Combining multiple approaches and omics technologies to detect circulating tumor cells (CTCs): a real clinical application in patients with early-stage non-small-cell lung cancer (NSCLC)?

Lung cancer is the main cause of cancer-related death worldwide, with non-small cell lung cancer (NSCLC) accounting for 80%-85% of lung cancer^[1]. Unfortunately, most NSCLC patients are diagnosed at an advanced stage^[2], with an overall 5-year survival rate of only 20%-25% across all stages^[3]. To date, solitary pulmonary nodules can be further evaluated radiologically through second-level imaging techniques to assess key parameters such as lesion size and location^[4].

The National Lung Screening Trial, first conducted in the United States in 2011, demonstrated that the implementation of low-dose computed tomography (LDCT) screening significantly increased the detection rate of early-stage lung cancer, thereby reducing the proportion of patients diagnosed at advanced stages^[5]. Conventional diagnostic methods for lung cancer include chest X-ray, computed tomography (CT), bronchoscopy, and sputum cytology^[6].

Once diagnosed, treatment of NSCLC is often challenging due to high tumor heterogeneity, which promotes therapy resistance and contributes to poor prognosis; therefore, prevention and early detection are key points in effective cancer management. In this context, the 2010 TNM classification introduced a new stage, cM 0 (i+), where the "i +" designation indicates the absence of clinical or radiological evidence of distant metastases but the presence of circulating tumor cells (CTCs) detected microscopically in blood, bone marrow, or tissues outside the regional lymph nodes. These CTCs are typically smaller than 0.2 mm and are found in patients without overt signs or symptoms of metastasis^[7].

CTCs were first described in 1869 by pathologist Thomas Ashworth, who identified them in the bloodstream of a deceased cancer patient. CTCs are rare in peripheral blood (occurring at a frequency of approximately 1 per 10^6-7 leukocytes), and may exist as single cells or in clusters known as circulating tumor microemboli, which can also contain immune cells and platelets^[8,9].

Building on this definition, Jiang et al. reported that blood CTC levels are significantly higher in advanced-stage NSCLC patients compared to those with early-stage disease^[10]. Therefore, in advanced or metastatic NSCLC, the detection of CTCs can serve as a valuable tool for dynamic monitoring of post-treatment response to distinguish patients at low or high risk of recurrence^[11].

However, Wan et al. found that CTCs can also be detected in early-stage (stage I) NSCLC patients (P < 0.01)^[12]. This raises the question: in the context of radiomics, could CTC detection be incorporated into NSCLC screening as a diagnostic and prognostic tool?

The multicenter cohort study AIR^[13] showed that CTC counting in patients with chronic obstructive pulmonary disease (COPD) can help differentiate malignant from benign nodular lesions identified on LDCT. Supporting this, Sun et al. integrated CTC counting using an automated fluorescence microscope with comparative imaging from 18F-FDG PET/CT^[14]. This approach employed multiple probes (CK8/18/19, EpCAM, Vimentin, and CD45) to quantify and subtype CTCs, illustrating how LDCT and t 18F-FDG PET/CT can be complemented by molecular diagnostics.

In early-stage NSCLC, is it worth considering whether the presence of CTCs in peripheral blood might bypass routine imaging and represent distant micrometastasis that typically remain undetectable until advanced stages? CTCs may exist in proliferative or quiescent states, and exhibit either epithelial or mesenchymal characteristics^[15]. Some also show cancer stem cell-like features, which are believed to contribute to tumor recurrence and metastasis due to their ability to survive in circulation and initiate new tumor growth^[16].

This biological heterogeneity, including the persistence of quiescent CTCs in various tissues even after tumor resection, presents significant challenges for researchers. Identifying biomarkers that can detect all CTC subtypes with high sensitivity and specificity remains difficult. Currently known serum tumor markers used for lung cancer diagnosis - such as carcinoembryonic antigen (CEA), cytokeratin fragment-19 (CYFRA21-1), and neuron-specific enolase (NSE) - often yield inconclusive results due to limited specificity^[17].

Tissue biopsy is currently the gold standard for cancer diagnosis^[18]. However, liquid biopsy is emerging as a minimally invasive and equally valid alternative that may be considered for future guideline inclusion. IN patients with advanced NSCLC, several liquid biopsy markers derived from peripheral blood have been reported in the literature^[19]. The most commonly used markers for dynamic and post-treatment monitoring include circulating tumor DNA (ctDNA), CTCs, microRNAs (miRNAs) and extracellular vesicles (EVs). The scope of liquid biopsy has recently expanded to other physiological fluids, such as cerebrospinal fluid, pleural fluid, urine, sputum, and saliva^[20].

While CTC count is an established prognostic tool in certain cancers, including breast, prostate, and colorectal cancer, the prognostic value of CTCs in lung cancer remains uncertain^[21]. CTCs are released from primary or metastatic lung tumors and enter the bloodstream. Based on the hypothesis that early hematogenous dissemination of CTCs may reflect the risk of disease progression, advanced disease, or overall prognosis, here we focus on the current clinical utility of CTC detection in patients with early-stage NSCLC - a setting in which no standardized detection method is currently available.

Previous studies have investigated CTC detection in cancer patients, noting their short plasma half-life (1-2.4 h)^[22]. Many authoritative articles have comprehensively reviewed existing CTC detection techniques [Table 1]^[23-26]. Notably, Tanaka et al. first used the CellSearch system to detect CTCs in patients with suspected or confirmed early-stage lung cancer^[27]. However, the system demonstrated limited specificity in distinguishing malignant from non-malignant conditions.

