Clinical applications of circulating tumor cell detection: challenges and strategies

Early screening of high-risk populations

Current clinical evidence suggests that cancer cells may have entered the bloodstream before patients exhibit symptoms. As a result, many anticipate using CTCs for screening high-risk populations [12], 13]. Unfortunately, sufficient evidence-based medicine currently does not support this application. Although individual case reports hint at the potential value of CTCs in this context [14], to truly implement them in clinical practice, apart from requiring long-term follow-up studies on large cohorts to obtain reliable, evidence-based medicine, there is an urgent need for more sensitive detection technologies to emerge.

Accurate staging of confirmed patients

The current clinical routine TNM staging system primarily evaluates the status of the primary tumor (Tumor, T), lymph nodes (Node, N), and distant metastasis (Metastasis, M) in cancer patients, providing significant guidance for treatment. However, according to reports in the literature, the detection rate of CTCs in early breast cancer, as determined by conventional pathology and imaging methods, ranges from 2 to 55 % [15]. This data suggests that even without evidence of distant metastasis, many patients already risk metastasis, making early treatment strategies potentially inappropriate. Considering this scenario, the American Joint Committee on Cancer (AJCC) introduced a new category, cM0 (i+), between MO (no distant metastasis) and Ml (distant metastasis) in its 2010 Cancer Staging Manual, marking the first time such a distinction was made in the TNM classification system, thus making the treatment more precise for patients [16].

Monitoring of postoperative recurrence and metastasis in early-stage patients

Even after undergoing adjuvant therapy post-surgery, 20–30 % of early-stage patients may experience recurrence and metastasis within years [17]. As the disease progresses in stages, if dynamic monitoring were initiated immediately following patients’ completion of their post-surgical adjuvant therapy, it might be possible to detect signs of recurrence and metastasis before they become visually apparent, allowing for earlier intervention and more control over treatment. Although there is currently a lack of evidence-based medicine supporting this approach, the potential application of CTCs in this phase is expected to expand significantly as future detection technologies improve.

Prognostic judgment of patients with advanced disease

For patients with advanced disease, the treatment strategy primarily focuses on palliative care, with greater emphasis on improving the quality of life for the patient. At this stage, CTC detection allows for more precise prognosis assessment, aiding personalized treatment, making it one of the most extensively studied areas in current CTC application research [18], [19], [20], [21], [22]. The most influential study was the prospective, multicenter registry clinical trial conducted by CellSearch system in the United States [22], which established a threshold of 5 CTCs per 7.5 mL in breast cancer and confirmed that CTCs are an independent predictor of both progression-free survival and overall survival. This work has been further validated in other breast cancer studies [17], 23] and has had a profound impact on CTC research in other cancer types, including colorectal cancer [24], [25], [26], prostate cancer [27], 28], non-small cell lung cancer [29], [30], [31], and small cell lung cancer [32], [33], [34], all of which have yielded similar findings.

Efficacy evaluation and efficacy prediction

Tumor markers and imaging tests are conventional methods for evaluating patient efficacy. However, the former has limited reference value due to individual patient differences. Moreover, the latter has a certain lag in time, typically requiring two treatment cycles (6–8 weeks) to reach a definitive conclusion. Previous literature reports have identified that the number of CTCs remains ≥5 after 1–2 treatment cycles as a standard for assessing whether a patient benefits from the treatment [35]. Compared to tumor markers, the evaluation of CTCs is more consistent with clinical outcomes, and compared to imaging, it provides more timely assessments, allowing for accurate evaluations by the end of the first course of treatment (3–4 weeks), which is particularly meaningful for patients undergoing expensive treatments [36], 37]. Although imaging methods remain the primary standard for evaluating patient efficacy, it is shown [38] that CTC can provide helpful corrections and supplements to traditional imaging evaluations [39], and the combination of the two can provide richer patient information, thus ensuring that patients can receive more rational treatment. Regarding the prognostic judgment and efficacy evaluation of advanced patients, a large amount of evidence-based medical evidence has been accumulated, which is the most classical clinical application of CTC [40], 41].

Late-stage patients undergo targeted therapy after the detection of relevant biomarkers to predict efficacy, such as EGFR mutations and ALK fusions in lung cancer [42], HER2 amplification in breast cancer [43], and KRAS mutations in colorectal cancer, among others [44]. In clinical practice, tumor tissue samples are the optimal source for these tests. However, late-stage patients may not always provide samples from the time of diagnosis or have enough material for testing, in which case CTCs can be a beneficial supplement. The aspect is that CTC reflects the patient’s real-time condition, and on the other hand, because CTC is the most dangerous cell in the patient’s body, treating these cells will bring more benefits to the patient. However, unlike histological testing, which has been repeatedly practiced and proven in clinical settings, the criteria for interpreting CTC tests must be better established and require validation and optimization through more clinical data.

