Mapping lung squamous cell carcinoma pathogenesis through in vitro and in vivo models

Lung cancer is the main cause of cancer death worldwide, with lung squamous cell carcinoma (LUSC) being the second most frequent subtype. Preclinical LUSC models recapitulating human disease pathogenesis are key for the development of early intervention approaches and improved therapies. Here, we review advances and challenges in the generation of LUSC models, from 2D and 3D cultures, to murine models. We discuss how molecular profiling of premalignant lesions and invasive LUSC has contributed to the refinement of in vitro and in vivo models, and in turn, how these systems have increased our understanding of LUSC biology and therapeutic vulnerabilities

Epithelial cell migration as a potential therapeutic target in early lung cancer.

Lung cancer is the most lethal cancer type worldwide, with the majority of patients presenting with advanced stage disease. Targeting early stage disease pathogenesis would allow dramatic improvements in lung cancer patient survival. Recently, cell migration has been shown to be an integral process in early lung cancer ontogeny, with preinvasive lung cancer cells shown to migrate across normal epithelium prior to developing into invasive disease. TP53 mutations are the most abundant mutations in human nonsmall cell lung cancers and have been shown to increase cell migration via regulation of Rho-GTPase protein activity. In this review, we explore the possibility of targeting TP53-mediated Rho-GTPase activity in early lung cancer and the opportunities for translating this preclinical research into effective therapies for early stage lung cancer patients

<i>Lrig1</i> expression identifies airway basal cells with high proliferative capacity and restricts lung squamous cell carcinoma growth

Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression.Together, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes

Two cell line models to study multiorganic metastasis and immunotherapy in lung squamous cell carcinoma

There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines in vivo and assessed their response to immune checkpoint inhibition. The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.This work was supported by FIMA, Centro de Investigacion Biomedica en Red de Cancer (CIBERONC) (grant number: CB16/12/00443), Fundacion Cientifica Asociacion Espanola Contra el Cancer (grant number: GCB14-2170), Fundacion Ramon Areces, Instituto de Salud Carlos III and the European Regional Development Fund (ERDF, A way to make Europe) (grant numbers: PI19/00098; PI19/00230; PI20/00419), Fundacion Roberto Arnal Planelles and an IASLC Fellowship funding (K.V.); D.S. was supported by the Juan de la Cierva-Incorporacion program, Spanish Ministry of Science and Innovation (grant number: IJCI-2016-27595); E.R. was supported by a FPU, Spanish Ministry of Education ( grant number: FPU17/01168); M.E. was supported by PFIS, Spanish Ministry of Health, M.L. was supported by a Junior Investigator grant from AECC

Progress towards non-small-cell lung cancer models that represent clinical evolutionary trajectories

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Although advances are being made towards earlier detection and the development of impactful targeted therapies and immunotherapies, the 5-year survival of patients with advanced disease is still below 20%. Effective cancer research relies on pre-clinical model systems that accurately reflect the evolutionary course of disease progression and mimic patient responses to therapy. Here, we review pre-clinical models, including genetically engineered mouse models and patient-derived materials, such as cell lines, primary cell cultures, explant cultures and xenografts, that are currently being used to interrogate NSCLC evolution from pre-invasive disease through locally invasive cancer to the metastatic colonization of distant organ sites

Premalignant Progression in the Lung: Knowledge Gaps and Novel Opportunities for Interception of Non-Small Cell Lung Cancer. An Official American Thoracic Society Research Statement

Rationale: Despite significant advances in precision treatments and immunotherapy, lung cancer is the most common cause of cancer death worldwide. To reduce incidence and improve survival rates, a deeper understanding of lung premalignancy and the multistep process of tumorigenesis is essential, allowing timely and effective intervention before cancer development. Objectives: To summarize existing information, identify knowledge gaps, formulate research questions, prioritize potential research topics, and propose strategies for future investigations into the premalignant progression in the lung. Methods: An international multidisciplinary team of basic, translational, and clinical scientists reviewed available data to develop and refine research questions pertaining to the transformation of premalignant lung lesions to advanced lung cancer. Results: This research statement identifies significant gaps in knowledge and proposes potential research questions aimed at expanding our understanding of the mechanisms underlying the progression of premalignant lung lesions to lung cancer in an effort to explore potential innovative modalities to intercept lung cancer at its nascent stages. Conclusions: The identified gaps in knowledge about the biological mechanisms of premalignant progression in the lung, together with ongoing challenges in screening, detection, and early intervention, highlight the critical need to prioritize research in this domain. Such focused investigations are essential to devise effective preventive strategies that may ultimately decrease lung cancer incidence and improve patient outcomes

