Prostate cancer (PrCa) cells form differentiated spheroids or "prostaspheres" in three-dimensional cell culture (3D culture) when embedded in extracellular matrix such as MatrigelTM, rat tail collagen, or hydrogel. While this has been pioneered and explored widely, e.g. in breast cancer, the characterization of prostate cancer cells in 3D culture has been seriously lagging behind. In this recent study, the authors have now systematically investigated the differentiation potential of over 30 prostate cancer cell lines in 3D culture. This included all of the classic PrCa lines, some of them established several decades ago, as well as a panel of more recently established PrCa lines, in vitro immortalized and transformed cell lines, plus primary prostate epithelial cells. This panel of cell lines results in a range of phenotypes and morphologies in 3D cultures, recapitulating many aspects of prostate cancer in vivo, ranging from acinar morphogenesis, branching, and tumor cell invasion. In particular, the formation of cell-cell contacts and the invasive features are not faithfully recapitulated by traditional 2D monolayer cultures. Additionally, 3D settings clearly demonstrate that many cell lines, but also primary cells, are highly heterogeneous, resulting in structures side-by-side with strikingly different morphologies.
Most of the transformed PrCa cell lines readily formed spheroids with defective epithelial integrity (mass- or grape-like spheroids, lacking proper cell polarization and without a lumen), a hallmark of cancers. In contrast, some of the most aggressive lines (e.g. PC3, DU145) formed well-differentiated structures, despite the fact that these cells are highly malignant and harbor many cancer-specific mutations and gene losses/amplifications. Spheroids from these cell lines essentially showed all features of functionally intact spheroids, and were almost indistinguishable from those of normal and non-transformed cells. However, the normal-like differentiation capacity of such PrCa spheroids in 3D culture was only temporary: these structures spontaneously transformed themselves into stellate, undifferentiated cell masses, indicative of tumor cell invasion and aggressive growth. Our data powerfully demonstrate that the tumorigenic and aggressive phenotype of tumor cell lines strongly depends on the tumor cell environment, and that signals from the extracellular matrix may override the tumor phenotype (and genotype) in favor of normal differentiation patterns, which are still functional.
This experimental system also provided us with the opportunity to test small molecule inhibitors targeting key pathways involved in cell growth, and invasion specifically. Compounds blocking the PI3Kinase and AKT pathways (phosphatidylinositole tris-phosphate kinase; protein kinase B) were the most specific drugs targeting tumor cell invasion with minimal effects on cell growth and proliferation. Furthermore, some PrCa lines undergo rapid growth arrest when embedded in MatrigelTM, indicating that these lines may not be resistant to the strong growth-inhibitory signals from the laminins in the surrounding tumor environment. In contrast to the temporary repression of invasive properties however, the induction of cellular senescence by ECM was permanent.
All of the invasive tumor cells highly expressed numerous markers for epithelial-to-mesenchymal transition (EMT), indicating that these invasive lines may have undergone an EMT - possibly in vitro. However, the expression of most of these EMT markers (e.g. Vimentin, Fibronectin, tenascin C, etc.) was consistently observed in both 2D culture and also in the well-differentiated spheroids in 3D culture. Expression of EMT markers and transcriptional regulators related to EMT (e.g. Snail and Slug; SNAI1 and SNAI2) were remarkably stable and did not significantly increase when these spheroids transformed themselves into stellate, invasive cell masses. This may indicate that EMT is necessary to provide tumor cells with basic invasive properties, but may not be sufficient to support invasive processes as such. It also demonstrates that it is difficult to make reliable conclusions about tumor cell invasiveness based on 2D cell culture experimentation. Our data show that PrCa cells can display an extreme degree of plasticity, and have the capacity to form strikingly different phenotypes with the potential of mutual transformation from one into the other; largely controlled by extracellular signals and growth conditions. This may eventually have consequences for our understanding of invasive processes, the formation of micro-metastases, and tumor cell migration in lymphatic and blood systems.
In summary, the analysis of PrCa cell lines in three-dimensional cell culture, even such as the PC3 and DU145 lines which have been studied for over 30 years, can still reveal fundamental insights into tumor cell biology that are completely elusive in traditional monolayer conditions.
Written by Härmä V, Virtanen J, Mäkelä R, Happonen A, Mpindi JP, Knuuttila M, Kohonen P, Lötjönen J, Kallioniemi O, and Nees M as part of Beyond the Abstract on . This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations, etc., of their research by referencing the published abstract.
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