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.

Ecto-5′-nucleotidase (CD73) is a membrane-bound enzyme, which catalyzes the conversion of adenosine monophosphate to adenosine. CD73 has been postulated to play an important role in carcinogenesis, as adenosine promotes tumor progression and CD73-expressing cancer cell lines are more aggressive. However, other studies have shown that activated adenosine receptors may also inhibit cell proliferation. This study investigated the clinical significance of CD73 expression in breast cancer. The study group included 136 consecutive stage I-III breast cancer patients treated between 2001 and 2008 at 2 institutions. CD73 expression was examined by immunohistochemistry (IHC) on tissue microarrays, using antihuman mouse monoclonal antibody. Survival curves were generated by the Kaplan-Meier method and compared using the log-rank test. CD73 staining was expressed as the score calculated by multiplying the staining intensity (0=negative, 1=weak, 2=intermediate, 3=strong) and percentage of positive cells (0% to 100%). The median score among all samples was 100. Positive CD73 staining (defined as score equal or higher than 100) occurred in 74% of the cases. No correlation was found between CD73 expression and grading, tumor size, lymph node status, histologic type, estrogen receptor, or progesterone receptor status. Positive CD73 expression strongly correlated with longer disease-free survival (hazard ratio=0.26; 95% confidence interval, 0.1-0.66; P=0.0044) and overall survival (hazard ratio =0.24; 95% confidence interval, 0.07-0.85; P=0.027). Multivariate analysis for disease-free survival revealed correlation with tumor size and CD73 status. Elevated CD73 expression in breast cancer can predict a good prognosis. However, the actual role of CD73 in cancerogenesis remains unclear and requires further analysis.

Background: Cancer-associated fibroblasts (CAFs) have been shown to support tumor development in a variety of cancers. Different markers were applied to classify CAFs in order to elucidate their impact on tumor progression. However, the exact mechanism by which CAFs enhance cancer development and metastasis is yet unknown. Methods: Alpha-smooth muscle actin (α-SMA) was examined immunohistochemically in intratumoral CAFs of nonmetastatic breast cancers and correlated with clinicopathological data. Four CAF cell lines were isolated from patients with luminal breast cancer (lumBC) and classified according to the presence of α-SMA protein. Conditioned medium (CM) from CAF cultures was used to assess the influence of CAFs on lumBC cell lines: MCF7 and T47D cells using Matrigel 3D culture assay. To identify potential factors accounting for promotion of tumor growth by α-SMA^high CAFs, nCounter PanCancer Immune Profiling Panel (NanoString) was used. Results: In luminal breast cancer, presence of intratumoral CAFs expressing high level of α-SMA (13% of lumBC group) correlated with poor prognosis (p = 0.019). In in vitro conditions, conditioned medium obtained from primary cultures of α-SMA-positive CAFs isolated from luminal tumors was observed to enhance growth of lumBC cell line colonies in 3D Matrigel, in contrast to CM derived from α-SMA-negative CAFs. Multigene expression analysis indicated that osteopontin (OPN) was overexpressed in α-SMA-positive CAFs in both clinical samples and in vitro models. OPN expression was associated with higher percentage of Ki67-positive cells in clinical material (p = 0.012), while OPN blocking in α-SMA-positive CAF-derived CM attenuated growth of lumBC cell line colonies in 3D Matrigel. Conclusions: Our findings demonstrate that α-SMA-positive CAFs might enhance tumor growth via secretion of OPN.

Breast cancer metastasis is the leading cause of cancer deaths in women and is difficult to combat due to the long periods in which disseminated cells retain a potential to be re-activated and start the relapse. Assessing the number and molecular profile of circulating tumor cells (CTCs) in breast cancer patients, especially in early breast cancer, should help in identifying the possibility of relapse in time for therapeutic intervention to prevent or delay recurrence. While metastatic breast cancer is considered incurable, molecular analysis of CTCs still have a potential to define particular susceptibilities of the cells representing the current tumor burden, which may differ considerably from the cells of the primary tumor, and offer more tailored therapy to the patients. In this review we inspect the routes to metastasis and how they can be linked to specific features of CTCs, how CTC analysis may be used in therapy, and what is the current status of the research and efforts to include CTC analysis in clinical practice.

Circulating tumour cells (CTCs) can provide valuable prognostic information in a number of epithelial cancers. However, their detection is hampered due to their molecular heterogeneity, which can be induced by the epithelial-mesenchymal transition (EMT) process. Therefore, current knowledge about CTCs from clinical samples is often limited due to an inability to isolate wide spectrum of CTCs phenotypes. In the current work, we aimed at isolation and molecular characterization of CTCs with different EMT status in order to establish their clinical significance in early breast cancer patients. We have obtained CTCs-enriched blood fraction from 83 breast cancer patients in which we have tested the expression of epithelial, mesenchymal and general breast cancer CTCs markers (MGB1/HER2/CK19/CDH1/CDH2/VIM/PLS3), cancer stem cell markers (CD44, NANOG, ALDH1, OCT-4, CD133) and cluster formation gene (plakoglobin). We have shown that in the CTCs-positive patients, epithelial, epithelial-mesenchymal and mesenchymal CTCs markers were detected at a similar rate (in 28%, 24% and 24%, respectively). Mesenchymal CTCs were characterized by the most aggressive phenotype (significantly higher expression of CXCR4, uPAR, CD44, NANOG, p < 0.05 for all), presence of lymph node metastases (p = 0.043), larger tumour size (p = 0.023) and 7.33 higher risk of death in the multivariate analysis (95% CI 1.06–50.41, p = 0.04). Epithelial-mesenchymal subtype, believed to correspond to highly plastic and aggressive state, did not show significant impact on survival. Gene expression profile of samples with epithelial-mesenchymal CTCs group resembled pure epithelial or pure mesenchymal phenotypes, possibly underlining degree of EMT activation in particular patient’s sample. Molecular profiling of CTCs EMT phenotype provides more detailed and clinically informative results, proving the role of EMT in malignant cancer progression in early breast cancer.

Tumor dissemination is one of the most-investigated steps of tumor progression, which in recent decades led to the rapid development of liquid biopsy aiming to analyze circulating tumor cells (CTCs), extracellular vesicles (EVs), and circulating nucleic acids in order to precisely diagnose and monitor cancer patients. Flow cytometry was considered as a method to detect CTCs; however, due to the lack of verification of the investigated cells’ identity, this method failed to reach clinical utility. Meanwhile, imaging flow cytometry combining the sensitivity and high throughput of flow cytometry and image-based detailed analysis through a high-resolution microscope might open a new avenue in CTC technologies and provide an open-platform system alternative to CellSearch^®, which is still the only gold standard in this field. Hereby, we shortly review the studies on the usage of flow cytometry in CTC identification and present our own representative images of CTCs envisioned by imaging flow cytometry providing rationale that this novel technology might be a good tool for studying tumor dissemination, and, if combined with a high CTC yield enrichment method, could upgrade CTC-based diagnostics.