Recent studies have highlighted CTCs role as potential prognostic, diagnostic and monitoring biomarkers [20]. CTCs provide a real tumor snapshot (DNA, RNA and proteins) ready to be analyzed and they might prove useful to perform ex vivo functional studies and cultures, as well [16]. The isolation of CTCs occurs through methods presenting a great variability in detection rates, sensitivity and specificity. There is only one technique approved by the U.S. Food and Drug Administration (FDA) for CTCs isolation: CellSearch™ (Veridex LLC), i.e. magnetic microspheres with anti-epithelial cell adhesion molecule antibodies (EpCAM). This technique has been approved for metastatic breast, colorectal or prostate cancer [21, 22]. Isolation of CTCs is very demanding in NSCLC, compared to other cancers since it is a tumor with high aggressiveness and invasiveness. In fact, the isolation of CTCs in NSCLC is less effective. CTCs can be lost as they undergo to epithelial-mesenchymal transition (EMT) and for the down-regulate of their epithelial markers during progression. Therefore, the initial EMT does not have the ability to resize the epithelial properties of cancer cells and it is difficult to discriminate those markers with existing techniques [23]. Several techniques can be used in NSCLC metastatic tumors such as the “CTC-chip” characterized by the use of antibodies to isolate CTCs: a technique that successfully detects CTCs in the blood with 50% of purity [24]. Other techniques take advantage of intrinsic properties, such as size (e.g., ISET; Rare Cell Diagnostics, Paris, France), deformability, or dielectric sensitivity and/or negative selection of white blood cells [25, 26]. Some mutations like EGFR, KRAS or the expression of MET have been detected in lung cancer. Their analysis becomes an additional method to complement the ctDNA test to facilitate monitoring of the patients response for a personalized therapy [27–30]. In a recent work, a technique called ISET was used to detect malignant circulating cells in patients with asymptomatic NSCLC [31]. However, several studies have enlightened the relevance of CTCs as prognostic and diagnostic sources of information. CTCs have been used as valid biomarkers to prevent lung tumors, as showed in chronic obstructive pulmonary disease [32]. Similarly, a study on early-stage NSCLC patients suggested that folate receptor positive CTCs could be used as diagnostic biomarkers [33]. However, despite the positive results regarding the value of CTCs as diagnostic biomarkers, there is no reliable evidence in the clinic in NSCLC [34]. In fact, new technologies to detect CTCs are now being developed (Figure 2). The methods currently in use are represented by erythrocyte lysis and cell centrifugation with long processing times and high costs. For these reasons, it is necessary to develop a system capable of limiting the loss of CTCs during isolation and increase the level of purity and establish a standard protocol to efficiently isolate vital CTCs suitable for in vitro expansion and ex vivo clinical applications [35]. A valid alternative to those systems is represented by nanotechnologies, with unique physical properties able to overcome the limits of traditional CTC detection methods [36].

ctDNA is a fraction of cfDNA which differs from normal circulating DNA and it is characterized by specific mutations in oncogenes or suppressor genes [14, 16]. Consequently, ctDNA is more concentrated in sick than in healthy subjects [37]. Cancer specific ctDNA provides molecular clues on the fragmented DNA of tumors and their specific mutations [35]. The ctDNA release mechanisms in the bloodstream are not yet clear, but it has been assumed that it can be released following apoptosis or cell lysis due to necrosis [38]. ctDNA is important because it can provide a picture of what are the mutations occurred to oncogenes or tumor suppressor genes, genetic amplification or epigenetic alterations of the patient [39]. Generally, ctDNA appears fragmented and mixed with non-tumor DNA at very low concentrations (0.01–1% cfDNA), thus making it difficult to quantify it in the early stage of the disease.

It is possible to discriminate ctDNA from cfDNA through ultra-sensitive analytical assays. The methods currently in use to analyze the extracted cfDNA are PCR, digital PCR (dPCR), ddPCR, the clamp-based peptide acid-based PCR test, peptide nucleic acid (PNA) Taqman dosage, microspheres, BEAMing, polymerization managed by pyrophosphorolysis and new NGS [40]. The NGS technique advanced technology can detect multiple mutations present through a single test in a very short time [41]. Sozzi et al. [17], showed a sensitive and specific real-time qPCR test, efficient for plasma DNA evaluation; it represents a new non-invasive approach to identify individuals with lung cancer or with the risk to develop the disease. Gautschi et al. [42], demonstrated that the plasma and serum DNA concentration was higher in NSCLS subjects than in healthy subjects.

