The presence of high expressing epithelial cell adhesion molecule (EpCAMhigh) circulating tumor cells (CTC) enumerated by CellSearch® in blood of cancer patients is strongly associated with poor prognosis. This raises the question about the presence and relation with clinical outcome of low EpCAM expressing CTC (EpCAMlow CTC). In the EU-FP7 CTC-Trap program, we investigated the presence of EpCAMhigh and EpCAMlow CTC using CellSearch, followed by microfiltration of the EpCAMhigh CTC depleted blood. Blood samples of 108 castration-resistant prostate cancer patients and 22 metastatic breast cancer patients were processed at six participating sites, using protocols and tools developed in the CTC-Trap program. Of the prostate cancer patients, 53% had ≥5 EpCAMhigh CTC and 28% had ≥5 EpCAMlow CTC. For breast cancer patients, 32% had ≥5 EpCAMhigh CTC and 36% had ≥5 EpCAMlow CTC. 70% of prostate cancer patients and 64% of breast cancer patients had in total ≥5 EpCAMhigh and/or EpCAMlow CTC, increasing the number of patients in whom CTC are detected. Castration-resistant prostate cancer patients with ≥5 EpCAMhigh CTC had shorter overall survival versus those with <5 EpCAMhigh CTC (p = 0.000). However, presence of EpCAMlow CTC had no relation with overall survival. This emphasizes the importance to demonstrate the relation with clinical outcome when presence of CTC identified with different technologies are reported, as different CTC subpopulations can have different relations with clinical outcome.
During intravasation, circulating tumor cells (CTCs) detach from the epithelium of origin and begin the epithelial‐to‐mesenchymal transition (EMT) process, where they lose epithelial features and pass through the endothelium to enter circulation. Although detachment from the extracellular matrix is a strong source of metabolic stress, which induces anoikis, CTCs can survive. Recently, the tumor suppressor liver kinase B1 (LKB1) has gained attention for its role as a proto‐oncogene in restoring the correct ATP/AMP ratio during metabolic stress. The aim of this study was to assess LKB1 expression in epithelial‐negative CTCs isolated from patients with metastatic breast cancer and to characterize its possible association with EMT and stemness features. Transcriptome analysis of EpCAM‐negative CTCs indicated that over 25% of patients showed enhanced LKB1 levels, while almost 20% of patients showed enhanced levels of an EMT transcription factor known as ZEB1. Transcriptome and immunofluorescence analyses showed that patients with enhanced LKB1 were correspondingly ZEB1 negative, suggesting complementary activity for the two proteins. Only ZEB1 was significantly associated with cancer stem cell (CSC) markers. Neither LKB1 nor ZEB1 upregulation showed a correlation with clinical outcome, while enhanced levels of stemness‐associated CD44 correlated with a lower progression‐free and overall survival. Ex vivo models showed that MDA‐MB‐231, a mesenchymal tumor cell line, grew in suspension only if LKB1 was upregulated, but the MCF‐7 epithelial cell line lost its ability to generate spheroids and colonies when LKB1 was inhibited, supporting the idea that LKB1 might be necessary for CTCs to overcome the absence of the extracellular matrix during the early phases of intravasation. If these preliminary results are confirmed, LKB1 will become a novel therapeutic target for eradicating metastasis‐initiating CTCs from patients with primary breast cancer.
e23061 Background: To reach distant sites, CTC overcome metabolic stress and acquire a mesenchymal phenotype. So we assessed the pro survival function of liver kinase B1 (LKB1) a cellular metabolic stress controller, in combination with the EMT transcription factor Zinc finger E-box binding homeobox factor 1 (ZEB1) and stem cell markers e.g. CD44 in EpCAM negative CTC. Tumor spheroids and targeted si-RNA knockdown of LKB1 were analyzed in two breast cancer cell lines to support transcriptomic data. Methods: CTC were isolated from whole blood of 27 metastatic breast cancer patients. 17 age matched healthy donors were negative control. All patients were analyzed with CellSearch system to detect EpCAM positive CTC, which were eliminated by immunodepletion (AdnaTest BreastCancerSelect Kit, TATAA Biocenter) together with CD45 positive white blood cells (Miltenyi Biotec). RNA transcripts were quantified by qPCR (TaqMan Gene Expression Assay, ThermoFischer Scientific) and the relative expression was calculated by the 2-DCtmethod. EpCAM negative CTC and spheroids were analyzed by immunofluorescence. Data were analyzed by non-parametric statistical tests. Results: According to the CellSearch system, most patients (74%, n = 20) were CTC negative. 20% of this sub-group showed enhanced levels of ZEB1, 15% of LKB1, and 40% of stem cell markers. ZEB1 and LKB1 were never co-expressed, while there was an almost significant association between enhanced ZEB1 and stem cell marker expression (p = 0.053). Immunofluorescence analysis on single CTC confirmed the transcriptomic result. MDA-MB 231 cell lines, normally negative for LKB1 and unable to grow in suspension, showed the ability to generate spheroids only if LKB1 expression was activated. Targeted knockdown of LKB1 confirmed these results. Conclusions: CellSearch system negative patients present a heterogeneous CTC population with enhanced levels of ZEB1, CD44, or LKB1. While ZEB1 and CD44 might be highly expressed in cells that already have entered EMT, LKB1 overexpression could represent the response to metabolic stress induced in the early phase of intravasation Hence, LKB1 might represent a novel therapeutic target to chase CTC and prevent metastasis.
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