas與FasL的交互作用，會使細胞內受質引發一系列的訊息傳遞，導致表現Fas的細胞凋亡。部分腫瘤細胞表面的FasL與免疫細胞表面的Fas結合後，可阻止免疫細胞侵入腫瘤組織，甚至引發免疫細胞的凋亡，以逃避免疫清除。人類腦神經膠質瘤細胞 (glioblastoma) 是快速生長的腦腫瘤細胞，槌頭型ribozyme對FasL具專一性，經轉染後可有效抑制腦神經膠質瘤細胞U-118MG的FasL表現。轉染Fas-L ribozyme的U-118MG細胞稱為U-118R，而轉染空載體的U-118MG細胞則作為載體控制組，稱之U-118V。轉染Fas-L ribozyme並不影響U-118R細胞形成腫瘤球體。經過混合培養後，Jurkat、BJAB、HL-60等免疫細胞皆附著於腫瘤球體表面，U-118V腫瘤球體結構被Jurkat與HL-60破壞，而U-118R腫瘤球體經混合培養後保持完整球狀結構，表示FasL的表現量影響腫瘤球體與免疫細胞之間的交互作用。利用Fas的競爭型抗體CH-11及抑制型抗體ZB-4的處理，有助於釐清FasL在腫瘤球體崩解中扮演的角色。腫瘤球體中的細胞與胞外基質結合而形成球狀架構，本實驗中發現FasL的表現量影響腫瘤球體中細胞黏附因子 (focal adhesion molecules) 的表現，並進一步調控細胞移動的能力。此外，FasL的表現量影響腫瘤球體matrix metalloproteinase (MMP) 的分泌，細胞黏附因子如何受到調控而影響細胞移動能力，以及MMP是否參與其中，需要進一步探討。本實驗結果中，推測FasL可能具有啟動免疫細胞毒殺腫瘤球體細胞的能力，另一方面則調控細胞的移動能力，有助於腫瘤細胞的移動及轉移。

Malignant cells in neoplastic tissue are organized in complex three-dimensional networks displaying nutrient and signal gradients, cell-cell contact, as well as cell-extracellular matrix interaction. Some tumor cell lines form multicellular tumor spheroids (MTS) in vitro when they are grown on an agar-medium base. The MTS culture system has been shown to provide new insights into tumor biology as well as organ differentiation. In this study, we used the MTS culture system to investigate the contribution of FasL for immune survaillance. Fas (CD95/APO-1) and its specific ligand, FasL (CD95L) are surface proteins of the tumor necrosis factor receptor superfamily whose interaction triggers a cascade of subcellular events resulting in apoptosis of the Fas-expressing targets including immune cells. The presence of FasL on tumor cells is thought to inactivate tumor-infiltrating lymphocytes. A hammerhead Fas-L-specific ribozyme (Fas-L ribozyme) has been used to suppress the FasL gene of U-118MG glioblastoma cells, which are rapidly growing brain tumors. U-118MG-derived cells carried Fas-L ribozyme named U-118R and carried control plasmid named U-118V. Transfection of Fas-L ribozyme did not affect the spheroid formation. Jurkat cells, BJAB cells and HL-60 cells attached well to the surface of spheroids. Only U-118V spheroids but not U-118R spheroids were destroyed by the presence of Jurkat or HL-60, demonstrating that different levels of FasL could affect the interaction between tumor cells and immune cells. To elucidate how tumor spheroids were destructed, the application of angonistic anti-Fas antibody CH-11 and antagonistic anti-Fas antibody ZB-4 demonstrated the effect between Jurkat cells and tumor spheroids. We further found that FasL modified the expression of focal adhesion molecules in spheroids, which contribute to the adhesion of cells to the extracellular matrix, and involved in the potential of cell migration. To clarify the cell migration ability of tumor spheroids, the possible involvement of focal adhesion molecules and matrix metalloproteinase (MMP) should be further investigated. Our results suggested that FasL might initiate the cytotoxic activity of immune cells against tumor spheroids, and also mediate cell migration of tumor spheroids.

Alderson M.R., Tough T.W., Davis-Smith T., Braddy S., Falk B., Schooley K.A., Goodwin R.G., Smith C.A., Ramsdell F., and Lynch D.H. Fas ligand mediates activation-induced cell death in human T lymphocytes. J. Exp. Med., 181, 71, 1995.

