膽道癌依照癌細胞生長的部位區分為肝內膽管癌與肝外膽管癌，無論是哪一型的膽管癌預後皆不甚理想。預後不理想的原因可能與膽道癌目前尚無有效的治療藥物有關，經影像醫學確診後，通常為手術切除癌症病灶，再配合化學藥物治療。此外，膽道癌通常伴隨淋巴轉移，但目前尚未完全解開發生淋巴轉移的原因。從我們的實驗中觀察到，當移除細胞培養基中的血清時，癌細胞內的Ｃ型血管內皮生長因子(VEGF-C)的蛋白質表現量會增加，隨著移除時間越長，蛋白的表現量增加越多。並且釋放出可溶性的Ｃ型血管內皮生長因子到培養基中，相較於未移除血清的組別在完全移除的組別中偵測到更高量的表現。在先前的報導中指出Ｃ型血管內皮生長因子與癌細胞的轉移相關，而最近的研究也表明Ｃ型血管內皮生長因子與血管內皮生長因子接受器結合後活化淋巴管增生因而發生淋巴轉移或其他器官轉移。因此，我們推測隨著腫瘤增長，越接近中心的腫瘤細胞越無法從腫瘤周邊的血管得到充足的血液支持，因而發展出對抗較差的腫瘤微環境的機制。我們選用肝內膽管癌細胞株（SNU-1079和HuH28）作為研究的模型。由我們的實驗數據可得知癌細胞透過降低Ｃ型血管內皮生長因子蛋白質的降解釋放較大量的因子到培養基中，促進了癌細胞的自身的遷徙能力。此外，我們也發現培養基中的Ｃ型血管內皮生長因子對於促進淋巴內皮細胞的遷徙能力和淋巴管增生有正向的相關。未來可以再加入肝內膽管癌細胞作為研究模型，以了解是否膽道癌細胞都有因應惡劣生存條件所發展出的生存機制。透過我們的研究，再結合臨床上的觀察，或許可以釐清為何膽道癌經常伴隨著淋巴轉移，將來亦可應用於膽道癌藥物的研究開發或是作為診斷轉移的生物標識。

Lymphatic system is important for the maintenance of fluid homeostasis, transportation of macro- molecules and immune cells trafficking. Most of lymphatic networks are formed during embryonic development; while neo-lymphangiogenesis occurs in certain pathological conditions, for instance inflammation and tumorigenesis. Expression of vascular endothelial growth factor-C (VEGF-C), one of the major mediators involved in lymphangiogenesis, in the cancer cells was noted in 30% of resected intra-hepatic cholangiocarcinomas (iCCA) that was associated with the presence of lymphatic invasion, lymph node metastasis and significantly inferior survival as compared to those tumors without VEGF-C expression cancer cells (Aishima, Modern Pathol 2008). However, the molecular mechanisms of regulating VEGF-C expression and its biological function in iCCA are seldom reported. In current study, we found all two tested iCCA cell lines (SNU1079 and Huh28) do not only express and secrete VEGF-C but also express VEGFR3 as detected by either Western blot or ELISA. The expression and secretion of VEGF-C in all three iCCA cells were enhanced by serum starvation in a dose (0% and 2% vs. 10%)- and largely time (0, 0.5, 1, 2, 6 and 24 hrs)-dependent manners. Of the latter, the increase of VEGF-C was noted as early as 0.5 hr and reaching plateau at 6 hrs after serum starvation. The serum starvation-induced increase of VEGF-C protein level was accompanied with a modest increase of mRNA level (<50% by quantitative RT-PCR) but a significantly prolonged VEGF-C half-life, 1-2.5 hrs in 10% serum vs. not reached at 6 hrs in 0% serum, after cycloheximide treatment. Serum starvation also facilitated the transwell migration activity of SNU1079 and Huh28, which could be partially alleviated by VEGF-C targeted, multi-tyrosine kinase inhibitor. More specific pharmacological and/or genomic approaches will be required to confirm the role of VEGF-C/VEGFR3 axis in enhancing iCCA migration under serum starvation condition. However, our findings suggest a novel mechanism of cancer cell-derived VEGF-C in promoting lymphatic invasion and lymph node metastasis in iCCA that deserves further exploration.

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