口腔鱗狀細胞癌(OSCC)是口腔頷面部常見的癌症，在全世界10大癌症排行第六名，在亞洲也約有8％到10％的人罹患此癌症。有文獻指出神經膠原母細胞瘤的癌類幹細胞可以分化成功能性的內皮細胞，而內皮細胞在血管新生過程中扮演重要角色，也有文獻發現腫瘤幹細胞在懸浮球體(tumor sphere)中含量較多。血管新生(angiogenesis)在腫瘤生長和轉移時必要的過程，近年來科學家們也發現血管新生和癌症的發展有密切的關係。目前在臨床上發現癌症病患仍會有抗藥性或許給予化療不見得有好轉，甚至不能完全治癒。因此，此篇研究目的想探討腫瘤類幹細胞與血管新生之間的交互作用是否是造成此現象的原因。
一開始先比較人類舌鱗狀細胞癌（SAS）的tumor sphere和單層細胞(monolayer)中幹細胞markers (如: CD133、Nanog、ALDH1A1和Oct4)的表現變化。這些marker在real-time PCR、西方點墨法(western blotting)以及免疫染色(immunostaining)的結果都可以發現懸浮球體的表現量高於單層細胞。之後利用angiogenesis PCR array分析並篩選upregulation的血管新生因子，此因子包括CXCL10（趨序趨化因子10）、CXCL3（趨序趨化因子3）、IL8（白細胞介素8）和TNF(腫瘤壞死因子）。另外也使用細胞分選 (cell sorting)篩選出CD133+ cells，並使用real-time PCR檢測發現CXCL3、IL8、TNF具有高度表現，另外我們也檢測內皮細胞分子標誌 Factor VIII的表現，其實驗結果證實CD133+ cells中Factor VIII表現量比CD133- cells來的高。
以上結果可證明懸浮球體確實具有癌症幹細胞特性。我們也鑑定一些有關腫瘤類幹細胞的血管生成因子（趨化因子）。此研究結果可能有助於在癌症治療上開發新的方法。

Oral squamous cell carcinoma (OSCC), the most common cancer in the oral and maxillofacial region, is the sixth most prevalent type of malignancy worldwide and account for approximately 8% to 10% of all cancers in Asia.
Some studies indicate that tumor sphere has more cancer stem-like (CSCs) cells. Angiogenesis is the propelling force for tumor growth and metastasis. Some cancer patients also accompany drug resistance during treatment, so they have poor prognosis after chemotherapy treatment. A portion of the patients can not be completely cured due to cancer metastasis. Therefore, many researchers highly suspect that cancer stem-like cells and angiogenesis may be the main reasons for this phenomenon.
First, we compared human tongue squamous cell carcinoma (SAS) tumor sphere and monolayer for expression of stem cell genes and related proteins, including CD133, Nanog, ALDH1A1 and Oct4, using quantitative RT-PCR, western blotting, and immunostaining to analyze these stem cell markers expression in tumor sphere. Next, we utilized angiogenesis PCR array to identify up-regulated angiogenic factors. We also confirmed the findings using real-time PCR on cell-sorted CD133+ cells to reconfirm the data.
The levels of CD133, Nanog, ALDH1A1 and Oct4 were up-regulated in tumor sphere compared with monolayer based on quantitative RT-PCR, western blotting and immunostaining. We demonstrated tumor sphere contained more cancer stem-like cell. The data of angiogenesis PCR array identified up-regulation of several angiogenic factors, including CXCL10 (C-X-C motif Chemokine 10), CXCL3 (C-X-C motif Chemokine 3), IL8 (Interleukin 8) and TNF (Tumor nerosis factor). Most of them are chemotactic factors. Cell sorting for CD133+ cells also confirm CXCL3, IL8 and TNF were more expressed in CD133+ cells. We were also examined endothelium cell marker Factor VIII and found that it was more expressed in CD133+ cells.
Our analysis indicated that tumor sphere have cancer stem-like cell characteristics. We identified some upregulated angiogenic factors (chemotactic factors) in cancer stem-like cells. The results may help to develop new cancer therapies.

Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J., and Clarke, M.F. (2003). Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100, 3983-3988.

Araki, K., Mizokami, D., Tanaka, N., Suzuki, H., Sato, S., and Shiotani, A. (2013). Targeted gene transfer into head and neck squamous cell carcinoma by nanosecond pulsed laser-induced stress waves. Lasers Med Sci.

