Cancer stem cells (CSCs) are a promising target for treating cancer, yet how CSC plasticity is maintained in vivo is unclear and is difficult to study in vitro. Here we establish a sustainable primary culture of Oct3/4( þ )/Nanog( þ ) lung CSCs fed with CD90( þ ) cancer-associated fibroblasts (CAFs) to further advance our knowledge of preserving stem cells in the tumour microenvironment. Using transcriptomics we identify the paracrine network by which CAFs enrich CSCs through de-differentiation and reacquisition of stem cell-like properties. Specifically, we find that IGF1R signalling activation in cancer cells in the presence of CAFs expressing IGF-II can induce Nanog expression and promote stemness. Moreover, this paracrine signalling predicts overall and relapse-free survival in stage I non-small cell lung cancer (NSCLC) patients. IGF-II/IGF1R signalling blockade inhibits Nanog expression and attenuates cancer stem cell features. Our data demonstrate that CAFs constitute a supporting niche for cancer stemness, and targeting this paracrine signalling may present a new therapeutic strategy for NSCLC.

The tumorous niche may drive the plasticity of heterogeneity and cancer stemness, leading to drug resistance and metastasis, which is the main reason of treatment failure in most cancer patients. The aim of this study was to establish a tumor microenvironment (TME)-based screening to identify drugs that can specifically target cancer stem cells (CSCs) and cancer-associated fibroblasts (CAFs) in the TME. Methods: Lung cancer patient-derived cancer cell and CAFs were utilized to mimic the TME and reproduce the stemness properties of CSCs in vitro and develop a high-throughput drug screening platform with phenotypical parameters. Limiting dilution assay, sphere-forming and ALDH activity assay were utilized to measure the cancer stemness characteristics. In vivo patient-derived xenograft (PDX) models and single-cell RNA sequencing were used to evaluate the mechanisms of the compounds in CSCs and CAFs. Results: The TME-based drug screening platform could comprehensively evaluate the response of cancer cells, CSCs and CAFs to different treatments. Among the 1,524 compounds tested, several drugs were identified to have anti-CAFs, anticancer and anti-CSCs activities. Aloe-emodin and digoxin both show anticancer and anti-CSCs activity in vitro and in vivo , which was further confirmed in the lung cancer PDX model. The combination of digoxin and chemotherapy improved therapeutic efficacy. The single-cell transcriptomics analysis revealed that digoxin could suppress the CSCs subpopulation in CAFs-cocultured cancer cells and cytokine production in CAFs. Conclusions: The TME-based drug screening platform provides a tool to identify and repurpose compounds targeting cancer cells, CSCs and CAFs, which may accelerate drug development and therapeutic application for lung cancer patients.

Cancer stem cells (CSCs) have been proposed to be responsible for chemoresistant, tumor recurrent, and metastasis. To culture and characterize the CSCs become urgent and may be helpful to develop novel diagnostic and therapeutic strategies. Accumulated evidences indicated the importance of CSCs in tumor formation and successfully isolated the CSCs from patients or cell lines with different cancer types; however, this has not been well explored in lung cancer. Not alike previous isolation through specific markers and quiescent condition, we cultured the lung cancer stem/initiating cells from patient with lung adenocarcinoma and the CSCs formed tumor sphere as fed by the surrounding stroma cells. These lung cancer stem/initiating cells were characterized with highly expressed levels of the stemness makers, nanog, oct4, sox2, and klf4 (53.3-, 37.7-, 13.0, and 4.4-folds changes) as comparing to the feeder cells, also, showed relative higher percentages of side population (27.4%), ALDH activity (14%), higher tumorigenesity compared to other lung cancer cell lines including adenocarcinoma (A549, H522, H23, Hop62, and EKVX), large cell carcinoma (Hop92), squamous cell carcinoma (H226), and bronchi alveolar carcinoma (H322M). These lung CSCs also showed higher drug resistance capacity represented by IC50 to chemotherapy drugs including docetaxel, cisplatin, vinorelbine distrate, and etoposide (13.0-, 1.55, 27.2- and 103.3-folds increased). Furthermore, we found that as less as 100 cells could generate adenocarcinoma formation both in orthotropic or subcutaneous models in severe combined immunodeficient (NOD/SCID) mice. Interestingly, the lung CSCs could spontaneous differentiate into epithelial types of cancer cells as removing the feeder cells in vitro or different phenotypes of adenocarcinoma cells in vivo. Transcriptomic analysis indicated that the Wnt, Notch, TGF-β and HGF signaling pathways are highly expressed in the lung CSCs and quickly down-regulated as removing the feeder cells. The gene expression profiles also provide the novel markers for selection CSCs from lung cancer cell lines and lung cancer patients. To sum up, we have cultured the cancer stem/initiating cells from lung cancer patient with the feeder cells, which providing the tumor microenvironment to maintain the stemness of the cancer stem cells. This model should be helpful not only for isolating and cultured the lung CSCs from patients, but also in developing the anti-cancer strategy targeting on lung CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 501. doi:10.1158/1538-7445.AM2011-501