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• W3083501712 abstract "•Alveolar epithelial progenitor cells are transcriptionally distinct upon KRAS expression•Alveolar epithelial organoids recapitulate early-stage lung adenocarcinoma•Oncogenic KRAS leads to loss of lineage identity in AT2 cells•Bulk, scRNA-seq, and proteomics data from murine and human KRAS mutant AT2 cells 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. 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. KRAS is one of the most frequently mutated oncogenes in epithelial cancers. Limited understanding of the biology of KRAS and its downstream effectors in epithelial cells likely contributes to the limited therapeutic targets for KRAS mutant cancers. Oncogenic KRAS is associated with poor prognosis and therapy resistance (Haigis, 2017Haigis K.M. KRAS Alleles: The Devil Is in the Detail.Trends Cancer. 2017; 3: 686-697Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Tumor cell line experiments revealed that the rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathways are activated upon overexpression of oncogenic KRAS, but pathway activation is distinct when oncogenic KRAS is expressed at physiological levels from its endogenous promoter (Tuveson et al., 2004Tuveson D.A. Shaw A.T. Willis N.A. Silver D.P. Jackson E.L. Chang S. Mercer K.L. Grochow R. Hock H. Crowley D. et al.Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects.Cancer Cell. 2004; 5: 375-387Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar; Zhu et al., 2014Zhu Z. Golay H.G. Barbie D.A. Targeting pathways downstream of KRAS in lung adenocarcinoma.Pharmacogenomics. 2014; 15: 1507-1518Crossref PubMed Scopus (18) Google Scholar). Oncogenic KRAS mutations are driving events in lung cancer and are present in 30% of lung adenocarcinomas (LUADs) (Collisson et al., 2014Collisson E.A. Campbell J.D. Brooks A.N. Berger A.H. Lee W. Chmielecki J. Beer D.G. Cope L. Creighton C.J. Danilova L. et al.Comprehensive molecular profiling of lung adenocarcinoma: The cancer genome atlas research network.Nature. 2014; 511: 543-550Crossref PubMed Scopus (2727) Google Scholar). Furthermore, expression of oncogenic KRASG12D is sufficient to initiate LUAD in genetically engineered mouse models (GEMMs) (Jackson et al., 2001Jackson E.L. Willis N. Mercer K. Bronson R.T. Crowley D. Montoya R. Jacks T. Tuveson D.A. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes Dev. 2001; 15: 3243-3248Crossref PubMed Scopus (1292) Google Scholar). Despite the significant effect of KRAS mutations in lung cancer, the effect of oncogenic KRAS on epithelial cells shortly after its activation, besides initiation of proliferation, has not been explored. Recent advances in technologies such as single-cell RNA sequencing (scRNA-seq) and organoids make it possible to study transcriptional changes that follow oncogenic KRAS activation with single-cell resolution in a controlled environment. Previously published lung tumor organoids were derived from tumor cell lines or from tumors (Kaisani et al., 2014Kaisani A. Delgado O. Fasciani G. Kim S.B. Wright W.E. Minna J.D. Shay J.W. Branching morphogenesis of immortalized human bronchial epithelial cells in three-dimensional culture.Differentiation. 2014; 87: 119-126Crossref PubMed Scopus (21) Google Scholar; Kim et al., 2019Kim M. Mun H. Sung C.O. Cho E.J. Jeon H.J. Chun S.M. Jung D.J. Shin T.H. Jeong G.S. Kim D.K. et al.Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening.Nat. Commun. 