Liquid Biopsy Snapshots of Key Phosphoproteomic Pathways in Lung Cancer Patients for Diagnosis and Therapy Monitoring

Abstract

Phosphorylation is a post-translational modification in proteins that changes protein conformation and activity for regulating signal transduction pathways. This mechanism is frequently impaired in lung cancer, resulting in permanently active constitutive phosphorylation to initiate tumor growth and/or reactivate pathways in response to therapy. We developed a multiplexed phosphoprotein analyzer chip (MPAC) that enables rapid (detection time: 5 min) and sensitive (LOD: 2 pg/μL) detection of protein phosphorylation and presents phosphoproteomic profiling of major phosphorylation pathways in lung cancer. We monitored phosphorylated receptors and downstream proteins involved in mitogen-activated protein kinase (MAPK) and PI3K/ AKT/mTOR pathways in lung cancer cell line models and patientderived extracellular vesicles (EV). Using kinase inhibitor drugs in cell line models, we found that the drug can inhibit the phosphorylation and/or activation of the kinase pathway. We then generated a phosphorylation heatmap by EV phosphoproteomic profiling of plasma samples isolated from 36 lung cancer patients and 8 noncancer individuals. The heatmap showed a clear difference between the noncancer and cancer samples and identify the specific proteins that are activated in the cancer samples. Our data also showed that MPAC could monitor immunotherapy responses by assessment of the phosphorylation states of the proteins, particularly for PD-L1. Finally, with a longitudinal study, we found that the phosphorylation levels of the proteins were indicative of a positive response to therapy. We believe that this study will lead to personalized treatment by providing a better understanding of the active and resistant pathways and will provide a tool for selecting combined and targeted therapies for precision medicine.

A Mesoporous Gold Sensor Unveils Phospho PD-L1 in Extracellular Vesicles as a Proxy for PD-L1 Expression in Lung Cancer Tissue

“…The findings in this study are supported in part by a previous study in a cohort of 35 patients that found a correlation between nonphosphorylated EV PD-L1 and TPS using a novel Simoa Epcam-PD-L1 EV detection assay. This test lacked the sensitivity of the EV pPD-L1 nanotechnology platform utilized in the study reported here nor was reproducibility confirmed with a validation cohort .…”

“…Additionally, as phosphorylation increases the hydrophilicity of proteins, we hypothesized that phosphorylated proteins dispersed more effectively in the solution, thereby facilitating increased interaction with the gold surface. Moreover, to minimize the biofouling and increase the affinity and specificity for accurate assessment of PD-L1 TPS, we focused on rigorous sample preparation methods consistent with our previously published reports. ,, SEC was employed for isolating EVs, and immunoprecipitation was performed to extract specific PD-L1 proteins from the EVs. This two-step approach effectively removed most contaminants that could lead to nonspecific binding, contributing to the reliability of our assays.…”

“…These cell lines were chosen based on their expression of distinct mutations in the epidermal growth factor receptor (EGFR) gene. EGFR mutations have been associated with increased cell surface expression of PD-L1 and alteration in the phosphorylation status of PD-L1. ,− The HCC827 cell line expresses mutation in EGFR tyrosine kinase receptor which constitutively leads to activating EGFR . This phosphorylation cascade also phosphorylates PD-L1, contributing to immune response suppression.…”

“…The isolation and enrichment of EVs were performed according to the previous methods. , Briefly, 500 μL of plasma EVs from lung cancer patient samples and healthy plasma controls (Red Cross, Australia) were isolated and concentrated through size exclusion columns Amicon Ultra-15, 50 kDa centrifugal filter (Merck, Germany). The EV concentrations were analyzed using NanoSight (NS300, Malvern Instruments, UK) according to a previous report .…”

“…To extract the target protein from patient sample EVs, magnetic immunoprecipitation was performed using Pierce Cross-Link Magnetic IP/Co-IP kit (Thermo Fischer Scientific, Australia) with phosphorylation-independent anti-PD-L1 antibody following previously published work. , Initially, anti-PD-L1 monoclonal antibody (#14-5983-82, Thermo Fisher Scientific, Australia) was coupled to Pierce protein A/G magnetic beads through 20X Coupling buffer (pH 7.2, 10 mM Na3 PO4, 150 mM NaCl), and the beads containing the complex were collected with a magnetic stand. Subsequently, the antibody-functionalized beads were cross-linked using disuccinimidyl suberate.…”