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In the last decade, three-dimensional (3D) cell culture technology has gained a lot of interest due to its ability to better recapitulate the in vivo organization and microenvironment of in vitro cultured cancer cells. In particular, 3D tumor models have demonstrated several different characteristics compared with traditional two-dimensional (2D) cultures and have provided an interesting link between the latter and animal experiments. Indeed, 3D cell cultures represent a useful platform for the identification of the biological features of cancer cells as well as for the screening of novel antitumor agents. The present review is aimed at summarizing the most common 3D cell culture methods and applications, with a focus on prostate cancer modeling and drug discovery.

Epithelial-to-mesenchymal transition (EMT) is a step-wise process observed in normal and tumor cells leading to a switch from epithelial to mesenchymal phenotype. In tumors, EMT provides cancer cells with a metastatic phenotype characterized by E-cadherin down-regulation, cytoskeleton reorganization, motile and invasive potential. E-cadherin down-regulation is known as a key event during EMT. However, E-cadherin expression can be influenced by the different experimental settings and environmental stimuli so that the paradigm of EMT based on the loss of E-cadherin determining tumor cell behavior and fate often becomes an open question. In this review, we aimed at focusing on some critical points in order to improve the knowledge of the dynamic role of epithelial cells plasticity in EMT and, specifically, address the role of E-cadherin as a marker for the EMT axis.

We aimed at analyzing the effect of the 3D-arrangement on the expression of some genes and proteins which play a key role in pancreatic adenocarcinoma (PDAC) progression in HPAF-II, HPAC and PL45 PDAC cells cultured in either 2D-monolayers or 3D-spheroids. Cytokeratins 7, 8, 18, 19 were differently expressed in 3D-spheroids compared to 2D-monolayers. Syndecan 1 was upregulated in HPAF-II and PL45 3D-spheroids, and downregulated in HPAC. Heparanase mRNA levels were almost unchanged in HPAF-II, and increased in HPAC and PL45 3D-spheroids. Hyaluronan synthase (HAS) 2 and 3 mRNA increased in all 3D-spheroids compared to 2D-monolayers. CD44 and CD44s were expressed to a lower extent in HPAF-II and HPAC 3D-spheroids. By contrast, the CD44s/v3 and the CD44s/v6 ratio increased in HPAC and PL45 3D-spheroids, compared to 2D-monolayers. The expression of MMP-7 was strongly upregulated in 3D-spheroids. STAT3 was similarly expressed 3D-spheroids or 2D-monolayers, while pSTAT3 was almost undetectable in 2D-monolayers and strongly upregulated in 3D-spheroids. These results suggest that 3D-spheroids represent a cell culture model that allows the characterization of PDAC cell phenotype, adding new information that contributes to a better understanding of the biology and behavior of PDAC cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell lines, characterized by different EMT-related phenotypes, cultured in 2D monolayers and 3D spheroids. We observed that the YAP/p-YAP ratio was reduced in HPAC and MIA PaCa-2 cell lines and remained unchanged in BxPC-3 cells when cultured in a 3D setting. CTGF and CYR61 gene expression were down-regulated in all PDAC 3D compared to 2D cultures, without any significant effect following actin cytoskeleton inhibition by Cytochalasin B (CyB) treatment. Moreover, LATS1 mRNA, indicating the activation of the Hippo pathway, was not influenced by CyB and differed in all PDAC cell lines having different EMT-related phenotype but a similar pattern of CTGF and CYR61 expression. Although the role of YAP modulation in response to mechanical cues in cancer cells remains to be completely elucidated, our results suggest that cell arrangement and phenotype can determine variable outcomes to mechanical stimuli in PDAC cells. Moreover, it is possible to speculate that YAP and Hippo pathways may act as parallel and not exclusive inputs that, converging at some points, may impact cell behavior. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) in the tumor microenvironment modulates the cancer cell phenotype, especially in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by an intense desmoplastic reaction. Because the epithelial-to-mesenchymal transition (EMT), a process that provides cancer cells with a metastatic phenotype, plays an important role in PDAC progression, the authors aimed to explore in vitro the interactions between human PDAC cells and ECM components of the PDAC microenvironment, focusing on the expression of EMT markers and matrix metalloproteinases (MMPs) that are able to digest the basement membrane during tumor invasion. EMT markers and the invasive potential of HPAF-II, HPAC, and PL45 cells grown on different ECM substrates (fibronectin, laminin, and collagen) were analyzed. While N-cadherin, αSMA, and type I collagen were not significantly affected by ECM components, the E-cadherin/β-catenin complex was highly expressed in all the experimental conditions, and E-cadherin was upregulated by collagen in PL45 cells. Cell migration was unaffected by fibronectin and delayed by laminin. In contrast, collagen significantly stimulated cell migration and the secretion of MMPs. This study's results showed that ECM components impacted cell migration and invasive potential differently. Collagen exerted a more evident effect, providing new insights into the understanding of the intricate interplay between ECM molecules and cancer cells, in order to find novel therapeutic targets for PDAC treatment. [ABSTRACT FROM AUTHOR]

