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Circulating tumor cell

Circulating tumor cells (CTCs) are rare malignant cells that detach from primary tumors or metastatic sites, intravasate into the bloodstream or , and circulate throughout the body, serving as key mediators of cancer and systemic disease progression. First observed in , these cells represent a non-invasive "liquid " source for tumor profiling, though only a small fraction survive circulation to form distant metastases due to , immune surveillance, and anoikis. CTCs exhibit significant heterogeneity in and genotype, often undergoing to enhance invasiveness and stem-like properties, such as expression of and ALDH1, which correlate with poor in cancers like and . Biologically, CTCs interact dynamically with the blood microenvironment, including platelets and neutrophils that shield them from immune detection and promote survival, while clusters of CTCs—formed by homotypic or heterotypic aggregation—show increased metastatic potential compared to cells. Recent advances in -cell sequencing (scRNA-seq) have revealed CTC heterogeneity, identifying pathways like Wnt signaling and variants in CTCs, as well as oligoclonal origins in metastatic clusters. This molecular diversity enables CTCs to evade therapies and adapt to new niches, underscoring their role in tumor evolution. Detection of CTCs remains challenging due to their rarity (1–10 per mL of blood amid billions of hematopoietic cells), but methods have evolved from EpCAM-based immunomagnetic capture (e.g., FDA-approved CellSearch system) to label-free approaches like size-based , microfluidic , and charge-based nanoprobes exploiting the negative surface charge on cancer cells. High-throughput techniques, including diagnostic combined with , now yield thousands of CTCs from 1–2 L of blood, facilitating genomic and proteomic analysis. Clinically, elevated CTC counts (≥5 per 7.5 mL blood) predict worse survival in ( 6.25) and guide therapy monitoring, such as detecting EGFR mutations or PD-L1 expression for selection. These applications position CTCs as vital for early , , and precision across solid tumors.

Overview and Biology

Definition and Origin

Circulating tumor cells (CTCs) are defined as rare cancer cells that detach from primary tumors or metastatic sites, intravasate into the bloodstream, and circulate systemically, retaining the potential to extravasate and establish distant metastases. These cells represent a critical intermediary in the metastatic cascade, distinguishing them from stationary tumor masses by their motility and survival in the vascular environment. CTCs primarily originate from epithelial malignancies, such as carcinomas of , , and , where they arise through the epithelial-mesenchymal transition (), a process enabling tumor cells to lose and adhesion while gaining mesenchymal traits like invasiveness and resistance to . During EMT, epithelial markers such as E-cadherin are downregulated, and mesenchymal markers like and N-cadherin are upregulated, facilitating detachment from the tumor and entry into circulation. Although most extensively studied in epithelial cancers, CTCs can also derive from non-epithelial tumors, including sarcomas and melanomas, though detection in these contexts often requires alternative markers due to the absence of epithelial antigens. Unlike (ctDNA), which consists of acellular, fragmented DNA shed from apoptotic or necrotic tumor cells, CTCs are intact, nucleated cells capable of proliferation and , providing a more direct representation of viable tumor biology. Similarly, CTCs differ from tumor-derived exosomes, which are extracellular vesicles lacking cellular integrity and unable to form metastases independently.

