## 3 Circulating Tumor Cells--Relevance to Skin Cancers
### Advances in CTCs Detection  
As mentioned before, the lack of precision in CTC detection is one of the main factors why this technique has not yet been applied in daily clinical practice. It should be noted, however, that during recent years important improvements were made in this direction. CellSearch(r) (Menarini Silicon Biosystems, Florence, Italy)is currently the only existing FDA-approved device, able to accurately enumerate and detect CTCs by a standardized method [43]. The principle of CellSearch(r) is based on cell surface molecule detection, mainly epithelial-based adhesion molecule (EpCAM) expression that is relatively specific for CTCs and not found in WBC and RBC [44]. The evident limitation of the technique is revealed when trying to detect and separate EpCAM negative and EpCAM low CTCs, which remain normally undetected in classical CellSearch(r) protocol. In this context, several groups of researchers tried to optimize the technique, adding a second step within the separation process. For example, [PERSON] et al. [45] combined CellSearch(r) with another platform based on filtration and fluorescent staining, increasing the outcome of cell separation in lung and breast cancer. A different approach, epitope independent, seems to be also effective in CTC separation. Two studies showed satisfactory cell detection by epitope-independent platforms in high-risk head and neck cancer [46] and prostate cancer [47], enlarging the possibilities of CTC separation, which can be based also on other physical-chemical characteristics compared to only cell surface molecules examination. Advances in CTC detection are observable in many CTC technologies, some of the most relevant ones also for skin cancer research being detailed below. In order to counterattack the extremely low number of CTCs normally found in one sample, improvements related to cell sample enrichment were made. First, a good separation rate can be obtained by including physical characteristics, such as size, density, electric charges, and deforma  
Figure 1: Schematic representation of tumor dissemination: CTCs are released in the circulatory system via several entry sites; in the blood, there is a high CTCs heterogeneity; different extravasation sites for CTCs regarding tumor type. Reprinted by permission from Springer Nature Ltd.: Nat. Rev. Cancer 2019, 19, 553–567, doi.org/10.1038/s41568-019-0180-2. Copyright (2019) [37]. bility, within the separation process. Microfiltration was demonstrated to increase CTC's detection rate, some devices achieving an average capture efficiency of 84% [48]. The major drawback of this approach is the high degree of heterogeneity of the final sample, which contains impure or mixed origin CTCs. Furthermore, the increased fragility of CTCs makes physical separation methods undesired, damaged, or clustered CTCs being improper for further analysis. Electrical characteristics can play important roles in CTC segregation, with the method of dielectrophoresis gaining more and more ground, representing also an important part of this review (see below). Similarly, another label-free, non-invasive approach developed and extensively employed by [PERSON] et al. [49] is based on photoacoustic detection of CTCs. Known as Cytophone, this platform was successfully employed both in vitro and in vivo, however not reaching large-scale deployment yet. A more refined method of cell sorting is based on the affinity of antigens to specific antibodies used in by different platforms. As CellSearch(r) already demonstrated, through this protocol, one is able to obtain high purity CTC samples, however, still with existing limitations. The use of several specific antibodies combined with clever device design is a promising direction for CTCs detection. Lastly, as microfluidic platforms become highly specialized, it is possible to tailor microfilters of varying sizes, flexibility, and geometry, in order to analyze blood samples within a short time, with a high recovery rate and high throughput [50].