Phenotypic heterogeneity in small cell lung cancer models in vitro and in vivo

Small cell lung cancer (SCLC) accounts for around 15% of new cases and 25% of deaths from lung cancer, making it the most aggressive subtype. This is due to the fact that tumours quickly develop resistance to therapy and metastasise early to the brain, liver and bone marrow. SCLC is normally characterised as a neuroendocrine tumour which secretes hormone precursors. Pro-opiomelanocortin (POMC) is primarily produced as a component of the stress pathway but neuroendocrine lung tumours can secrete high levels of unprocessed POMC into the circulation. Given the well recognised neuroendocrine origin of SCLC, the prevalence of epithelial biomarkers highlights the ambiguity of the phenotype and origin of SCLC. We proposed that POMC could be a novel biomarker to identify patients with SCLC and those with more aggressive, metastatic tumours. Results from a panel of SCLC cell lines, show that 40% of cell lines secreted POMC. All cell lines were positive for cytokeratin 18 and there was distinct heterogeneous cytokeratin staining in cell lines. There was also heterogeneity when looking at other markers of both neuroendocrine and epithelial origin.In vivo, circulating POMC correlated strongly with tumour growth in a subcutaneous xenograft model. POMC was elevated 11 fold compared to a SCLC xenograft model derived from a non-POMC-secreting cell line (H526). POMC was also a sensitive biomarker of tumour response to varying doses of radiotherapy and of tumour relapse after complete regression post-radiotherapy. Unfortunately, metastases could not be detected in the brains and livers of these xenografted animals even when several alternative methods of tumour inoculation were used. However there was evidence of aggressive invasion of cells with a dual neuroendocrine and epithelial phenotype through the surrounding fibrotic sheath and muscle of all the primary tumours.Phenotypic characterisation of the xenograft tumour showed that cells appeared to homogeneously express both neuroendocrine and epithelial markers and did not express mesenchymal markers. However, post-irradiation the tumours appeared to have a distinct heterogeneous expression of POMC. These results suggest that irradiation treatment could cause a phenotypic shift which could have implications in tumour progression and metastasis.Epithelial to mesenchymal transition (EMT) has been linked to tumour metastasis in a number of epithelial cancers. When DMS 79 SCLC cells were treated with a known stimulus of EMT, transforming growth factor ? (TGF-? ), the mesenchymal marker N-cadherin was upregulated along with a decrease in cell proliferation. These changes are both characteristic of an EMT. However, there was no change in the epithelial nature of these cells and no obvious change in morphology. We suggest the cells are undergoing a partial transition and that this transition could possibly be involved in SCLC metastasis.In summary, the analysis of neuroendocrine features in SCLC, using POMC as a biomarker, has provided new evidence for a dual neuroendocrine and epithelial phenotype both in vitro and in vivo. Circulating POMC can also identify tumour development, progression and regression after irradiation treatment. In addition, we have highlighted the dual phenotype of invasive cells and the difficulty in studying metastasis of human SCLC in a mouse model.