High-plex immunofluorescence single cell analysis of fibrotic murine lung on tissue microarrays

Background: Sequential multiplex immunofluorescent technology1 allows simultaneous detection of multiple markers on the same slide2, giving a high throughput and significantly facilitates and accelerates in situanalysis of tissues in target validation and discovery. Aim of the study To develop a sequential multiplex immunofluorescence on tissue microarray (TMA) generated from fibrotic Bleomycin (BLM) model and control lung. Selected biomarkers will be evaluated either by image cytometry-based method of cell clusterization or thresholding quantification. Methods: C57BL/6 mice were challenged oropharyngeal aspiration (OA)at day 0 and day 4 with BLM (1 mg/kg). Fibrosis progression and pharmacological response to Nintedanib (60 mg/kg) was evaluate by Micro CT and histology (7, 14, 21 days). Twenty antibodies were applied on the same TMA and analyzed as high throughput single cell assay. Results: The phenotype classification within the fibrosis time course compared to control, showed differential clusters. As regards to the airway epithelial cells, these lose their normal spatial profile and are actively involved in the constitution of fibrotic foci. Nintedanib treatmentmodulates: Collagen I, Cytokeratin, Vimentin and α-SMA. Conclusions: A detailed analysis of co-expression, with a limited number of antibodies, helps to clarify the cell subtypes act in different phases of fibrosis progression;moreover the quantification of individual markers allows to highlight the drug effect in high throughput. Bolognesi, M. M. et al. J Histochem Cytochem 2017; 65:431–444 Giltnane, J. M. & Rimm, D. L. Nat Clin Pract Oncol 2004; 1:104-11