Pre-clinical lung squamous cell carcinoma mouse models to identify novel biomarkers and therapeutic interventions

Primary lung carcinoma or lung cancer (LC) is classified into small-cell or non-small-cell (NSCLC) lung carcinoma. Lung squamous cell carcinoma (LSCC) is the second most common subtype of NSCLC responsible for 30% of all LCs, and its survival remains low with only 24% of patients living for five years or longer post-diagnosis primarily due to the advanced stage of tumors at the time of diagnosis. The pathogenesis of LSCC is still poorly understood and has hampered the development of effective diagnostics and therapies. This review highlights the known risk factors, genetic and epigenetic alterations, miRNA biomarkers linked to the development and diagnosis of LSCC and the lack of therapeutic strategies to target specifically LSCC. We will also discuss existing animal models of LSCC including carcinogen induced, transgenic and xenograft mouse models, and their advantages and limitations along with the chemopreventive studies and molecular studies conducted using them. The importance of developing new and improved mouse models will also be discussed that will provide further insights into the initiation and progression of LSCC, and enable the identification of new biomarkers and therapeutic targets

Aberrant basal cell clonal dynamics shape early lung carcinogenesis

Preinvasive squamous lung lesions are precursors of lung squamous cell carcinoma (LUSC). The cellular events underlying lesion formation are unknown. Using a carcinogen-induced model of LUSC with no added genetic hits or cell type bias, we find that carcinogen exposure leads to non-neutral competition among basal cells, aberrant clonal expansions, and basal cell mobilization along the airways. Ultimately, preinvasive lesions develop from a few highly mutated clones that dominate most of the bronchial tree. Multi-site sequencing in human patients confirms the presence of clonally related preinvasive lesions across distinct airway regions. Our work identifies a transition in basal cell clonal dynamics, and an associated shift in basal cell fate, as drivers of field cancerization in the lung

Defining the role of LRIG1 dependent EGFR signalling on airway homeostasis and lung cancer development

Aberrations of EGFR signalling drive cancer development. In squamous cell lung cancer, EGFR is overexpressed. LRIG1 is a negative regulator of EGFR and patient pre-invasive squamous cell lung cancer samples show LRIG1 loss, suggesting involvement in early disease pathogenesis. In skin and gut homeostasis, LRIG1 regulates stem cells. In the upper airway, basal cells act as stem cells and are the putative origin of squamous cell lung cancer. I hypothesise LRIG1 has a key role in the airway homeostasis and its loss tilts this towards pre-invasive squamous cell lung cancer development. Lrig1 EGFP-ires-CreERT2 mice delineated airway LRIG1 expression. Flow sorted LRIG1-positive and -negative murine basal cells were used in 2D and 3D colony-forming, spheroid and proliferation assays. A murine squamous cell lung cancer model was set up through application of N-Nitrosotris-(2-chloroethyl)urea (NTCU). Pre-invasive lesions and tumour development were compared between wild-type (WT), LRIG1-heterozygous and LRIG1-null animals. Human basal cells obtained from bronchoscopy were sorted according to LRIG1 expression and used directly in colony-forming assays or maintained in primary culture to assess the effect of shRNA knockdown of LRIG1. LRIG1 is expressed by 50% of airway basal cells. LRIG1-expressing murine basal cells exhibit increased colony-forming capacity (p=0.0133), spheroid formation (p=0.0020) and proliferation (p=0.0011) compared to LRIG1-negative cells. Similarly, LRIG1-expressing human airway basal cells isolated from endobronchial brush biopsy samples exhibit increased colony-forming capacity (p=0.0067) and proliferation (p=0.0153). Topical application of NTCU to mice recapitulates the development of human pre-invasive and squamous cell lung cancer lesions after 23 weeks. Results show lesions in LRIG1-null mice to be larger than those of WT animals. shRNA knockdown of LRIG1 in cultured human airway basal cells alters cell phenotype, leading to increased colony-forming efficiency and greater proliferation at cell confluence. LRIG1 has an important role in stem cell homeostasis of the human and murine airway epithelium. Loss of LRIG1 promotes lesion development in a murine squamous cell lung cancer mouse model and alters behaviour of human epithelial cells in culture, indicating a potential target for the treatment of squamous cell lung cancer in humans