The Microwell-mesh: A high-throughput 3D prostate cancer spheroid and drug-testing platform

Abstract

Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major limitation in the development of new therapies is the prediction of drug efficacy using in vitro models. Classic in vitro 2-dimensional (2D) cell monolayer cultures are hypersensitive to anti-cancer drugs. As a result, there has been a surge in the development of platforms that enable three dimensional (3D) cultures thought to better replicate natural physiology and better predict drug efficacy. A deficiency associated with most 3D culture systems is that their complexity reduces the number of replicates and combination therapies that can be feasibly evaluated. Herein, we describe the use of a microwell platform that utilises a nylon mesh to retain 3D micro-tumours in discrete microwells; termed the Microwell-mesh. The Microwell-mesh enables the manufacture of ~150 micro-tumours per well in a 48-well plate, and response to anti-tumour drugs can be readily quantified. Our results demonstrate that 3D micro-tumours, unlike 2D monolayers, are not hypersensitive to Docetaxel or Abiraterone Acetate, providing a superior platform for the evaluation of sequential drug treatment. In summary, the Microwell-mesh provides an efficient 3D micro-tumour platform for single and sequential drug screening.

Figure 1

Drug treatment protocol. (a) For androgen deprivation, cell cultures where seeded in standard FBS supplemented medium on day 0 and replaced with CSS medium 24 hours later (day 1) to starve cells of androgen. On day 3, Abiraterone Acetate or Enzalutamide was added to cell cultures. Cultures were terminated on day 5 and analysed using the specified methods. (b) A single treatment of Docetaxel was performed after 24 hours of seeding the cells (day 1). Drug response was then assessed following three days of exposure to Docetaxel (day 4).

Figure 2

Microwell platform manufacture and establishment of 3D micro-spheroid culture. (a) The traditional open-top microwell platform results in dislodged spheroids following culture media exchange (scale bar = 500 µm). (b) Schematic illustration and bright field images show PDMS discs with and without the mesh which can be inserted in 48-well tissue culture plates and cells aggregated after 24 hours of seeding cell suspension (scale bar = 200 µm). (c) Schematic illustration of cell seeding using traditional open-top microwells (top) and Microwell-mesh being microwells modified with a 36 µm mesh-top (bottom) large enough to allow single cells to pass through, but small enough to retain spheroids within discrete microwells.

Figure 3

AlamarBlue assay optimization in Microwell plates. No significant change in the linearity of the AlamarBlue assay after 3 hours incubation with 3% of AlamarBlue reagent when the fluorescence was acquired in situ (a) or when the reaction product was transferred to a black 96-well plate (b).

Figure 4

Characterisation of prostate cancer cell lines in 3D micro-tumour culture. (a) Prostate cancer (C42B and LNCaP) and prostate myofibroblasts (WPMY-1) cell lines were cultured in the 3D platform (600 cells/spheroid) and the growth of cell spheroids was assessed by DNA quantification and spheroid diameter measurement. DNA quantification data represents the mean value of four replicate cultures. A minimum of 50 spheroids were measured per time point for diameter measurement. (b) Confocal images of spheroids (600 cells/spheroid) stained with Ki67 (red) and nuclear stain (DAPI; blue) were acquired on day 1 and 7 of culture. Scale bar = 100 µm.

Figure 5

Monolayer and micro-tumour behaviour of C42B and LNCaP cell lines in androgen deprived conditions. (a) C42B (Top) and LNCaP (Bottom) cells were seeded in expansion culture medium for 24 hours followed by medium exchange to androgen-depleted medium (CSS) for a further 48 hours. Abiraterone Acetate was then added to the culture medium at the indicated concentrations for an additional 48 hours. AlamarBlue, Cell Titre-Glo 3D Cell Viability and PicoGreen assays were then performed to assess metabolic activity, ATP quantity, and DNA quantity, respectively. All results are represented as a percentage of the FBS-containing culture medium control values (data was collected from three independent experiments, each having four technical replicate cultures). Statistical significance was calculated by two-way ANOVA compared to the corresponding zero value (**P < 0.0001 and *P < 0.01) or compare 2D and 3D values with the same drug treatment (Ψ P < 0.005). (b) Metabolic activity (AlamarBlue assay) and DHR123 staining of LNCaP monolayers at specified Abiraterone Acetate concentrations. Results represented as the mean fluorescence values of four individual samples normalized to control culture values. Statistical significance was performed using one-way ANOVA (*** P<0.001). Side panel represents the cellular morphology of LNCaP cells at the indicated Abiraterone Acetate concentrations (µM). Scale bar = 200 µm.

Figure 6

C42Band LNCaP Docetaxel drug response. C42B and LNCaP cells in 2D and 3D cultures were treated with Docetaxel in the indicated concentrations for 72 hours followed by metabolic activity and DNA content measurements. All results are represented as a percentage of the vehicle control values (data was collected from three independent experiments each had four replicate cultures n = 4). Statistical significance was calculated by two-way ANOVA compared to the corresponding values in 2D cultures (***P < 0.0001).

Figure 7

Sequential Docetaxel treatment and prostate cancer cell recovery. (a) Sequential treatment of Docetaxel was performed at the indicated time points (red arrows). Each treatment for 72 hours was followed by metabolic activity measurement (blue arrows) and drug removal (purple arrows) at day zero and 6. In addition, cell recovery assessment was also performed at days 1, 3, 6 and 7 for single treatments and at days 6 and 7 for sequential treatments. (b) 2D and 3D cultures of C42B and LNCaP cells were treated with 5 nM Docetaxel following the sequential treatment protocol and the metabolic activity of the survived cells were assessed at the indicated time points using AlamarBlue assay. All results are represented as a percentage of the vehicle control values (Two independent experiments each had four replicate cultures n = 4).