Reporter assays introduce a reporter gene into cells and detect the product of the reporter gene when the gene is transcribed and translated by the cell. The reporter can include a protein tag or an enzyme such as luciferase. In this example, the LC3 protein, widely used as a marker of autophagic activity, is tagged with HiBiT, a small 11a.a. peptide tag. Cells expressing the Autophagy LC3 HiBiT Reporter are treated with inducers or inhibitors of autophagy. At the end of the experiment, cells are lysed and total LC3 is quantitated by adding the NanoGlo® HiBiT Lytic Detection Reagent, which includes the complementing LgBiT protein and NanoLuc® substrate.
The Autophagy LC3 HiBiT Reporter was introduced into HEK293 cells, and the reporter cells were grown in both monolayer and 3D spheroids. Does the LC3 HiBiT Reporter signal accurately reflect reporter levels in 3D spheroids?
Adaptation for 3D Models
In this assay, to detect and quantify the tagged protein, the cells must be fully lysed to recover the tagged protein from all the cells. In order to facilitate lysis of the 3D spheroids, the protocol was adapted to increase the shaking time and reagent processing time after addition of the lytic reagent. The 2-minute shaking plus 10-minute incubation for monolayers was increased to 30 minutes of spheroid shaking and incubation.
The performance of the Autophagy LC3 HiBiT Reporter Assay System in 3D models was verified by comparing the recovery of the reporter to the ATP content in the spheroid model, wherein ATP is a useful surrogate for cell number. Different size spheroids were created from the HEK293 reporter cells by seeding increasing cell number in Corning® Ultra-Low Attachment 96-well plates. After 4 days of culture, spheroids of a wide range of sizes were generated for subsequent assay of autophagy reporter levels with the NanoGlo® HiBiT Lytic Detection Reagent. In parallel plates, a similar set of spheroids was processed with CellTiter-Glo® 3D to determine ATP content.
Figure 5. Reporter recovery from HEK293 spheroids. Spheroids were generated by seeding varying amounts of HEK293 reporter cells in Corning® Ultra-Low Attachment plates for 4 days. After 4 days, parallel spheroids were processed with either CellTiter-Glo® 3D or the Nano-Glo® HiBiT Lytic Detection Reagent, using a modified protocol of 30 minutes shaking and incubation.
To determine the relationship between reporter and ATP levels, the signals from the two assays were plotted against each other (Figure 5). The relationship between the two assays is linear, meaning that increases in ATP levels (i.e., increases in Day 4 cell number) correlate with a proportional increase in detectable autophagy reporter levels. These results verify the quantitative recovery and reporter detection by the Nano-Glo® HiBiT Lytic Detection Reagent, with the extended shake time, when applied to Autophagy LC3 HiBiT Reporter cell spheroids.
Once the autophagy reporter assay with increased shake time was verified to perform effectively in 3D cell spheroids, the assay system could be reliably used to evaluate autophagic activity in treated cell spheroids. Will cells grown in a 3D model respond differently to inhibitors or stimulators of autophagy compared to cells grown in a monolayer? In Figure 6, the three particular compounds tested in HEK293 Autophagy LC3 HiBiT Reporter cells behaved similarly in monolayer vs. spheroid culture models. The autophagy stimulator produced similar degradation of the autophagy reporter in both cell models, while both autophagy inhibitors prevented reporter degradation in monolayer and spheroid formats. Perhaps the most notable difference was an apparent decrease in bafilomycin A1 potency in spheroids as compared to monolayer cells.
Figure 6. Compound testing in 2D and 3D models. HEK293 Autophagy LC3 HiBiT Reporter cells were used to form spheroids (right) or grown in monolayer (left). Monolayer or spheroids were treated with increasing concentrations of a known autophagy stimulator (alone) or by increasing concentrations of two autophagy inhibitors in the presence of a fixed concentration of the autophagy stimulator. The resultant autophagy reporter levels were measured with the monolayer protocol or modified protocol for 3D structures. Results were normalized to luminescence of the untreated control for each sample type.