engleski

Let's go 3D: the future of prostate cancer models

Tissues are three-dimensional (3D) entities as well as the tumor that arises within them. Therefore, to study the complexity of tumor model it is necesary to develop more realistic model than classical 2D monolayers used so far. Even though traditionally used for decades, because of vast benefits they provide, 2D cell cultures do not meet the demands in terms of being able to effectively mimic complex cellular signaling, angiogenesis, invasion and metastasis present in cancer. Moreover, 2D monolayer represents overly simplified extracellular matrix (EMC) which has been proven unsuccessful in indentifying preventative anti-cancer treatments. The aim of this research was to establish a platform for development of multicellular 3D spheroid models of prostate cancer which can easily be produced and maintained in our laboratory setting. More precisely, by producing such a model we hoped to elucidate the tumor-stromal cell interaction in prostate cancer, which is of great importance for prostate cancer research but it was difficult to study in 2D environment. One of the relevant pathways in prostate cancer progression is the Hedgehog-GLI (HH-GLI) signaling pathway which is crucial for normal embryonic development, stem cell maintenance and tissue homeostasis in adult organisms but it seems hyperactivated in various tumors, including prostate cancer. The final goal is to better understand the differences in HH-GLI signaling pathway in co-culture of prostate cancer and stromal cells while comparing three- dimensional (spheroid) models with two-dimensional (adherent) setting used so far. We have produced 3D models (spheroids) of prostate cancer, using adenocarcinoma prostate cell line LNCaP and cancer-associated prostate fibroblasts WPMY-1 as a monoculture or co-culture by the means of hanging drop system. Cells were grown separately for 72 hours depending on the cell line, at 37 °C, 95% relative humidity in a 5% CO2 and air atmosphere. Afterwards, each cell line was counted at density of 1x106/ml, labeled with membrane-integrating fluorescent dyes and mixed in diverse ratios before setting them into hanging drops for spheroid formation. These spheroids were subsequently trypsinized and analyzed on the MUSE Cell Analyzer to measure cell ratios and cell viability, in order to establish optimal experimental conditions (cell number per sphere, duration of cultivation). Furthermore, spheroids were subjected to the cryosectioning. We collected each spheroid by pipetting and embedded them in the CryoFix gel, followed by freezing in liquid nitrogen vapor. Frozen blocks were cut on the cryotome and preserved at -80°C until staining. Cryosections of 3D spheroids were then stained by immunofluorescent staining for HH-GLI pathway proteins important for the prostate cancer development (SHH, PTCH, GLI1, GLI2, GLI3) and epithelial/mesenchymal markers (E-cadherin, vimentin) to determine the localization of proteins in the 3D model and the HH-GLI pathway activity in tumor vs. stromal cells. We established a simple, cost effective and straightforward protocol for development of multicellular 3D spheroid models of prostate cancer which can easily be produced and maintained in our laboratory setting. Cancer and stromal cells are forming spheroids of different shapes and sizes. Immunofluorescent staining for HH-GLI pathway proteins and epithelial/mesenchymal markers showed correlation with previous expression analyses of the key genes in question. It is visible that stromal cells are changing their characteristics to the higher extent compared to the cancer cell lines. Results obtained so far are pointing out the importance of interaction between the cancer cells and cancer associated fibroblast in the prostate cancer progression.

Prostate cancer ; HH-GLI signaling pathway ; spheroids

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