Organoids Model Transcriptional Hallmarks of Oncogenic KRAS Activation in Lung Epithelial Progenitor Cells

Abstract

Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.

Keywords: KRAS; alveolar; developmental programs; early-stage lung cancer; iPSC; loss of differentiation; organoid; single-cell RNA sequencing; stage IA lung adenocarcinoma; tumor progression.

Figure 1.. ScRNA-Seq of distal lung epithelium reveals distinct transcriptional clusters of KRAS^G12D activated cells during early tumorigenesis. See also ^{Figure S1}.

(A) Experimental strategy to analyze epithelial populations during early-stage LUAD in vivo using scRNA-Seq. (B) (C) Clustering of transcriptomes using UMAP. Cells are colored based on (B) Louvain clusters or (C) Batch ID. (D) Batch contributions to each Louvain cluster with number of cells indicated. (E) Log expression of lung epithelial cell marker genes in each Louvain cluster. (F)(G)(H)(I) Z-scores of indicated signatures in Louvain clusters 0 and 1. Dashed line marks median of reference sample.

Figure 2.. Inducible organoids rapidly recapitulate in vivo tumor progression and form tumors upon transplantation. See also ^{Figure S2}.

(A) Experimental strategy to grow air liquid interphase (ALI) organoid cultures in growth factor reduced (GFR) Matrigel. (B) Representative whole-well brightfield (BF) and YFP-channel images of organoid cultures. Images were stitched together to show whole wells. (C) Representative H+E stained organoid slides. Arrows: pleomorphic cells. Arrowheads: giant, multinucleated cells. Scale bar = 25 μm. (D)(E) Quantification of KI67+ cells per organoid on (D) day 7 and (E) day 14 of organoid culture based on IF staining. Each dot represents one organoid. (F, G, H) H+E staining of mouse lungs that were transplanted with organoid-derived cells. Scale bar lower magnification = 100 μm. Scale bar higher magnification = 25 um. P-values were determined using the Mann-Whitney rank test. n.s.=p≥0.05, *=p<0.05, **=p<0.005, ***=p<0.0005.

Figure 3.. KRAS^G12D activated cells in organoids lose AT2 differentiation markers and express developmental lung markers. See also ^{Figure S3}.

(A) Experimental strategy to grow air liquid interphase (ALI) organoid cultures to perform RNA-Seq. (B) Venn diagram showing the overlap of the top 100 differentially expressed genes in KY-CRE and KPY-CRE compared to their respective -Emp controls. (C) Log2 fold change expression of selected genes compared to their control from RNA-Seq results. (D) Representative pictures of IF staining on day 7 of organoid culture. Scale bar = 100 μm. (E) Quantification of SPC+ cells per organoid on day 7 of organoid culture. Each dot represents one organoid. (F) Representative pictures of IF staining on day 7 of organoid culture. Scale bar = 25 μm. P-values were determined using the Mann-Whitney rank test. n.s.=p≥0.05, ***=p<0.0005

Figure 4.. KRAS^G12D expressing organoid cells are transcriptionally distinct and transition to a developmental-like state. See also ^{Figure S4}.

(A) Experimental strategy to grow air liquid interphase (ALI) organoid cultures followed by scRNA-Seq. (B)(C) Clustering of transcriptomes using UMAP. Cells are colored based on (B) Louvain clusters or (C) Batch ID. (D) Batch contributions to each Louvain cluster with number of cells indicated. (E)(F)(G)(H) Z-scores of indicated signatures in each Louvain cluster. Dashed line marks marks median of reference sample. (I)(J) Log2 expression of indicated genes. Dashed line marks median expression of the reference sample. (K) Z-score of indicated signature in each Louvain cluster. Dashed line marks median of reference sample. (L) RNA velocity analysis of KRAS^G12D organoid scRNA-Seq dataset. Louvain clusters are shown on the left. Sox9 expression is visualized on the right. P-values were determined using a Mann-Whitney rank test *** = p-value > 0.001, ** = p-value > 0.01.

Figure 5.. Human iAT2s downregulate differentiation and maturation markers and upregulate progenitor markers upon KRAS^G12D expression. See also ^{Figure S5}.

(A) Schematic of AAVS1 locus with integrated dox inducible KRAS^G12D. (B) Experimental strategy and timeline to grow and analyze KRAS^G12D inducible iAT2. DOX=doxycycline (1μg/ml), pi, p2, p3 = passage 1, 2, 3. (C) Volcano plots indicating differential protein (left) and phosphoprotein (right) expression between dox induced and control iAT2s. (D) Top 10 upregulated pathways in dox induced compared to control iAT2s based on phosphoproteomics analysis. (E) FACS analysis of iAT2s over three passages following the initiation of dox vs. control vehicle (DMSO) treatment. Mean fluorescence intensity (MFI) of tdTomato is indicated. (F) Log expression of indicated genes. Log expression of indicated genes. P-values were determined using the MAST single-cell test. *p<0.05. (G) Log expression of indicated gene signatures. P-values were determined using a Welch Two Sample t-test. *p<0.05. (H) Log expression of indicated genes. P-values were determined using the MAST single-cell test. *p<0.05.

Figure 6.. Differentiation and maturation markers are downregulated in AT2 cells from human early stage LUAD. See also ^{Figure S6}.

(A) Experimental strategy to obtain cells from human early stage IA LUAD for scRNA-Seq. (B)+(C) Louvain clustering of transcriptomes of non-immune LUAD cell types and matching normal lung tissue. Cells are colored based on (B) Louvain clusters or (C) Sample ID. (D) Batch contributions to each Louvain cluster shown in 6B with number of cells indicated. (E) Violin plots showing gene expression values of selected genes in annotated clusters shown in 6B. (F) Z-score of gene signature comprised of AT2 signature genes shared between mouse and human from the Panglao database. Dashed line marks y=0. (G) Transcriptional comparison of KRAS LUAD models. Correlation heatmap of individual cells of the organoid scRNA-Seq data (x-axis) and z-normalized gene signatures (y-axis). Cells are ordered based on correlation distance calculation. Louvain clusters are annotated.