In the realm of fundamental research, CTCs serve as a crucial material source for exploring pivotal issues such as the molecular mechanisms underlying epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) [36], 45], 46], the phenotypic characteristics and functional identification of circulating tumor stem cells [47], the elucidation of resistance mechanisms and drug sensitivity analysis [48], distinguishing markers for CTC dormancy and fate determination [49], the causes and action mechanisms of CTMs [50], and so forth. Addressing these questions will aid in our understanding of the specific roles of CTCs in tumor progression, facilitate the optimization and improvement of CTC detection methods, and provide essential foundations for future drug development or prevention strategies.

Higher enrichment efficiency

The subsequent information will be more comprehensive and trustworthy only by capturing as many complete populations of CTCs as possible. However, a significant proportion of late-stage patients cannot detect CTCs or have insufficient numbers for downstream analysis, indicating room for improvement in the enrichment efficiency of detection methods. Researchers are also attempting to omit the enrichment step, which requires more sensitive digital RT-PCR techniques or high-resolution image scanning technologies, which currently need to be more widely applied [59]. The factors influencing enrichment efficiency can vary; thus, the corresponding solutions may differ.

Firstly, as the most commonly used enrichment method, positive sorting relying solely on the EpCAM leads to suboptimal) enrichment efficiency. A significant reason for this lies in the occurrence of epithelial-to-mesenchymal transition (EMT) during tumor metastasis, where cells lose their epithelial characteristics and gain mesenchymal traits, making them prone to be overlooked due to reduced or absent expression of EpCAM [60]. In response to this problem, some techniques have attempted to use combinations of antibodies or employ CD45-negative sorting. Others have opted for non-affinity-independent methods that do not rely on antigen expression. However, the heterogeneity of CTC populations may exceed our expectations, and each enrichment method has limitations. As a result, a recent trend has been the combination of multiple enrichment approaches to complement each other’s strengths and further enhance efficiency, albeit at the potential cost of prolonged processing steps that might affect CTC viability. Strategies for enriching CTCs in the future will need a more comprehensive understanding of their biological properties [59].

Another possible reason for the inefficiency of enrichment could be rough handling. For instance, in the classic CellSearch system, the enrichment and fluorescent labeling steps occur within a 15 mL centrifuge tube, with repeated washing steps that inevitably lead to unnecessary cell loss. A more advanced microfluidic chip offers a promising solution to this problem. Typically, these chips are about the size of a standard microscope slide and can process 1–2 mL of blood samples. They have a reduced risk of sample loss and significantly lower reagent costs [61], 62].

Detecting low blood volume can also impact detection effectiveness, primarily due to the limited number of CTCs. Additionally, the volume of blood sampled is usually only about 0.1 % of the total blood volume in an average human, leading to sampling errors that affect comparability between batches. Increasing the blood volume for detection is not feasible as it would be difficult for patients to accept and would add significant burdens to subsequent processing, making it hard to enhance detection throughput. In this context, in vivo detection might be a potential solution, such as the CellCollector technology, which involves inserting EpCAM-coated metal wires into a patient’s elbow vein for CTC collection, resulting in a processed blood volume of up to 1,500 mL after 30 min, significantly exceeding the volume used in external detection, with a higher detection rate than traditional Cellsearch systems [63]. Moreover, animal experiments suggest that the number of CTCs obtained through continuous external circulation enrichment is notably higher than that from single-sample results [64], 65]. However, the safety and reliability of this operation in humans still require further validation.

Specialized validation strategies

The significant presence of leukocytes in peripheral blood, which remains high even after CTC enrichment, requires specific methods for CTC identification. Among the existing methods, phenotypic identification is an immediate test but may yield false positives and negatives; functional identification offers better specificity but is time-consuming, making it unsuitable for routine clinical application. Recently, researchers have proposed using a glucose uptake experiment to identify CTCs, which operate on a principle similar to PET-CT, distinguishing between benign and malignant cells based on metabolic differences. This method combines the immediacy of conventional phenotypic identification with the ability to reflect the functional characteristics of the target cells, suggesting promising future applications if supported by more clinical data [66]. This approach also suggests that the development of specific identification strategies needs to fully account for the biological properties of CTCs, which is one of the essential responsibilities of basic CTC research.

Automation and standardization

Automation and standardization primarily encompass sample processing and image scanning analysis. In the realm of sample processing, this involves the development and operation of automated equipment, locking in operational procedures and technical parameters (such as centrifugation speed, incubation time, and volume of samples added), standardizing reagents and consumables (including volume, concentration, antibody clone numbers, material, manufacturer), implementing comprehensive quality control and troubleshooting strategies, among others. These steps are essential to mitigate artificial factors and ensure the comparability of test results. Numerous new technologies are making strides in these areas with promising outcomes. However, the automated image scanning analysis system still needs to improve for many testing methodologies. Relying solely on manual operations and analyses is time-consuming and labor-intensive, often leading to missed detections. A reliable image scanning analysis system necessitates high-performance microscopes to enhance image quality and requires meticulous optimization and locking of Huorescently labeled antibody concentrations along with corresponding exposure times. It demands the development of accompanying software and algorithms for automatic focus adjustment of microscopes, precise location of target cells, intelligent recognition, and interpretation, among other tasks [59]. This area represents a significant challenge in the future development of CTC detection technology, requiring deep interdisciplinary integration across fields such as biology, optics, mechanics, software development, and artificial intelligence to achieve breakthroughs.

