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Review

Liquid Biopsy and Dielectrophoretic Analysis—Complementary Methods in Skin Cancer Monitoring

by
Thomas Gabriel Schreiner
1,2,*,
Ina Turcan
1,
Marius Andrei Olariu
1,
Romeo Cristian Ciobanu
1 and
Maricel Adam
1
1
Faculty of Electrical Engineering and Information Technology, Gheorghe Asachi Technical University of Iasi, 21–23 Professor Dimitrie Mangeron Blvd., 700050 Iasi, Romania
2
Faculty of Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, 16 Universitatii Street, 700115 Iasi, Romania
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(7), 3366; https://doi.org/10.3390/app12073366
Submission received: 14 February 2022 / Revised: 15 March 2022 / Accepted: 22 March 2022 / Published: 25 March 2022
(This article belongs to the Special Issue New Challenges in Skin Cancer)
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Abstract

:
The incidence and prevalence of skin cancers is currently increasing worldwide, with early detection, adequate treatment, and prevention of recurrences being topics of great interest for researchers nowadays. Although tumor biopsy remains the gold standard of diagnosis, this technique cannot be performed in a significant proportion of cases, so that the use of alternative methods with high sensitivity and specificity is becoming increasingly desirable. In this context, liquid biopsy appears to be a feasible solution for the study of cellular and molecular markers relevant to different types of skin cancers. Circulating tumor cells are just one of the components of interest obtained from performing liquid biopsy, and their study by complementary methods, such as dielectrophoresis, could bring additional benefits in terms of characterizing skin tumors and subsequently applying personalized therapy. One purpose of this review is to demonstrate the utility of liquid biopsy primarily in monitoring the most common types of skin tumors: basal cell carcinoma, squamous cell carcinoma, and malign melanoma. In addition, the originality of the article is based on the detailed presentation of the dielectrophoretic analysis method of the most important elements obtained from liquid biopsy, with direct impact on the clinical and therapeutic approach of skin tumors.