2019; 10: 3991Crossref PubMed Scopus (112) Google Scholar; Sachs et al., 2019Sachs N. Papaspyropoulos A. Zomer-van Ommen D.D. Heo I. Böttinger L. Klay D. Weeber F. Huelsz-Prince G. Iakobachvili N. Amatngalim G.D. et al.Long-term expanding human airway organoids for disease modeling.EMBO J. 2019; 38: e100300Crossref PubMed Scopus (187) Google Scholar) and, therefore, do not model the events in early-stage tumorigenesis. Efforts have been made to model all stages of cancer progression with organoids in non-lung tissues (Drost et al., 2015Drost J. van Jaarsveld R.H. Ponsioen B. Zimberlin C. van Boxtel R. Buijs A. Sachs N. Overmeer R.M. Offerhaus G.J. Begthel H. et al.Sequential cancer mutations in cultured human intestinal stem cells.Nature. 2015; 521: 43-47Crossref PubMed Scopus (504) Google Scholar; Li et al., 2014Li X. Nadauld L. Ootani A. Corney D.C. Pai R.K. Gevaert O. Cantrell M.A. Rack P.G. Neal J.T. Chan C.W.M. et al.Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.Nat. Med. 2014; 20: 769-777Crossref PubMed Scopus (211) Google Scholar; Matano et al., 2015Matano M. Date S. Shimokawa M. Takano A. Fujii M. Ohta Y. Watanabe T. Kanai T. Sato T. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.Nat. Med. 2015; 21: 256-262Crossref PubMed Scopus (561) Google Scholar; Seino et al., 2018Seino T. Kawasaki S. Shimokawa M. Tamagawa H. Toshimitsu K. Fujii M. Ohta Y. Matano M. Nanki K. Kawasaki K. et al.Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression.Cell Stem Cell. 2018; 22: 454-467.e6Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). We demonstrated previously that primary murine lung progenitor cells survive in vitro activation of oncogenic KRAS in organoid cultures (Zhang et al., 2017aZhang H. Brainson C.F. Koyama S. Redig A.J. Chen T. Li S. Gupta M. Garcia-de-alba C. Paschini M. Herter-sprie G.S. et al.Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2.Nat. Commun. 2017; 8: 1-14PubMed Google Scholar). However, the specific effect of oncogenic KRAS on transcriptional states was not studied in any of these reports. To facilitate study of oncogenic KRAS-induced changes, we analyzed data from an early-stage KrasG12D GEMM, in-vitro-induced KrasG12D alveolar epithelial progenitor (AT2) cell-derived murine lung organoids, in-vitro-induced KRASG12D human lung organoids derived from induced pluripotent stem cells (iPSCs), and lesions from stage IA LUAD patients, all at single-cell resolution. Characterization of the data revealed that a reduction in AT2 cell lineage marker gene expression is an early consequence of oncogenic KRAS. Our organoid systems are tools to rapidly and accurately model LUAD progression in vitro, and our datasets useful resources for the cancer research community. We used scRNA-seq to define transcriptional changes in distal epithelial cell populations during early-stage LUAD in the yellow fluorescent protein (YFP) reporter containing KrasLSL-G12D; Rosa26LSL-YFP (KY) LUAD GEMM (Jackson et al., 2001Jackson E.L. Willis N. Mercer K. Bronson R.T. Crowley D. Montoya R. Jacks T. Tuveson D.A. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes Dev. 2001; 15: 3243-3248Crossref PubMed Scopus (1292) Google Scholar). KY mice were infected with an adenovirus 5 vector containing Cre recombinase driven by the ubiquitous cytomegalovirus (CMV) promoter (Ad5-CMV-Cre) (Figure 1A). After 7 weeks, we observed small clusters of YFP+ cells consistent with atypical adenomatous hyperplasia (Figure S1A). Viable, epithelial cell adhesion molecule (EPCAM) positive, recombined (CD31−/CD45−/EPCAM+/YFP+ [YFP+]) and non-recombined (CD31−/CD45−/EPCAM+/YFP− [YFP−]) cells were collected using fluorescence-activated cell sorting (FACS) (Figure S1B). We used 10X Genomics scRNA-seq to examine gene expression during early-stage LUAD and analyzed the data using ScanPy (Wolf et al., 2018Wolf F.A. Angerer P. Theis F.J. SCANPY: large-scale single-cell gene expression data analysis.Genome Biol. 2018; 19: 15Crossref PubMed Scopus (601) Google Scholar). After pre-processing, we focused on clusters containing more than 100 cells, leaving four clusters for further analysis (Figures 1B, S1C, and S1D; STAR