Mechanisms of Dissemination

Circulating tumor cells (CTCs) disseminate from primary tumors through a multistep metastatic that involves detachment, entry into the bloodstream, survival during circulation, and eventual colonization of distant sites. This process begins with intravasation, where tumor cells breach the endothelial barrier to enter the vascular system, often facilitated by , which confers motility and invasiveness by downregulating epithelial markers like E-cadherin and upregulating mesenchymal ones such as . Intravasation can occur actively through EMT-driven invasion or passively via tumor-induced vascular damage, with the , including matrix metalloproteinases, playing a critical role in degradation. Once in circulation, CTCs face harsh conditions, representing less than 0.01% of cells and exhibiting a short of approximately 1-2 hours due to rapid clearance. To survive from flow, anoikis (detachment-induced ), and immune , CTCs undergo , acquiring a epithelial-mesenchymal that enhances stemness, plasticity, and resistance to . This plasticity allows evasion of immune clearance by downregulating antigens and expressing immunosuppressive molecules like PD-L1. CTCs also form protective interactions with components; platelets aggregate around them to shield against forces and killer cells, while neutrophils promote survival through like and adhesion molecules such as VCAM1. CTC clusters, comprising multiple cells, further bolster survival by reducing anoikis susceptibility compared to single CTCs. Extravasation marks the exit of surviving CTCs from circulation to potential metastatic sites, involving rolling, , and transmigration across the . is mediated by (e.g., α4β1, αVβ3) that bind components, while such as guide homing by binding on CTCs, promoting firm arrest on endothelial cells. Successful requires mesenchymal-epithelial transition (MET) to restore colonization potential, enabling CTCs to invade surrounding tissue. Prior to arrival, CTCs or tumor-derived exosomes can precondition distant organs by forming a pre-metastatic niche, recruiting bone marrow-derived cells and remodeling the to create a supportive environment for seeding.

Classification

Single CTCs

Single circulating tumor cells (CTCs) are classified into subtypes based on their phenotypic states, primarily reflecting the extent of epithelial-mesenchymal transition () they have undergone. These include epithelial CTCs, mesenchymal CTCs, and hybrid CTCs, each characterized by distinct marker expressions and biological properties. Epithelial CTCs retain epithelial characteristics, expressing markers such as EpCAM and E-cadherin, which maintain and are commonly detected using EpCAM-based assays. These cells exhibit lower invasiveness and metastatic potential compared to other subtypes, often predominating in early-stage cancers where EMT is minimal. However, their detection can be straightforward with standard epithelial markers, though challenges arise if partial EMT leads to marker downregulation. Mesenchymal CTCs, resulting from full EMT, express mesenchymal markers like vimentin and N-cadherin, conferring enhanced motility, invasiveness, and stem-like properties that correlate with higher metastatic aggressiveness. They are more prevalent in advanced-stage cancers, reflecting tumor progression and to circulatory . Detection of these cells is hindered by the loss of epithelial markers like EpCAM, necessitating alternative strategies beyond conventional methods. Hybrid CTCs display phenotypic plasticity by co-expressing both epithelial (e.g., EpCAM, E-cadherin) and mesenchymal (e.g., , N-cadherin) markers, enabling dynamic state transitions that enhance adaptability and colonization efficiency. This heterogeneity within single cells underscores their role in , as the intermediate state allows for reversible shifts between phenotypes, complicating isolation but amplifying their prognostic significance.

CTC Clusters

Circulating tumor cell (CTC) clusters are multicellular aggregates consisting of two or more tumor cells circulating in the bloodstream, typically ranging from 2 to 50 cells per cluster. These clusters form through homotypic adhesions between tumor cells, mediated by molecules such as cadherins and plakoglobin, or heterotypic interactions involving non-tumor cells like platelets that shield the aggregate from and immune detection. They originate from cohesive budding as oligoclonal groups directly from the . Compared to single CTCs, clusters exhibit markedly enhanced properties that promote , including a 23- to 50-fold increased metastatic potential relative to single CTCs in models, independent of tumor subtype. This heightened efficiency stems from resistance to , facilitated by cell-cell signaling pathways such as , which sustains cluster integrity and viability during dissemination. Clusters also display stem-like traits, characterized by hypomethylation of pluripotency genes like OCT4 and , elevated expression of , and enhanced self-renewal capacity, contributing to their aggressive behavior. CTC clusters are rare, comprising less than 5% of all detected CTCs in patient blood, with frequencies as low as 2-3% in prostate cancer. They are particularly enriched in breast and prostate cancers, where their presence correlates with advanced disease and poor prognosis. Unlike the genetic heterogeneity often observed in single CTCs, clusters tend to show greater homogeneity among constituent cells, enabling coordinated invasion and colonization. Additionally, clusters evade immune surveillance more effectively than single CTCs, through mechanisms such as platelet cloaking and upregulated PD-L1 expression, which suppress T-cell activity and foster an immunosuppressive niche.