1. Introduction

Skin cancer is the most frequent type of cancer in the White population [1], its incidence and prevalence recording a continuous growth during recent years [2]. Divided into two different types, malignant melanoma and the more heterogeneous group of non-melanoma skin cancers (NMSC), the two entities differ when referring to their risk factors, genetics, pathological, clinical, and therapeutic particularities [3]. The group of NMSC includes several types of skin cancer, with the most common types, representing almost 99% of the group, being basal cell carcinoma and squamous cell carcinoma [4], also included in this review.
Basal cell carcinoma (BCC), a slow growing locally invasive epidermal tumor, is by far the most frequent type of skin cancer [5]. More commonly affecting the Caucasian population, according to the latest available data, BCC incidence has been estimated to reach 4.3 million cases each year in the Unites States alone [6]. Furthermore, the numbers are on an ascending pathway, as the situation in Europe showed an important rise in incidence (over 5% annually) during the last 10 years. This epidemiological trend is expected to continue in the near future, on the one hand, due to more frequent and efficient screening and diagnosis, but also due to increased exposure to UV radiation and the growing aging population [7]. Although having a low mortality risk due to very low malignancy and metastatic rare, BCC can cause significant morbidity due to its destructive local spread [8]. High prevalence together with the accessibility of skin examination have allowed researchers to obtain detailed information about BCC’s pathogenesis, clinical presentation, and histopathology. The complexity of the disease is noticed when trying to summarize its pathogenesis, with several environmental (UV radiation, UV-sensitive phenotype) and genetic risk factors (genetic polymorphisms) coming into interplay [9]. Additionally, BCC heterogeneous histological classification, its immunohistochemical and genetic profile bring relevant [10], however frequently incomplete, data for the therapeutic approach.
Squamous cell carcinoma (SCC), the second most frequent cancer in humans, accounts for the majority of the remaining cases of NMSC [11]. In trend with the enhanced incidence and prevalence of BCC, epidemiological data on SCC estimates a doubling of its incidence in European countries by 2030 [12]. Although SCC usually exhibits a benign clinical behavior, it can be locally invasive and have a significantly higher risk of metastasis compared to BCC, caring in that case poor survival prognosis [13]. SCC arises from dysplastic epidermal keratinocytes, the malignant proliferation being sustained by several risk factors, including immunosuppression, previous history of actinic keratosis (AK), and chronic sun exposure [14].
Both BCC and SCC have been intensively studied in recent years, and a huge amount of new data related to pathogenesis and treatment is emerging. Although still incompletely elucidated, the underlying mechanism of the neoplastic process is better understood nowadays thanks also to a better knowledge of the molecular and genetic factors involved [15]. Subsequently, a fast development within the therapeutic field can also be observed, in addition to surgical intervention, with the emergence in current clinical practice of new agents that inactivate key molecules in the malignant process. PTCH1 drug efflux antagonist [16] and Smoothened (SMO) inhibitors [17] used in BCC therapy, together with Anti-EGFR agents [18] and Janus kinase (JAK) inhibitors suited for SCC pharmacological treatment are just a couple of examples of currently available drugs in the NMSC therapeutic approach.
Malign melanoma (MM), one of the most aggressive cancers in humans, is becoming one of the most common malignancies worldwide [19]. Recording a continuous increase in incidence, MM is the cause of more than 90% of skin-disease-related deaths [20]. The imperative need for early detection and fast treatment led to intensive research focused on the better control of the risk factors and a more detailed understanding of the neoplastic process. In line with NMSC, sun exposure, particularly UV radiation, remains one of the most important risk factors [21]. The molecular basis is of great importance, and key events, such as TP53 gene mutations, are opening new, personalized therapeutic perspectives. The activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma; several compounds, such as WIP1 inhibitor and MDM2-p53 binding antagonists, are showing promising results [22]. The detection of specific mutations, such as the V600E mutation in the BRAF gene, has recently permitted the use of more precision treatment with specific inhibition therapy [23].
Despite advances in understanding the pathophysiology of the malignant process and the treatment of skin cancers, an important limitation remains regarding the diagnosis. Currently, skin biopsy followed by histopathological examination is the only gold standard diagnosis method accepted [24]; however, one must acknowledge its limitations. There are several techniques available, including punch, shave, incisional and excisional biopsy, the decision to choose one over the others being based on the type of lesion, site of lesion, and also on the proficiency of the dermatologist [25]. Regardless of the chosen approach, technique-related complications might occur, with pain, bleeding, scarring, and infection being the most frequently encountered. Moreover, the localization and wrong timing of the possible malign skin lesion makes the biopsy lose its sensitivity and specificity. In this context, complementary diagnostic methods are preferred, with the technique known as “liquid biopsy” increasingly gaining ground during recent years [26]. One of the main reasons for its popularity relies upon the possibility to study several cellular and molecular components from only small amounts of sample; circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are some of the most studied particles.
In line with the current development of new diagnostic techniques, this narrative review aims to offer fresh insights on the role of liquid biopsy in skin cancer detection and monitoring, a highly discussed topic nowadays, which is not yet fully elucidated. After describing the impact of this method on the clinical and therapeutic aspects of BCC, SCC, and MM, the authors focus mainly on the role of CTCs in the early detection and disease monitoring of these three most common skin cancers, reviewing also the utility of other relevant particles, such as ctDNA or exosomes. Finally, dielectrophoretic analysis is presented as a promising complementary diagnostic method, opening new perspectives for electrical and electrochemical impedance measurements to become a veritable screening and monitoring tool in daily clinical practice.

2. Liquid Biopsy and Its Utility in Skin Cancer Monitoring

Liquid biopsy, a non or minimally invasive technique, is considered nowadays a revolutionary method capable of opening previously unexpected perspectives in the oncological and dermatological fields [27]. The most important advantage of this technique relies upon the possibility of relatively easy detection and isolation of several relevant biomarkers, such as circulating tumor cells (CTCs), circulating tumor DNA, and exosomes, compared to conventional tissue biopsy [28]. Due to recent technological and molecular advances, mainly in the microfluidic domain, the components obtained via liquid biopsy can now be more efficiently purified and analyzed [29].
As skin cancer molecular profile is essential for a targeted therapeutic approach, the detection and isolation of tumor cells are mandatory in guiding the therapy. Although liquid biopsy is currently not completely adopted in daily clinical practice, it is still a useful modality of cell obtaining and subsequent characterization, especially when classic tissue biopsy is difficult to obtain or has led to unreliable results [30]. Several strong points of liquid biopsy make this technique appealing for clinical use, the most relevant characteristics compared to the classical approach being summarized in Table 1.
One of the most important benefits of liquid biopsy is the possibility of serial monitoring. Samples can be prevailed at any time during patient therapy and follow-up, with the dynamic follow-up of tumor evolution (especially linked to the short half-life of ctDNA) being close to the principles of personalized medicine, when fine adjustments of drug therapy are advisable according to patient response. While initially, only the study of CTCs was possible from samples collected via liquid biopsy, it is possible now to perform an extended analysis of many components released by the tumor in body fluids (mainly blood). Cell-free circulating nucleic acids, such as DNA, mRNA, micro-RNA, and long non-coding RNA, are becoming of great importance, together with the study of “tumor-educated platelets” and vesicles, such as exosomes. In the next part, the authors focus mainly on the detection and characterization of the CTCs relevant to skin tumors, only briefly summarizing the emerging role of other components separated through liquid biopsy.

