Research progress of CTC, ctDNA, and EVs in cancer liquid biopsy

Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and extracellular vehicles (EVs) have received significant attention in recent times as emerging biomarkers and subjects of transformational studies. The three main branches of liquid biopsy have evolved from the three primary tumor liquid biopsy detection targets—CTC, ctDNA, and EVs—each with distinct benefits. CTCs are derived from circulating cancer cells from the original tumor or metastases and may display global features of the tumor. ctDNA has been extensively analyzed and has been used to aid in the diagnosis, treatment, and prognosis of neoplastic diseases. EVs contain tumor-derived material such as DNA, RNA, proteins, lipids, sugar structures, and metabolites. The three provide different detection contents but have strong complementarity to a certain extent. Even though they have already been employed in several clinical trials, the clinical utility of three biomarkers is still being studied, with promising initial findings. This review thoroughly overviews established and emerging technologies for the isolation, characterization, and content detection of CTC, ctDNA, and EVs. Also discussed were the most recent developments in the study of potential liquid biopsy biomarkers for cancer diagnosis, therapeutic monitoring, and prognosis prediction. These included CTC, ctDNA, and EVs. Finally, the potential and challenges of employing liquid biopsy based on CTC, ctDNA, and EVs for precision medicine were evaluated.


Introduction
Cancer is a highly lethal ailment that poses a significant threat to human existence.Key components of cancer prevention, detection, and therapy encompass the assessment of the effectiveness of cancer treatment, the surveillance of patient's post-treatment, and the provision of timely alerts regarding the risk of tumor metastasis and recurrence.There is no accurate biomarker for early cancer diagnosis due to the complexity of tumor incidence and tumor heterogeneity, Despite the development of multiple technologies aimed at the early detection of tumor biomarkers from diverse clinical samples and the significant research conducted in this field (1).Technological developments have led to alternate methods of tumor liquid biopsy, which have seen significant success and promise (2).The current understanding in the scientific community is that a tumor can release many components, such as tumor cells proteins, extracellular vehicles (EVs), and nucleic acids, into the peripheral circulation (3).Since the original identification of tumor cells in patient peripheral blood in the 1860s, significant advancements have been made in separating circulating tumor cells (CTCs) from various blood cell types (4).Furthermore, in 1996, the identification of microsatellite alterations in the circulating tumor DNA of individuals with tumors demonstrated a precise correlation with the microsatellite changes noticed in the primary tumor (5).In 2011, the concept of liquid biopsy based on CTC detection was proposed (4), and after that, the technology of using ctDNA to obtain tumor biological information was included in the liquid biopsy's scope (5).In 2015, the Massachusetts Institute of Technology Review named liquid biopsy one of the top ten breakthrough technologies of the year.Afterward, liquid biopsy entered a rapid development stage, and detection technologies such as extracellular vehicles (EVs) (6) and tumor-educated platelets (7) were also included in the scope of liquid biopsy.A method of in vitro diagnosis called liquid biopsy uses body fluids as the test material to obtain tumor biological information relative to tissue biopsy.Liquid biopsy, which obtains a sample of bodily fluids such as saliva, blood, and cerebrospinal fluid, has gained broad attention due to its little invasion and has increased prospects for cancer detection and continuous monitoring (6).
Each of the three techniques in liquid biopsy has its own strengths.CTCs detection monitors the trend of changes in the type and number of CTCs by capturing CTCs present in the peripheral blood in order to monitor tumor dynamics in real time, assess treatment efficacy, and enable real-time individual therapy.ctDNA is a fragment of DNA that is necrotic, apoptotic, or normally secreted by tumor cells into the bloodstream, and carries information about cancer-related genetic mutations, and can be used for early detection of cancer when the it can be used for early detection of cancer because it can be detected when mutations occur at the molecular level of the tumor.ctDNA is capable of fusion and remapping in different types of cancers (8).ctDNA has a number of clinical applications, for example, sequential ctDNA assays can be used to efficiently monitor patients and detect tiny residual lesions, which can help in the early detection of disease progression and the adjustment of the Adjuvant therapeutic regimens for ovarian cancer treatment (9).Plasma preoperative ctDNA testing in ovarian cancer patients is expected to serve as a biomarker for tumor staging and prognosis prediction (10).Early changes in circulating tumor DNA (ctDNA) predict treatment response in patients with metastatic KRAS-mutant colorectal cancer (mCRC) (11).ctDNA and radiated tumor volume identify patients with resected early-stage non-small-cell lung cancers who are at risk of recurrence (12).EVs have been linked to driving malignant cell behavior, including stimulating tumor cell growth, suppressing immune responses, inducing angiogenesis, facilitating tumor cell migration, and establishing metastasis, making them particularly attractive as cancer biomarkers (13).EVs contain cargoes of miRNAs,mRNAs, and proteins.It has been shown that microRNAs carried by EVs are associated with colorectal cancer (14), hepatocellular carcinoma (15), lung cancer (16), oral cancer (17), and ovarian cancer (18).The contents of EVs such as mRNA can promote epithelial-mesenc hymal transition and chemoresistance in colorectal cancer (19).Therefore, not only CTCs can be used for tumor liquid biopsy, but also ctDNA,EVs can be used for cancer detection.
In this comprehensive analysis, we thoroughly examine the latest developments in the rapidly evolving domain of liquid biopsy.Our main objective is to explore the potential uses of liquid biopsy techniques in cancer clinical diagnosis, therapy monitoring, and prognosis prediction.These techniques encompass circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and extracellular vesicles (EVs).We also discussed the benefits and drawbacks of the most recent CTC, ctDNA, and EV detection, capture, and downstream analysis technologies.The application of comprehensive liquid biopsy combined with CTC, ctDNA, and EVs detection in cancer monitoring.Finally, we discussed liquid biopsy's potential applications and difficulties in precision medicine.Figure 1 shows CTCs、ctDNA, and EVs detection techniques and their clinical applications.is extremely rare, with approximately one billion blood cells possibly being CTC.This poses significant technical challenges to isolate CTCs (23) effectively.Two counting processes comprise most CTC detection processes: CTC enrichment and separation and CTC identification.The term "CTC enrichment and separation technology" describes the process of concentrating and separating CTC from a large number of blood cells in peripheral blood based on one or more distinctive properties of CTC.CTCs were separated through biomarker-driven cell capture, employing various enrichment techniques rooted in biophysical and other characteristics, including immunogenicity, positive and negative enrichment, as well as enrichment strategies relying on size, density, dielectric properties, and deformability (14) (Table 1).CTC identification methods mainly include cell morphology identification, immunofluorescence staining, Fluorescence in situ hybridization, PCR etc (Table 2).CellSearch ® is the first FDAapproved CTC separation technique that uses antibody-labeled magnetic nanoparticles to select cells that express EpCAM and fluorescence microscopy to identify keratin and DAPI positive CD45 negative cells (31).The isolation methods of CTCs are shown in Table 1.
After enriching and separating CTC in the blood, effective methods must be combined for analysis and identification.The detection of CTC has evolved from counting to analysis and type, single-cell sequencing, and cell function analysis due to the ongoing development of CTC enrichment, detection, and analysis methods.By directly examining the mechanisms of drug resistance, migration, and recurrence in each CTC, single-cell genomics and transcriptome analysis can aid in early cancer detection and provide better recommendations for early cancer diagnosis.It can also help to personalize treatment.These benefits include discovering the dynamic changes of tumors, analyzing tumor growth rules, and more (32).Table 2 lists CTC identification techniques.

