Tumor Cell Heterogeneity in Small Cell Lung Cancer (SCLC): Phenotypical and Functional Differences Associated with Epithelial-Mesenchymal Transition (EMT) and DNA Methylation Changes

Krause, Alexander; Ahrens, Theresa D.; Yalcin, Arzu; Plönes, Till; Wehrle, Julius; Taromi, Sanaz; Wollner, Stefan; Follo, Marie; Brabletz, Thomas; Mani, Sendurai A.; Claus, Rainer; Hackanson, Björn; Burger, Meike

doi:10.1371/journal.pone.0100249

Cited by 62 publications

(65 citation statements)

References 40 publications

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“…However, in a recent study Krohn et al demonstrated that several SCLC cell lines comprise a small subpopulation of adherent cells which express high levels of the mesenchymal markers vimentin and fibronectin and very low levels of the epithelial markers E-cadherin and Zona Occludens 1 (ZO-1). 35 Although primary tumors and CTCs present EMT features in most tumors, distant metastases are generally epithelial in morphology. In 2002, Jean Paul Thiery, proposed the reversible EMT metastasis model in which tumor cells activate EMT to invade, while, upon arriving at distant sites, they undergo a reversion process, or MET, to form epithelial metastases.…”

Section: Resultsmentioning

confidence: 99%

Small cell lung cancer: Circulating tumor cells of extended stage patients express a mesenchymal-epithelial transition phenotype

Hamilton

Hochmair

Rath

et al. 2016

Cell Adhesion & Migration

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Small cell lung cancer (SCLC) is distinguished by aggressive growth, early dissemination and a poor prognosis at advanced stage. The remarkably high count of circulating tumor cells (CTCs) of SCLC allowed for the establishment of permanent CTC cultures at our institution for the first time. CTCs are assumed to have characteristics of cancer stem cells (CSCs) and an epithelial-mesenchymal transition (EMT) phenotype, but extravasation of tumors at distal sites is marked by epithelial features. Two SCLC CTC cell lines, namely BHGc7 and BHGc10, as well as SCLC cell lines derived from primary tumors and metastases were analyzed for the expression of pluripotent stem cell markers and growth factors. Expression of E-cadherin and β-Catenin were determined by flow cytometry. Stem cell-associated markers SOX17, α-fetoprotein, OCT-3/4, KDR, Otx2, GATA-4, Nanog, HCG, TP63 and Goosecoid were not expressed in the 2 CTC lines. In contrast, high expression was found for HNF-3β/FOXA2, SOX2, PDX-1/IPF1 and E-cadherin. E-cadherin expression was restricted to the 2 CTCs and 2 cell lines derived from pleural effusion (SCLC26A) and bone metastases (NCI-H526), respectively. Thus, these SCLC CTCs established from extended disease SCLC patients lack expression of stem cell markers which suppress the epithelial phenotype. Instead they express high levels of E-cadherin consistent with a mesenchymal-epithelial transition (MET or EMrT) and form large tumorospheres possibly in response to the selection pressure of first-line chemotherapy. HNF-3β/FOXA2 and PDX-1/IPF1 expression seem to be related to growth factor dependence on insulin/IGF-1 receptors and IGF-binding proteins.

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Section: Resultsmentioning

confidence: 99%

Small cell lung cancer: Circulating tumor cells of extended stage patients express a mesenchymal-epithelial transition phenotype

Hamilton

Hochmair

Rath

et al. 2016

Cell Adhesion & Migration

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“…More generally, the transition from a neuroendocrine state to a state where neuroendocrine differentiation is decreased but neuronal differentiation is increased may be related to the exceptional plasticity of SCLC cells (reviewed in (Yuan et al , 2019)). Epithelial-to-mesenchymal transition (EMT) is thought to contribute to migration, metastasis, and resistance to treatment in many cancer contexts and may play a role in SCLC (Allison Stewart et al , 2017, Krohn et al , 2014, Canadas et al , 2014, O’Brien-Ball and Biddle, 2017, Singh and Settleman, 2010). Vascular mimicry (or epithelial-to-endothelial transition (EET) (Yuan et al , 2019)) may also contribute to tumor growth and response to treatment in SCLC (Williamson et al , 2016).…”

