Antipeptide Immunocapture with In-Sample Calibration Curve Strategy for Sensitive and Robust LC-MS/MS Bioanalysis of Clinical Protein Biomarkers in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Despite huge promises, bioanalysis of protein biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for clinical applications is still very challenging. Here, we describe a sensitive and robust LC-MS/MS assay to quantify clinical protein biomarkers in FFPE tumor sections using automated antipeptide antibody immunocapture followed by in-sample calibration curve (ISCC) strategy with multiple isotopologue reaction monitoring (MIRM) technique. ISCC approach with MIRM of stable isotopically labeled (SIL) peptides eliminated the need for authentic matrices for external calibration curves, overcame the matrix effects, and validated the quantification range in each individual sample. Specifically, after deparaffinization, rehydration, antigen retrieval, and homogenization, the protein analytes in FFPE tumor tissues were spiked with a known concentration of one SIL peptide for each analyte, followed by trypsin digestion and antipeptide immunocapture enrichment prior to MIRM-ISCC-based LC-MS/MS analysis. This approach has been successfully used for sensitive quantification of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) in 15 representative FFPE tumor samples from lung, colorectal, and head and neck cancer patients. Except for one sample, PD-L1 and PD-1 in all samples were quantifiable using this assay with concentrations of 27.85-798.43 (amol/µg protein) for PD-L1 and 16.96-129.89 (amol/µg protein) for PD-1. These results were generally in agreement with the immunohistochemistry (IHC) data but with some exceptions. This approach demonstrated the feasibility to quantify low abundant protein biomarkers in FFPE tissues with improved sensitivity, specificity, and robustness and showed great potential as an orthogonal analytical approach to IHC for clinical applications.

Development and validation of an in vitro 3D model of NASH with severe fibrotic phenotype.

Nonalcoholic steatohepatitis represents a significant and rapidly growing unmet medical need. The development of novel therapies has been hindered in part, by the limitations of existing preclinical models. There is a strong need for physiologically relevant in vivo and in vitro liver fibrosis models that are characterized by better translational predictability. In this study, we used the InSphero 3D InSightTM three-dimensional (3D) human liver microtissue (3D-hLMT) system prepared by co-culturing primary human hepatocytes with hepatic stellate cells, Kupffer cells and endothelial cells to develop a model of NASH with a severe fibrotic phenotype. In our model, palmitic acid (PA) induced a robust proinflammatory and profibrogenic phenotype in the 3D-hLMT. PA significantly increased several markers of the inflammatory and profibrotic process including gene expression of collagens, α-sma, tissue inhibitor of matrix metalloprotease 1 (timp1) and the stellate cell activation marker pdgfrß as well as secreted CXCL8 (IL8) levels. We also observed TGFß pathway activation, increase in active collagen synthesis and significant overall increase in tissue damage in the 3D-hLMTs. Immunohistochemistry analysis demonstrated the upregulation of collagen, cleaved caspase 3 as well as of the PDGFRß protein. We further validated the model using a phase 3 clinical compound, GS-4997, an apoptosis signal-regulating kinase 1 (ASK-1) inhibitor and showed that GS-4997 significantly decreased PA induced profibrotic and proinflammatory response in the 3D-hLMTs with decreases in apoptosis and stellate cell activation in the microtissues. Taken together we have established and validated an in vitro 3D-hLMT NASH model with severe fibrotic phenotype that can be a powerful tool to investigate experimental compounds for the treatment of NASH.