Tobacco chemical-induced mouse lung adenocarcinoma cell lines pin the prolactin orthologue proliferin as a lung tumour promoter

Abstract

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro, and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers’ LADC, including carcinogen-specific Kras point mutations (KrasQ61R in urethane- and KrasQ61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease.

Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

“…Interestingly, all hyperplasias and tumors of GFP;CCSP.CRE mice contained GFP-labeled airway cells that did not express the club cell marker CCSP anymore, but had acquired expression of the alveolar epithelial markers SFTPC with or without LYZ2 (Figure 1D, Figure 1—figure supplements 12–15, and Figure 1—figure supplement 13—source data 1). Identical results were recapitulated using single urethane hits to GFP;CCSP.CRE, GFP;SFTPC.CRE, and GFP;LYZ2.CRE mice backcrossed >F12 to the susceptible FVB strain, which result in human LUAD-like mutations including Kras Q61R (Westcott et al, 2015; Vreka et al, 2018; Kanellakis et al, 2019) (Figure 1D and Figure 1—figure supplements 16–19). Collectively, these data support that airway cells contribute to chemical-induced LUAD, shifting from airway to alveolar marker expression during carcinogenesis.…”

An in vivo inflammatory loop potentiates KRAS blockade

“…To exclude the possibility of developmental effects of knockout mice, we total-body irradiated (900 Rad) Ccr2 -/- mice and performed adoptive BMT from WT or Ccr2 -/- donors, as described previously [14, 18]. For this experiment, WT and Ccr2 -/- mice back-crossed > F12 to the FVB strain were used together with syngeneic FULA1 cells ( Kras Q61R ) to obtain results with another cell line harboring a different Kras mutation and a broad mutation spectrum relevant to human KRAS -mutant LADC [17]. Again, daily ip saline or deltarasin treatments were started when tumors > 100 mm 3 were established.…”

“… (A) Total-body irradiated (900 Rad) Ccr2 -/- mice received adoptive BMT from WT or Ccr2 -/- donors (all back-crossed > F12 to the FVB strain). After one month allowed for chimeric bone marrow reconstitution, chimeras received 106 syngeneic FULA1 cells ( Kras Q61R ) sc [17]. Daily ip saline or deltarasin (15 mg/Kg in saline) treatments were started when tumors > 100 mm 3 were established (arrow).…”

“…NCI-H358, NCI-H358M, NCI-H460, NCI-H520, NCI-H1299, NCI-H1944, NCI-H3122 (referred to hereafter omitting NCI), EKVX, A549, LLC, B16F10, and PANO2 cell lines were from the National Cancer Institute (Frederick, MD); MC38 cells were a gift from Dr. Timothy S. Blackwell (Vanderbilt University, Nashville, TN) and AE17 cells from Dr. YC Gary Lee (University of Western Australia, Perth, Australia) [13–15]. FULA1 ( FVB urethane-induced lung adenocarcinoma 1) and CULA ( C57BL/6 urethane-induced lung adenocarcinoma) cell lines were isolated from the lungs of FVB and C57BL/6 mice, respectively, harboring primary lung adenocarcinomas induced by urethane [13,16,17]. Human and murine cell lines were cultured, respectively, in Roswell Park Memorial Institute (RPMI)-1640 and Dulbecco’s Modified Eagle Medium (DMEM), both supplemented with 10% FBS and 100 IU/mL penicillin/streptomycin, and were maintained in a humidified incubator at 37 °C with 95% air–5% CO 2 .…”

“…Isogenic cell line doublets stably expressing sh C or sh Kras (LLC, MC38, and AE17 cells) and p C or p Kras G12C (PANO2 and B16F10 cells) were generated as described elsewhere [13]. Benign samples including whole murine lungs, tracheal epithelial cells (TEC; cultured out from murine tracheas), and bone marrow-derived macrophages (BMDM; cultured from murine bone marrow by weekly incubation with 20 ng/mL M-CSF) and mast cells (BMMC; cultured from murine bone marrow by monthly incubation with 100 ng/mL IL-3 plus KITL) were prepared as described elsewhere [14, 17, 18]. Cellular RNA was isolated using Trizol (Thermo Fisher) followed by RNAeasy column purification and genomic DNA removal (Qiagen, Hilden, Germany).…”