Balic, M., Lin, H., Young, L., Hawes, D., Giuliano, A., McNamara, G., Datar, R.H., and Cote, R.J. (2006). Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res 12, 5615-5621.

Bao, S., Wu, Q., McLendon, R.E., Hao, Y., Shi, Q., Hjelmeland, A.B., Dewhirst, M.W., Bigner, D.D., and Rich, J.N. (2006a). Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-760.

Bao, S., Wu, Q., Sathornsumetee, S., Hao, Y., Li, Z., Hjelmeland, A.B., Shi, Q., McLendon, R.E., Bigner, D.D., and Rich, J.N. (2006b). Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res 66, 7843-7848.

Bergers, G., Hanahan, D., and Coussens, L.M. (1998). Angiogenesis and apoptosis are cellular parameters of neoplastic progression in transgenic mouse models of tumorigenesis. Int J Dev Biol 42, 995-1002.

Bjerkvig, R., Johansson, M., Miletic, H., and Niclou, S.P. (2009). Cancer stem cells and angiogenesis. Semin Cancer Biol 19, 279-284.

Botchkina, I.L., Rowehl, R.A., Rivadeneira, D.E., Karpeh, M.S., Jr., Crawford, H., Dufour, A., Ju, J., Wang, Y., Leyfman, Y., and Botchkina, G.I. (2009). Phenotypic subpopulations of metastatic colon cancer stem cells: genomic analysis. Cancer Genomics Proteomics 6, 19-29.

Calabrese, C., Poppleton, H., Kocak, M., Hogg, T.L., Fuller, C., Hamner, B., Oh, E.Y., Gaber, M.W., Finklestein, D., Allen, M., et al. (2007). A perivascular niche for brain tumor stem cells. Cancer Cell 11, 69-82.

Campisi, J. (2003). Cancer and ageing: rival demons? Nat Rev Cancer 3, 339-349.

Canis, M., Lechner, A., Mack, B., Zengel, P., Laubender, R.P., Koehler, U., Heissmeyer, V., and Gires, O. (2012). CD133 is a predictor of poor survival in head and neck squamous cell carcinomas. Cancer Biomark 12, 97-105.

Carmeliet, P. (2005). Angiogenesis in life, disease and medicine. Nature 438, 932-936.

Chang, C.C., Hsu, W.H., Wang, C.C., Chou, C.H., Kuo, M.Y., Lin, B.R., Chen, S.T., Tai, S.K., Kuo, M.L., and Yang, M.H. (2013). Connective tissue growth factor activates pluripotency genes and mesenchymal-epithelial transition in head and neck cancer cells. Cancer Res.

Chen, K., Huang, Y.H., and Chen, J.L. (2013). Understanding and targeting cancer stem cells: therapeutic implications and challenges. Acta Pharmacol Sin.

Chen, S.F., Chang, Y.C., Nieh, S., Liu, C.L., Yang, C.Y., and Lin, Y.S. (2012). Nonadhesive culture system as a model of rapid sphere formation with cancer stem cell properties. PLoS One 7, e31864.

Chen, Y.J., Lin, S.C., Kao, T., Chang, C.S., Hong, P.S., Shieh, T.M., and Chang, K.W. (2004). Genome-wide profiling of oral squamous cell carcinoma. J Pathol 204, 326-332.

Chiou, S.H., Yu, C.C., Huang, C.Y., Lin, S.C., Liu, C.J., Tsai, T.H., Chou, S.H., Chien, C.S., Ku, H.H., and Lo, J.F. (2008). Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma. Clin Cancer Res 14, 4085-4095.

Clarke, M.F., Dick, J.E., Dirks, P.B., Eaves, C.J., Jamieson, C.H., Jones, D.L., Visvader, J., Weissman, I.L., and Wahl, G.M. (2006). Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66, 9339-9344.

Clay, M.R., Tabor, M., Owen, J.H., Carey, T.E., Bradford, C.R., Wolf, G.T., Wicha, M.S., and Prince, M.E. (2010). Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase. Head Neck 32, 1195-1201.

Collins, A.T., Berry, P.A., Hyde, C., Stower, M.J., and Maitland, N.J. (2005). Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65, 10946-10951.

Curley, M.D., Therrien, V.A., Cummings, C.L., Sergent, P.A., Koulouris, C.R., Friel, A.M., Roberts, D.J., Seiden, M.V., Scadden, D.T., Rueda, B.R., et al. (2009). CD133 expression defines a tumor initiating cell population in primary human ovarian cancer. Stem Cells 27, 2875-2883.