Prevalence

Frequency in Circulation

Circulating tumor cells (CTCs) are present at extremely low frequencies in the bloodstream, making their detection challenging amid billions of normal blood cells. In healthy individuals, CTCs are typically absent, with detection rates near zero using standard assays. In patients with early-stage, localized cancers, CTC counts are generally below 1 per mL of blood, often detectable in only 20-25% of cases when using a threshold of more than 1 CTC per 7.5 mL. In contrast, metastatic cancer patients exhibit higher frequencies, ranging from 1 to 10 CTCs per mL of peripheral blood, though counts can vary widely from 0 to over 23,000 per 7.5 mL in some samples. CTC frequencies are commonly measured in CTCs per mL of or standardized volumes like 7.5 mL, as processed by commercial systems such as CellSearch. Thresholds for positivity vary by context and assay; for instance, the CellSearch system defines unfavorable prognosis in metastatic , , or at ≥5 CTCs per 7.5 mL of blood. These counts are reported relative to the total nucleated cell population in blood, where CTCs represent approximately 1 per 10^5 to 10^7 peripheral blood mononuclear cells in metastatic settings, underscoring their rarity against millions of leukocytes per mL. Frequencies are notably higher in advanced metastatic disease compared to localized stages, reflecting increased tumor shedding. Temporal fluctuations influence CTC enumeration, including diurnal variations where counts peak during , potentially due to rhythmic hormonal changes like levels. Post-treatment, CTC numbers often decrease significantly, with rapid declines correlating to therapeutic response in various cancers. However, EpCAM-based enumeration methods, such as those in CellSearch, introduce biases by underdetecting CTCs that have undergone epithelial-to-mesenchymal transition and lost EpCAM expression, potentially leading to underestimation of true frequencies.

Factors Influencing Prevalence

The prevalence of circulating tumor cells (CTCs) varies significantly based on disease-specific characteristics, such as cancer type, stage, and tumor burden. In epithelial cancers like and malignancies, CTC detection rates are relatively high, often exceeding 50% in advanced cases, due to the tumors' propensity for vascular and . In contrast, primary tumors such as gliomas exhibit lower CTC prevalence compared to epithelial cancers, with detection rates varying widely (e.g., 20–80% across studies depending on the detection method), attributed in part to the blood-brain barrier limiting intravasation. CTC numbers increase with disease progression, particularly in metastatic stages, where from the primary site enhances shedding into the bloodstream. Similarly, higher tumor burden correlates with elevated CTC levels, as larger tumor volumes promote greater release of cells through disrupted vasculature and increased proliferative activity. Patient-specific factors also modulate CTC prevalence, including vascular access, , and regimens. CTC counts may be higher when blood is drawn from central venous sites compared to peripheral sites due to closer proximity to tumor , though the specific effects of insertion on CTC require further study. , often elevated in cancer patients due to tumor-associated cytokines or comorbidities like , facilitates CTC release by promoting endothelial permeability and interactions with immune cells such as neutrophils. cycles can dynamically alter CTC shedding; for instance, certain agents induce transient spikes in CTC numbers by damaging tumor vasculature, while repeated cycles may reduce prevalence through tumor debulking. Recent studies from 2025 highlight the role of epithelial-mesenchymal transition (EMT) status in elevating CTC prevalence, with EMT-positive CTCs showing increased intravasation efficiency and survival in circulation, leading to higher detectable numbers in patients with aggressive disease phenotypes. In , hormone-related differences further influence CTC levels, as receptor-positive tumors may exhibit lower CTC shedding compared to hormone receptor-negative subtypes, potentially due to hormonal modulation of and . Unique environmental and therapeutic factors, including circadian rhythms and anti-angiogenic treatments, also affect CTC prevalence. Circadian rhythms regulate CTC shedding, with peak dissemination often occurring during rest phases at night, driven by fluctuations in hormones like and glucocorticoids that influence and tumor cell motility. Anti-angiogenic therapies, such as , reduce CTC intravasation by normalizing tumor vasculature and limiting hypoxia-induced cell release, thereby decreasing overall prevalence despite potential increases in single-cell survival.