ctDNA
Apoptosis and necrosis of human cells will release fragmented DNA into body fluid, called cfDNA, which includes the DNA of tumor cells and the DNA of normal cells; the latter is called ctDNA.Cancer patients' cell-free DNA has been found to include ctDNA, allowing for the analysis of the genetic abnormalities that characterize each patient's cancer (22).Over the past 10 years, research has shown considerable promise as a possible clinical tool to promote personalized therapy.Most ctDNA fragments are double-stranded and have lengths between 160 and 200 base pairs, or roughly the same as a nucleosome (33).Numerous aspects of the tumor, including its location, metastasis, size, tumor status, vascular infiltration and staging, impact the amount of ctDNA that enters the bloodstream.As a result, there is a wide variation in the proportion of ctDNA (32).Free DNA released by blood cells and another somatic cell may produce an interference phenomenon that must be effectively distinguished.Therefore, distinguishing ctDNA from normal cfDNA is essential to reflect the disease status (34,35) accurately.
ctDNA can provide tumor-specific genetic variation (Point mutation, insertion and deletion, rearrangement, fusion, etc.) and epigenetics (methylation, etc.) and copy number variation information; its application value in the whole process management of tumor patients has been recognized and has been written into several clinical diagnosis and treatment guidelines.The main obstacles facing ctDNA detection technology are increasing the sensitivity of detection technology and suppressing background noise signals because ctDNA has been isolated and analyzed using a variety of techniques, including Tagged-amplicon deep sequencing (TAm-Seq), cancer personalized profiling by deep sequencing (CAPP-Seq), whole genome bisulfite sequencing (WGBS-Seq), and droplet digital polymerase chain reaction (ddPCR) sequencing technologies.
Tumor Circulating Among the new liquid biopsy technologies, DNA analysis is the most advanced, although meaningful comparisons between studies are made difficult by the range of ctDNA assays.In addition to being beneficial for calculating copy number variants and discovering possibly rare mutations, ddPCR is only useful for screening for known mutations (36).DdPCR (32) is one of the most useful and applicable methods today for analyzing   (16,25).Whole genome sequencing (WGS) is a comprehensive approach that examines a tumor's complete genomic content, which can greatly aid therapeutic decision-making and reveal novel therapy plans, unusual mutations, and invisible oncogenes.However, WGS is constrained by difficulties with quality control, ethics, cost, and timing (39).To identify frequent or rare disease-related abnormalities, whole exome sequencing (WES) characterizes and analyzes all currently reported tumor mutations.However, it might have a lower sensitivity than other methods (36,40).Table 3 lists the benefits and drawbacks of the various detection strategies.