3. Circulating Tumor Cells—Relevance to Skin Cancers

3.1. From Origin to Metastasis—CTCs as Key Players of the Process

Although not a new concept, CTCs have returned to the attention of researchers in recent years as a result of the improvements in liquid biopsy techniques. Only recently, according to several studies, CTCs have become veritable biomarkers in a multitude of cancers [31,32], either for the early detection of the malignancy, but more frequent for disease monitoring and therapeutic prognosis [33].
CTCs constitute a very small fraction of cells detected in peripheral blood, compared to exceedingly higher numbers of white blood cells (WBCs) and red blood cells (RBCs). These are cancer cells, originating in the early phases of cancers from the primary tumor (even before metastatic lesions occur) and during the later stages also from metastatic lesions, circulating through the bloodstream to different target organs. In addition to the small number of CTCs in the sample (<10 cells/mL of blood), another aspect that makes the detection difficult is the short half-life of only 1–2.4 h [34]. Moreover, the high heterogeneity of CTCs is also well known; despite having the same tissue or tumor origin, two CTCs may have different molecular profiles and express different surface receptors (i.e., clusters of differentiation) [35]. Despite the abovementioned evident limitations, CTCs are the intermediate stages in metastasis [36], making them an interesting topic for oncologic research.
The metastatic process, observable frequently in MM and also relevant for SCC, has a complex, multiple stage course, CTCs playing a major role. After the detachment from the primary tumor, CTCs are involved in the first step of intravasation, travelling with the blood flow to specific sites. Then, CTCs extravasate to bone marrow or other organs, finally disseminating and metastasizing locally (see Figure 1) [37].
Summarizing the available literature, CTCs seem to be of interest in three different directions related to skin cancer patient management. First, CTCs were thought to help in the early detection of skin malignancies [38]; however, as a screening method, because of multiple detection and characterization limitations, there is further need for technique improvements. On the other hand, CTC’s study was highly successful when performing treatment follow-ups. In this regard, the study conducted by Hong et al. [39] showed the potential role of the RNA-based scoring of melanoma CTCs in providing relevant data for the early monitoring of response to specific targeted therapies. The prediction of malignant recurrence after tumor excision is an essential step in secondary prevention, with CTCs analysis gaining more ground in tumor dynamic follow-up, early assessment of therapeutic efficacy, risk of relapse [40], and real-time monitoring of the apparition of treatment resistance [41]. Lastly, CTCs might offer valuable insight related to skin cancer prognosis, with clinicians being able to make early assessments and correlations with tumor burden modifications [42].

3.2. 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® (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® 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® protocol. In this context, several groups of researchers tried to optimize the technique, adding a second step within the separation process. For example, de Wit et al. [45] combined CellSearch® 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 deformability, 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 Galanzha 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® 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]. The integration of other nanotechnologies in the classical design allows the possibility of further functionalities for a more controllable release and separation of cells.

