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A4348 - Multiplexed In Situ Hybridization Gives New Insight into the Pathogenesis of IPF
Author Block: N. Juul1, M. Nagendran1, P. Harbury2, T. Desai3; 1Pulmonary and Critical Care Medicine, Stanford University, Palo Alto, CA, United States, 2Biochemistry, Stanford University, Palo Alto, CA, United States, 3Pulmonary and Critical Care Medicine, Stanford University, Stanford, CA, United States.
Rationale. Available therapies for IPF affect homeostatic pathways and as such have severe side effects and minimal effect on the course of disease. Current animal models do not recapitulate the defining characteristics of IPF. Therefore, to elucidate the upstream cellular and molecular mechanisms of the disease and develop new treatments for IPF we must look in human tissue. The fibrotic cascade begins at different times in countless locations around the lung, a feature referred to as “temporal heterogeneity”, and this may be the key to its tractability. Because of this temporal heterogeneity, a small section of lung from an IPF patient can contain the complete spectrum of lesions, from normal to end-stage, creating a record of its pathogenesis. By correlating molecular abnormalities with lesions at a range of stages, we can infer and distinguish the mechanisms of disease initiation and progression.
Methods. Recently, our lab co-invented a technique called Proximity Ligation In Situ Hybridization (PLISH), a multiplexed in situ hybridization technology that is quantitative for single cells in intact tissue. Localizing the source and receipt of cell signaling is achieved through simultaneously visualizing transcripts of ligands and downstream signals with canonical cell-type markers. Doing this in situ allows the retention of the temporal dimension of the tissue that is lost in dissociative approaches such as single cell RNA sequencing. We have been able to combine PLISH with immunofluorescence and traditional staining techniques such as picrosirius red collagen staining to give further functional protein context to our findings. Combining the ability to erase and re-assay tissue with automation allows for efficient large-scale quantification and analysis.
Results. Multiplexed in situ hybridization has brought about several early revelations that impact our understanding of IPF. Transcripts of surfactant protein C (SPC), seen exclusively in AT2 cells in control lung, are seen not only in luminal cells but also in cells buried in socked-in fibrosis. The cells lining honeycomb cysts, thought to originate from airway epithelia, show transcription for SPC and cadherin, suggesting a potential alveolar epithelial origin. SPC appeared to also colocalize with Col1a1, the transcript for type I collagen, in some cells.
Conclusions. Using PLISH in lesions across the spectrum of temporal heterogeneity in IPF tissue will create a transcriptional record of its pathogenesis within representative single cell types. This technique will allow pinpointing the timing and localization of the multitude of signaling pathways at play in IPF.