Almeida E.A.C., Ilic D., Han Q., Huack C.R., Jin F., Kawakatsu H., Schlaepfer D.D., and Damsky C.H. Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH2-terminal kinase. J. Cell Biol., 149, 741-754, 2000.

Aoki K., Kurooka M., Chen J-J., Petryniak J., Nabel E.G., and Noel G. J. Extracellular matrix interacts with soluble CD95L: retention and enhancement of cytotoxicity. Nat. Immunol., 2, 333-337, 2001.

Arai H., Chen S.Y., Bishop D.K., and Nabel G.J. Inhibition of the alloantibody response by CD95 ligand. Nature Med., 3, 843, 1997.

Arai H., Gordon D., Nabel E.G., and Nabel G.J. Gene transfer of Fas ligand induces tumor regression in vivo. Proc. Natl. Acad. Sci. USA, 94, 13862, 1997.

Beningo K.A., Dembo M., Kaverina I., Small J. V., and Wang Y.L. Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. J. Cell. Biol., 153, 881-887, 2001.

Biancone L., Martino A.D., Orlandi V., Conaldi R.G., Toniolo A., and Camussi G. Development of inflammatory angiogenesis by local stimulation of Fas in vivo. J. Exp. Med., 186, 147, 1997.

Budd R.C. Death receptors couple to both cell proliferation and apoptosis. J. Clin. Invest., 109, 437-442, 2002.

Chen Y.L., Wang J.Y., Chen S.H. and Yang B.C. Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behaviour. Br. J. Cancer, 87, 359-365, 2002.

Chio C.C., Chen Y.S., Lin S.J. and Yang B.C. Down-regulation of Fas-L in glioma cells by ribozyme reduces cell apoptosis, tumor-infiltrating cells, and liver damage but accelerates tumor formation in nude mice. Br. J. Cancer, 85, 1185-1192, 2001.

Davies P.F. Multiple signaling pathways in flow-mediated endothelial mechanotransduction: PYK-ing the right location. Arterioscler Thromb. Vasc. Biol., 22, 1755-1757, 2002.

Du Q.S., Ren X.R., Xie Y., Wang Q., Mei L. and Xiong W.C. Inhibition of PYK2-induced actin cytoskeleton reorganization, PYK2 autophosphorylation and focal adhesion targeting by FAK. J. Cell Sci., 114, 2977-2987, 2001.

Friedl P. and Wolf K. Tumor-cell invasion and migration: diversity and escape mechanisms. Nature Review, 3, 362-374, 2003.

Goldbrunner R.H., Bernstein J.J., and Tonn J.C. ECM-mediated glioma cell invasion. Microscopy research and technique, 43, 250-257, 1998.

Griffith T.S., Yu X., Herndon J.M., Green D.R., and Ferguson T.A. CD95-induced apoptosis of lymphocytes in an immune privileged site induces immunological tolerance. Immunity, 5, 7, 1995.

Hahne M., Rimoldi D., Schroter M., Romero P., Schreier M., French L.E., Schneider P., Bornand T., Fontana A., Lienard D., Cerottini J.C., and Tschopp J. Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for tumor immune escape. Science, 274, 1363, 1996.

Hotary K.B., Allen E.D., Brooks P.C., Datta N.S., Long M.W., and Weiss S.J. Membrane type 1 matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix. Cell, 114, 33-45, 2003.

Hsu S.C., Gavrilin M.A., Tsai M.H., Han J., and Lai M.Z. p38 mitogen-activated protein kinase is involved in Fas ligand expression. J. Biol. Chem., 274, 25769-25776, 1999.

Huack C.R., Sieh D.J., Hsia D.A., Loftus J.C., Gaarde W.A., Monia B.P., and Schlaepfer D.D. Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcimona cells. Cancer Res., 61, 7079-7990, 2001.

Huang C., Rajfur Z., Borchers C., Schaller M.D., and Jacobson K. JNK phosphorylates paxillin and regulates cell migration. Nature, 424, 219-223, 2003.

Hu B., Guo P., Fang Q., Tao H.Q., Wang D., Nagane M., Huang H.J.S., Gunji Y.J., Nishikawa R., Alitalo K., Cavenee W.K., and Cheng S.Y. Angiopoietin-2 induces human glioma invasion through the activation of matrix metalloprotease-2. Proc. Natl. Acad. Sci. USA, 100, 8904-8909, 2003.