3.3. From CTCs to Other Important Components Obtained via Liquid Biopsy

As liquid biopsy has been refined, researchers have turned their attention to other components isolated from peripheral blood samples, which might be of interest for the metastatic process (see Table 2). Thereby, another recently described component obtained from liquid biopsy samples are “tumor educated platelets” (TEPs). TEPs are thought to play central roles in the systemic and local response to tumor growth. These subtypes of platelets have altered RNA profiles [51]. One distinct feature is the capacity of TEPs to be directly and indirectly modulated by tumor cells and the tumor microenvironment. The main pathway remains direct RNA ingestion, with subsequent queue-specific splice events that confer specific RNA signatures according to external tumoral stimuli [52]. With studies current in their infancy, the promising results obtained in the field of sarcoma diagnosis [53] indicate that the interactions between platelets and skin tumor cells could present an interesting potential for diagnosis and monitor also for MM or NMSC.
Exosomes, playing important roles in cell crosstalk [54], are also of interest when considering the detection of tumors and tumor-related alterations. This type of extracellular vesicles has an endocytic origin and a variable size between 30 and 100 nm [55]. Similar to CTCs, exosomes are highly heterogeneous; several detection and isolation procedures mainly based on surface membrane markers from the group of CD molecules (CD63, CD9, and CD81) have been described [56]. Continuous improvements in isolation techniques are essential for a better characterization of exosomes; the now available physical methods, such as density gradient centrifugation and ultracentrifugation, are limited in separating a high purity sample [57]. The roles of exosomes in MM are just beginning to be determined. One important discovery is the presence of melanoma-derived exosomes in MM patients, which promote the growth and metastasis of melanoma. Macrophages are one of the main targets, melanoma-derived exosomes inducing a mixed M1 and M2 polarization, towards a protumor macrophage activation phenotype [58]. Other immune cells are also modulated by melanoma-derived exosomes, with CD8+ T lymphocytes being inhibited mainly by exosomes derived from the B16F0 melanoma cell line [59]. Regarding natural-killer cells that are associated with anti-tumoral immune response, exosomes derived from the NK-92 cell line seem to exert cytotoxic effects against melanoma [60], suggesting that the development of similar nano-vesicles could be a potential therapy direction.
One of the smallest compounds obtained via liquid biopsy, but relevant for the molecular profiling of skin cancers are circulating cell-free DNA (ccfDNA) and miRNAs. ccfDNA, although proposed as potential skin cancer biomarkers, do not present the necessary sensitivity and specificity, with only the circulating DNA derived directly from the tumor (ctDNA) being of interest for further analysis. CtDNA has a short half-life of around 2 h in peripheral circulation, an advantage when trying to do dynamic, real-time monitoring of tumors. Although different ctDNA levels were observed in primary versus metastasized cancers, or with respect to the examined biological fluid (blood, urine, and cerebrospinal fluid), one limitation of this potential new biomarker is the absence of well-defined cut-off values [61]. Special technical considerations must also be taken into account, as the importance of preanalytical steps from sampling to DNA extraction that are crucial for a reliable ctDNA detection was reviewed recently [62]. To date, several technical strategies are available to identify specific mutations in ctDNA, most of them being polymerase chain reaction (PCR)-based and next generation sequencing (NGS)-based approaches. We mention both targeted sequencing techniques, such as Amplicon sequencing (AmpliSeq) [63] and Targeted error correction sequencing (TEC-Seq) [64], together with nontargeted sequencing approaches including whole-exome sequencing (WES) [65] and whole-genome sequencing (WGS) [66]. Although having high sensitivity and specificity and showing promising preliminary results in cancer-related clinical trials, including melanoma research [67], these techniques are limited to the detection of specific known mutations, thus not making a breakthrough in the daily clinical practice yet. Indeed, the use of ctDNA in skin cancers monitoring is still in its infancy. As currently available techniques are not sensitive enough to be used in early MM screening, researchers have turned their attention to advanced MM monitoring and prognosis. We mention the study conducted by Sacco et al. [68] where ctDNA was used as a powerful tool to obtain relevant prognostic information in surgically resectable melanomas, while testing during treatment allowed the assessment of systemic therapy response and resistance mechanisms identification.
Lastly, the potential powerful roles of micro RNAs (miRNAs) in cancers should not be neglected, as these are the most abundant RNA molecules found in peripheral blood circulation, in close connection to exosomes and TEPs. Regulating protein expression after transcription, miRNAs play a crucial role in the development and progression of skin cancer, as specific patterns of miRNAs in specific skin cancer types could be used as diagnostic markers [69]. The majority of the recent research has focused on the role of miRNA in malignant melanoma. Several studies have already demonstrated the impact of different miRNAs, such as miR-21, miR-125b, miR-150, miR-155, miR-205, and miR-211, in skin melanoma oncogenesis [70], while the impact of miRNAs in NMSC remains less characterized. The already existing preliminary data on the association between miRNA expression profiles and premalignant and/or malignant skin lesions [71] demonstrates the capability of phenotype- and stage-related tumor miRNAs to become reliable biomarkers in the near future. In order to integrate miRNAs into the daily clinical workflow, several advances remain mandatory. The current technologies comprising quantitative PCR (qPCR) or microarray platforms are still a challenge, with the uneconomical running costs and the requirements of highly trained personnel being currently the main limitations [72]. Improvement in pre-analytical handling of samples and the use of next-generation sequencing are thus mandatory for better potential results, as in the case of ctDNA analysis.

