Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines.
Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines.
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DOI：
10.1186/s12935-021-01898-9
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发表时间：
2021-04-13
影响因子：
5.8
通讯作者：
Madjd Z
中科院分区：
文献类型：
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作者：
Gheytanchi E;Naseri M;Karimi-Busheri F;Atyabi F;Mirsharif ES;Bozorgmehr M;Ghods R;Madjd Z
结直肠癌的复发和转移通常归因于肿瘤干细胞(CSCs)，它是一种具有耐药能力的小亚群肿瘤细胞。因此，有必要发展合适的模型来研究CSCs的生物学和建立有效的治疗策略。因此，我们的目标是评估两个广泛使用和重要的结直肠癌细胞系HT-29和Caco-2产生球体的能力及其详细的形态和分子特征。在无血清和无附着条件下，采用悬滴和强制漂浮的方法制备结直肠癌球体，并用扫描电子显微镜(SEM)观察其形态特征。然后，通过连续球体形成能力的分析、关键干性基因(KLF4、OCT4、SOX2、NANOG、C-MYC)的实时聚合酶链式反应以及潜在的CRC-CSCs表面标志(CD166、CD44和CD133)的表达，比较CSCs在球体和贴壁细胞中的富集性。检测EMT相关基因(Vimentin、SNAIL1、Twist1、N-cadherin、E-cadherin、ZEB1)和多药耐药基因(ABCB1、ABCC1、ABCG2)的表达水平。尽管具有不同的形态特征，但这两个细胞系都形成了富含CSCs的球体，表明它们能够形成连续球体，茎系基因SOX2、C-myc、NANOG和OCT4在HT-29中显著上调，在Caco-2球体中SOX2、C-myc和KLF4的表达显著上调(p值 <； 0.05)，并且与亲本细胞相比，CRC-CSC标记的表达增加(p值 <； 0.05)。此外，HT-29球体显示ABCB1和ABCG2的表达显著增加(p值 = 0.02)。促EMT基因ZEB1、Twist1、E-钙粘素和SNAIL1在HT-29球体中显著上调(p值 = 0.03)，SNAIL1和Vimentin在Caco-2球体中显著上调(p值 <； 0.05)，N-钙粘附素在两种球体中表达下调。在HT-29和Caco-2(首次不使用特殊支架/生化)球体中丰富了CSC相关特征，表明球体培养是一种健壮、可重复、简单和经济有效的模型，可以模拟体内肿瘤的复杂性，包括自我更新、耐药和侵袭，用于体外研究CRC-CSCs。
Relapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics. CRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-cadherin, E-cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated. Although with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value < 0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value < 0.05). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 (p-value = 0.02). The significant up-regulation of promoting EMT genes, ZEB1, TWIST1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value = 0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value < 0.05) and N-cadherin down-regulation in both spheroids were observed. Enrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.