Influence of TP53 and CDH1 genes in hepatocellular cancer spheroid formation and culture: a model system to understand cancer cell growth mechanics

Abstract

BackgroundSpheroid based culture methods are gaining prominence to elucidate the role of the microenvironment in liver carcinogenesis. Additionally, the phenomenon of epithelial-mesenchymal transition also plays an important role in determining the metastatic potential of liver cancer. Tumor spheroids are thus important models to understand the basic biology of liver cancer.MethodsWe cultured, characterized and examined the formation of compact 3-D micro-tumor spheroids in five hepatocellular carcinoma (HCC) cell lines, each with differing TP53 mutational status (wt vs mutant vs null). Spheroid viability and death was systematically measured over a course of a 10 day growth period using various assays. We also examined the TP53 and E-cadherin (CDH1) mRNA and protein expression status in each cell line of the 2-D and 3-D cell models.ResultsA novel finding of our study was the identification of variable 3-D spheroid morphology in individual cell lines, ranging from large and compact, to small and unstable spheroid morphologies. The observed morphological differences between the spheroids were robust and consistent over the duration of spheroid culture growth of 10 days in a repeatable manner. Highly variable CDH1 expression was identified depending on the TP53 mutational status of the individual HCC cell line, which may explain the variable spheroid morphology. We observed consistent patterns of TP53 and CDH1 expression in both 2-D and 3-D culture models.ConclusionsIn conclusion, we show that 3-D spheroids are a useful model to determine the morphological growth characteristics of cell lines which are not immediately apparent in routine 2-D culture methods. 3-D culture methods may provide a better alternative to study the process of epithelial-mesenchymal transition (EMT) which is important in the process of liver cancer metastasis.

Exosomes derived from 3D-cultured MSCs improve therapeutic effects in periodontitis and experimental colitis and restore the Th17 cell/Treg balance in inflamed periodontium

“… 80 Recent studies have shown that as a key transcription factor of CDH1 that participates in the formation of MSC spheroids, 67 , 81 the TP53 status is crucial in determining the morphology and size of the spheroidal structures. 82 Hence, when DPSCs form aggregates under low-attachment conditions through enhanced cadherin binding induced by TP53, TP53 may simultaneously increase the expression of miR-1246, leading to its increased level in exosomes. Another suggested mechanism acts on the epigenetic level.…”

Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells

“…Differential E-cadherin expression levels have been associated with altered spheroid formation in head and neck carcinoma cell lines 15 . Differential E-cadherin expression was also associated with compact spheroid formation in hepatocellular carcinoma cell lines 16 , 17 and in renal cell carcinoma 18 . In addition spheroid models were used to identify cooperative roles for E-cadherin and the desmosome proteins DSG2 and DSC2 in colon and breast carcinoma cell lines 19 .…”

<p>Genetic And Epigenetic Regulation Of E-Cadherin Signaling In Human Hepatocellular Carcinoma</p>

“…One SNP array analysis of 125 HCC patients revealed that 20.8% of TP53 mutations were inactivating mutations 52. Recently, it was found that TP53 mutant cell lines showed a smaller cell size in a hepatocellular cancer spheroid formation test, which resulted from the lower CDH1 expression levels 53. Moreover, evidence demonstrated the mutant-p53 could affect the expression of Twist1 and Smad interacting protein 1 (SIP1) and further regulate E-cadherin in adrenocortical carcinoma 54.…”