Carcinoma cells undergo epithelial-mesenchymal transition (EMT); however, contributions of EMT heterogeneity to disease progression remain a matter of debate. Here, we addressed the EMT status of ex vivo cultured circulating and disseminated tumor cells (CTCs/DTCs) in a syngeneic mouse model of metastatic breast cancer (MBC). Epithelial-type CTCs with a restricted mesenchymal transition had the strongest lung metastases formation ability, whereas mesenchymal-type CTCs showed limited metastatic ability. EpCAM expression served as a surrogate marker to evaluate the EMT heterogeneity of clinical samples from MBC, including metastases, CTCs, and DTCs. The proportion of epithelial-type CTCs, and especially DTCs, correlated with distant metastases and poorer outcome of patients with MBC. This study fosters our understanding of EMT in metastasis and underpins heterogeneous EMT phenotypes as important parameters for tumor prognosis and treatment. We further suggest that EpCAM-dependent CTC isolation systems will underestimate CTC numbers but will quantify clinically relevant metastatic cells.

EpCAM (epithelial cell adhesion molecule) was discovered four decades ago as a tumor antigen on colorectal carcinomas. Owing to its frequent and high expression on carcinomas and their metastases, EpCAM serves as a prognostic marker, a therapeutic target, and an anchor molecule on circulating and disseminated tumor cells (CTCs/DTCs), which are considered the major source for metastatic cancer cells. Today, EpCAM is reckoned as a multi-functional transmembrane protein involved in the regulation of cell adhesion, proliferation, migration, stemness, and epithelial-to-mesenchymal transition (EMT) of carcinoma cells. To fulfill these functions, EpCAM is instrumental in intra- and intercellular signaling as a full-length molecule and following regulated intramembrane proteolysis, generating functionally active extra- and intracellular fragments. Intact EpCAM and its proteolytic fragments interact with claudins, CD44, E-cadherin, epidermal growth factor receptor (EGFR), and intracellular signaling components of the WNT and Ras/Raf pathways, respectively. This plethora of functions contributes to shaping intratumor heterogeneity and partial EMT, which are major determinants of the clinical outcome of carcinoma patients. EpCAM represents a marker for the epithelial status of primary and systemic tumor cells and emerges as a measure for the metastatic capacity of CTCs. Consequentially, EpCAM has reclaimed potential as a prognostic marker and target on primary and systemic tumor cells.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by outstanding molecular heterogeneity that results in severe therapy resistance and poor clinical outcome. Inter- and intratumoral heterogeneity in epithelial-mesenchymal transition (EMT) was recently revealed as a major parameter of poor clinical outcome. Here, we addressed the expression and function of the therapeutic target epidermal growth factor receptor (EGFR) and of the major determinant of epithelial differentiation epithelial cell adhesion molecule (EpCAM) in clinical samples and in vitro models of HNSCCs. We describe improved survival of EGFRlow/EpCAMhigh HNSCC patients (n = 180) and provide a molecular basis for the observed disparities in clinical outcome. EGF/EGFR have concentration-dependent dual capacities as inducers of proliferation and EMT through differential activation of the central molecular switch phosphorylated extracellular signal–regulated kinase 1/2 (pERK1/2) and EMT transcription factors (EMT-TFs) Snail, zinc finger E-box-binding homeobox 1 (Zeb1), and Slug. Furthermore, soluble ectodomain of EpCAM (EpEX) was identified as a ligand of EGFR that activates pERK1/2 and phosphorylated AKT (pAKT) and induces EGFR-dependent proliferation but represses EGF-mediated EMT, Snail, Zeb1, and Slug activation and cell migration. EMT repression by EpEX is realized through competitive modulation of pERK1/2 activation strength and inhibition of EMT-TFs, which is reflected in levels of pERK1/2 and its target Slug in clinical samples. Accordingly, high expression of pERK1/2 and/or Slug predicted poor outcome of HNSCCs. Hence, EpEX is a ligand of EGFR that induces proliferation but counteracts EMT mediated by the EGF/EGFR/pERK1/2 axis. Therefore, the emerging EGFR/EpCAM molecular cross talk represents a promising target to improve patient-tailored adjuvant treatment of HNSCCs.

BackgroundBrown adipose tissue [BAT] metabolism in vivo is vital for the development of novel strategies in combating obesity and diabetes. Currently, BAT is activated at low temperatures and measured using 2-deoxy-2-18F-fluoro-D-glucose [18F-FDG] positron-emission tomography [PET]. We report the use of β3-adrenergic receptor-mediated activation of BAT at ambient temperatures using (R, R)-5-[2-[2,3-(3-chlorphenyl)-2-hydroxyethyl-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate, disodium salt [CL316,243] (a selective β3-adrenoceptor agonist) and measured by 18F-FDG PET/computed tomography [CT].MethodsControl and CL316,243-treated (2 mg/kg) male Sprague-Dawley rats were administered with 18F-FDG for PET/CT studies and were compared to animals at cold temperatures. Receptor-blocking experiments were carried out using propranolol (5 mg/kg). Dose effects of CL316,243 were studied by injecting 0.1 to 1 mg/kg 30 min prior to 18F-FDG administration. Imaging results were confirmed by autoradiography, and histology was done to confirm BAT activation.ResultsCL316,243-activated interscapular BAT [IBAT], cervical, periaortic, and intercostal BATs were clearly visualized by PET. 18F-FDG uptake of IBAT was increased 12-fold by CL316,243 vs. 1.1-fold by cold exposure when compared to controls. 18F-FDG uptake of the CL-activated IBAT was reduced by 96.0% using intraperitoneal administration of propranolol. Average 18F-FDG uptake of IBAT increased 3.6-, 3.5-, and 7.6-fold by doses of 0.1, 0.5, and 1 mg/kg CL, respectively. Ex vivo 18F-FDG autoradiography and histology of transverse sections of IBAT confirmed intense uptake in the CL-activated group and activated IBAT visualized by PET.ConclusionOur study indicated that BAT metabolic activity could be evaluated by 18F-FDG PET using CL316,243 at ambient temperature in the rodent model. This provides a feasible and reliable method to study BAT metabolism.