EVs
EVs are membrane vesicles containing lipid bilayer secreted by cells into the extracellular space (30-2,000 nm), which nearly all cells actively secrete (6).Extracellular vesicles (EVs) can be divided into three classes according to the International Society for Extracellular Vesicles' (MISEV's) recommendations, depending on their size: microvesicles, apoptotic bodies, and tiny extracellular vesicles.The classification of these EVs is determined by their respective dimensions (49).It is reported that EVs derived from cancer cells are important in changing the tumor microenvironment and promoting tumor progression (50).They also carry distinctive molecules from parental cells, including proteins, miRNAs, mRNAs, lncRNAs, DNA, and lipids, indicating the state of the disease at the time.Surface markers mostly include CD63, CD81, CD9, TSG101, and HSP27 (51).Multiple studies have shown that EVs may be reliably found in a variety of bodily fluids, including blood (44), saliva (45), urine (46), bronchoalveolar fluid (47), breast milk (48), and semen (49).As a result, EV cargo targeting enables us to evaluate essential molecular data concerning illness status (50).In recent years, there have been numerous methods for the detection of exosome contents, such as RT-PCR ( 52),genome sequencing (53),and proteomics (54).Table 4 lists the detections of exosomal contents.Table 5 lists the comparison of miRNA, mRNA and protein detection methods in EVs.
EVs are an important research direction in liquid biopsy; the value of its potential applications has been continually tapped.Repeatable EV separation and enrichment will enable the evaluation of their biological functions.However, the key obstacles in this sector are the separation and purification of EVs, which are heterogeneous in function, size, content, and source ( 6).An important step in experimental research is effective extraction.Size exclusion chromatography (SEC), polymerization, the process of precipitation immunoaffinity chromatography, and microfluidics-based methods are the primary methods used to isolate and refine extracellular vesicles (EVs) (51, 52).For different purposes and objectives, different separation methods are utilized.However, polymer precipitation, size-based isolation, immunoaffinity capture and ultracentrifugation are routinely used.Every approach and strategy is employed based on EVs' size and origins.Each has advantages and disadvantages.The advantages and disadvantages of the various separation methods are listed in Table 6.

Importtant areas of clinical applications of CTCs、ctDNA and EVs
CTCs, ctDNA, and extracellular vesicles (EVs) are now used in clinical settings as substitute biomarkers for various solid tumors, and numerous research have been conducted, indicating enormous potential in therapeutic applications.

Clinical applications of CTCs
The increased demand for CTCs as precision oncology biomarkers.CTC detection is an appealing, non-invasive method for cancer diagnosis.CTC detection has important value in early diagnosis, efficacy determination, prognosis evaluation, and treatment monitoring of tumors.CTC analysis makes real-time monitoring, further analysis, and identification of protein, RNA,

RT-qPCR
Quantification by standard curve method or internal reference method Through the two steps of reverse transcription and PCR, the content of specific substances in exosomes can be detected with high specificity and sensitivity; if the efficiency of reverse transcription is high and the efficiency of PCR amplification is stable, then the sensitivity of RT-qPCR will be increased; if the primers are welldesigned and can specifically bind to the target sequences, and if the PCR amplification conditions can effectively inhibit non-specific amplification, then the specificity of RT-qPCR will be improved.Low-abundance proteins can be detected, and relative and absolute protein quantification can be performed; through mass spectrometry and other techniques, proteins and their modifications expressed in exosomes can be detected, with high sensitivity, and small differences in expression in exosomes can be detected; through the selection of appropriate protein markers and mass spectrometry analysis methods, interference with non-target cells can be avoided, and a high degree of specificity is achieved.
Extraction and purification of exosomes is relatively difficult and susceptible to interference from other proteins and substances.Proteomics generates a large amount of data, which requires professional bioinformatics personnel to interpret and analyze.(54,58) and DNA molecules possible by effectiveness, non-invasiveness, and high repeatability (58,59).Many clinical studies have evaluated the potential of CTCs to use blood samples from known cancer diagnosis patients for cancer testing (Table 7).