Section: Discussionmentioning

confidence: 99%

Axon-like protrusions promote small cell lung cancer migration and metastasis

Yang

Cai

et al. 2019

Preprint

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14Metastasis is the main cause of death in cancer patients but remains a poorly understood 15 process. Small cell lung cancer (SCLC) is one of the most lethal and most metastatic types of 16 human cancer. SCLC cells normally express neuroendocrine and neuronal gene programs but 17 accumulating evidence indicates that these cancer cells become relatively more neuronal and less 18 neuroendocrine as they gain the ability to metastasize. Here we show that mouse and human 19 SCLC cells in culture and in vivo can grow cellular protrusions that resemble axons. The 20 formation of these protrusions is controlled by multiple neuronal factors implicated in 21 axonogenesis, axon guidance, and neuroblast migration. Disruption of these axon-like 22 protrusions impairs cell migration in culture and inhibits metastatic ability in vivo. The co-option 23 of developmental neuronal programs is a novel molecular and cellular mechanism that 24 contributes to the high metastatic ability of SCLC. 25 65 cells that have transitioned to a more neuronal cell state. 66 5 68 To investigate SCLC migration, we developed an assay in which SCLC cells, which 69 classically grow in culture as floating spheres or aggregates, are grown as a monolayer under 70 Matrigel ((Denny et al., 2016) and Methods). Unexpectedly, we noticed that cells from some 71 SCLC cell lines (N2N1G, 16T, 6PF) derived from the Rb f/f ;p53 f/f (DKO) and Rb f/f ;p53 f/f ;p130 f/f 72 (TKO) genetically engineered mouse models form long cellular protrusions into cell-free spaces 73 ( Figure 1A-B). To determine whether these structures specifically project into cell-free areas or 74 they also exist within monolayers, we cultured a minor fraction of fluorescently-labeled, RESULTS 67 SCLC cells can form long cellular protrusions in culture and in vivo 75GFP positive SCLC cells with control SCLC cells. We found that SCLC cells also form protrusions 76 when they are in close contact with surrounding cancer cells (Figure S1A). Similar mixing 77 experiments performed in subcutaneous allografts also documented the growth of protrusions by 78 SCLC cells in vivo (Figure 1C-D). Finally, similar structures also extend from SCLC micro-79 metastases in the liver in the autochthonous TKO mouse model and after intravenous 80 transplantations of SCLC cells (Figure S1B-C). 81Human SCLC patient-derived xenografts (PDXs) recapitulate many important features of the 82 human disease (e.g. (Gardner et al., 2017, Saunders et al., 2015). To label rare cancer cells 83 within human SCLC PDXs and identify whether they had protrusions in unperturbed tumors, we 84 used DiI tracing. DiI is a lipophilic dye that diffuses within cell membranes and has been widely 85 employed to label projections from individual neurons (Heilingoetter and Jensen, 2016, Mufson 86 et al., 1990). Protrusions from SCLC cells were easily identifiable in two out of three PDX 87 models ( Figure 1E-F). In the 2D monolayer culture system, not all human SCLC cell lines 88 formed protrusions, but NCI-H446 cells formed long protru...

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“…However, in the current study, as well as several previous studies, we identified the complexity of lung CSCs and thus highlight the difficulties that need to be dealt with before proceeding towards clinical application 18 . The presence of intra-tumor heterogeneity and inter-tumor phenotypic heterogeneity have been long described in general for cancer, particularly lung cancer 33 - 35 . Thus, it is likely that the specificity and sensitivity of markers used for lung CSC identification will vary with tumor type (adeno, squamous, small cell), stage, genetic signature and other phenotypic determinants.…”

Section: Discussionmentioning

confidence: 99%

Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma

Nishino¹,

Ozaki²,

Hegab³

et al. 2017

J. Cancer

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Background: Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines.Methods: We examined several lung adenocarcinoma cell lines for the ability of CD44v and/or ALDH expression to enrich for cells with CSC characteristics such as in vitro differential proliferation rate, chemotherapeutic-resistance, tumorsphere formation, and in vivo tumorigenicity. We also compared their in vivo secondary tumor formation, and histological characteristics of their xenograft tumors, and examined their expression of PD-L1, EGFR, xCT, and reactive oxygen species (ROS).Results: Both CD44vhigh/ALDHhigh and CD44vhigh/ALDHlow cells were enriched in cells with CSC characteristics, with the CD44vhigh/ALDHlow cells being more proliferative and more resistant to chemotherapeutics, whereas CD44vhigh/ALDHhigh cells were more efficient in forming tumorspheres in vitro, in making primary xenograft tumors, and in propagating secondary tumors in vivo. Applying stricter sorting gates to select for cells with the highest CD44v/ALDH expression caused the CD44vhigh/ALDHlow cells to lose their high proliferation rates and chemotherapeutic resistance ability, but enriched for the tumorsphere-forming cells among the CD44vhigh/ALDHhigh and CD44vhigh/ALDHlow cells. CD44vhigh expression was associated with PD-L1 and xCT expression in both H1650 and HCC827 cells. This association was not modified by ALDH expression in the H1650 cell line. However, in the HCC827 cell line, ALDH expression was negatively associated with PD-L1 and positively associated with xCT expression.Conclusion: Lung adenocarcinoma cells with high CD44v expression are enriched for CSCs. Addition of ALDH as an enrichment marker bestowed some CSCs characteristics to CD44vhigh/ALDHlow cells and others to CD44vhigh/ALDHhigh cells. We propose that lung adenocarcinoma contains different CSCs, each of them endowed with different CSC characteristics.

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Tumor Cell Heterogeneity in Small Cell Lung Cancer (SCLC): Phenotypical and Functional Differences Associated with Epithelial-Mesenchymal Transition (EMT) and DNA Methylation Changes

Cited by 62 publications

References 40 publications

Small cell lung cancer: Circulating tumor cells of extended stage patients express a mesenchymal-epithelial transition phenotype

Small cell lung cancer: Circulating tumor cells of extended stage patients express a mesenchymal-epithelial transition phenotype

Axon-like protrusions promote small cell lung cancer migration and metastasis

Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma

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