Dalerba, P., Dylla, S.J., Park, I.K., Liu, R., Wang, X., Cho, R.W., Hoey, T., Gurney, A., Huang, E.H., Simeone, D.M., et al. (2007). Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A 104, 10158-10163.

Du, L., Wang, H., He, L., Zhang, J., Ni, B., Wang, X., Jin, H., Cahuzac, N., Mehrpour, M., Lu, Y., et al. (2008). CD44 is of functional importance for colorectal cancer stem cells. Clin Cancer Res 14, 6751-6760.

Eramo, A., Lotti, F., Sette, G., Pilozzi, E., Biffoni, M., Di Virgilio, A., Conticello, C., Ruco, L., Peschle, C., and De Maria, R. (2008). Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 15, 504-514.

Felthaus, O., Ettl, T., Gosau, M., Driemel, O., Brockhoff, G., Reck, A., Zeitler, K., Hautmann, M., Reichert, T.E., Schmalz, G., et al. (2011). Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines. Biochem Biophys Res Commun 407, 28-33.

Ferrara, N. (2002). VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer 2, 795-803.

Folkman, J. (1971). Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182-1186.

Frank, N.Y., Schatton, T., and Frank, M.H. (2010). The therapeutic promise of the cancer stem cell concept. J Clin Invest 120, 41-50.

Fujita, A., Furutama, D., Tanaka, T., Sakai, R., Koyama, A., Hanafusa, T., Mitsuhashi, T., and Ohsawa, N. (2002). In vivo activation of the constitutive androstane receptor beta (CARbeta) by treatment with dehydroepiandrosterone (DHEA) or DHEA sulfate (DHEA-S). FEBS Lett 532, 373-378.

Gabellini, C., Trisciuoglio, D., Desideri, M., Candiloro, A., Ragazzoni, Y., Orlandi, A., Zupi, G., and Del Bufalo, D. (2009). Functional activity of CXCL8 receptors, CXCR1 and CXCR2, on human malignant melanoma progression. Eur J Cancer 45, 2618-2627.

Galli, R., Gritti, A., Bonfanti, L., and Vescovi, A.L. (2003). Neural stem cells: an overview. Circ Res 92, 598-608.

Gunes, C., and Rudolph, K.L. (2013). The role of telomeres in stem cells and cancer. Cell 152, 390-393.

Hueng, D.Y., Sytwu, H.K., Huang, S.M., Chang, C., and Ma, H.I. (2011). Isolation and characterization of tumor stem-like cells from human meningiomas. J Neurooncol 104, 45-53.

Kaur, B., Khwaja, F.W., Severson, E.A., Matheny, S.L., Brat, D.J., and Van Meir, E.G. (2005). Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis. Neuro Oncol 7, 134-153.

Kawamoto, H., Yuasa, T., Kubota, Y., Seita, M., Sasamoto, H., Shahid, J.M., Hayashi, T., Nakahara, H., Hassan, R., Iwamuro, M., et al. (2010). Characteristics of CD133(+) human colon cancer SW620 cells. Cell Transplant 19, 857-864.

Kerbel, R.S. (2008). Tumor angiogenesis. N Engl J Med 358, 2039-2049.

Klonisch, T., Wiechec, E., Hombach-Klonisch, S., Ande, S.R., Wesselborg, S., Schulze-Osthoff, K., and Los, M. (2008). Cancer stem cell markers in common cancers - therapeutic implications. Trends Mol Med 14, 450-460.

Lapidot, T., Sirard, C., Vormoor, J., Murdoch, B., Hoang, T., Caceres-Cortes, J., Minden, M., Paterson, B., Caligiuri, M.A., and Dick, J.E. (1994). A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367, 645-648.

Laquer, V., Hoang, V., Nguyen, A., and Kelly, K.M. (2009). Angiogenesis in cutaneous disease: part II. J Am Acad Dermatol 61, 945-958; quiz 959-960.

Le Tourneau, C. (2010). [Molecularly targeted therapy in head and neck cancer]. Bull Cancer 97, 1453-1466.

Lee, J., Kotliarova, S., Kotliarov, Y., Li, A., Su, Q., Donin, N.M., Pastorino, S., Purow, B.W., Christopher, N., Zhang, W., et al. (2006). Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9, 391-403.

