Investigating NanoLuc-EGFR engineered cell lines for real-time monitoring of EGFR protein dynamics in live cells

Abstract

Evaluating the steady-state protein level of the EGFR in live cells presents significant challenges compared to measuring its kinase activity. Traditional testing methods, such as immunoblotting, ELISA, and immunofluorescence assays, are generally restricted to fixed cells or cell lysates. Despite their utility, these methods are cumbersome and provide only intermittent snapshots of EGFR levels at specific time points. With emerging trends in drug development shifting toward engineering novel agents that promote protein degradation, rather than simply inhibiting kinase activity, a tool that enables real-time, quantitative detection of drug effects in live cells could catalyze advances in the field. Such an innovation would expedite the drug development process, enhancing the translation of research findings into effective, patient-centered therapies. The NanoLuc-EGFR cell line, created through CRISPR genome editing, allows for the continuous tracking and analysis of EGFR protein levels and their degradation within live cells. This approach provides quantitative monitoring of protein dynamics in real time, offering insights that go beyond absolute protein levels to include aspects such as protein stability and degradation rate. Using this cell line model, we observed that AT13387 and H84T BanLec induce EGFR degradation in A549-HiBiT cells, with the results confirmed by immunoblotting. In contrast, Erlotinib, Osimertinib, and Cetuximab inhibit EGFR phosphorylation without altering total EGFR levels, as validated by the HiBiT luciferase assay. The NanoLuc-EGFR cell line marks a significant advancement in understanding protein regulation and serves as an instrumental platform for investigating targeted therapies that modulate protein kinases, especially those that induce protein degradation.

Keywords: EGFR; HiBiT; Kinase inhibitors; Nano-luciferase; Protein degradation.

Figure 1.. Development and Optimization of EGFR Detection Substrate Amount and Confirmation in HiBiT cells.

(A) The schematic presents the split NanoLuc luciferase assay in A549 cells for detecting both surface and total EGFR expression. The 11 amino acid HiBiT tag, visualized as a blue triangle, is fused to the N-terminus of EGFR. Upon introduction of the complementary 156 amino acid LgBiT subunit, pre-conjugated with the luciferase substrate furimazine, the HiBiT and LgBiT subunits interact to reconstitute the active enzyme, which catalyzes a luminescent reaction. The presence of a lytic agent causes permeabilization of the cell membrane, depicted by a dotted plasma membrane, allowing LgBiT to access and bind with intracellular HiBiT-tagged EGFR. This results in bioluminescence that reflects total EGFR levels within the cells. Substrate optimization: One thousand A549 parental and HiBiT cells per well were plated in a 96-well plate and allowed to grow overnight. The following day, the media were replaced with FBS-free media, and the cells were incubated for 1 hour. The substrate was diluted in a buffer, and 50 μL of the diluted substrate was added to each well and incubated for 10 minutes. The resulting average surface luminescence (B) and the total luminescence (C) from at least three experiments were recorded using a GloMax Discover system and plotted along with the standard deviation.

Figure 2.. Optimization of Cell Numbers for EGFR Detection. (A-B) Cell Density Optimization:

Cells were plated in a 96-well plate at densities ranging from 0 to 8000 cells per well. Surface EGFR levels and total EGFR levels were quantified as described in Figure 1. (C) Concurrently, total viable cell counts were determined using the Titer-Glo assay., Results are presented as an average of at least three experiments +/− standard deviation (A, B, and C) and (D) 0.6 million A549-HiBiT EGFR-expressing cells were allowed to adhere and grow overnight. The next day, the cells were counted and aliquoted into 8 tubes with varying numbers (0 to 60,000) and were then processed for immunoblotting to detect EGFR levels. The EGFR band was detected in lysates prepared from 15,000 cells and above.

Figure 3.. Validation of EGFR degradation by AT13387

(A) Dose-response relationship: 1000 A549-HiBiT cells were plated in a 96-well plate and treated with 0–1000 nM AT13387; luminescence was measured post 24-hour incubation. (B) Time-course analysis: 1000 A549-HiBiT cells were treated with 60 nM AT13387, and luminescence was measured periodically over 24 hours to assess the temporal effect of AT13387 on EGFR. (C) 0.6 million A549-HiBiT cells were seeded in a 60 mm dish and treated with AT13387 ranging from 0–1000 nM the following day. After 24 hours, cell lysates were collected for immunoblotting with specific antibodies. (D) Average EGFR levels of an immunoblotting data were quantified using ImageJ and plotted (error bars represent the SD from the average of three, separate experiments).

Figure 4.. Validation of EGFR degradation by H84T BanLec

(A) Dose-Response with H84T BanLec: One thousand A549-HiBiT cells were plated in a 96-well plate. The following day, cells were exposed to various concentrations of H84T BanLec (0–100 μg/ml). Readings were acquired at 24 hours. (B) Validation of EGFR degradation: 0.6 million A549-HiBiT cells were seeded in 60 mm dishes and treated with various concentrations of H84T BanLec (0–100 μg/ml). Cell lysates were prepared at 24 hours and subjected to immunoblotting using designated antibodies. (C) Average EGFR levels from the immunoblotting data were quantified using ImageJ and plotted; error bars represent the standard deviation (SD) from the average of three separate experiments.

Figure 5.. Effects of Erlotinib, Osimertinib, and Cetuximab on Bioluminescence

(upper panels). 1,000 A549-HiBiT cells were plated in a 96-well plate. The following day, the cells were exposed to various concentrations of Osimertinib (0–1 μM) (A), Erlotinib (0–10 μM) (B), or Cetuximab (0–300 μg/mL) (C). Bioluminescence from the surface or the entire cells was acquired at 24 hours. Data from three independent experiments are plotted. Validation of the Treatment Effect on EGFR and pEGFR (lower panels). 0.6 million A549-HiBiT cells were seeded in 60 mm dishes and treated with Osimertinib, Erlotinib, or Cetuximab as described above. Cell lysates were prepared at 24 hours and subjected to immunoblotting for pEGFR and EGFR. GAPDH was used as the loading control.

Figure 6.. Confirmation of EGFR Steady State and EGFR Phosphorylation Using Immunofluorescence.

A549-HiBiT-EGFR cells were seeded on coverslips in a 100 mm dish and treated with the following compounds: AT13387 (300 nM), H84T BanLec (100 μg/mL), Erlotinib (10 μM), Osimertinib (1 μM) or Cetuximab (300 μg/mL). The coverslips were collected after 24 hours of treatment. EGFR and phospho-EGFR were detected using anti-EGFR and anti-phospho-EGFR antibodies, respectively. DAPI was used to stain the nuclei. A semi-quantitative analysis (+, ++, +++) was performed through visual analysis of 50 cells in 3 random fields and shown below the corresponding panel (scale bars = 50 μm).