Methods). Cluster 1 (C1) was comprised primarily of YFP+ cells and C0 of YFP− cells, whereas C2 and C3 had equivalent contributions from YFP+ and YFP− cells (Figures 1C and 1D). Expression of the AT2 cell markers Sftpc and Lyz2 was highest in C0 and C1, of the ciliated cell markers Foxj1 and Cd24a in C2, and of the club cell markers Scgb1a1 and Scgb3a2 in C3 (Figure 1E). Although YFP+ and YFP− cells were present in C2 and C3, only C0 and C1 with elevated AT2 cell marker expression formed transcriptionally distinct YFP− and YFP+ clusters (Figures 1B–1D). Correlation analysis of all clusters revealed that C0 and C1 had some degree of similarity, whereas C2 and C3 were more distinct (Figure S1E). AT2 cells have been proposed previously as the LUAD cells of origin (Lin et al., 2012Lin C. Song H. Huang C. Yao E. Gacayan R. Xu S.-M. Chuang P.-T. Alveolar type II cells possess the capability of initiating lung tumor development.PLoS ONE. 2012; 7: e53817Crossref PubMed Scopus (63) Google Scholar; Xu et al., 2012Xu X. Rock J.R. Lu Y. Futtner C. Schwab B. Guinney J. Hogan B.L.M. Onaitis M.W. Evidence for type II cells as cells of origin of K-Ras-induced distal lung adenocarcinoma.Proc. Natl. Acad. Sci. USA. 2012; 109: 4910-4915Crossref PubMed Scopus (158) Google Scholar) and are the only lung epithelial cell type that forms a transcriptionally distinct cluster upon KRASG12D expression. Hence, we focused our studies on the consequences of KRAS activation in AT2 cells. To test whether the transcriptional changes in YFP+ C1 agree with previously published data, we calculated Z scores using gene signatures we expected to be elevated in YFP+ C1. Consistent with published observations, KRAS and nuclear factor κB (NF-κB) target gene signatures were elevated in C1 cells, as was a proliferation signature, indicating that the cluster is transcriptionally primed to proliferate (Figures 1F–1H; Table S1; Barbie et al., 2009Barbie D.A. Tamayo P. Boehm J.S. Kim S.Y. Moody S.E. Dunn I.F. Schinzel A.C. Sandy P. Meylan E. Scholl C. et al.Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1.Nature. 2009; 462: 108-112Crossref PubMed Scopus (1276) Google Scholar; Bild et al., 2006Bild A.H. Yao G. Chang J.T. Wang Q. Potti A. Chasse D. Joshi M.B. Harpole D. Lancaster J.M. Berchuck A. et al.Oncogenic pathway signatures in human cancers as a guide to targeted therapies.Nature. 2006; 439: 353-357Crossref PubMed Scopus (1549) Google Scholar; Meylan et al., 2009Meylan E. Dooley A.L. Feldser D.M. Shen L. Turk E. Ouyang C. Jacks T. Requirement for NF-kappaB signalling in a mouse model of lung adenocarcinoma.Nature. 2009; 462: 104-107Crossref PubMed Scopus (412) Google Scholar; Travaglini et al., 2019Travaglini K.J. Nabhan A.N. Penland L. Sinha R. Gillich A. Sit R.V. Chang S. Conley S.D. Mori Y. Seita J. et al.A molecular cell atlas of the human lung from single cell RNA sequencing.bioRxiv. 2019; https://doi.org/10.1101/742320Crossref Scopus (0) Google Scholar). Next, we performed differential expression (DE) analysis to identify genes, transcription factors (TFs), and co-factors (TFCs) that define C0 and C1 (Figures S1F and S1G; Tables S1 and S2). We found that the lung fate TF Nkx2-1 and the AT2 cell identity TF Etv5 were enriched in C0 (Morrisey and Hogan, 2010Morrisey E.E. Hogan B.L.M. Preparing for the first breath: genetic and cellular mechanisms in lung development.Dev. Cell. 2010; 18: 8-23Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar; Zhang et al., 2017bZhang Z. Newton K. Kummerfeld S.K. Webster J. Kirkpatrick D.S. Phu L. Eastham-Anderson J. Liu J. Lee W.P. Wu J. et al.Transcription factor Etv5 is essential for the maintenance of alveolar type II cells.Proc. Natl. Acad. Sci. USA. 2017; 114: 3903-3908Crossref PubMed Scopus (36) Google Scholar). In contrast, the proto-oncogene Myc (Chen et al., 2018Chen H. Liu H. Qing G. Targeting oncogenic Myc as a strategy for cancer treatment.Signal Transduct. Target. Ther. 2018; 3: 5Crossref PubMed Scopus (217) Google Scholar; Dang, 2012Dang C.V. MYC on the path to cancer.Cell. 2012; 149: 22-35Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar; Poole and van Riggelen, 2017Poole C.J. van Riggelen J. MYC—master regulator of the cancer epigenome and transcriptome.Genes (Basel). 