Detection and Isolation

Biological Methods

Biological methods for circulating tumor cell (CTC) detection primarily rely on immunoaffinity principles, which exploit the binding of antibodies to specific surface markers expressed on CTCs, such as (EpCAM) and cytokeratins, or tumor-specific antigens like HER2 and MUC1. These techniques enable the selective capture and enrichment of CTCs from blood samples by leveraging antigen-antibody interactions, often combined with downstream verification steps like or molecular assays. Immunoaffinity approaches are categorized into positive selection, which directly targets CTCs using antibodies against epithelial or tumor markers, and negative depletion, which removes non-target cells such as leukocytes via antibodies against CD45 to enrich the CTC fraction indirectly. Positive selection is widely favored for its specificity in epithelial-origin tumors, though negative depletion can capture a broader range of CTC phenotypes. The CellSearch system represents a cornerstone FDA-approved immunoaffinity platform for CTC enumeration, utilizing coated with anti-EpCAM antibodies to capture CTCs from 7.5 mL of blood, followed by and staining for cytokeratins (positive), CD45 (negative), and (nuclear). This method achieves a detection sensitivity of approximately 70% in patients with , where CTC counts of five or more per 7.5 mL correlate with poorer . However, its reliance on EpCAM limits detection of epithelial-to-mesenchymal transition ()-CTCs, which downregulate EpCAM during dissemination, potentially underestimating CTC burden by missing mesenchymal-like cells. The AdnaTest complements such platforms by employing immunomagnetic beads coated with antibodies against EpCAM and MUC1 for positive selection, followed by mRNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) to detect tumor-associated transcripts like GA733-2, MUC-1, and HER2. This approach enhances molecular characterization post-capture, with studies showing improved CTC detection rates in metastatic when combined with CellSearch. Limitations across these biological methods, particularly EpCAM dependency, have prompted 2025 advancements in multi-marker panels that incorporate antibodies against additional targets such as and to better detect heterogeneous CTC populations undergoing . These panels aim to overcome marker loss, improving overall capture efficiency in diverse cancers like and .

Physical Methods

Physical methods for isolating circulating tumor cells (CTCs) exploit inherent biophysical differences between CTCs and surrounding components, such as , , deformability, and electrical properties, without relying on specific biomarkers. CTCs generally exhibit larger diameters, ranging from 12 to 40 μm, compared to leukocytes (typically under 12 μm) and erythrocytes (7-8.5 μm), enabling size-based separation. differences allow CTCs, which often have densities between 1.045 and 1.070 g/mL, to be distinguished from other cells during . Electrical properties, including higher membrane in CTCs, further facilitate label-free enrichment through dielectrophoretic forces. These approaches offer broad capture of heterogeneous CTC populations, including those undergoing epithelial-mesenchymal transition (), but often achieve lower specificity than biological methods due to potential co-isolation of non-target cells like activated leukocytes. Density gradient centrifugation represents one of the earliest and simplest physical techniques, utilizing media like Ficoll-Paque (density ~1.077 g/mL) to separate mononuclear cells, including CTCs, from denser erythrocytes and granulocytes. This method achieves substantial enrichment, with OncoQuick—a commercial variant—demonstrating up to 632-fold leukocyte depletion in blood samples spiked with tumor cells. However, it requires subsequent steps for CTC and may lose smaller CTCs. Size-based builds on this by passing blood through membranes with micropores tailored to CTC dimensions, such as the 6.5-8 μm pores in ScreenCell devices, which yield recovery rates of 74-91% for epithelial and mesenchymal CTCs from various cancers. The Isolation by Size of Epithelial Tumor cells (ISET) system, using 8 μm pores, similarly captures CTCs with high efficiency while minimizing cell damage, though it can clog with high samples. Microfluidic platforms enhance precision and throughput by leveraging hydrodynamic forces, such as inertial focusing, to align and separate cells based on size and deformability. The CTC-iChip, for instance, integrates inertial focusing with deterministic lateral displacement to process at rates up to 10^7 cells per second, achieving over 95% recovery of viable CTCs while depleting >99.99% of leukocytes. The Parsortix system employs microfluidic slits (6.5 μm gap) to capture CTCs via size and deformability, with FDA-cleared PC1 cassettes reporting harvest rates of 63.5-76.2% for and other lines, and the ability to process up to 80 mL of per run. This epitope-independent design excels at isolating mesenchymal CTCs, which may evade antigen-based capture. Image-based methods like Maintrac further refine physical selection by analyzing cell size and morphology in real-time via automated after , enabling non-destructive enumeration of vital CTCs with intact morphology in 100% of patient samples tested. These physical methods prioritize high throughput—often processing milliliter volumes rapidly—but trade off with purity, typically requiring downstream validation to distinguish CTCs from contaminants. A key advantage is their capacity to capture diverse CTC subtypes, including EMT-shifted cells resistant to targeted therapies. As of 2025, advances in acoustic separation have emerged, using acoustophoresis to exploit differences in acoustic contrast factors between CTCs and blood cells. In-line acoustofluidic devices achieve >98% recovery of cancer cells from undiluted while depleting ~90% of erythrocytes, offering gentle, scalable enrichment with 14-43-fold purity gains at normal levels, suitable for point-of-care applications.

