Department of Surgery, Tulane University, New Orleans, LA, United States 70112; ; University of Mississippi Cancer Institute, Jackson, MS, United States 39216.
Chin J Cancer Res. 2013 Apr;25(2):134-42. doi: 10.3978/j.issn.1000-9604.2013.03.02.
Colon cancer stem cells (CSCs) are implicated in colorectal cancer carcinogenesis, metastasis, and therapeutic resistance. The identification of these cells could help to develop novel therapeutic strategies. Doublecortin-like kinase 1 (DCLK1) has been viewed as a marker for gastrointestinal stem cells that fuel the self-renewal process, however others view them as a marker of Tuft cells or as an enteroendocrine subtype. The purpose of this study was to use a colon cancer cell line to identify and characterize the stem-like characteristics of the DCLK1+ cell population.
To enrich stem-like cells, HCT116 cells (derived from colon adenocarcinomas) were cultured using serum-free media to form spheres under both normal oxygen and hypoxia condition. DCLK1 transcript expression in the adherent parental cells and spheroids was quantified using quantitative real time reverse transcription- polymerase chain reaction [(q)RT-PCR]. DCLK1 protein expression was determined using flow cytometry. Self-renewal capability from adherent parental cells and spheroids was determined using extreme limiting dilution analysis (ELDA).
Under both normal oxygen and hypoxia condition, the adherent parental cells were composed of cells that express low levels of DCLK1. However, spheroids exhibited an increased frequency of cells expressing DCLK1 on both mRNA and protein levels. Cells derived from spheroids also possess stronger self-renewal capability.
The higher fraction of DCLK1+ cells exhibited by spheroids and hypoxia reflects the stem-like characteristics of these cells. DCLK1 may represent an ideal marker to study and develop effective strategies to overcome chemo-resistance and relapse of colon cancer.
结肠癌细胞（CSCs）被认为参与了结直肠癌的发生、转移和治疗耐药性。这些细胞的鉴定可能有助于开发新的治疗策略。双皮质素样激酶 1（DCLK1）被认为是胃肠道干细胞的标志物，这些干细胞为自我更新过程提供动力，然而，其他人认为它们是 Tuft 细胞的标志物，或作为肠内分泌亚型的标志物。本研究的目的是使用结肠癌细胞系鉴定和表征 DCLK1+细胞群体的类干细胞特征。
为了富集类干细胞，HCT116 细胞（源自结肠腺癌）在无血清培养基中培养，在正常氧和低氧条件下形成球体。使用实时定量逆转录聚合酶链反应（qRT-PCR）定量测定贴壁亲本细胞和球体中 DCLK1 转录物的表达。使用流式细胞术测定 DCLK1 蛋白表达。使用极端极限稀释分析（ELDA）从贴壁亲本细胞和球体确定自我更新能力。
在正常氧和低氧条件下，贴壁亲本细胞由表达低水平 DCLK1 的细胞组成。然而，球体在 mRNA 和蛋白水平上均表现出 DCLK1 表达增加的频率。源自球体的细胞也具有更强的自我更新能力。
球体和低氧条件下 DCLK1+细胞的较高比例反映了这些细胞的类干细胞特征。DCLK1 可能代表研究和开发有效策略以克服结肠癌化疗耐药性和复发的理想标志物。
