The occurrence of either regional or distant metastases is an indicator of poor prognosis for cancer patients. The mechanism of their formation has not yet been fully uncovered, which limits the possibility of developing new therapeutic strategies. Nevertheless, the discovery of circulating tumor cells (CTCs), which are responsible for tumor dissemination, and cancer stem cells (CSCs), required for tumor growth maintenance, shed light on the metastatic cascade. It seems that CTCs and CSCs are not necessarily separate populations of cancer cells, as CTCs generated in the process of epithelial-mesenchymal transition (EMT) can bear features characteristic of CSCs. This article describes the mechanisms of CTC and CSC formation and characterizes their molecular hallmarks. Moreover, we present different types of EMT occurring in physiological and pathological conditions, and we demonstrate its crucial role in providing CTCs with a CSC phenotype. The article delineates molecular changes acquired by cancer cells undergoing EMT that facilitate metastasis formation. Deeper understanding of those processes is of fundamental importance for the development of new strategies of early cancer detection and effective cancer treatment approaches that will be translated into clinical practice.

Mouse models indicate that metastatic dissemination occurs extremely early; however, the timing in human cancers is unknown. We therefore determined the time point of metastatic seeding relative to tumour thickness and genomic alterations in melanoma. Here, we find that lymphatic dissemination occurs shortly after dermal invasion of the primary lesion at a median thickness of ~0.5 mm and that typical driver changes, including BRAF mutation and gained or lost regions comprising genes like MET or CDKNA2, are acquired within the lymph node at the time of colony formation. These changes define a colonisation signature that was linked to xenograft formation in immunodeficient mice and death from melanoma. Thus, melanoma cells leave primary tumours early and evolve at different sites in parallel. We propose a model of metastatic melanoma dormancy, evolution and colonisation that will inform direct monitoring of adjuvant therapy targets.

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IntroductionCirculating tumor cells (CTCs) that present mesenchymal phenotypes can escape standard methods of isolation, thus limiting possibilities for their characterization. Whereas mesenchymal CTCs are considered to be more malignant than epithelial CTCs, factors responsible for this aggressiveness have not been thoroughly defined. This study analyzed the molecular profile related to metastasis formation potential of CTC-enriched blood fractions obtained by marker unbiased isolation from breast cancer patients without (N−) and with lymph nodes metastases (N+).Materials and MethodsBlood samples drawn from 117 patients with early-stage breast cancer were enriched for CTCs using density gradient centrifugation and negative selection with anti-CD45 covered magnetic particles. In the resulting CTC-enriched blood fractions, expression of CK19, MGB1, VIM, TWIST1, SNAIL, SLUG, HER2, CXCR4 and uPAR was analyzed with qPCR. Results were correlated with patients' clinicopathological data.ResultsCTCs (defined as expression of either CK19, MGB1 or HER2) were detected in 41% (20/49) of N− and 69% (34/49) of N+ patients (P = 0.004). CTC-enriched blood fractions of N+ patients were more frequently VIM (P = 0.02), SNAIL (P = 0.059) and uPAR-positive (P = 0.03). Positive VIM, CXCR4 and uPAR status correlated with >3 lymph nodes involved (P = 0.003, P = 0.01 and P = 0.045, respectively). In the multivariate logistic regression MGB1 and VIM-positivity were independently related to lymph node involvement with corresponding overall risk of 3.2 and 4.2. Moreover, mesenchymal CTC-enriched blood fractions (CK19−/VIM+ and MGB1+ or HER2+) had 4.88 and 7.85-times elevated expression of CXCR4 and uPAR, respectively, compared with epithelial CTC-enriched blood fractions (CK19+/VIM− and MGB1+ or HER2+).ConclusionsTumors of N+ patients have superior CTC-seeding and metastatic potential compared with N- patients. These differences can be attributed to VIM, uPAR and CXCR4 expression, which endow tumor cells with particularly malignant phenotypes.