The CellSearch® CTC test (Menarini-Silicon Biosystems, Bologna, Italy) is currently the only FDA-validated method for the detection of CTCs in the blood of patients with breast, colorectal, and prostate cancer^[28,29]. Although the CellSearch® system is known to effectively detect CTCs^[30], its performance is not always reliable in NSCLC due to the frequent downregulation of epithelial markers, such as EpCAM, and the phenotypic heterogeneity of NSCLC CTCs^[31]. These limitations make it challenging to develop a universally sensitive and specific assay for NSCLC that meets FDA validation standards, unlike in breast, colorectal, and prostate cancers^[32].

Rossi et al. conducted a pilot study using a novel morphological measurement approach to detect viable CTCs from pleural lavage samples in individual NSCLC patients^[33]. The samples were processed using the CellSearch system. Single-cell light scattering analysis served as the basis for a machine learning (ML) model to characterize the biophysical properties of individual CTCs. Single-cell techniques^[34], including mass spectrometry (MS) analysis, preserve the original biological properties of isolated CTCs. However, the rarity of these cells and their weak signal intensity pose challenges for analysis^[35].

Briefly, the CellSearch® system is an antigen-based isolation method. In the initial step, EpCAM⁺CTCs are enriched using immunomagnetic beads conjugated with anti-EpCAM antibodies. This is followed by fluorescent labeling with anti-cytokeratin (CK) antibodies and anti-CD45-allophycocyanin antibodies to exclude leukocytes^[36]. The immunomagnetically labeled cells are retained in a magnetic field and analyzed using the CellTracks Analyzer II® (Menarini © Silicon Biosystems)^[37].

CTCs can be isolated from blood or pleural effusion relatively easily^[38]. Most CTC isolation technologies rely on the detection of epithelial cell surface markers (EpCAM). However, mesenchymal markers - like vimentin - are also expressed by white blood cells, complicating detection. This may limit the ability to identify heterogeneous CTCs, particularly those with stem-like phenotypes or those in varying cellular states (dormant vs proliferative).

According to He et al., a dual-label approach using anti-EpCAM and anti-oncofetal chondroitin sulfate (ofCS) rVAR2-coated magnetic nanoparticles (NC@silica-SA) could provide a more sensitive method for identifying CTCs in the blood of NSCLC patients, employing the FETCH separation system^[39]. Previously, Salanti et al. demonstrated the specificity of ofCS, the target of rVAR2, which is absent in healthy lung tissue but present in tumor tissue^[40].

Sun et al. reported the presence of CD45⁺EpCAM⁺ cells in tumor tissues and malignant pleural fluid of NSCLC patients^[41]. Additionally, the proportion of CD45⁺EpCAM⁺ cells in the peripheral blood mononuclear cells (PBMCs) of patients was significantly higher than in healthy volunteers (patients: 0.244 ± 0.353 vs. healthy volunteers: 0.012 ± 0.004). Combining the detection of CD45⁺EpCAM⁺ cells in circulation with common serum markers such as CEA and CYFRA21-1 may enhance early-stage NSCLC screening.

Despite the variety of available CTC detection technologies, differences in sensitivity and specificity often hinder the ability to obtain adequate sampling volumes of CTCs^[42,43]. Microfluidic devices can rapidly process large blood volumes, making them suitable for detecting rare CTCs. These devices reduce the volume of blood required for analysis, lower reagent costs, and can be designed to be portable and automated. Furthermore, combining different microfluidic techniques, such as size-based separation with immunoaffinity capture, can improve both capture efficiency and the purity of mesenchymal CTCs. Table 2 compares the pros and cons of Cellsearch with those of microfluidics.

As an example, Ye et al. designed a microfluidic chip featuring well-arranged micropillars within the channel^[44]. This device enables the separation of leukocytes from blood samples using CD45 antibodies, removal of smaller cells via a nanofiltration membrane, and classification of isolated CTCs through a fluorescence staining system. Furthermore, biomarker-independent strategies employing surface-charged superparamagnetic nanoprobes are emerging as effective tools for capturing diverse CTC subpopulations^[45-48].

Recently, surgical specimens of primary lung tumors have been used to establish lung cancer organoids (LCOs)^[49,50]. Several studies have also reported innovative methodologies for isolating CTCs to generate CTC-derived organoids^[51-55], as described by De Renzi et al. in small cell lung cancer (SCLC) patients^[56]. Hamilton et al. demonstrated that a high CTC count is significantly associated with poorer prognosis in SCLC^[57]. These 3D patient-derived organoids replicate the heterogeneity and structural complexity of tumors - including intratumoral diversity and interactions with the microenvironment - and, importantly, can be expanded in vitro or in vivo in mice. However, only a few such studies have been conducted in NSCLC patients. A summary of key studies on CTC-derived organoids or xenografts in lung cancer is provided in Table 3.

In conclusion, NSCLC CTCs often evade detection by traditional epithelial markers such as EpCAM, due to their downregulation and phenotypic heterogeneity. This makes it challenging to develop a universally sensitive and specific assay that meets FDA validation standards, unlike those available for breast, colorectal, and prostate cancers.

We believe that relying on a single biomarker and/or a single detection technology may result in false-negative findings and does not align with the principles of personalized medicine. The lack of reliable blood-based biomarkers remains a major barrier to the diagnosis of NSCLC, in both early and advanced stages. Despite their potential, CTCs are not yet routinely used in clinical practice due to limitations such as low sensitivity, high cost, and a lack of standardization. To advance precision medicine in lung cancer, it is essential to invest in pilot studies that evaluate the real-world clinical application of CTCs in early-stage NSCLC patients. Additionally, retrospective studies involving cohorts of patients with both early- and advanced-stage disease could help move the field forward. Gaining a deeper understanding of the tumor microenvironment - before the onset of metastasis - may enable the development of multimodal diagnostic strategies for early-stage NSCLC.