Capturing single cells

We are increasingly aware that CTCs constitute a heterogeneous population, with only a fraction of cells exhibiting stem cell phenotypes or metastatic proliferation potential. If the entire population is analyzed in a lump, the information from these cells could be lost. To prevent this, single-cell analysis techniques that can identify each cell’s characteristics are essential [67], 68]. Currently, single-cell sequencing technologies used in tumor tissues, such as those from IOX genomics, assign a unique “barcode” to each cell on a slide, enabling high-throughput detection of thousands of cells [69]. However, due to the relatively low abundance of CTCs, these technologies are unsuitable, partly because they are expensive per cell and partly because of the limited number of cells affecting their differentiation. For single-cell analysis of CTCs, the first step involves capturing and recovering individual cells, which can be achieved through manual aspiration under a microscope, microdissection on a slide, determining coordinates for target cells using “traps” or “microhooks,” or sorting cells into liquid flows or single-cell microparticles based on fluorescence or charge signals. After obtaining the single cells, whole-genome amplification (WGA) is required, commonly performed using multiple displacement amplification or multiple annealing and looping-based amplification cycles [70]. In summary, the technical barriers for CTC single-cell analysis are high, typically requiring high-performance microscopes for cell confirmation, which is time-consuming and labor-intensive and does not quickly increase detection throughput. Currently, it is more commonly used in primary research fields.

Cultivation of CTCs in vitro

The in vitro culture of CTCs is a crucial approach for their functional analysis, which is essential for studying tumor metastasis and resistance mechanisms [48]. The outcomes of such studies can, in turn, drive improvements and enhancements to CTC detection technologies. However, establishing in vitro culture is complex. Even human primary tumors can only be propagated continuously in cell lines in rare instances. For CTCs, this challenge is even more pronounced because the establishment of in vitro culture for CTCs must meet two non-negotiable prerequisites simultaneously. Firstly, there needs to be appropriate technology capable of enriching a sufficient number of viable CTCs, with previous literature reporting successful in vitro cultures of CTCs at counts of 1,109 and 509 per 7.5 mL of peripheral blood [71]. Many existing CTC detection technologies fall short of achieving this. Secondly, a suitable culturing condition must be created that effectively stimulates and supports the growth of CTCs in vitro. To date, however, we lack the necessary understanding of the specific details of these culturing conditions.

For the first criterion, the most contradictory aspect is that the best methods for capturing CTCs either require cell fixation or expose cells to high shear forces, both of which reduce cell viability; conversely, the methods that best preserve cell viability often do not perform well in terms of efficiency and effectiveness when enriching for CTCs. Balancing cell viability with effective capture of CTCs is thus a challenging issue that detection technologies for CTCs must confront. The second criterion typically involves adding growth factors to stimulate epithelial cell growth, using methods such as adherent culture, serum-free suspension culture, and co-culture with feeder cells. While these methods are feasible, overall success rates are generally low and heavily dependent on the number and viability of enriched CTCs [71]. Recent reports indicate that organoid culture can be used to culture CTCs, which can maintain long-term cell survival under conventional culture conditions with the addition of growth factors by adding unique extracellular matrix materials. This method could be a promising direction for future CTC culture approaches [72].

Evidence-based medicine and regulatory approval

The primary reason for the limited clinical application of CTC detection is the need for evidence-based medical data, making it difficult to gain regulatory approval. It includes systematic evaluations, meta-analyses, animal studies, observational studies, diagnostic test accuracy studies, case reports, and expert opinions. The only system that US and Chinese regulatory bodies have approved is the CellSearch system, not just due to its standardized operational procedures and high comparability of results but, more importantly, because it facilitates multicenter clinical research. These studies are meticulously designed with well-structured follow-up data, providing high-level evidence that significantly guides clinical practice. However, many new CTC detection technologies are deficient in clinical research, merely demonstrating the essential performance of related technologies through testing on specific patients, which is far from accurate clinical application.

Furthermore, more universally accepted criteria for real-time companion diagnostics using CTCs need to be established, and future efforts will require large-scale clinical studies to establish and continuously refine these standards [71]. Conducting these multicenter clinical studies is challenging and lengthy, posing a significant burden on any CTC technology development company. However, this challenge is inevitable for the entire industry as it is necessary to accumulate sufficient and reliable evidence-based medical data to achieve clinical acceptance.