Early diagnosis
Studies have shown that CTCs can diagnose or detect clinically related cancers early.CTC, for example, has been identified in individuals with early stages (stage I-IIIA) cancer of the breast (60,61); more than one form of CTC was found in 20% of stage I disease patients, 26.8% of stage II disease patients, and 26.7% of stage III disease patients (62).CTC has been detected using cell search technology in patients with non-metastatic colorectal cancer (including stage I and II) and non-metastatic prostate cancer (159,160).According to Barriere et al. (65), 41% of T1-stage breast cancer patients and 47% of axillary lymph node-negative patients had CTC.A study conducted by Thery et al. (66) found that the incidence of positive circulating tumor cells (CTCs) was 21% in lymph node-negative breast cancer and 24% in instances of lymph node-positive breast cancer cases.Using the EpCAM-based NanoVelcro CTC chip, CTC can be detected in 60% of patients with stage II diseases, and positive CTC distinguishes pancreatic ductal adenocarcinoma patients from non-adenocarcinoma pancreatic disease patients (161).A meta-analysis comprising 18 prospective studies has demonstrated that a positive CTC (circulating tumor cell) result serves as a valuable biomarker for forecasting adverse overall survival outcomes in individuals diagnosed with early-stage non-small cell lung cancer (NSCLC) (68).In another study, 98 suspected prostate cancer patients were predicted to undergo biopsy diagnosis before biopsy, and Clinically significant cancer was highly associated with CTC detection utilizing the Parsortix isolation technique (99).
Collectively, these findings demonstrate that CTCs can be identified ahead of primary tumors in imaging investigations, highlighting the fact that the greatest obstacle facing the use of CTCs in early cancer diagnosis is, in fact, their paucity and isolation.The PROLIPSY examination, as exemplified by the study identifier NCT04556916, along with analogous examinations related to breast cancer (NCT03511859), n on -small cell lung cancer (NCT02380196), colorectal cancer (CRC; NCT05127096), and pancreatic cancer (PANCAID) (70), represent a subset of the numerous clinical trials exploring the application of CTCs (circulating tumor cells) in the early detection of various cancers.

Prognosis and treatment monitoring
CTCs have been examined extensively for their prognostic usefulness and are now recognized as an independent prognostic factor.CTCs have been found to have prognostic significance in various malignancies, including breast, prostate, colorectal, small cell, and non-small cell lung cancers (71)(72)(73)(74), and their discovery in the bloodstream has been connected to various diseases.In addition to the only clinical CTC testing system approved by the FDA, CellSearch, there are other CTC testing systems, such as CanPatol and CTC chips (162).
The examination's primary focus is the CTC count, with a positive threshold of ≥ 5, typically representing a poor prognosis.The persistence of CTC following therapy is associated with a poorer prognosis, and studies have demonstrated that changes in the quantity of CTC provide better predictive information than baseline CTC status (76,77).The number of CTCs present before beginning neoadjuvant therapy hurts survival, according to a meta-analysis of 2239 breast cancer patients from 21 trials (246) (60).Furthermore, patient CTC analysis can aid in predicting low residual disease and late disease recurrence because CTCs can be seen 7-9 weeks before the disease's clinical manifestation (163) and provide tools for early cancer detection (164,165).A prognostic factor for a lower survival rate is an increased CTC count, or a failure to eliminate CTC during treatment (80, 81), and reduction or clearance of CTC count is associated with good treatment response (166).In addition, evaluating the abundance of CTC clusters can significantly improve the prognostic value of patients receiving treatment (167).CTC clusters indicate a bad prognosis and elevated baseline CTC levels are linked to decreased survival rates (168).Studies have shown that the molecular phenotype of CTC, including HER2 (159), CD47, and PD-L1, has significant prognostic value (85).CTC has been utilized as a helpful biomarker for assessing the effectiveness of cancer treatment in numerous clinical trials (99,161), assisting clinical doctors in personalized treatment and drug resistance selection during tumor progression.CTCs can be used to track the progress of metastatic disease in patients with breast and colorectal cancer and prognosis, and a current NIH-sponsored clinical trial (NCT02973204) is looking into the utility of CTCs as clinical support tools in hepatocellular carcinoma (169).