4. The Role of Dielectrophoresis in the Characterization of Skin Tumor Cells

4.1. Theoretical Background

The sorting and identification of various target particles, including tumor cells, has undergone an important evolution in recent years, with different methods now available. Depending on the employed external stimuli, there is a wide range of cell manipulation techniques currently in use, including dielectrophoresis (DEP). DEP is a non-invasive manipulation method suitable for measurements at the micro- and nanoscale level, with a great applicability potential in a large variety of bioparticles, including several types of tumor cells [73], CTCs [74], DNA [75], RNA [76] and other small particles, such as exosomes [77].
The applicability of DEP in skin tumor cell characterization relies on both the suitable technical aspects of this method for fragile living cells and the electrochemical properties of the particles. First described by Pohl [78] in the early 1950s, this method is currently living a veritable renaissance when thinking of its widely used application in the oncological field. The technique is based on the controlled motion of dielectrically polarized materials suspended in a fluid, which change their spatial and electric load position under a nonuniform electric field. The phenomenon, noticed in both direct and alternating current, is known as positive DEP (pDEP), if the object is displaced toward the stronger electric field [79], or negative DEP (nDEP), when the studied particle is displaced off the field [80]. In order to be a good candidate for DEP, the particle does not need to be electrically charged a priori. Under the presence of the nonuniform electric field gradient, the target particle will act as an electric dipole, so that the electric charges will segregate [81]. In addition to the particle/cell characteristics, DEP is also strongly related to the electrical properties of the suspension media, and the interfacial polarization mechanism expressed mathematically through the Maxwell−Wagner (MW) equation plays an important role [82].
F D E P → = 2 π R 3 ε m R e f C M ˜ ∇ E r m s → 2
The use of DEP principles and upgraded microfluidic devices are suitable for cell sorting; theoretically, tumor cells have different responses to healthy ones in different electrical field gradients. As described in the above equation, DEP force depends on cellular factors, such as the electrical properties of the cell’s membrane and cytoplasm, and cell’s size, but also on extracellular factors, mainly suspense solution conductivity. The role of the Clausius–Mossotti (CM) factor in determining one particle’s electrical behavior is detailed in other recent reviews [83,84].
Skin tumor cells broadly imitate the behavior of other tumor cells in the variable electric field. Cancer cells have different morphological properties than normal cells, thereby manifesting different electrical behavior (Figure 2). MM and NMSC cells have irregular shapes and sizes, structurally altered membranes, including folds and ruffles, modifications of the nucleus, and reduced cytoplasm/nucleus ratio. According to previous research [85], membranes’ folding factor is a relevant parameter for characterizing the electrical behavior of tumor cells, as larger membrane surface area compared to normal variants alter measurements results by increasing the cell capacitance. Ionic conduction at the cellular level is also relevant when measurements are done in a broader spectrum, cytoplasm resistance decreasing with increased applied voltage as some studies suggest [86]. Cell viability interferes also with DEP results, this feature being particularly of interest when studying cancer cells. According to literature data, the efficacy assessment of antitumor drugs can be done via DEP, as anticancer medication lowers cell viability, thereby decreasing its impedance and most probably deteriorating its membrane and cytoplasm [87].
The differences between malignant and benign skin cells also consist of distinct molecular profiles, as recent reviews suggest [15]. Thus, mutations in TP53, CDKN2A, NRAS, BRAF, or KIT genes have already been demonstrated to play a significant role in malignant melanoma, becoming targets for newly developed therapies [88,89]. CDKN2A, TP53, and NRAS mutations were also detected in SCC [90,91], explaining the effectiveness of novel MM-employed therapeutic approaches also in SCC. Regarding BCC, the PTCH1, GLI1, SMO, and E2F5 mutations were detected, with specific inhibitors and antagonists being already available in clinical practice, while other molecules are still under investigation within clinical trials [92]. It is clear, however, that there are still many unknown gene mutations involved in skin cancers, as their potential influence on the dielectric properties of skin cells remains to be demonstrated.
Finally, recent research increasingly emphasizes the importance of buffer solutions in DEP measurements, as the liquid medium is highly important for the cell’s behavior in the nonuniform electrical current field, as stated in Maxwell–Wagner equation. On the one hand, electrolyte ionic composition is essential for tumor cell trapping. Low conductivity media, containing a lower amount of salts, are preferred, as lower extracellular ionic concentrations induce stronger polarization in the cell’s cytoplasm [93]. Low buffer conductivity is helpful in both pDEP and nDEP for cell discrimination, also reducing the possibility of a possible Joule heating effect [94]. On the other hand, extracellular media is directly influencing living cells’ viability. Especially in frail skin tumor cells and CTCs, the choice of an appropriate suspension medium is all the more relevant, the loss of cell viability leading to erroneous final results [95]. There is an obvious need to develop better suspense solutions for biological particles, both cell-friendlier and more suitable for DEP.

