Alveolar epithelial type 1 cells serve as a cell of origin for lung adenocarcinoma with distinct molecular and phenotypic presentation

Abstract

Lung adenocarcinoma (LUAD) is the most common subtype of cancer arising in the distal lung. LUAD encompasses several pathologic subtypes, each with differing clinical outcomes and biological behaviors. However, the molecular and cellular underpinnings of the different subtypes are largely unknown. Understanding which cell populations in the distal lung contribute to LUAD could provide insights into the marked heterogeneity in pathologic features, clinical presentation and responses to therapy of LUAD. Differential expression analysis of lung adenocarcinoma transcriptomes from The Cancer Genome Atlas revealed distinct alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cell signatures within human LUAD with significantly different survival outcomes between tumors expressing AT2 and AT1 gene signatures, suggesting AT1 cells might contribute to a subset of LUAD cases. To address this, we tested the ability of AT1 cells to give rise to LUAD following induction of KrasG12D, a known oncogenic driver of human LUAD. Activation of KrasG12D in Gram-domain containing 2 (Gramd2)+ AT1 cells gave rise to multiple LUAD lesions, primarily of papillary histology. In contrast, activation of KrasG12D in surfactant protein C (Sftpc+) AT2 cells resulted in LUAD lesions of lepidic histology. Immunohistochemistry established that Gramd2:KrasG12D lesions were of primary lung origin and not metastatic events. Spatial transcriptomic profiling revealed distinct pathway alterations within Gramd2- and Sftpc-derived LUAD. Immunofluorescence confirmed differences observed in the spatial transcriptomic analysis in expression patterns and distribution of cell-specific markers depending on cell of origin, while universal upregulation of the Krt8 intermediate cell state marker was observed. Our results are consistent with Gramd2+ AT1 cells serving as a putative cell of origin for LUAD and suggest that LUAD may be a collection of adenocarcinomas that share a common location within the distal lung but arise from different cells of origin.

GRAMD2+alveolar type I cell plasticity facilitates cell state transitions in organoid culture

“…GFP signal in tamoxifen-induced Gramd2 creERT2;mTmG lung sections extensively overlapped with several known AT1 cell markers, including AQP5, PDPN and HOPX. Interestingly, not all AQP5 + , PDPN + or HOPX + AT1 cells in Gramd2 creERT2;mTmG mice showed colocalization with GFP ( Figure 1B-1D ) (20). Higher magnification images further demonstrated colocalization of membranous GFP staining with PDPN + and HOPX + AT1 cells ( Figure 1E-1H ,) specifically under tamoxifen-induced CreERT2 activation conditions compared to no tamoxifen treatment ( Figure 1I, 1J ).…”

“…AT1 cell function and plasticity have been far less studied than that of AT2 cells, largely due to the limited specificity of ‘classical’ AT1 cell markers for AT1 cell lineage tracing and fragility of AT1 cells which limits their isolation for in vitro studies. To address the need for additional specific AT1 cell markers, we identified a new lung-specific AT1 cell marker, GRAMD2 (19) through cross-species transcriptomic profiling across human, mouse and rat, and generated a new transgenic mouse line, Gramd2 CreERT2 (20). Here, we further characterized the AT1 cell specificity of GRAMD2 by crossing Gramd2 CreERT2 to Rosa mTmG reporter mice for lineage labeling of AT1 cells in distal lung epithelium and utilized these mice to investigate AT1 cell plasticity in vitro .…”

“…Activation of Kras, a known oncogenic driver, in HOPX + cells can produce tumors with adenocarcinoma histology (13, 15). Our recent paper utilizing the Gramd2 CreERT2 model to activate Kras G12D oncogenic driver expression in GRAMD2 + AT1 cells (20), showed that the resulting tumors had varied histology, including precancerous atypical adenomatous hyperplasia (AAH), lepidic, and papillary adenocarcinoma, which may reflect the AT1 transitional cell state responses to the stress induced by Kras G12D activation. This is supported by observations in GRAMD2 + AT1 LUAD, where IF staining showed tumors were SCGB3A2 + and KRT8 + (20), markers which were observed in our 3-D organoid co-culture of GRAMD2 + AT1 cells undergoing cellular fate transitions.…”

“…We recently developed Gramd2 CreERT2 ;mTmG mice by crossing Gramd2 CreERT2 with mTmG reporter mice. Our initial characterization demonstrated green fluorescent protein (GFP) expression predominantly in AT1 cells, suggesting that these mice may be valuable for further exploring AT1 cell biology in health and disease (20).…”

“…We recently developed Gramd2 CreERT2 ;mTmG mice by crossing Gramd2 CreERT2 with mTmG reporter mice. Our initial characterization demonstrated green fluorescent protein (GFP) expression predominantly in AT1 cells, suggesting that these mice may be valuable for further exploring AT1 cell biology in health and disease (20). In this study, we extensively characterized the specificity of tamoxifen-mediated Gramd2 -driven GFP expression in lungs of Gramd2 CreERT2;mTmG mice and evaluated phenotypic plasticity of GFP + AT1 cells during 3-dimensional (3-D) culture using immunostaining and single cell RNAseq (scRNAseq).…”