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FIGURE 2 from Activation of KrasG12D in Subset of Alveolar Type II Cells Enhances Cellular Plasticity in Lung Adenocarcinoma

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posted on 2023-11-24, 14:40 authored by Priyanka Chaudhary, Xia Xu, Guangfang Wang, Jacob P. Hoj, Rishi R. Rampersad, Marie-Liesse Asselin-Labat, Stephanie Ting, William Kim, Pablo Tamayo, Ann Marie Pendergast, Mark W. Onaitis

Double-positive cells in Sftpc-CreER; LSL-KrasG12D-induced lung adenocarcinoma are tumor-propagating cells and forms tumors upon transplantation. A, Experimental outline for growing cells in 3D organoid culture. B, FACS scatter plot showing single (type I or type II) and double-positive population sorted from Sftpc-CreER; LSL-KrasG12D; TdTomato mice. Type I (TdTm−Pdpn+), type II (TdTm+ Pdpn−), and double-positive (type I/II+; TdTm+Pdpn+) fractions were sorted. C, Brightfield images of 3D organoid cultures from different indicated sorted populations on day 14. Colony number (D) and colony size (E) was quantified using ImageJ software. F, 3D organoid culture from double-positive cells on day 14 in red channel. G, Immunofluorescent image on day 14 of organoid culture showing double-positive cells differentiate into type I and type II cells. Ki67 (H) and TTF1 (I) staining of 3D organoid culture. J, Representative H&E staining of tumors sections which were obtained after subcutaneous transplantation of double-positive (type I/II+) cells. Right panel shows higher magnification of boxed area in left panels. K, Representative IHC images of tumor sections for TTF1. Right panel shows higher magnification of boxed area in left panels. L, Representative immunofluorescent image for in red channel showing Tdtomato expression in tumor obtained after transplantation. M, Representative IF images for Sftpc and Rage staining. Right panel shows higher magnification of boxed area in left panels. Results shown in B, F, G, H, I, J, K, L, and M are representative of three independent experiments. D and E represent data from three independent experiments. Error bars represent the mean ± SEM values and significance is defined as *, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001.

Funding

HHS | NIH | NCI | National Cancer Institute (NCI)

VA | Veterans Affairs San Diego Healthcare System (VASDHS)

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ARTICLE ABSTRACT

We have previously identified alveolar type II cell as the cell-of-origin of KrasG12D-induced lung adenocarcinoma using cell lineage–specific inducible Cre mouse models. Using gain-of-function and loss-of-function genetic models, we discovered that active Notch signaling and low Sox2 levels dictate the ability of type II cells to proliferate and progress into lung adenocarcinoma upon KrasG12D activation. Here, we examine the phenotype of type II cells after Kras activation and find evidence for proliferation of cells that coexpress type I and type II markers. Three-dimensional organoid culture and transplantation studies determine that these dual-positive cells are highly plastic and tumor initiating in vivo. RNA sequencing analysis reveals that these dual-positive cells are enriched in Ras/MAPK, EGFR, and Notch pathways. Furthermore, the proliferation of these cells requires active Notch signaling and is inhibited by genetic/chemical Sox2 upregulation. Our findings could provide new therapeutic strategies to target KRAS-activated lung adenocarcinomas. Identification of progenitor like tumor-initiating cells in KRAS-mutant lung adenocarcinoma may allow development of novel targeted therapeutics.