Clinical applications of ctDNA
With advantages such as being non-invasive, sensitive, comprehensive, and dynamic, ctDNA has shown great clinical

Early diagnosis
ctDNA detection has also shown great promise in the early identification of cancer, opening up new avenues for developing highly targeted therapeutic therapies for cancer patients (174).Future clinical trials may further explore predictive biomarkers found using ctDNA analysis (168).The ctDNA of cancer patients can be examined for genetic changes such as mutations, loss of heterozygosity, microsatellite instability, methylation, and copy number variations (CNVs) (85, 169).These molecular hallmarks of tumors serve as useful biomarkers for cancer diagnosis, staging, and treatment.Cancers of the breast, colon, pancreas, and esophagus were all found to have increased ctDNA levels in their advanced stages compared to their preliminary stages, and this was true regardless of tumor-specific molecular characteristics (175).

Prognosis and treatment monitoring
CtDNA collection from blood is non-invasive and repeatable over time compared to conventional tumor biopsy.It can monitor  tissue, which may be further examined in these patients.In order to prognostically and predictively stratify individuals based on their DNA, ctDNA assays may be used (175).In a prospective study of 69 patients with advanced NSCLC, those with high ctDNA levels fared significantly worse overall than those with low ctDNA levels and progression-free survival.Higher ctDNA than baseline indicates a poor prognosis (177).In various large patient cohorts, including prospective screening cohorts in high-risk groups for colorectal cancer, Luo and associates examined the methylation patterns on ctDNA.Methylation-based diagnostic scores were found and validated to distinguish colorectal cancer patients from healthy control groups in addition to prognosis ratings linked to patient survival (177).Additionally, tumor cells from various sources may have different methylation profiles, enabling ctDNA analysis to reveal location information (129).Cell-free DNA (ctDNA) analysis has the potential to provide transcriptional information that could aid in the early diagnosis of cancers like prostate and colorectal by inferring transcription factor binding.Furthermore, ctDNA can predict tumor immune infiltration and response to systemic treatment following tumor recurrence after radical hepatectomy and help solve monitoring decisions and post-recurrence treatments (129), and tumor immune infiltration and responsiveness to systemic treatment after tumor recurrence after ctDNA can predict radical hepatectomy.

Clinical Applications of EVs
Exosome biology in disease is a relatively new area of research.EVs provide a rich source of biomarkers for the diagnosis and prognosis of disease.Tumor EVs are largely used in cancer because identifying cancer-predictive biomarkers in them can assist in increasing the early tumor diagnosis specificity and sensitivity.They have also made some headway in treating conditions affecting the heart, lungs, and central nervous system (53), and their application is increasingly being used to treat conditions affecting the liver (180) (180), kidney (181), and lung (181), among others.Due to their exceptional characteristics for transporting functional cargoes to diseased cells, exosomes can be therapeutic carriers at both the basic and clinical levels.

Diagnostic potential of EVs
EVs are desirable as minimally invasive liquid biopsies because they are present in all biological fluids, are substantially concentrated in biofluids, and are secreted by all cells.According to some research, there is a small amount of DNA in EVs; this can be used to detect mutations in serum EVs associated with cancer (180,182).EVs may include particular miRNAs or miRNA sets with diagnostic or prognostic value for cancer identification.Since EVs include miRNAs produced differently in cancer cells than normal cells, they may have a high diagnostic value and aid in early detection.Bladder and prostate cancer have been associated with increased levels of exosomal miR-21 in the urine, whereas glioblastomas, colorectal, colon, liver, breast, ovarian, and esophageal cancer have all been connected to increased levels of circulating exocrine miR-21.Tumor-suppressing miRNAs like miR-1 4 6a and miR-3 4a are associated with hematological, liver, breast, colon, and pancreatic cancer (183,184).Colorectal cancer can be diagnosed with miR-638 (185), while exosomes generated from breast cancer cells are greatly enriched in miR-1246 and miR-21 (186).Furthermore, those experiencing acute myocardial infarction and heart failure have shown increased secretory microRNAs linked to cardiovascular ailments.MicroRNAs such as miR-499, miR-133, miR-208, miR-192, miR-194, and miR-34a (86)(87)(88)187) belong to this group.There is great potential for the clinical identification of disorders of the central nervous system based on the unique expression patterns of miR-21, miR-29, miR-219, LRP6, REST1, and caveolin1 in exosomes (89-92).Combining different microRNAs may increase EV miR characteristics' diagnostic and prognostic value, which are constantly associated with cancer diagnosis and prognosis (93,94).Breast cancer patients' circulating exosomes have been studied for their surface proteins (96), and their phosphoproteins have been suggested to have diagnostic potential (95).Also, lung cancer patients have significant EV protein CD151 expression (97).

