Background: Bronchial pre-malignant lesions (PMLs) are the putative precursors for bronchial squamous cell carcinoma. PMLs represent a spectrum of histologies, from low-grade lesions (hyperplasia, metaplasia) to high-grade lesions (dysplasia, carcinoma in situ). The majority of these lesions will regress or remain stable without clinical intervention while a subset of lesions will progress to invasive carcinoma. We performed single-cell RNA sequencing (scRNAseq) of these lesions to elucidate the cross-talk between epithelial, stromal, and immune populations in lesions of increasing histological grade. Methods: Thirty lesions from seventeen participants were biopsied via bronchoscopy. Cells were sorted by CD45+/- FACS gating and sequenced with the Cel-Seq2 protocol. Celda was used to bi-cluster genes into modules and cells into clusters. Cells were filtered by mitochondrial percentage (%mito < 50%), minimum UMI counts (nUMI > 300), and doublet detection. Cell types were labeled by marker gene expression. Results: After filtering low quality cells, we analyzed 4,382 cells. We observed expected smoking related shifts in epithelial cell type proportions, including an increase in secretory cells (χ2 = 31.39, p = 2.11 X 10-8) and a decrease in ciliated cells (χ2 = 4.83, p = 0.028) among current smokers. Distinct differences in expression of transcriptional modules were observed between KRT5+ (basal) cells from different histologic grades. Basal cells from high grade lesions expressed smoking detoxification and cell cycle gene programs, while low grade lesion basal cells expressed differentiation gene programs. We also identified a group of cells from CIS lesions involved in an epithelial-to-mesenchymal transition, marked by an increase in SPARC and COL4A1 expression and a decrease in CDH1 expression. Subpopulations of immune cells identified include macrophages, CD4/8+ T, B, dendritic cells, and natural killer cells. Several clusters of CD4+ and CD8+ T cells displayed an exhausted phenotype, marked by the expression of PD-1, CTLA4, LAG3, and TIGIT. Samples with high grade histology (dysplasia, carcinoma in situ) were enriched in CD4+ Tregs and myeloid cells compared to low grade histology samples (hyperplasia, metaplasia), which were enriched in Natural Killer and cytotoxic CD8+ T cells (χ2 = 298.95, p = 0.001). Discussion: Our results suggest that changes in specific transcriptional programs are associated with the transition of epithelial cells to more invasive states and that changes in immune populations are associated with increasing histological grade. These signatures can suggest novel avenues for chemoprevention and cancer interception. Citation Format: Conor Shea, Lukas Kalinke, Kitty De Jong, Kate Gowers, Diane Ding, Sherry Zhang, Gang Liu, Jack Cunningham, Ipsita Dey-Guha, Mark Hennon, Sai Yendamuri, Christopher Stevenson, Avrum Spira, Mary E. Reid, Marc E. Lenburg, Sam M. Janes, Jennifer E. Beane, Sarah A. Mazzilli, Joshua D. Campbell. Epithelial, stromal, and immune changes associated with lung squamous premalignant lesion severity identified by single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2201.

Abstract Background: Sampling the bronchial airway during bronchoscopies in screening populations at high risk for lung cancer has increased detection of squamous bronchial pre-malignant lesions (PML). The majority of these lesions will regress or remain stable without any clinical intervention. However, a subset of lesions will progress to invasive malignancy. In this work, we investigate the gene expression changes in epithelial cell states and immune cell proportion shifts associated with premalignant histologic stage using single-cell RNA sequencing to begin to identify molecular features associated with PML regression or progression. Methods: Cells (n=3,325) were isolated from 11 endobronchial biopsies from 9 patients, where biopsy histology range from normal, dysplasia, carcinoma in situ (CIS) and squamous cell carcinoma. Immune cells (CD45+) and epithelial cells (CD45− EPCAM+) were sorted into separate 96-well plates using fluorescence activated cell sorting (FACS) and sequenced using CEL-Seq2 single cell RNA seq protocol. Results: After filtering low quality cells, we profiled 2,998 cells (1,052 CD45+ and 1,946 CD45−) with an average of 1,190 genes per cell. Within the epithelial cells, we focused on basal cells as they are the airway progenitor cells. We observed distinct differences in expression of KRT5+ (basal) cells between different histologic grades, where KRT5+ cells present in a hyperplasic lesion expressed multiple secretory cell markers suggesting they were transitioning to a secretory cell phenotype and KRT5+ cells from a CIS lesion from a current smoker expressed higher levels of smoking inducible genes, such as GSTM1 and CYP1A1. Lastly, KRT5+ cells from a lung squamous carcinoma tumor expressed higher levels of many genes related to cell cycle progression. Among the immune populations, low-grade PMLs with non-dysplastic histology were enriched for CD8+ T cells, whereas higher-grade PMLs and an invasive tumor were enriched for myeloid cells. Conclusions: To date, we have begun to identify histology-associated cellular and molecular profiles in bronchial premalignancy and early-stage carcinoma. Future resampling of these patients and expansion of cases will allow us to discover biomarkers associated with lesion progression and molecular targets for lung cancer interception. Citation Format: Conor Shea, Lukas Kalinke, Kitty De Jong, Kate Gowers, Diane Ding, Sherry Zhang, Gang Liu, Jack Cunningham, Ipsita Dey-Guha, Mark Hennon, Sai Yendumuri, Christopher Stevenson, Steven Dubinett, Avrum Spira, Marc Lenburg, Samuel Janes, Mary Reid, Jennifer Beane, Sarah Mazzilli, Joshua D. Campbell. Cellular and molecular changes associated with lung squamous premalignant lesion severity identified by single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2212.