4.2. Clinical Applications

Among CTCs and skin tumor cells detection methods, DEP emerged as a potential alternative technique for currently available conventional approaches. Some major strong points of DEP remain the possibility to perform label-free particle discrimination [96] and to isolate single cells [97]. Via DEP, cells’ evolution in real time can be monitored, based solely on the cell’s electrical properties [98]. Moreover, DEP has higher specificity compared to other alternative approaches, such as density gradient centrifugation [99].
Despite DEP’s incommensurable advantages, a series of drawbacks (low selectivity and limited reproducibility) has limited this technique to reach large scale deployment. Table 3 highlights the most important advantages and limitations of DEP, in a pragmatic comparison to other cellular detection/separation methods.
Strictly related to skin cancers, DEP was used first in melanoma cells more than 30 years ago, however, with limited further studies until the present. Table 4 summarizes the most relevant up-to-date results regarding the utility of DEP in human and different animal skin cells analysis.
Human skin cancer cells have specific electrical properties that facilitate separation when using DEP. One important aspect revealed since the earliest experiments [109], is the role of melanin content in modulating melanoma cells’ behavior in a non-uniform electrical field. Modifications in cell structure due to normal ageing or destructive changes under different drug therapies will alter the cell movement under DEP, facilitating or impeding single cell separation.
Another important aspect related to DEP is the continuous improvement of the technique, during the last couple of years, new technological breakthroughs changing also the classical DEP approach. One of the most used DEP techniques is the electrode-based DEP (eDEP) that employs metallic electrodes positioned within a micro-channel, having direct contact with the measured particles and the suspending medium [115]. The obvious disadvantage of this method is the higher risk of biological sample deterioration, as high frequencies charging is used. A simple solution to limit this negative effect is the use of insulator-based dielectrophoresis (iDEP), especially when manipulating fragile skin tumor cells. As the micro-electrodes are placed within an insulating structure, higher electric fields can be applied, offering a higher degree of selectivity of biological samples while maintaining a chemically inert environment [116]. Considering another DEP technique where particle contact is minimal, contactless DEP is a good example. The generation of a nonuniform electric field is possible in the absence of direct contact between electrodes and target particles, which is adequate when manipulating fragile biological cells [117]. Innovations regarding electric waves were also included in some DEP devices, traveling wave DEP consisting of a moving electrical field produces by the signals of shifting electrical phases. Not many studies used this kind of technique, but we mention the work of Cen et al. [118] where traveling wave DEP was employed along with other particle separation techniques in order to manipulate human malignant cells. Finally, a fine-tunning potent technique is optically induced DEP, where, compared to classical DEP, the examiner can easily and quickly create or modify an electrode layout through the control of optical patterns, by acting as a virtual electrode. This method was successfully applied in microfluidic systems for the isolation and purification of rare cell species in clinical samples, such as CTCs [119] and CTC clusters [120], in a higher performance manner than their conventional counterparts.
More recently, another promising approach consists of combining DEP with other methods of particle separation, such as magnetophoresis [121], photophoresis [122], and dielectrophoretic impedance measurement (DEPIM) [123]. Regarding skin tumor cells, the study conducted by Kelp et al. [114] demonstrated the efficacy of combining DEP with metasurface-enhanced infrared reflection spectroscopy in order to detect and characterize A431 cells. These encouraging results in malignant cells originating from different cancerous tissues can be a good starting point for further research based on the field of skin cancer cells, including CTCs and other relevant tumor particles, based on DEP techniques.

