Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines

Elmira Gheytanchi,Zahra Madjd,Roya Ghods,Feridoun Karimi-Busheri,Marzieh Naseri,Ensie Sadat Mirsharif,Mahmood Bozorgmehr,Fatemeh Atyabi

doi:10.1186/s12935-021-01898-9

Abstract

BackgroundRelapse 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.MethodsCRC 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.ResultsAlthough 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.ConclusionEnrichment 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.

Highlights

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
Most of the challenges in cancer treatment such as treatment failure, tumor aggressiveness, relapse, metastasis and poor prognosis are related to CSCs characteristic, most of which are attributed to epithelial to mesenchymal transition (EMT) potential, stemness signaling pathways regulating pluripotency, as well as the high expression of ATP-binding cassette (ABC) transporter genes in this subpopulation [11,12,13,14,15,16,17,18]
Spheroid formation in non-adherent condition on poly-HEMA coated dishes at different cell densities was applied for generation of spheroids from HT-29 and Caco-2 adherent cells

Summary

Introduction

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. CSCs are referred to as a sub-population of tumor cells with stem cell-like properties including self-renewal and multi-lineage differentiation capacity which are resistant to conventional therapies [6,7,8,9]. Most of the challenges in cancer treatment such as treatment failure, tumor aggressiveness, relapse, metastasis and poor prognosis are related to CSCs characteristic, most of which are attributed to epithelial to mesenchymal transition (EMT) potential, stemness signaling pathways regulating pluripotency, as well as the high expression of ATP-binding cassette (ABC) transporter genes in this subpopulation [11,12,13,14,15,16,17,18]. Signal transduction pathways and microenvironmental signals in CSCs are considered the main therapeutic aspects linked to stem-cell biology that can be targeted [13, 22, 23]

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