Therapeutic potential of EVs
Currently actively exploring EVs or as carriers for drug payloads as therapeutic agents (6).Compared to liposomes, injected exosomes can efficiently enter other cells and deliver functional cargo with minimal immune clearance when administered exogenously in mice (181,188,189).Tumor stroma-derived exosomes have also been associated with cancer chemotherapy resistance (183,190), and thus targeting specific functions of exosomes can enhance response to therapy.EVs have the potential to serve as a targeted medication carrier and benefit from the advantages of natural drug delivery due to their capacity to cross biological barriers like the blood-brain barrier.Chinese herbal medicine Western medicine uses paclitaxel (PTX) (98), curcumin (100), berry anthocyanins (101), -element ( 102) triptolide (103), and compound Buyanghuanwu Decoction (104).Catalase (CAT) (106), doxorubicin (105), and others (53).Additionally, EVs can employ gene therapy techniques and transport gene therapy materials like DNA and RNA.For instance, oligonucleotides can mute particular genes to treat various human diseases, such as cancer or neurological disorders (107, 108).MicroRNAs (miRNAs) have been found to efficiently contribute to the degradation of target mRNA and the suppression of gene expression in receptor cells; as a result, EVs have been developed for CNS disorders and tumors to transport miRNA or small interfering RNA (siRNA) payloads.For example, preclinical tests using EVs to deliver miRNA or siRNA payloads focused on anti-cancer treatment and exploratory brain targeting in rodents with breast cancer (184), glioma (185)and pancreatic cancer (186,187).Additionally, ligand enrichment on modified EVs can direct EVs to particular cell types or trigger or inhibit signal transduction events in receptor cells (113,114).

Comprehensive liquid biopsy
With the advent of precision medicine, researchers are looking into liquid biopsy to monitor tumor growth in real time and direct treatment accordingly.Molecular residual disease (MRD), also called a minimum residual disease or, in some circumstances, quantifiable residual disease, refers to solid tumors that, following therapy, cannot be detected by conventional imaging or other laboratory techniques.However, detecting tumor molecular abnormalities through liquid biopsy methods such as ctDNA and CTC indicates the persistence and clinical progression of the tumor (191).According to studies, liquid biopsies can detect metastatic diseases at least 4 years before they are clinically detected, indicating that thorough liquid biopsy evaluation of CTC combined with ctDNA monitoring MRD can provide very important information for treating and treating patients with breast cancer (192).Clinical studies have also shown the combination of CTC and ctDNA to improve the sensitivity and specificity of MRD detection in hepatic carcinoma (193).A new study examined the prognostic significance of combining CTC and ctDNA analyses.The results showed that CTC and total cfDNA levels were separately and together related to PFS and OS in MBC (194).In addition, combining ctDNA analysis based on Guardant360 NGS with CTC counting in MBC can help determine the site of metastasis (195).In a recent pilot study, 16 patients with metastatic urothelial cancer were compared using matched CTC and ctDNA samples, demonstrating that CTC and ctDNA offered complementing information (196).Therefore, CTC and ctDNA can be jointly tested for clinical tumor MRD monitoring services.There are two techniques in the field of ctDNA MRD, tumor native and tumor agnostic.Table 8 shows the advantages and disadvantages of these two techniques.
In recent years, it has been discovered that EVs miRNAs can act as vital information intermediaries for cancer cells and other cells, having a significant impact on the pathological development of tumors (205) and being potentially appropriate from a material aspect for different medication delivery and therapeutic uses (206).EVs can assess drug resistance, diagnose early cancers, and provide a prognosis.The key to understanding cancer may be found in the miRNAs found in EVs.A potential novel approach to treating lung cancer may involve preventing the production and release of EVs.EVs can be employed for additional study of ctDNA and miRNA discovered in The stability of nucleic acid molecules is successfully maintained by EVs, protecting ctDNA from degradation (17).ExoDX Lung (ALK), the first cancer diagnosis product ever released by EVs Diagnostics, was introduced in 2016.Real-time screening for EML4-ALK mutations in non-small cell lung cancer patients is made possible by the device's EV detection technique, which can concurrently detect EVs RNA and ctDNA.According to the information supplied by EVs diagnostic (53), ExoDx lung (ALK), which can be utilized to assist physicians in determining if patients are amenable to targeted therapy with ALK inhibitors, has 88% sensitivity and 100% specificity in identifying non-small cell lung cancer.This is particularly true for those who cannot or do not want to perform a tissue biopsy.
Long-term high cancer mortality rates have motivated domestic and international research teams to create effective cancer therapy strategies.Liquid biopsy aims to collect biomarkers from biological tissues that are not solid in order to assess the disease status.It can sample and analyze different biogenic substances such as CTCs, nucleic acids, proteins or EVs in bodily fluids such as blood or urine (207).Currently, researchers are considering combining CTCs, ctDNA, RNA, miRNA, protein, and lipid EV cargos in diagnosing and prognosticating cancer and other diseases.A thorough understanding of the whole course of disease for each cancer patient might be provided if these technologies are combined to boost the positive and negative predictive value.Table 9 lists the applications of CTCs, ctDNA and EVs in the diagnosis, prognosis and therapeutic monitoring of different diseases.