It is clear that the quantification of cfDNA could be exploited as an additional screening together with those currently in use including chest CT scans and cytological/histological examination, because it is more promising and effective in the prognosis and diagnosis of carcinoma. Currently, in addition to the NGS technique, Cancer Personal Profiling through deep Sequencing (CAPP-Seq) also represents a valid tool; it is an even more sensitive approach, capable of eliminating wild-type background DNA from normal cells and detecting multiple classes of somatic mutations. Therefore, it appears useful to monitor treatment response, residual disease and progression before radiological approaches [43]. Although cfDNA shows an important prognostic and diagnostic value, clinical application is still limited due to the lack of standardization.

Exosomes are extracellular vesicles measuring between 30–100 nm. Almost all cell subtypes are able to secrete those endocytosis generated vesicles, that’s why it is possible to find them in different body fluids like pleural and cerebrospinal fluid, saliva, sperm and of course plasma and serum [44]. Exosomes contain different cellular product such as proteins, DNA, mRNAs, and miRNAs. Exosomes are crucial intermediaries between cells in a variety of diseases, including cancer, but also in physiological circumstances [45]. These vesicles are involved in tumor growth, progression, drug resistance and in the preparation of metastatic niche [16, 46]. Different techniques are described to help with their isolation. It is possible to extract exosomes from the above-mentioned fluids by density centrifugation or ultracentrifugation, transmission microscopy, and protein markers exploitation (CD9, CD63, tetraspanin proteins). Extracellular vesicular matrix or immunobead precipitation [16, 47] are also described as isolation methods. Exosomes with tumor origin can contain oncogenic material, causing cancerous transformations in receiving cells. An example is the transfer of EGFR to vascular endothelial cells, together with their own vascular endothelial growth factor (VEGF) [48]. The extraction of miRNAs from biological fluids could give some information about somatic mutations, junction variants, or valuable knowledge on protein and gene expression using enzyme-linked immunosorbent assay (ELISA) or Western Blot analysis together with NGS and real time polymerase chain reaction (RT-PCR) profiling. miRNAs contained inside exosomes are physically protected from RNAse degradation and their concentration in the bloodstream is generally higher. miRNAs are studied extensively due to their potential use diagnostic biomarkers in pulmonary adenocarcinoma [49]. Under this light, the most promising exosomal miRNAs are miR-21, miR-146, miR-17-3p, miR-210, miR-106a, miR-203, miR-214, miR-155, miR-212, miR-191, and miR-192. miR-23a, in particular, is involved in lung cancer progression by suppressing the development of propylhydroxylase, increasing the accumulation of hypoxia-inducible factor 1-α, facilitating the transendothelial passage of malignant cells [50]. miR-151a-5p, miR-30a-3p, miR-100, miR-200b-5p, miR154 and miR-629 were also highlighted as diagnostic biomarkers important to discriminate pulmonary granuloma and adenocarcinoma [51]. A recent study using miRNA-seq showed a unique expression pattern shared between squamous cell carcinoma (SCC) and stage I adenocarcinoma patients. In particular, an exosomal prophylactic profile of tumor-derived miRNA was analyzed showing 80.65% and 83.33% sensitivity and 91.67% and 90.32% specificity for adenocarcinoma and SCC diagnosis, suggesting their potential role in NSCLC early diagnosis as non-invasive and high sensitive biomarker [52]. The exosomes in lung cancer serum patients also mediate EMT and metastasis in healthy cells by increasing vimentin expression [53]. Moreover, exosomes surface membrane proteins like CD317, CD91 and EGFR might represent potential tumor makers [54]. miRNAs are believed important in tumor profiling and crucial for early diagnosis, treatment response, and cancer recurrence marker and may predict the overall survival, as well.