The tumor microenvironment influences cancer cell behavior in relation to tumor progression, as well as cell proliferation and invasion. Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense desmoplastic reaction and extracellular matrix (ECM) components in the tumor microenvironment are involved in a crosstalk between tumor cells, stromal fibroblasts and ECM components, influencing tumor cell behavior. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and the ECM of the microenvironment by culturing PDAC cells on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell phenotype and the proteins occurring in the desmoplastic tissue. For this purpose, we analyzed some epithelial-to-mesenchymal transition (EMT) markers and the migration and invasive potential in human HPAF-II, HPAC and PL45 PDAC cells cultured on collagen type I (COL), laminin (LAM) and fibronectin (FN). Interestingly, the expression of E-cadherin was not significantly affected, but some differences were revealed by the wound healing assay. In fact, migration of HPAFII and PL45 cells was decreased on FN and LAM, and increased on COL, compared to control cells grown on plastic (NC). By contrast, HPAC was very rapid and unaffected by the substrate. SDS-zymography showed that COL induced a strong upregulation of MMP-2 activity in HPAF-II and HPAC cells, and of MMP-9 in HPAF-II and PL45 cells, compared to NC. These preliminary results suggest that ECM components could differently affect PDAC migration and invasion, possibly depending on the differentiation grade. The characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is one of the most common male neoplasm in the western world, being the most commonly diagnosed non-skin cancer and the second leading cause of cancer death. Various potential risk factors exist for the initial triggering events, including exposure to infectious agents, such as the human Polyomavirus BK (BKV). BKV is a good candidate as risk factor of PCa because it naturally infects the human reno-urinary tract, it establishes latency, and encodes oncoproteins that interfere with tumor suppressors pathways, thus altering the normal progression of cell cycle. Previous studies suggested a potential association between BKV and PCa, revealing that the prevalence of BKV was significantly higher in cancer than in control tissues, with a significant association between viral expression and cancer. However, this hypothesis is controversial because BKV is not restricted to tumor tissues but also infects healthy individuals in a high percentage. Moreover, an in vitro model of BKV infection in prostate cells is not available to understand the role for BKV in pathogenesis of PCa. Our aims were to determine whether BKV a) could infect normal epithelial prostate cells, b) affects cell phenotype and c) affects the phenotype of human prostate tumor cell line PC3. For this purpose normal epithelial prostate cell line RWPE-1 and prostate cancer cells PC3 were infected with BKV for 21 days. Cell proliferation, epithelial-to-mesenchymal markers (EMT) and invasion potential were analyzed by, respectively, MTT, immunofluorescence and SDS-zymography. Our results show that cell proliferation was increased or decreased by BKV, respectively, in RWPE-1 and PC3 cells. BKV induced E-cadherin downregulation and vimentin expression in both control and BKV-infected cells RWPE-1, suggesting that uninfected cells underwent EMT. Matrix metalloproteinase-2 and 9 activity was increased in RWPE-1 cells after BKV infection. By contrast, BKV did not significantly modified the phenotype of PC3 cells. These preliminary results suggest that normal epithelial prostate cells RWPE-1 and PC3 are susceptible and permissive to BKV infection. However, RWPE-1 cells exhibit some phenotype modifications related to EMT, possibly induced by the papilloma virus used to obtain their immortalization, thus suggesting that further experiments will be necessary to define if they represent a good experimental model to study prostate cancer. [ABSTRACT FROM AUTHOR]