5. Conclusions and Future Research Directions

With growing incidence and prevalence, skin cancers will remain a global health burden many years to come, and fast, cost-effective, and specific diagnoses are thus mandatory. In addition to the improvements related to conventional tissue biopsy, the development of novel complementary techniques, such as liquid biopsy, is a must, especially in cases when the classic approach is unavailable. The role of CTCs along with other relevant particles (exosomes, ctDNA, miRNA) detected in the peripheral blood of skin cancer patients is already a successful method for disease monitoring and prognosis after therapy. However, continuous research has to be conducted in order for the cells and particles obtained through liquid biopsy to be relevant biomarkers for early cancer detection.
Another useful technique in cell separation and characterization is DEP, with its newer variants (travelling wave DEP, optically induced DEP) offering higher performances compared to the conventional approach. Although during the last decade, DEP has demonstrated its functionality also in skin cancer cells detection, there are still many unanswered questions and unexplored usage. Firstly, there is an urgent need to standardize the values regarding crossover frequencies for different types of skin tumor cells. Secondly, large-scale implementation of the technique will allow researchers to elaborate specific protocols, including regulations from the time of sample collection to the use of the most appropriate buffer solution and translation into clinical and therapeutic relevance. Finally, the ultimate goal when discussing about liquid biopsy and DEP would be their contribution to early skin cancer detection, as reliable screening techniques.

Author Contributions

T.G.S. and R.C.C.: Conceptualization, Data acquisition, Analysis and Interpretation of Data, Writing—Original draft preparation; I.T. and M.A.O.: Analysis and Interpretation of Data. M.A.: Supervision, Writing—Reviewing and Editing. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CCCDI–UEFISCDI, within PNCDI III program, project ID: ERANET-EURONANOMED-INTREPIDUS-1, contract no. 193/2020.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data and materials supporting the results of the present study are available in the published article.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

BCCbasal cell carcinoma
CTCscirculating tumor cells
ctDNAcirculating tumor deoxyribonucleic acid
DEPdielectrophoresis
miRNAmicro ribonucleic acid
MMmalignant melanoma
NGSnext-generation sequencing
NMSCnon-melanoma skin cancers
PCRpolymerase chain reaction
SCCsquamous cell carcinoma

