Background

Epithelial ovarian cancer remains the deadliest gynecological malignancy with an estimated five-year survival rate below 29% for patients with advanced stage disease, which is when most patients are first diagnosed (Torre et al., 2018). At these later stages, ovarian cancer cells have metastasized to distant sites across the peritoneum. This spread of ovarian cancer across the peritoneal cavity is facilitated by the passive dissemination of tumor cells directly into the peritoneal ascites fluid. In the malignant ascites, tumor cells can be found as both single cells and as multicellular clusters, also commonly referred to as “spheroids”. Formation of spheroid aggregates, which are enriched in cancer stem cells (Liao et al., 2014; Kenda Suster and Virant-Klun, 2019), are key to the progression of ovarian cancer, as these ensure maintenance of intercellular survival signaling among cells detached from the primary tumor (Tan et al., 2006; Al Habyan et al. 2018). In addition, cancer stem-like cells in spheroids display metabolic flexibility. They shift their metabolism based on nutrient availability and metabolic demands, potentially allowing tumor cells to survive in ascites and invade the neighboring peritoneal organs (Anderson et al.., 2013; Kim et al.., 2020; Ghoneum et al., 2020). To advance our understanding of ovarian cancer metastatic progression and associated changes in tumor metabolism, it is important to develop methods that allow us to study the metabolic changes in anchorage-independent tumor spheroids, which mimic tumor cell clusters found in malignant ascites of patients.

The following protocol utilizes ultra-low attachment (ULA) plates to generate multi-cellular tumor cell spheroid clusters in order to study their metabolic phenotype using the Seahorse XFp (Agilent) extracellular flux assay platform. Spontaneous clustering of tumor cells in anchorage independence is facilitated by the lack of ability to attach to the hydrophilic neutrally charged hydrogel coating of ULA plates. Compared to cells cultured in conventional attached monolayer conditions on plastic surfaces, spheroids more closely reflect the metastatic tumor cells found in anchorage-independent multicellular clusters in malignant ascites. This makes spheroids a good in vitro model to better simulate the in vivo behavior of tumor cells and to study their unique characteristics related to cell survival, drug sensitivity, hypoxia, and tumor metabolism. During optimization of this protocol, we determined that uniform spheroid formation is integral to minimizing variability in extracellular flux measurements. The use of ULA platforms with small well sizes, such as 96 or 384 wells per plate, permits the generation of uniformly sized spheroids by controlling the seeding density. The use of bioprinting in combination with magnetic nanoparticles has been investigated as an alternate method for metabolic studies of 3D tumor-fibroblast co-cultures (Noel et al., 2017). However, for cell types that spontaneously cluster into spheroids in response to anchorage independence, the ULA culture platform represents a relatively straightforward approach to generate uniform multicellular clusters, without the need for further manipulation.

The Seahorse extracellular flux analyzers specifically measure oxygen consumption (OCR) and extracellular acidification rates (ECAR), allowing for the quantification of mitochondrial respiration and an indirect indication of glycolysis, respectively. Several past studies have interrogated mitochondrial function in 3D cultures using single well assays and measured the response to single or a limited number of metabolic stress compounds, with the need to independently determine the rates of oxygen consumption and extracellular acidification (Rodríguez-Enríquez et al., 2008; Schafer et al., 2009; Mandujano-Tinoco et al., 2013; Bloch et al., 2014; Marin-Hernandez et al., 2017). We have optimized the transfer of spheroids from 96 well ULA plates to the Seahorse cell culture plates and the use of this platform for assessment of spheroid bioenergetics, allowing for multi-well analysis and sequential addition of pharmacological agents to manipulate OCR/ECAR. Below are described the use of the mitochondrial stress test and the glycolysis stress test, together with how spheroid culturing and transfer is best achieved. Using substrate limited media and pharmacological approaches, the assay is easily modified to gain further insight into the specific metabolic activity of spheroids. Although not discussed in detail below, iterations of the extracellular flux assay include the palmitate oxidation stress test, which is a modified assay that determines the capacity of cells to oxidize palmitate in the absence or limitation of other exogenous carbon substrates. We refer the reader to the Seahorse Agilent website for alternate assays to assess substrate utilizations. The protocol was developed using the Agilent Seahorse XFp Metabolic Analyzer, and can be applied to the Seahorse XF96 format, as these two platforms utilize cell culture plates with similar surface areas and media volumes. It can also be adapted for the larger surface area of the XF24 platform wells. Hence, the method explained in this paper can be used as a master template for performing not only the mentioned mitochondrial and glycolysis stress tests, but also altered extracellular flux assays with 3D anchorage-independent tumor cell spheroids.

The method outlined below was originally designed for the study of ovarian cancer spheroids cultured in anchorage-independent conditions, which mimic tumor cell clusters commonly found in malignant ascites of ovarian cancer patient (Kim et al., 2020). It can be adapted to other cancer cell lines, primary cells, stem cell cultures, or cell types where 3D cell culture represents a more patho/physiologically relevant culture condition.