After red cells, platelets are the most abundant cells within the bloodstream. Platelets can be considered immune system “scanning soldiers”, sensing the presence of bacteria, cross-communicating with lymphocytes, and partially regulating immune cell extravasation [55]. They have a significant role in several pathological processes like EMT, which increases the cancer cells ability to penetrate and invade distant tissues exploiting the circulation; they also create a neovascularization supportive environment by stimulating the expression of proangiogenic factor secretion such as platelet-derived growth factor (PDGF), VEGF, and basic fibroblast growth factor (bFGF) [56, 57], inducing tumor cell apoptosis and anoikis and epithelial-mesenchymal switch in tumor cells by direct physical interaction and release of transforming growth factor beta (TGFβ) molecules [58]. Platelets act protecting circulating tumor cells from the normal immune response, through several growth and proangiogenic factors release [59] significantly contributing to metastatization [60]. They are highly involved in tumor microenvironment and important factors in cancer biology as they contribute to tumorigenesis and progression, and to response to therapy. Moreover, platelets form a cell–fibrin–platelet-aggregate surrounding CTCs providing mechanical protection [61]. TEPs can be considered non-invasive biomarkers suitable for RNAs panels determination. In fact, spliced TEP RNA surrogate signatures might give specific information about the presence, localization and cancer molecular phenotype. Platelets carry high amount of genetic material: RNAs, such as miRNAs, pre-mRNAs, mRNAs, circular RNAs (cirRNA), long noncoding RNAs, and mitochondrial DNA [62]. Certainly, platelet activation can induce subsequent pre-mRNA splicing and protein translation [63]. Platelets count and size give information about the putative tumor presence; in fact, a high count is associated to increased mortality in different tumors such as malignant mesothelioma, gynaecological malignancies and lung, kidney, gastric, colon cancers rectum and breast [64]. In NSCLC, tumor-derived platelet factor 4 [PF4, chemokine (C-X-C motif ) ligand 4 (CXCL4)] [65] has been reported to promote megacarbonite-mediated platelet production in the bone marrow and an RNA panel was altered in TEPs in metastatic NSCLC patients [66]. Spliced RNA profiles are easily identifiable by minimal amounts of platelet RNA (100–500 picograms) through the thromboSeq platform, a methodology based on RNA sequencing. This allowed the metastatic carcinoma patients individuation with accuracy between 84% and 96% and identified surrogate signatures of spliced RNAs, associated with the molecular subtype of the tumor tissue, such as EGFR and KRAS mutations and HER2 and MET amplification [67]. An algorithm, called swarm-intelligence, has recently been implemented and iteratively optimizes the RNA panel to select a set of biomarkers [68]. This system allows the diagnosis of advanced stage NSCLC with an accuracy of 89% in an independent validation cohort. Nilsson et al. [69], through RT-PCR noticed echinoderm microtubule-associated protein-like 4 (EML4)-ALK rearrangements in platelets derived from NSCLC patients blood with 100% specificity and 65% sensitivity. EML4-ALK+ platelets patients had a remarkably lower progression-free survival: 3.8 months, compared with 16 months showed by patients with EML4-ALK-platelets. Moreover, 30 months monitoring of EML4-ALK rearrangements in patients platelets revealed the occurrence of crizotinib resistance onset two months earlier than radiologic disease progression [69]. Sheng et al. [70], carried out a support vector machine network analysis on RNA-seq data derived from 402 NSCLC patient platelets, compared with 231 healthy donors. Target genes were 48, and several modules appeared to play crucial roles in this tumor onset and progression, like Wiskott-Aldrich Syndrome Protein Family Member 1 (WASF1), Arginine and Serine Rich Coiled-Coil 1 (RSRC1), Protein Kinase AMP-Activated Non-Catalytic Subunit Beta 2 (PRKAB2), Pyruvate Dehydrogenase E1 Subunit Beta (PDHB), Myosin Light Chain 9 (MYL9), Tropomyosin 2 (TPM2), and Protein Phosphatase 1 Regulatory Subunit 12C (PPP1R12C). In a recent study, Integrin Subunit Alpha 2b (ITGA2B), platelet and megakaryocytes transmembrane glycoprotein, involved in platelet aggregation [71] and in thrombasthenia, was identified as NSCLC platelet RNA marker in a test and an independent validation cohort [72]. TEPs RNA analysis may be complementary to currently in use non-invasive screenings to enhance the early stage cancer detection, characterization and monitoring in order to make therapy more efficient.