Conclusions and perspectives
Precision therapy is individualized pharmacological treatment based on specific characteristics of tumors (17).Tumor heterogeneity, variations in gene expression, and polymorphisms may all affect a patient's response to medication differently.Precision medicine and molecular diagnosis have developed quickly due to current diagnostic technology (208).Table 10 looks at the prospects of CTCs, ctDNA and EVs in tumor diagnostics in terms of cost, operability, simplicity and industrialization.Despite potential and challenges, liquid biopsies are at the forefront of this revolutionary approach, and the future of precision oncology is bright.It is also a relatively recent method to assist clinical decision-making for preventing, identifying, and treating human cancer.
Although CTC analysis has several clinical limitations, it has the potential to deliver more thorough and accurate information on tumors.Individual differences and the extremely low CTC content in peripheral blood necessitate further research into effective CTC

Small-cell lung cancer
CTCs detection can provide additional information for the diagnosis of small cell lung cancer by capturing tumor cells in the circulating blood.
Particularly in cases where the nature of a lung nodule is difficult to clarify or where there is a need for earlier information about changes in the disease, CTCs detection can be used as a rapid, non-invasive method to help doctors determine whether a small cell lung cancer is present.
The number and characteristics of CTCs can provide information about the prognosis of small cell lung cancer.
The effectiveness of treatment can be assessed by detecting changes in the number and characteristics of CTCs at regular intervals during the course of treatment.
( 1. Blood contains high levels of tumor-associated DNA, therefore, the MRD concept is mainly applied to hematologic malignancies.2. Because not all cancer patients can detect ctDNA in their blood, ctDNA testing has certain limitations, and ctDNA is mainly used to diagnose cancer by comparing the differences in DNA sequences, if a patient is infected with other viruses or microorganisms, then the body may also produce cfDNA, which will increase the cost of ctDNA testing and may also reduce the cost of the test.This will increase the cost of ctDNA testing to a certain extent, and may also decrease the accuracy of the test.
3. The current ctDNA test is not mature enough to be used in clinical practice.
4. ctDNA data in MRD are promising for testing in clinical applications, but most studies providing supporting evidence are small and limited in scope and need to be validated using large cohort studies. 5.No method for diagnosing early-stage cancer with ctDNA testing has been approved by the FDA. 6.The ability to release ctDNA into the plasma may vary from tumor to tumor, and therefore insufficient amounts of ctDNA may result in false negatives.7.Under the influence of tumor heterogeneity and drug selection, the monitoring points of recurrent tumors may disappear, thus affecting the application of MRD technology in the process of tumor monitoring.8. ctDNA MRD positivity at different stages may vary with increasing sample size.Therefore, if a limited number of samples are collected, the positivity rate will decrease.(193,194,(197)(198)(199)(200)(201)(202)(203)(204)(205) tumor agnosticism 1.When we perform MRD testing, the number of specific variants we focus on is very small due to the limited total number of gene copies in plasma samples, which may not accurately reflect tumor characteristics.
2. Since ctDNA is usually DNA fragments released by apoptotic or necrotic tumor cells, it is unknown whether these fragments specifically reflect the status of the tumor itself, and therefore, even less so for MRD detection.
3. Whether positive ctDNA-MRD results work to improve clinical outcomes or whether ctDNA-MRD can be used to more accurately guide adjuvant therapy is unknown.
1. Certain cancers may be missed.2. Tumor agnosticism lacks scientific basis to explain the mechanism of tumor occurrence and development.This viewpoint cannot provide effective guidance for the prevention and treatment of tumors.3. Neglecting individual differences: tumor agnosticism ignores the individual differences of each patient and is unable to formulate personalized treatment plans according to the patient's specific situation.This may lead to inaccuracy and insufficiency of treatment and affect the treatment effect.4. Lack of effective treatments: Since tumor agnosticism believes that tumors are unknowable, effective treatments cannot be found.This may lead to patients missing the best time for treatment, aggravating their conditions and even endangering their lives.
(205, 206) enrichment approaches to increase the reliability of CTC detection (23).Secondly, it is not possible to determine the appropriate CTC detection time.The morphological, genetic, and functional properties of CTCs differ due to various timeframes and treatment modalities, which impacts their therapeutic application (17).Therefore, the monitoring method for CTC is significantly challenged by its rarity and heterogeneity, which limits its clinical application to a certain extent.In addition, there are unanswered questions in CTC biology, unresolved concerns regarding the application of CTC biology in clinical settings (227) and so on.as a possible biomarker for cancer detection.However, their clinical limits are also impacted by the number of EVs, separation tools, and purity (210).Even though researchers from both home and abroad have made some headway in exploring different methods for separation and purification, EV research and application are still constrained by cost and efficiency; as a result, developing a trustworthy EV extraction method will be advantageous for the clinical application of EVs (211).
In conclusion, a successful combination of these technologies may be useful for tumor research, notably for in-depth analysis and possible therapeutic applications.This is true despite the advantages and disadvantages of each technology.Blood is one of the windows for studying health and diseases.If the information in organs and blood is transformed into biological data and linked to the health trajectory of the human body, it will greatly benefit human health.Leroy E. Hood's team found metastatic biomarkers of breast cancer, pancreatic cancer, lung cancer and hematopoietic system malignancies from the proteomic changes of plasma samples of health project participants, which advanced the diagnosis of metastatic cancer by more than 10 months (228).He has always been a proposer and practitioner of 4P medicine, distinguished by participative, personalized preventative, and predictive medicine.The core premise of 4P medicine is to identify early markers of disease transformation (229).Many of these biomarker techniques, including liquid biopsy as represented by CTCs (214), are now being researched、ctDNA (5) and EVs (65).Tumor formation and incidence are complex processes, and the underlying laws are not fully understood.One significant example is the early detection of cancers, which allows localized treatment choices to eradicate primary cancer and improves survival while lowering the likelihood of recurrence.Whether it is tissue biopsy or liquid biopsy represented by CTC, ctDNA, and EVs, it is a way to gain a deeper knowledge and understanding of tumors.Suppose these methods are integrated according to the concept of integrative medicine, and comprehensive detection and integrated analysis are carried out as far as possible when conditions permit the development of a more targeted treatment plan.In that case, it may help patients benefit from the survival time and quality of life.
Fast detection speed; Simultaneous multi-channel detection; High sensitivity as it can detect small changes on individual cells; High specificity as it can recognize and differentiate between specific cell types by labeling cells with specific antibodies or fluorescent dyes.The staining and labeling of cells using the principle of antibody specific binding to cell surface antigens has a high degree of specificity.The detection of multiple markers of CTCs can be achieved through different antibody combinations and staining methods with high sensitivity, enabling more comprehensive identification and analysis of CTCs.

