Targeted MRM assay quantifies androgen receptor variants in castration-resistant prostate cancer cell lines and PDX models

The development of castration-resistant prostate cancer (CRPC) is often driven by the emergence of Androgen Receptor variants (AR-Vs). These AR-Vs, particularly AR-V7, lack the ligand-binding domain, making them constitutively active transcription factors that bypass current AR-targeted endocrine therapies. This mechanism renders standard treatments ineffective, highlighting an urgent need for robust methods to identify and quantify specific AR-V proteins to guide patient stratification and develop more effective therapies.

Researchers developed a targeted multiple reaction monitoring (MRM) assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify total AR proteins via the AR N-terminal domain (AR-Nterm), the well-characterized AR splice variant AR-V7, a putative AR-V12, and isoforms with an exon 3 duplicated junction. The assay was validated using three AR-positive PCa cell lines (22Rv1, VCaP16, LNCaP), two AR-negative cell lines (PC3, DU145), and a patient-derived xenograft (PDX model) of CRPC. Endogenous target peptides were quantified following tier-2 targeted mass spectrometry "fit-for-purpose" guidelines.

The analytical performance of the MRM assay was thoroughly characterized, demonstrating a linearity range from 4.15 fmol to 4000 fmol. Precision, accuracy, selectivity, stability, ion suppression, and repeatability studies confirmed its robustness. The overall coefficient of variation (CV) was consistently < 20% across seven independent validation experiments, indicating high reproducibility. Endogenous AR-V peptides were accurately quantified using calibration curves, achieving acceptable linear regression coefficients (R2 > 0.95).

Four targets, including AR-Nterm and 3 AR-variants, were successfully identified and quantified in both the PCa cell lines and the PDX pre-clinical model, confirming the assay's capability to detect these critical biomarkers. This establishes a reliable method for profiling AR-V protein expression.