The tumor-associated extracellular matrix (ECM) provides critical biochemical micro-environment cues, as well as an essential structural scaffold, for solid tumors to survive and grow (see Pickup et al. 2014 for review). With a view to enabling more translational and turnkey 3D in vitro assays for cancer biology, we have developed and optimized techniques for seeding, growing and automatically quantifying the properties of multiple tumor spheroids in ECMs using real-time live-cell analysis. 

Matrigel (Corning) was dispensed across a range of volumes (20 – 50 L) and concentrations (1 – 5 mg mL-1) into flat-bottomed 96-well TC micro-plates to form a solidified base layer. Tumor cells (A549, MCF-7, SKOV-3, MDA-MB-231) were seeded on top (1 – 2K cells per well), and in some experiments a full ECM sandwich was created by addition of a further volume of Matrigel (2 – 25%, ~ 0.2 – 5 mg mL-1). Using a custom autofocusing method, phase contrast, bright-field and fluorescence images (10x) were captured every 6h for 7 days from within the cell incubator (IncuCyte® S3 live-cell analyzer). Typically, 20 – 80 spheroids were analysed in each well. All four cell types formed multiple cell aggregates within the first 3 days, ranging in diameter from 30 – 80 m. A549, SKOV-3 and MCF-7 multi-spheroids grew as round aggregates while MDA-MB-231 spheroids displayed stellate branching characteristic of an invasive morphology, At Matrigel volumes < 40 L or concentrations < 3 mg mL-1, cells penetrated to the base of the plate and grew as ‘flat 2D’ cultures. The number, area and average size of the spheroids could be computed over time non-invasively and without the use of fluorescent labels using a novel bright-field image analysis algorithm. Once formed, A549, SKOV-3, MCF-7 and MDA-MB-231 multi-spheroids increased 3.0-, 1.6-, 3.8- and 3.3-fold in size over 4 days, respectively. Treatment of A549 multi-spheroids with the DNA enzyme topo-isomerase inhibitor camptothecin (1 M) inhibited growth with comparable spheroid size at day 0 and day 7 post treatment (average brightfield area 1.4 x104 m2). Using fluorescent protein reporters for apoptosis (Annexin V) and cell viability (IncuCyte CytoTox Green) we could verify camptothecin-induced cell death (fluorescence values 149±16% of control (Annexin V) and 243±51% of control (CytoTox). A concomitant decrease of stably expressed RFP (to 3±1% of control) was observed.

The combination of protocol developments, novel image acquisition/analysis algorithms and cell health reporters creates an integrated solution for measuring growth and vitality of multiple small spheroids in a relevant and 3D bio-matrix over time. This approach should be applicable to primary- and patient–derived organoid tumor samples as well as cancer cell lines.

Pickup, MW, Muow, JK, Weaver, MW (2014), EMBO Rep. 15(12): 1243–1253