Imai A., Takagi A., Takagi H., and Tamaya T. Evidence for fight compling of gonadotropin-releasing hormone receptor to stimulated Fas ligand expression in reproduction tract tumors: possible mechanism for hormone control of apoptotic cell death. J. Chin. Endocrinol. Metab., 83, 427, 1998.

Kang S.M., Schneider D.B., Lin Z., Hanahan D., Dichek D.A., Stock P.G., and Baekkeskov S. Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction. Nature Med., 3, 738, 1997.

Kelm J.M., Timmins N.E., Brown C.J., Fussenegger M., and Nielsen L.K. Biothchnology and Bioengineering, 83, 173-180, 2003.

Korff T. and Augustin H.G. Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J. Cell Biol., 143, 1341-1352, 1998.

Kurooka M., Nuovo G.J., Caligiuri M.A., and Nabel G.J. Cellular localization and function of Fas ligand (CD95L) in tumors. Cancer Res., 62, 1261-1265, 2002.

Kuzuya M. and Iguchi A. Role of matrix metalloproteinases in vascular remodeling. J. Athe. Thro., 10, 275-279, 2003.

Lakka S.S., Gondi C.S., Yanamandra N., Dinh D.H., Olivero W.C., Gujrati M., and Rao J.S. Synergistic down-regulation of urokinase plasminogen activator receptor and matrix metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion, angiogenesis, and tumor growth. Cancer Res., 63, 2454-2461, 2003.

Lee J. and Jocobson K. The composition and dynamics of cell-substratum adhesions in locomoting fish keratocytes. J. Cell Sci., 110, 2833-2844, 1997.

Li H.L. and Yuan J.Y. Deciphering the pathway of life and death. Cur. Opin. Cell. Biol., 11, 261-266, 1999.

Liu S., Calderwood D.A. and Ginsberg M.H. Integrin cytoplasmic domain-binding protein. J. Cell Sci., 113, 3563-3571, 2000.

McCourt M., Wang J.H., Sookhai S., and Redmond H.P. Proinflammatory mediators stimulate neutrophil-directed angiogenesis. Arch. Surg., 134, 1325, 1999.

McLean G.W., Avizienyte E. and Frame M.C. Focal adhesion kinase as a potential target in oncology. Expert Opin. Pharmacother., 4(2), 227-234, 2003.

Mizejewski G.J. Role of integrins in cancer: survey of expression patterns. P.S.E.B.M., 222, 124-138, 1999.

Morford L.A., Boghaert E.R., Brooks W.H., and Roszman T.L. Insulin-like growth factors (IGF) enhance three-dimensional (3D) growth of human glioblastomas. Cancer Lett., 115, 81-90, 1997.

Nagata S. Fas and Fas ligand: a death factor and its receptor. Adv. Immunol., 57, 129, 1994.

Nagata S., Suda T. Fas and Fas ligand: lpr and gld mutations. Immunol. Today, 16, 39, 1995.

Nagata S. Apoptosis by death factor. Cell, 88, 355, 1997.

Nagata K.I., Puls A., Futter C., Aspenstrom P., Schaefer E., Nagata T., Hirokawa N. and Hall A. The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3. EMBO J., 17, 149-158, 1998.

Nagata S. Fas ligand-induced apoptosis. Annu. Rev. Genet., 33, 29, 1999.

Niehans G.A., Brunner T., Frizelle S.P., Liston J.C., Salero C.T., Knapp D.J., Green D.R. and Kratzke R.A. Human lung carcinomas express Fas ligand. Cancer Res., 57, 1007, 1997.

O’Connell J., O’Sullivan G.C., Collins K., and Shanahan F. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J. Exp. Med., 184, 1074, 1996.

O’Connell J., Bennett M.W., O’sullivan G.G., Roche D., Kelly J., Collins J.K., and Shanahan F. Fas counter-attack the best form of tumor defense? Nature Med., 5, 267, 1999.

Ochsenbein A.F., Sierro S., Odermatt B., Pericin M., Karrer U., Hermans J., Hemmi S., Hengartner H. and Zinkernagel R.M. Role of tumor localization, second signals and cross priming in cytotoxic T-cell induction. Nature, 411, 1058-1064, 2001.

Oktay M., Wary K.K., Dans M., Brige R.B., and Giancotti F.G. Integrin-mediated activation of focal adhesion kinase is required for signaling to Jun NH2-terminal kinase and progression through the G1 phase of the cell cycle. J. Cell Biol., 145, 1461-1469, 1999.