2017; 8: 142Crossref PubMed Scopus (55) Google Scholar) and Id1, a TF shown to promote non-small cell lung cancer (NSCLC) cell proliferation and metastasis, were upregulated in C1 (Antonângelo et al., 2016Antonângelo L. Tuma T. Fabro A. Acencio M. Terra R. Parra E. Vargas F. Takagaki T. Capelozzi V. Id-1, Id-2, and Id-3 co-expression correlates with prognosis in stage I and II lung adenocarcinoma patients treated with surgery and adjuvant chemotherapy.Exp. Biol. Med. (Maywood). 2016; 241: 1159-1168Crossref PubMed Scopus (6) Google Scholar; Cheng et al., 2011Cheng Y.J. Tsai J.W. Hsieh K.C. Yang Y.C. Chen Y.J. Huang M.S. Yuan S.S. Id1 promotes lung cancer cell proliferation and tumor growth through Akt-related pathway.Cancer Lett. 2011; 307: 191-199Crossref PubMed Scopus (27) Google Scholar; Pillai et al., 2011Pillai S. Rizwani W. Li X. Rawal B. Nair S. Schell M.J. Bepler G. Haura E. Coppola D. Chellappan S. ID1 facilitates the growth and metastasis of non-small cell lung cancer in response to nicotinic acetylcholine receptor and epidermal growth factor receptor signaling.Mol. Cell. Biol. 2011; 31: 3052-3067Crossref PubMed Scopus (57) Google Scholar). Moreover, Foxq1, a TF found to be increased in NSCLC tumor tissue compared with paired adjacent tissue, was elevated (Li et al., 2020Li L. Xu B. Zhang H. Wu J. Song Q. Yu J. Potentiality of forkhead box Q1 as a biomarker for monitoring tumor features and predicting prognosis in non-small cell lung cancer.J. Clin. Lab. Anal. 2020; 34: e23031Crossref PubMed Scopus (1) Google Scholar), and Etv4, a TF expressed during lung development (Herriges et al., 2015Herriges J.C. Verheyden J.M. Zhang Z. Sui P. Zhang Y. Anderson M.J. Swing D.A. Zhang Y. Lewandoski M. Sun X. FGF-Regulated ETV Transcription Factors Control FGF-SHH Feedback Loop in Lung Branching.Dev. Cell. 2015; 35: 322-332Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and Klf4, important for inducing pluripotency in cells (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (16833) Google Scholar), had elevated expression in C1. Hence, upon KRASG12D expression, AT2 cells downregulate TF/TFCs that maintain AT2 cell identity, whereas factors known to promote cancer growth, important for developmental processes, and induce pluripotency have increased expression. We tested whether the expression of these TF/TFCs correlated with a transition to a less differentiated state, as often observed in late-stage cancers. Indeed, a signature consisting of 46 murine AT2 cell marker genes (Franzén et al., 2019Franzén O. Gan L.-M. Björkegren J.L.M. PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data.Database (Oxford). 2019; 2019: baz046Crossref PubMed Scopus (93) Google Scholar) was significantly lower in C1 compared with C0 (Figure 1I; Table S1). It has been shown recently that primary human LUAD contains cells that express multiple lineage-specific signatures (Laughney et al., 2020Laughney A.M. Hu J. Campbell N.R. Bakhoum S.F. Setty M. Lavallée V.P. Xie Y. Masilionis I. Carr A.J. Kottapalli S. et al.Regenerative lineages and immune-mediated pruning in lung cancer metastasis.Nat. Med. 2020; 26: 259-269Crossref PubMed Scopus (62) Google Scholar). Therefore, we looked for “lineage infidelity” in our early-stage GEMM data. We found that C1 had lower expression of the AT2 cell markers Sftpc, Lyz2, and Etv5, consistent with loss of AT2 cell identity. Strikingly, the alveolar type 1 (AT1) markers Aqp5 and Pdpn and the club cell markers Scgb1a1 and Scgb3a2 were upregulated, indicating transcriptional priming for other lung epithelial cell types. Furthermore, Ly6a (SCA1), a marker of lung stem cells in mice (Kim et al., 2005Kim C.F. Jackson E.L. Woolfenden A.E. Lawrence S. Babar I. Vogel S. Crowley D. Bronson R.T. Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer.Cell. 2005; 121: 823-835Abstract Full Text Full Text PDF PubMed Scopus (1715) Google Scholar) and tumor-propagating cells in the KrasLSL-G12D/+; p53fl/fl (KP) lung cancer model (Curtis et al., 2010Curtis S.J. Sinkevicius K.W. Li D. Lau A.N. Roach R.R. Zamponi R. Woolfenden A.E. Kirsch D.G. Wong K.K. Kim C.F. Primary tumor genotype is an important determinant in identification of lung cancer propagating cells.Cell Stem Cell. 2010; 7: 127-133Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) was also upregulated in some C1 cells (Figure S1H). Finally, we performed Gene Ontology (GO) analysis on differentially expressed genes in C0 and C1 to identify pathways altered in AT2 cells after KRAS activation. In total, we found 8 common, 73 C0-specific and 160 C1-specific enriched GO pathways (Figure S1I; Table S2). Unique terms in C1 included “NIK/NF-κB signaling” (NIK: NF-κB-inducing kinase), consistent with our finding (Figure 1G), and terms that indicate upregulated ribosome biogenesis and translation. Unique terms in C0 included cholesterol, alcohol, and lipid metabolism pathways, suggesting that these processes have an essential role in AT2 cell biology. To better understand transcriptional programs that follow KRASG12D activation, we developed an in vitro organoid system that allowed us to rapidly model changes in primary lung AT2 cells shortly after induction of oncogenic KRAS. We hypothesized that KrasG12D activation alone mimics an early tumor stage phenotype, whereas additional loss of the tumor suppressor Tp53 models a more advanced stage, as is the case in GEMMs (Jackson et al., 2001Jackson E.L. Willis N. Mercer K. Bronson R.T. Crowley D. Montoya R. Jacks T. Tuveson D.A. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes Dev. 2001; 15: 3243-3248Crossref PubMed Scopus (1292) Google Scholar, Jackson et al., 2005Jackson E.L. Olive K.P. Tuveson D.A. Bronson R. Crowley D. Brown M. Jacks T. The differential effects of mutant p53 alleles on advanced murine lung cancer.Cancer Res. 2005; 65: 10280-10288Crossref PubMed Scopus (372) Google Scholar). We generated organoids by dissecting lungs of adult KY, KrasLSL-G12D/+; p53fl/fl; Rosa26LSL-YFP (KPY), and Rosa26LSL-YFP(Y) control mice and used FACS to isolate AT2 cells (CD45−/CD31−/EPCAM+/SCA1−) (Kim et al., 2005Kim C.F. Jackson E.L. Woolfenden A.E. Lawrence S. Babar I. Vogel S. Crowley D. Bronson R.T. Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer.Cell. 2005; 121: 823-835Abstract Full Text Full Text PDF PubMed Scopus (1715) Google Scholar; Lee et al., 2014Lee J.-H. Bhang D.H. Beede A. Huang T.L. Stripp B.R. Bloch K.D. Wagers A.J. Tseng Y.-H. Ryeom S. Kim C.F. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis.Cell. 2014; 156: 440-455Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar; Figure 2A). Cells were infected with the Ad5-CMV-Cre (CRE) virus in vitro and cultured with stromal cells in our 3D organoid air-liquid interface (ALI) co-culturing system described previously (Lee et al., 2014Lee J.-H. Bhang D.H. Beede A. Huang T.L. Stripp B.R. Bloch K.D. Wagers A.J. Tseng Y.-H. Ryeom S. Kim C.F. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis.Cell. 2014; 156: 440-455Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, Lee et al., 2017Lee J.-H. Tammela T. Hofree M. Choi J. Marjanovic N.D. Han S. Canner D. Wu K. Paschini M. Bhang D.H. et al.Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6.Cell. 2017; 170: 1149-1163.e12Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Upon Cre expression, almost all organoids were YFP+, suggesting high Cre induction efficiency (Figure 2B). Histological analysis revealed that our tumor organoid model recapitulated in vivo tumor progression. Hematoxylin and eosin (H&E)-stained sections of organoids demonstrated that Y-CRE control organoids maintained normal nuclei, whereas the nuclei of KY-CRE and KPY-CRE cells became enlarged and abnormal, with giant multinucleated cancer cells in KPY organoids (Figure 2C). This observation is reminiscent of documented in vivo tumor cell phenotypes in the KrasLSL-G12D/+ and KP mouse models (Jackson et al., 2001Jackson E.L. Willis N. Mercer K. Bronson R.T. Crowley D. Montoya R. Jacks T. Tuveson D.A. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes Dev. 2001; 15: 3243-3248Crossref PubMed Scopus (1292) Google Scholar, Jackson et al., 2005Jackson E.L. Olive K.P. Tuveson D.A. Bronson R. Crowley D. Brown M. Jacks T. The differential effects of mutant p53 alleles on advanced murine lung cancer.Cancer Res. 2005; 65: 10280-10288Crossref PubMed Scopus (372) Google Scholar). Next, we interrogated the effect of KRASG12D on proliferation. On day 7 of organoid culture, there was no significant difference in the percentage of KI67+ cells per organoid between the Y-CRE control and KY-CRE, whereas there were 1.3-fold and 1.6-fold increases in KPY-CRE organoids compared to Y-CRE and KY-CRE, respectively (Figures 2D and S2A). On day 14, most of the Y-CRE control organoids stained negative for KI67, whereas KY-CRE and KPY-CRE organoids still contained cells that stained positive for KI67 (Figures 2E and S2B). Thus, organoids from all three genotypes contained a high number of proliferating cells on day 7, but although most of the cells in the control organoids had stopped proliferating by day 14, cells in KY and KPY organoids continued to proliferate. To test whether KRASG12D-expressing organoids form tumors in vivo, we performed orthotopic transplantation assays. We transplanted single-cell suspensions from Y-CRE control, KY-CRE, and KPY-CRE organoids into the lungs of bleomycin-injured mice (n = 4, n = 6, and n = 4, respectively). After 4 weeks, we evaluated tumor formation by histology. The lungs of Y-CRE control-transplanted mice did not show any signs of aberrant epithelial cell growth or tumor formation (Figure 2F). In contrast, in KY-CRE- and KPY-CRE-transplanted lungs, we found tumors that contained cells with pleomorphic features and giant cancer cells in KPY-CRE-transplanted lungs, comparable with observations in the organoid cultures (Figures 2G and 2H). Immunofluorescence (IF) staining for YFP confirmed that these tumor lesions contained transplanted cells (Figures S2C and S2D). Hence, cells derived from our in-vitro-induced tumor organoids formed tumors within 4 weeks, dramatically reducing the time required to model lung cancer in vivo compared with traditional GEMMs. To further investigate transcriptional changes following KRASG12D activation, we performed RNA-seq on cells from our organoid cultures. KY- and KPY-derived AT2 cells received the Ad5-CMV-Empty virus (Emp, control), no virus (control), or CRE (Figure 3A). Because we sought to reveal transcriptional changes that follow KRASG12D activation but not proliferation, we analyzed the organoids on day 7 of organoid culture, when proliferation was observed in all organoid types. After 7 days in culture, single-cell suspensions were enriched for epithelial cells by FACS for EPCAM+ cells (Figure S3A). 