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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].
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].
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Figure 2. Skin cancer cells vs. normal cells—relevance for dielectrophoretic analysis.
Figure 2. Skin cancer cells vs. normal cells—relevance for dielectrophoretic analysis.
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Table
Table 1. Liquid biopsy versus tissue biopsy in skin cancers—advantages and limitations.
Table 1. Liquid biopsy versus tissue biopsy in skin cancers—advantages and limitations.
Liquid BiopsyTissue (Conventional) Biopsy
Advantages of liquid biopsy over conventional biopsy
Easy to obtain (blood sample)Most often easy to obtain (localization dependent)
Non- or minimally invasiveInvasive
Virtually no clinical complicationsLow risk of complications (pain, bleeding, infection)
Possibility to highlight intra- and inter-tumor heterogeneity, and multiple tumor sitesLocalized analysis (no intra- or inter-tumor heterogeneity
Applicable to serial monitoringNot applicable to serial monitoring
Unmodified/fresh DNA studyPossibility of DNA study (possible technique-related DNA alterations)
Limitations of liquid biopsy compared to conventional biopsy
Limited use in clinical practiceCurrently gold standard approach
Utility under investigation
Limited histopathological analysis to CTCsHistopathological analysis and staging
Table
Table 2. CTCs and other relevant particles obtained via liquid biopsy.
Table 2. CTCs and other relevant particles obtained via liquid biopsy.
CTCsTEPsExosomesctDNAmiRNAs
AdvantagesMorphological characterization;
Functional analysis;
Possibility of cellular RNA analysis;
Interest/correlation with tumor diagnosis, prediction, prognosis		Abundant;
Relatively simple isolation technique;
Possibility of tumor RNA analysis		Potential correlation with tumor drug-resistance and metastasis;
Possibility of tumor DNA, RNA analysis		Good representation of tumor heterogeneity;
Relatively simple isolation technique;
Useful in tumor monitoring and prognosis		Released by different structures (TEPs, exosomes);
Stable in peripheral blood and other fluids
LimitationsExtreme rarity;
Fragility;
Heterogeneity		No standardized approach;
Time consuming		No effective enrichment method;
Heterogeneity		Rarity;
Fragility;
Contamination with normal cell DNA;
No functional analysis		High variability;
Unspecific to cancer type
Table
Table 3. Pros and cons of dielectrophoresis vs. other CTCs detection and separation methods.
Table 3. Pros and cons of dielectrophoresis vs. other CTCs detection and separation methods.
MethodProsConsReferences
DEPNon-invasive detection in liquid biopsy;
Both label-free and biomarker-based discrimination;
Single-cell isolation possibilities;
Cells’ evolution monitoring in real-time based on electrical properties		Low selectivity;
Limited reproducibility		[73,96,97,98]
Density gradient centrifugationLabel-free;
Simultaneous isolation of
more than two types of cells		Low specificity;
Physical stress—destructive for the CTCs		[99]
Gas chromatographyHighly reproducible;
High peak capacity molecules analysis		Use of high temperature (destructive for the sample);
Limited to volatile compounds		[100]
Mass spectrometryAccurate determination of mass;
High throughput		Destructive for the sample;
Less reproducible		[101]
Liquid chromatographyAdequate for a broad range of molecules;
High peak capacity molecules analysis		Limited throughput;
Potential longer separation time		[102]
Capillary electrophoresisMeasurements possible on
small samples;
Automated method (offers the possibility to process larger batches)		Sensitivity and resolution limits;
Increased immunofixation rate		[103]
Immunomagnetic separationHigh detection sensitivity (high purity) and effectiveness;
High-throughput		Limitation related to surface markers;
Antibody-mediated method		[104,105]
Fluorescence-activated cell sorting (FACS)High-throughput;
Simultaneous isolation of more than two types of cells		High costs in processing and pre-processing steps;
Skilled staff required		[106]
Raman spectroscopyHigh specificity;
Little sample preparation required		Sensitive and highly optimized instrumentation;
Use of high temperature
(Destructive for the sample)		[107]
Photoacoustic flow cytometryLabel-free;
Quantitative flow cytometry imaging		Limitation related to sample size and device parameters[108]
Table
Table 4. Clinical studies of DEP in skin cancers.
Table 4. Clinical studies of DEP in skin cancers.
Skin Tumor TypeMain FindingsReference
Human malign melanocytesCells’ behavior in DEP is markedly dependent upon pigmentation (melanin content), age, and drug treatment (chlorpromazine)[109]
Melanoma Colo38 cellsSeparation of melanoma Colo 38 cells from other cancer cells using levitation principle in software-controlled DEP[110]
Mouse melanoma B16F10 cellsDEP has the ability to differentiate between two malignant cells of the same origin, based on their melanin content[111]
Human skin fibroblast and human malignant melanocytesDistinguishing healthy and cancer cells using the microfluidic chamber DEP device[112]
A375 human skin cancer cellsAssessment of the effects of various parameters of nanosecond pulsed electric fields combined with multi-walled carbon nanotubes on skin cancer cells viability[113]
A431 human skin cancer cellsSeparation of A431 cells from liquid samples based on their DEP parameters[114]
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Schreiner, T.G.; Turcan, I.; Olariu, M.A.; Ciobanu, R.C.; Adam, M. Liquid Biopsy and Dielectrophoretic Analysis—Complementary Methods in Skin Cancer Monitoring. Appl. Sci. 2022, 12, 3366. https://doi.org/10.3390/app12073366

AMA Style

Schreiner TG, Turcan I, Olariu MA, Ciobanu RC, Adam M. Liquid Biopsy and Dielectrophoretic Analysis—Complementary Methods in Skin Cancer Monitoring. Applied Sciences. 2022; 12(7):3366. https://doi.org/10.3390/app12073366

Chicago/Turabian Style

Schreiner, Thomas Gabriel, Ina Turcan, Marius Andrei Olariu, Romeo Cristian Ciobanu, and Maricel Adam. 2022. "Liquid Biopsy and Dielectrophoretic Analysis—Complementary Methods in Skin Cancer Monitoring" Applied Sciences 12, no. 7: 3366. https://doi.org/10.3390/app12073366

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