Opdenakker G., Van den Steen P.E., and Van Damme J. Gelatinase B: a tuner and amplifier of immune functions. Trends Immunol., 22, 571, 2001.

Ozanne B.W., McGarry L., Spence H.J., Johnston I., Winnie J., Meagher L., Stapleton G. Transcriptional regilation of cell invasion: AP-1 regulation of a multigenic invasion programme. Eur. J. Cancer, 36, 1640-1648, 2000.

Pfaff M. and Jurdic P. Podosomes in osteoclast-like cells: structural analysis and cooperative roles of paxillin, proline-rich tyrosine kinase 2 (Pyk2) and integrin V3. J. Cell Sci., 114, 2775-2786, 2001.

Saas P., Walker P.R., Hahne M., Quiquerez A.L., Schnuriger V., Perrin G., Franch L., VanMeir E.G., Tribolet N.T., Schopp J., and Dietrich P.Y. Fas ligand xpression by astrocytoma in vivo: maintaining immune privilege in the brain? J. Clin. Invest., 99, 1173, 1997.

Sabelko-Downes K.A. and Russell J.H. The role of Fas ligand in vivo as a cause and regulator of pathogenesis. Cur. Opin. Immunol., 12, 330-335, 2000.

Schaller M.D. Paxillin: a focal adhesion-associated adaptor protein. Oncogene, 20, 6459-6472, 2001.

Seino K.I., Kayagaki N., Tsukada N., Fukao K., Yagita H., and Okumura K. Transplantation of CD95 ligand-expressing grafts. Influence of transplantation site and difficulty in protecting allo- and xenigrafts. Transplantation, 64, 1050, 1997.

Shiraki K., Tsuji N., Shioda T., Isselbacher K.J. and Takahashi H. Expression od Fas ligand in liver metastases of human colonic adenocarcinomas. Proc. Natl. Acad. Aci. USA, 94, 6420, 1997.

Strand S., Hofmann W.J., Hung H., Muller M., Otto G., Strand D., Martani S.M., Stremmel W., Krammer P.H., and Galle P.R. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells- a mechanism of immune evasion? Nature Med., 2, 1361, 1996.

Van Kempen L.C. and Coussens L.M. MMP9 potentiates pulmonary metastasis formation. Cancer Cell, 2, 251, 2002.

Walker P.R., Saas P. and Dietrich P.Y. Role of Fas ligand (CD95L) in immune escape: the tumor cells strikes back. J. Immunol., 158, 4521, 1997.

Walker P.R., Saas P., and Dietrich P.Y. Tumor expression of Fas ligand (CD95L) and the consequences. Curr. Opin. Immunol., 10, 564, 1998.

Webb D.J., Donais K., Whitmore L.A., Thomas S.M., Turner C.E., Parsons J.T. and Horwitz A.F. FAK-src signaling through paxillin, ERK and MLCK regulates adhesion disassembly. Nature Cell Biol., 6, 154-161, 2004.

Xiong W.C., Macklem M. and Parsons J.T. Expression and characterization of splice variants of PYK2, a focal adhesion kinase-related protein. J. Cell Sci., 111, 1981-1991, 1998.

Yang B.C., Wang Y.S., Wang C.H., Lin H.H., Tang M.J., Yang T.L. Transient apoptosis elicited by insulin in serum-starved glioma cells involves Fas/Fas-L and Bcl-2. Cell Biol. Int., 23, 533, 1999.

Yuhas J.M., Li A.P., Martinez A.O., and Ladman A.J. A simplified method for production and growth of multicellular tumor spheroids. Cancer Res., 37, 3639-3643, 1977.

Yuhas J.M., Tarleton A.E., and Molzen K.B. Multicellular tumor spheroid formation by breast cancer cells isolated from different sites. Cancer Res., 38, 2486-2491, 1978.

Yuhas J.M. and Tarleton A.E. Dormancy and spontaneous recurrence of human breast cancer in vitro. Cancer Res., 38, 3584-3589, 1978.

Zhao J, Zheng C. and Guan J.L. Pyk2 and FAK differentially regulate progression of the cell cycle. J. Cell Sci., 113, 3063-3072, 2000.

Zeytun A., Hassuneh M., Nagarkatti M., and Nagarkatti P.S. Fas-Fas ligand-based interation between tumor cells and tumor specific-cytotoxic T lymphocytes: a lethal two-way street. Blood, 90, 1952, 1997.