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

Cancer Cell International, Jun 2016

Background Spheroid 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. Methods We 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. Results A 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. Conclusions In 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.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

http://www.cancerci.com/content/pdf/s12935-016-0318-1.pdf

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

Pomo et al. Cancer Cell Int Influence of TP53 and CDH1 genes in hepatocellular cancer spheroid formation and culture: a model system to understand cancer cell growth mechanics Joseph M. Pomo 0 Robert M. Taylor 0 Rama R. Gullapalli 0 1 0 Department of Pathology, University of New Mexico , Room 308, MSC06-4840, Albuquerque, NM 87131 , USA 1 Department of Chemi- cal and Biological Engineering, University of New Mexico , Room 333A, MSC08-4640, Albuquerque, NM 87131 , USA Background: Spheroid 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. Methods: We 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. Results: A 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. Conclusions: In 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. Hepatocellular carcinoma; Epithelial-mesenchymal transition; Tumor spheroids; TP53; CDH1 Background The incidence of hepatocellular carcinoma (HCC) has been steadily increasing in the past decade. HCC is currently one of the fastest growing cancers in the United States. The outcomes in cases of HCC are dismal, with a 5 year survival of less than 8 % for stage III and above [ 1, 2 ]. Globally, HCC is the fifth most common cancer and the third most deadly [2]. The majority of cases of HCC globally are due to Hepatitis viral infections, namely, Hepatitis B and C [ 2 ]. In the United States, the major cause of HCC is due to alcoholism, however, obesity is also a fast emerging risk factor in the United States [ 3, 4 ]. The tumor microenvironment plays an important role in modulating HCC tumor biology, as well the efficacy of chemotherapeutic treatments. Traditional research techniques of cancer biology utilize two-dimensional, monolayer cell cultures grown in flasks. However, the role of the spatial cell signaling cues of the tumor microenvironment in the formation and metastasis of tumors, including HCC, is being increasingly appreciated [ 5, 6 ]. Previous studies have noted differences in the gene expression patterns, morphological features, cell growth kinetics and metabolic rates of 3-D tumor spheroids compared to 2-D monolayer cultures [5]. 3-D tumor spheroids allow a controlled, stratified representation of the tumor microenvironment to mathematically model the influence of external perturbants such as chemotherapeutic drugs which are affected greatly by the tumor microenvironment [ 7 ]. It has been previously noted that 3-D representations of tumor spheroids are more resistant to radiotherapy and chemotherapy than 2-D cell cultures [ 7, 8 ]. This is similar to the patterns of radio and chemo-resistance noted in real clinical patient tumors. Thus, tumor spheroids represent a valuable model to examine issues related to radio and chemo resistance in patients. Additionally, it is possible to create complex tumor spheroid models by incorporating other cellular components such as fibroblasts or macrophages to create realistic models of the in  vivo tumor microenvironment [9]. The role of adhesion molecules such as integrins and E-cadherin (CDH1), in intra-cellular cancer cell signal transduction have been elucidated previously, including HCC [ 10–13 ]. The presence of cell–cell contacts as well as cell–matrix contacts play a crucial role in modulating the morphological and gene expression patterns of the cancer cells [ 10, 12 ]. The extra-cellular matrix (ECM) of a tumor microenvironment (...truncated)


This is a preview of a remote PDF: http://www.cancerci.com/content/pdf/s12935-016-0318-1.pdf

Joseph Pomo, Robert Taylor, Rama Gullapalli. Influence of TP53 and CDH1 genes in hepatocellular cancer spheroid formation and culture: a model system to understand cancer cell growth mechanics, Cancer Cell International, 2016, pp. 44, 16, DOI: 10.1186/s12935-016-0318-1