肺癌幹細胞株之無餵養細胞培養系統

Abstract

腫瘤幹細胞(Cancer stem cells, CSCs)是腫瘤細胞中，特別具有自我更新、腫瘤細胞分化、腫瘤起始能力以及抗藥性的亞群。因此，開發出針對腫瘤幹細胞的標靶藥物被認為是治療癌症新策略的關鍵。腫瘤幹細胞的維持主要是藉由在腫瘤微環境中腫瘤相關纖維母細胞作為餵養細胞，餵養細胞能壯大腫瘤幹細胞群體並促進腫瘤幹細胞相關基因的表現。腫瘤幹細胞與腫瘤相關纖維母細胞共培養系統可有助於腫瘤幹細胞標靶藥物的篩選。然而，須由病人檢體培養的腫瘤相關纖維母細胞，其來源受個體差異而影響腫瘤幹細胞及藥物篩選的效力與再現性。本研究中，目的是要建立一個無餵養細胞的腫瘤幹細胞培養系統。基於轉錄體學與細胞激素的蛋白質維陣列分析，我們從腫瘤幹細胞與腫瘤相關纖維母細胞共培養後收集到的細胞共培養液所含的細胞激素分析中，找出了幾個細胞激素，這些細胞激素存在於腫瘤微環境中，能有助於促進與維持腫瘤幹細胞的幹性基因。隨後，我們試圖從這些細胞激素中，以單獨及多個組合的方式，找到能維持腫瘤幹細胞特性的細胞激素組合。我們發現IGF-1結合IL-6可以顯著增加腫瘤球數和促進Nanog基因表達，這種組合也可以改善腫瘤幹細胞的透明質酸立體細胞培養盤。我們的結果說明通過使用IGF-1和IL-6細胞激素組合來維持腫瘤幹細胞特性的無餵養細胞培養系統是可行的，而此系統有助於建立腫瘤幹細胞的標靶藥物篩選系統。

Cancer stem cells (CSCs) are sub-populations of tumor cells with the properties of self-renewal, clonal tumor initiation ability, drug-resistance, and for tumor initiation. Hence, targeting CSCs has been suggested as the key for development of new treatment strategy to cure cancer. CSCs reside in the microenvironment, which are majorly supported via the cancer-associated fibroblasts (CAF), as the feeder cells. The feeder cells could enrich the CSCs population and promote the stemness-associated gene expression in the CSC populations. The co-culture system is helpful to establish a model for CSC-targeting drug screening. However, the sources of CAFs and the heterogeneity of such stromal cells might affect the CSC and the drug-screening efficacy and reproducibility. Here, we wanted to setup a feeder-free culture system for CSC. Base on the CSCs/CAFs co-culture system, we have identified the CSCs-dependent micro-environmental factors, which may enrich and maintain their “stemness” through identifying the cytokine profiling in the co-culture condition medium. Then, we tried to find the suitable combination of CSCs-dependent cytokines for establishing a feeder free culture system of lung CSCs. Base on the transcriptomic analysis and protein cytokine array, several candidate cytokines were performed to examine the individual and combinatory effects on maintaining the CSCs characteristics. ry to find the suitable combination of different cytokines for establishing a feeder free culture system of lung CSCs. Here, we found IGF-1 combined with IL-6 could significantly increase the tumorous sphere numbers and promote Nanog gene expression. Such combination could also improve the CSCs 3-D culture in the HA-chitosan Plates. Above all, our results show the promising to maintain CSCs in the feeder-free culture system by using IGF-1 and IL-6 combination. This model could be helpful for establish the drug screening system for targeting on CSCs.