Lee, S.H., Hong, H.S., Liu, Z.X., Kim, R.H., Kang, M.K., Park, N.H., and Shin, K.H. (2012). TNFalpha enhances cancer stem cell-like phenotype via Notch-Hes1 activation in oral squamous cell carcinoma cells. Biochem Biophys Res Commun 424, 58-64.

Leung, E.L., Fiscus, R.R., Tung, J.W., Tin, V.P., Cheng, L.C., Sihoe, A.D., Fink, L.M., Ma, Y., and Wong, M.P. (2010). Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS One 5, e14062.

Li, A., Varney, M.L., Valasek, J., Godfrey, M., Dave, B.J., and Singh, R.K. (2005). Autocrine role of interleukin-8 in induction of endothelial cell proliferation, survival, migration and MMP-2 production and angiogenesis. Angiogenesis 8, 63-71.

Li, C., Lee, C.J., and Simeone, D.M. (2009). Identification of human pancreatic cancer stem cells. Methods Mol Biol 568, 161-173.

Maiti, G.P., Mondal, P., Mukherjee, N., Ghosh, A., Ghosh, S., Dey, S., Chakrabarty, J., Roy, A., Biswas, J., Roychoudhury, S., et al. (2013). Overexpression of EGFR in Head and Neck Squamous Cell Carcinoma Is Associated with Inactivation of SH3GL2 and CDC25A Genes. PLoS One 8, e63440.

Major, A.G., Pitty, L.P., and Farah, C.S. (2013). Cancer stem cell markers in head and neck squamous cell carcinoma. Stem Cells Int 2013, 319489.

Martens-de Kemp, S.R., Dalm, S.U., Wijnolts, F.M., Brink, A., Honeywell, R.J., Peters, G.J., Braakhuis, B.J., and Brakenhoff, R.H. (2013). DNA-Bound Platinum Is the Major Determinant of Cisplatin Sensitivity in Head and Neck Squamous Carcinoma Cells. PLoS One 8, e61555.

Morrison, S.J., and Kimble, J. (2006). Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 441, 1068-1074.

Nishida, N., Yano, H., Nishida, T., Kamura, T., and Kojiro, M. (2006). Angiogenesis in cancer. Vasc Health Risk Manag 2, 213-219.

O'Brien, C.A., Pollett, A., Gallinger, S., and Dick, J.E. (2007). A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445, 106-110.

Okahara, S., Arimura, Y., Yabana, T., Kobayashi, K., Gotoh, A., Motoya, S., Imamura, A., Endo, T., and Imai, K. (2005). Inflammatory gene signature in ulcerative colitis with cDNA macroarray analysis. Aliment Pharmacol Ther 21, 1091-1097.

Patterson, D.M., Shohet, J.M., and Kim, E.S. (2011). Preclinical models of pediatric solid tumors (neuroblastoma) and their use in drug discovery. Curr Protoc Pharmacol Chapter 14, Unit 14 17.

Pentenero, M., Gandolfo, S., and Carrozzo, M. (2005). Importance of tumor thickness and depth of invasion in nodal involvement and prognosis of oral squamous cell carcinoma: a review of the literature. Head Neck 27, 1080-1091.

Plate, K.H., Breier, G., Weich, H.A., Mennel, H.D., and Risau, W. (1994). Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int J Cancer 59, 520-529.

Plate, K.H., Breier, G., Weich, H.A., and Risau, W. (1992). Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359, 845-848.

Prince, M.E., Sivanandan, R., Kaczorowski, A., Wolf, G.T., Kaplan, M.J., Dalerba, P., Weissman, I.L., Clarke, M.F., and Ailles, L.E. (2007). Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 104, 973-978.

Rak, J., Yu, J.L., Klement, G., and Kerbel, R.S. (2000). Oncogenes and angiogenesis: signaling three-dimensional tumor growth. J Investig Dermatol Symp Proc 5, 24-33.

Ren, F., Sheng, W.Q., and Du, X. (2013). CD133: A cancer stem cells marker, is used in colorectal cancers. World J Gastroenterol 19, 2603-2611.

Reya, T., Morrison, S.J., Clarke, M.F., and Weissman, I.L. (2001). Stem cells, cancer, and cancer stem cells. Nature 414, 105-111.

Ribatti, D. (2012). Cancer stem cells and tumor angiogenesis. Cancer Lett 321, 13-17.

