Abstract PR003: Multiplexed immune phenotyping and miRNA-ISH in bronchial premalignant lesions reveals a mechanism of immune suppression

Abstract Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma. Prior work has shown that among proliferative subtype PMLs, progression to or persistence of a high-grade histology is associated with downregulation of an immune-related gene module. Furthermore, microRNA-149 (miR-149) was identified as a candidate negative regulator of this expression program. In this study, we aim to investigate the spatial distribution of miR-149 and its association with the immune environment and PML progression/persistence in PMLs. We identified 19 paraffin embedded bronchial biopsies transcriptionally defined as belonging to the proliferative subtype obtained from 9 subjects. Three consecutive sections were stained with H&E for histology annotation, chromogenic miRNA in situ hybridization (miRNA-ISH) for miR-149 abundance and localization, and with 30 antibodies targeted to epithelial and immune populations using imaging mass cytometry (IMC) to perform phenotyping. We trained a robust pixel classifier to detect foci of miR-149 signal in miRNA-ISH and quantified the density of miR-149 signal. For the IMC data, we used Mesmer for cell segmentation. PhenoGraph clustering based on quantification of each antibody in each cell was used for cell population identification. Ten recurrent cellular neighborhoods were identified by K-means clustering. miR-149 density was greater in the epithelium compared with stromal tissue. The miR-149 density in dysplastic epithelium normalized to miR-149 in normal epithelium is higher in the progressive/persistent PMLs than regressive PMLs. Additionally, in progressive/persistent PMLs, we find lower average epithelial staining for the miR-149 target NLRC5 associated with a cellular neighborhood that is enriched for goblet cells and PDL1+MUC5B+ secretory cells. Cell composition analysis showed that progressive/persistent PMLs have lower CD8 T cell infiltrate in both the stroma and epithelium. These results suggest that increased miR-149 expression in progressive/persistent PMLs contributes to an immune suppressive environment characterized by a shift in secretory cell populations and T cells within the bronchial epithelium. Interventions targeting miR-149 expression may be a potential interception strategy for high-grade PMLs. Citation Format: Darren J. Chiu, Roxana Pfefferkorn, Emily Green, Boting Ning, Myrtha Constant, Marc Lenburg, Eric Burks, Sarah Mazzilli, Jennifer Beane. Multiplexed immune phenotyping and miRNA-ISH in bronchial premalignant lesions reveals a mechanism of immune suppression. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR003.