87% ± 7% and 95% ± 2% of EPCAM+ cells of the KY-CRE and KPY-CRE samples, respectively, were YFP+, further confirming the high efficiency of in vitro Cre induction. Next we performed RNA-seq on EPCAM+ cells. Sample-sample correlation analysis revealed that all control samples were highly correlated, whereas KY-CRE and KPY-CRE samples had high correlation and were transcriptionally distinct from the controls (Figure S3B). To perform DE analysis, we compared the CRE samples with their respective Emp controls (henceforth, KY-Differential [KY-Dif] and KPY-Differential [KPY-Dif]; Table S3). To determine genes that were altered by KRASG12D expression, we compared KY-Dif with KPY-Dif and found 1,206 genes that were shared upregulated and 1,464 genes that were shared downregulated (Figure S3C; Table S3). Because we saw downregulation of AT2 cell differentiation genes in our GEMM data, we investigated the expression of known AT2 cell markers and lung development genes. When we compared the top 100 up- and downregulated genes in our RNA-seq data, we found that Cd74 and Lyz2, two AT2 cell marker genes, were among the top shared downregulated genes (Figures 3B and 3C). Conversely, the developmental genes Hmga2 and Sox9 were upregulated. Furthermore, we found increased expression of Ly6a (SCA1), consistent with our findings in vivo (Figures S1H, 3B, and 3C). Moreover, we found that other known AT2 cell markers, Sftpc, Sftpd, and Nkx2-1, were significantly downregulated in organoids from both genotypes (Figure 3C). Next, we investigated whether these changes also occurred at the protein level. IF staining for surfactant protein C (SPC; Sftpc) showed that the percentage of SPC+ cells per organoid decreased 6.7-fold in KY-CRE and 20-fold in KPY-CRE compared with Y-CRE control organoids on day 7 (Figures 3D and 3E). On day 14, there was a 1.1-fold decrease in KY-CRE and a 1.6-fold decrease in KPY-CRE compared with Y control organoids (Figures S3E and S3F). Furthermore, staining for the lung epithelial marker NKX2-1 and the developmental marker HMGA2 was negatively correlated; individual cells that gained HMGA2 expression had reduced levels of NKX2-1 (Figure 3F). Thus, we demonstrated that transcriptional downregulation of AT2 cell markers and upregulation of developmental markers correlated with altered expression of the respective proteins. To further characterized our KY-CRE organoids, we performed scRNA-seq. As before, we characterized day 7 EPCAM+ cells from KY-CRE and KY-Emp organoids (Figures 4A and S4A). After filtering and preprocessing the data, we identified three clusters: C0org, C1org, and C2org (Figures 4B, S4B, and S4C). C1org was composed mostly of KY-Emp cells, representing the control cluster, whereas C0org and C2org mostly contained KY-CRE cells (Figures 4B–4D). Correlation analysis revealed that all three clusters were distinct and that C0org and C1org were negatively correlated (Figure S4D). As with our GEMM data, we checked the expression of previously published gene signatures upregulated in NSCLC. As expected, the KRAS activation signature was upregulated in C0org and C2org compared with control cluster C1org (Figure 4E; Table S1). The NF-κB activation signature was lower in C2org and higher in C0org compared with C1org, indicating that only one of the Cre clusters had upregulated NF-κB signaling (Figure 4F; Table S1). Interestingly, the proliferation signature was only elevated in C2org but not in C0org, indicating that only one of the Cre clusters had a higher proliferation signature than the control, despite high Kras activation signatures in both clusters (Figure 4G; Table S1). Next, we performed DE analysis followed by identification of TF/TFCs (Figures S4E and S4F; Tables S1 and S4). Similar to our GEMM data, control C1org had elevated expression of Etv5, providing additional evidence of loss of AT2 cell transcriptional identity. One TF highly expressed in both Cre clusters compared with the control was Foxq1, and C2org had high expression of Id1" @default.
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