Ricci-Vitiani, L., Lombardi, D.G., Pilozzi, E., Biffoni, M., Todaro, M., Peschle, C., and De Maria, R. (2007). Identification and expansion of human colon-cancer-initiating cells. Nature 445, 111-115.

Ricci-Vitiani, L., Pallini, R., Biffoni, M., Todaro, M., Invernici, G., Cenci, T., Maira, G., Parati, E.A., Stassi, G., Larocca, L.M., et al. (2010). Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 468, 824-828.

Salcedo, R., Resau, J.H., Halverson, D., Hudson, E.A., Dambach, M., Powell, D., Wasserman, K., and Oppenheim, J.J. (2000). Differential expression and responsiveness of chemokine receptors (CXCR1-3) by human microvascular endothelial cells and umbilical vein endothelial cells. FASEB J 14, 2055-2064.

Sawyers, C. (2004). Targeted cancer therapy. Nature 432, 294-297.

Schatton, T., Murphy, G.F., Frank, N.Y., Yamaura, K., Waaga-Gasser, A.M., Gasser, M., Zhan, Q., Jordan, S., Duncan, L.M., Weishaupt, C., et al. (2008). Identification of cells initiating human melanomas. Nature 451, 345-349.

Seaberg, R.M., and van der Kooy, D. (2003). Stem and progenitor cells: the premature desertion of rigorous definitions. Trends Neurosci 26, 125-131.

Shmelkov, S.V., St Clair, R., Lyden, D., and Rafii, S. (2005). AC133/CD133/Prominin-1. Int J Biochem Cell Biol 37, 715-719.

Singh, S.K., Clarke, I.D., Terasaki, M., Bonn, V.E., Hawkins, C., Squire, J., and Dirks, P.B. (2003). Identification of a cancer stem cell in human brain tumors. Cancer Res 63, 5821-5828.

Singh, S.K., Hawkins, C., Clarke, I.D., Squire, J.A., Bayani, J., Hide, T., Henkelman, R.M., Cusimano, M.D., and Dirks, P.B. (2004). Identification of human brain tumour initiating cells. Nature 432, 396-401.

Smalley, M., and Ashworth, A. (2003). Stem cells and breast cancer: A field in transit. Nat Rev Cancer 3, 832-844.

Smith, C. (2003). Hematopoietic stem cells and hematopoiesis. Cancer Control 10, 9-16.

Stupp, R., and Hegi, M.E. (2007). Targeting brain-tumor stem cells. Nat Biotechnol 25, 193-194.

Tysnes, B.B., and Bjerkvig, R. (2007). Cancer initiation and progression: involvement of stem cells and the microenvironment. Biochim Biophys Acta 1775, 283-297.

Uras, N., Oguz, S.S., Zergeroglu, S., Akdag, A., Polat, B., Dizdar, E.A., and Dilmen, U. (2012). CD31 and Factor VIII in angiogenesis of normal and pre-eclamptic human placentas. J Obstet Gynaecol 32, 533-536.

Wu, C., Wei, Q., Utomo, V., Nadesan, P., Whetstone, H., Kandel, R., Wunder, J.S., and Alman, B.A. (2007). Side population cells isolated from mesenchymal neoplasms have tumor initiating potential. Cancer Res 67, 8216-8222.

Wu, M.S., Wang, G.F., Zhao, Z.Q., Liang, Y., Wang, H.B., Wu, M.Y., Min, P., Chen, L.Z., Feng, Q.S., Bei, J.X., et al. (2013). Smac mimetics in combination with TRAIL selectively target cancer stem cells in nasopharyngeal carcinoma. Mol Cancer Ther.

Yan, B., Zhou, Y., Feng, S., Lv, C., Xiu, L., Zhang, Y., Shi, J., Li, Y., Wei, P., and Qin, Z. (2013). beta -Elemene-Attenuated Tumor Angiogenesis by Targeting Notch-1 in Gastric Cancer Stem-Like Cells. Evid Based Complement Alternat Med 2013, 268468.

Yang, Z.F., Ho, D.W., Ng, M.N., Lau, C.K., Yu, W.C., Ngai, P., Chu, P.W., Lam, C.T., Poon, R.T., and Fan, S.T. (2008). Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 13, 153-166.

Zhao, Y., Bao, Q., Renner, A., Camaj, P., Eichhorn, M., Ischenko, I., Angele, M., Kleespies, A., Jauch, K.W., and Bruns, C. (2011). Cancer stem cells and angiogenesis. Int J Dev Biol 55, 477-482.