Establishment and characterization of an in vitro 3D ovarian cancer model for drug screening assays

Abstract

The acquired drug chemoresistance represents the main challenge of the ovarian cancer treatment. In addition, the absence of an adequate in vitro model able to reproduce the native tumor environment can contribute to the poor success rate of preclinical studies of new compounds. Three-dimensional (3D) culture models have been recently used for drug screening purposes due to their ability to reproduce the main characteristics of in vivo solid tumors. Here we describe the establishment and characterization of 3D ovarian cancer spheroids using different adenocarcinoma tumor cell lines (SKOV-3 and OVCAR-3 cells) in two different 3D scaffold-free methods: forced-floating in ultra-low attachment (ULA) plates and hanging drop (HD). Spheroids were evaluated in both 3D cultures in order to establish the best condition to perform the drug response analysis with Paclitaxel, a common drug used to treat ovarian cancer. SKOV-3 and OVCAR-3 spheroids with the desired characteristics (roundness close to 1.0 and diameter in the 200-500 μm range) were obtained using both methods after addition of the methylcellulose (MC) in the culture medium (0.25% and 0.5%, w/v). We also observed the presence of microvilli on the surface of the spheroids, higher presence of apoptotic cells and higher drug resistance, when compared with 2D cultures. The 3D cultures obtained seem to provide more reliable results in terms of drug response than those provided by 2D monolayer culture. The forced floating method was considered more suitable and straightforward to generate ovarian cancer spheroids for drug screening/cytotoxicity assays.

Generation of a Three-Dimensional in Vitro Ovarian Cancer Co-Culture Model for Drug Screening Assays

“…This behavior was similar to that observed in our previous work with 3D ovarian cells monoculture. 15 The absence or lower cell growth of 3D cultures can be associated with an increased ability in recapitulating the in vivo ovarian tumor. During the ascites process, the spheroid-like structures acquire the ability to survive in an environment without a solid support.…”

“…16 The SKOV-3 cell density (12.5 £ 10 3 cells/mL) and MC concentration (0.25%, v/v) used were based on our previous results. 15 After cell seeding, the ULA plate was centrifuged (300 x g by 10 min).…”

“…14 In our previous work, we established and characterized a 3D ovarian cancer model using epithelial adenocarcinoma cells (SKOV-3 and OVCAR-3) cultured as spheroids in ultra-low attachment (ULA) and hanging-drop plates. 15 Herein, in an attempt to increase the complexity and the relevance of the model, we describe the establishment of 3D multicellular spheroids using SKOV-3 cells in co-culture with mesenchymal umbilical cells (MUC-9) and fibroblasts (CCD27-Sk) in ULA plates. We demonstrated that SKOV-3 cells co-cultured with MUC-9 and CCD-27Sk were able to form regular and compact spheroids with diameters ranging from 300 to 400 mm and with a roundness close to 1.0.…”

Mechanical Studies of the Third Dimension in Cancer: From 2D to 3D Model

“…OVCAR-3 cells were recently used to create spheroids by forced floating in ultra-low attachment plates and hanging drop methods to perform drug response analysis with paclitaxel, a common drug used to treat ovarian cancer. Spheroids maintained the cell density and higher apoptosis behavior of ovarian cancer, as well as higher resistance against paclitaxel treatment when compared with 2D culture, closely mimicking the in vivo response [ 90 ]. Successful spheroids can also be generated from patient-derived tissue samples, such as 3D spheroid suspension cultures from radical prostatectomy specimens to model organ-confined prostate cancer.…”

News and Views

“…Adequate in vitro models of ovarian cancer which reproduce the native tumour environment would be beneficial for screening new therapies or for basic studies into the molecular mechanisms involved in the disease. A study by Tofani and colleagues 1 has described the development and characterisation of 3-D in vitro models of ovarian cancer. To determine the most appropriate set of culture conditions, they used two adenocarcinoma tumor cell lines (SKOV-3 and OVCAR-3 cells), and established the 3-D cultures by using two different scaffold-free methods (forced-floating in ultra-low attachment (ULA) plates and the hanging drop (HD) method).…”