Dissecting the heterogeneity of circulating tumor cells in metastatic breast cancer: Going far beyond the needle in the Haystack

6noAlthough the enumeration of circulating tumor cells (CTC) defined as expressing both epithelial cell adhesion molecule and cytokeratins (EpCAM+/CK+) can predict prognosis and response to therapy in metastatic breast, colon and prostate cancer, its clinical utility (i.e., the ability to improve patient outcome by guiding therapy) has not yet been proven in clinical trials. Therefore, scientists are now focusing on the molecular characterization of CTC as a way to explore its possible use as a “surrogate” of tumor tissues to non-invasively assess the genomic landscape of the cancer and its evolution during treatment. Additionally, evidences confirm the existence of CTC in epithelial-to-mesenchymal transition (EMT) characterized by a variable loss of epithelial markers. Since the EMT process can originate cells with enhanced invasiveness, stemness and drug-resistance, the enumeration and characterization of this population, perhaps the one truly responsible of tumor recurrence and progression, could be more clinically useful. For these reasons, several devices able to capture CTC independently from the expression of epithelial markers have been developed. In this review, we will describe the types of heterogeneity so far identified and the key role played by the epithelial-to-mesenchymal transition in driving CTC heterogeneity. The clinical relevance of detecting CTC-heterogeneity will be discussed as well.openopenBulfoni, Michela; Turetta, Matteo; Del Ben, Fabio; Di Loreto, Carla; Beltrami, Antonio Paolo; Cesselli, DanielaBulfoni, Michela; Turetta, Matteo; Del Ben, Fabio; DI LORETO, Carla; Beltrami, Antonio Paolo; Cesselli, Daniel

Towards Posture and Gait Evaluation through Wearable-Based Biofeedback Technologies

In medicine and sport science, postural evaluation is an essential part of gait and posture correction. There are various instruments for quantifying the postural system’s efficiency and deter- mining postural stability which are considered state-of-the-art. However, such systems present many limitations related to accessibility, economic cost, size, intrusiveness, usability, and time-consuming set-up. To mitigate these limitations, this project aims to verify how wearable devices can be assem- bled and employed to provide feedback to human subjects for gait and posture improvement, which could be applied for sports performance or motor impairment rehabilitation (from neurodegenerative diseases, aging, or injuries). The project is divided into three parts: the first part provides experimen- tal protocols for studying action anticipation and related processes involved in controlling posture and gait based on state-of-the-art instrumentation. The second part provides a biofeedback strategy for these measures concerning the design of a low-cost wearable system. Finally, the third provides al- gorithmic processing of the biofeedback to customize the feedback based on performance conditions, including individual variability. Here, we provide a detailed experimental design that distinguishes significant postural indicators through a conjunct architecture that integrates state-of-the-art postural and gait control instrumentation and a data collection and analysis framework based on low-cost devices and freely accessible machine learning techniques. Preliminary results on 12 subjects showed that the proposed methodology accurately recognized the phases of the defined motor tasks (i.e., rotate, in position, APAs, drop, and recover) with overall F1-scores of 89.6% and 92.4%, respectively, concerning subject-independent and subject-dependent testing setups

Assessment of the mutational status of NSCLC using hypermetabolic circulating tumor cells

Molecular characterization is currently a key step in NSCLC therapy selection. Circulating tumor cells (CTC) are excellent candidates for downstream analysis, but technology is still lagging behind. In this work, we show that the mutational status of NSCLC can be assessed on hypermetabolic CTC, detected by their increased glucose uptake. We validated the method in 30 Stage IV NSCLC patients: peripheral blood samples were incubated with a fluorescent glucose analog (2-NBDG) and analyzed by flow cytometry. Cells with the highest glucose uptake were sorted out. EGFR and KRAS mutations were detected by ddPCR. In sorted cells, mutated DNA was found in 85% of patients, finding an exact match with primary tumor in 70% of cases. Interestingly, in two patients multiple KRAS mutations were detected. Two patients displayed different mutations with respect to the primary tumor, and in two out of the four patients with a wild type primary tumor, new mutations were highlighted: EGFR p.746_750del and KRAS p.G12V. Hypermetabolic CTC can be enriched without the need of dedicated equipment and their mutational status can successfully be assessed by ddPCR. Finally, the finding of new mutations supports the possibility of probing tumor heterogeneity

Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy

The seasonal change of PAHs in Svalbard surface snow

The Arctic region is threatened by contamination deriving from both long-range pollution and local human activities. Polycyclic Aromatic Hydrocarbons (PAHs) are environmental tracers of emission, transport and deposition processes. A first campaign has been conducted at Ny-Ålesund, Svalbard, from October 2018 to May 2019, monitoring weekly concentrations of PAHs in Arctic surface snow. The trend of the 16 high priority PAH compounds showed that long-range inputs occurred mainly in the winter, with concentrations ranging from 0.8 ng L−1 to 37 ng L−1. In contrast to this, the most abundant analyte retene, showed an opposite seasonal trend with highest values in autumn and late spring (up to 97 ng L−1), while in winter this compound remained below 3 ng L−1. This is most likely due to local contributions from outcropping coal deposits and stockpiles. Our results show a general agreement with the atmospheric signal, although significant skews can be attributed to post-depositional processes, wind erosion, melting episodes and redistribution

In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis

Background: Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data. Methods: This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45(pos) leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45(neg) cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively. Results: By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45(neg) subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45(neg) cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment. Conclusion: This study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies