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)