TABLE 1
Comparison of Methods for Separation of CTCs.

TABLE 3
Comparison of ctDNA detection methods.
Detection and analysis of ctDNA by deep sequencing can detect low concentrations of ctDNA with high sensitivity.Through specific primer and probe design, tumorassociated variants can be accurately detected, avoiding interference with non-target cells and improving the specificity of detection.

TABLE 3 Continued
UnknownMutations in a single base can be detected, and even mutation frequencies below 1% can be detected with extremely high sensitivity and resolution.Through genome-wide sequencing, the sequence information of each gene can be accurately determined, including mutations, insertions, deletions, etc., with high specificity.
With high sensitivity and specificity, it can detect small differences in exosomes; through massively parallel sequencing, molecular features such as gene mutations, fusions and methylation in exosomes can be detected, so as to identify and categorize exosomes; for the detection of exosomal contents, specific experimental design and technical parameters are usually used.

TABLE 5
Comparison of miRNA,mRNA and protein detection methods in EVs.

TABLE 6
comparison of EVs separation methods.

TABLE 9
CTCs, ctDNA and EVs in diagnosis, prognosis and treatment monitoring of different diseases.

TABLE 8
Advantages and disadvantages of tumorigenic and tumor agnosticism.

TABLE 9 Continued
TABLE 10 Outlook of CTCs, ctDNA and EVs in tumor diagnostics in terms of cost, operability, simplicity and industrialization.
(209)ibuted in body fluids, and the standardization of detection techniques is relatively easy.However, like CTCs, tumor patients' ctDNA content is extremely low, making missed detection possible and necessitating highly sensitive detection technology(17).After DNA amplification, it is now possible to directly, qualitatively and quantitatively examine ctDNA using well-established detection techniques such as NGS and digital PCR owing to the rapid improvement of DNA sequencing technology.The therapeutic application value of ctDNA has to be further validated through many pertinent clinical trials and standardize the detection process(209).EVs have generated much attention in liquid biopsy of cancer