Hybrid and Emerging Methods

Hybrid and emerging methods for circulating tumor cell (CTC) detection integrate biological and physical principles to achieve higher compared to single-modality approaches. These strategies often combine label-free physical separation, such as size, deformability, or electrical properties, with biological affinity capture or to enrich and identify CTCs while minimizing to viable cells. By leveraging , these hybrids reduce required blood volumes to as little as 1-5 mL, enabling frequent sampling in clinical settings. Epic Sciences employs a platform that begins with red blood cell and nucleated cell deposition on slides (physical enrichment), followed by immunofluorescent for epithelial markers and high-content automated to score CTCs without EpCAM bias. This method detects heterogeneous CTCs, including those undergoing epithelial-mesenchymal transition, with recovery rates exceeding 90% for spiked tumor cells in . Similarly, the Parsortix system uses size-based microfluidic separation (physical) to harvest CTCs, which are then immuno-stained for and to confirm identity, achieving capture efficiencies of 80-95% for cells larger than 8 μm while preserving viability for downstream . Dielectrophoresis (DEP)-based systems, such as ApoStream, exploit differences in cell dielectric properties for continuous-flow, label-free (physical), often combined with post-enrichment (biological) to verify CTCs, yielding purities up to 50% and viabilities over 80% without surface marker dependency. Nano-Velcro chips integrate nanostructured silicon substrates coated with anti-EpCAM antibodies, enabling high-affinity capture (biological) enhanced by chaotic mixing in (physical), with release via temperature-sensitive polymers for intact CTC recovery at efficiencies above 90%. Recent advancements include AI-enhanced imaging on platforms like RareCyte, where algorithms analyze multi-parameter fluorescent slides to automate CTC identification and improve consistency in detecting rare events down to 1 CTC/mL. detection via photoacoustic methods, such as photoacoustic , uses laser-induced acoustic signals from in CTCs for label-free, real-time monitoring in circulation without blood draw. Additionally, isolated CTCs are increasingly cultured as organoids to propagate patient-specific models with varying success rates across cancer types, facilitating drug screening and studies.

Characterization

Morphological Analysis

Morphological analysis of circulating tumor cells (CTCs) entails the microscopic examination of their physical structure and appearance following , serving to verify their tumor origin and evaluate structural attributes that distinguish them from normal blood cells. This process relies on visual criteria to identify CTCs, often integrating protocols to highlight cellular components and exclude contaminants. Common techniques involve for basic size and shape assessment, complemented by immunofluorescence microscopy to visualize detailed features like the nucleus-cytoplasm ratio. typically employs to label nuclear DNA, cytokeratins to indicate epithelial characteristics, and CD45 to mark and exclude leukocytes, enabling clear differentiation of CTCs as cytokeratin-positive, -positive, and CD45-negative cells. CTCs generally exhibit distinct morphological traits, including larger diameters typically ranging from 9 to 19 μm, exceeding most (6-12 μm), irregular nuclear contours, and instances of multi-nucleation indicative of abnormal division. In CTC clusters, structural variations appear as compact, tightly adhered formations or looser aggregates with less defined cell-cell junctions, reflecting differences in intercellular cohesion. A substantial proportion of CTCs in circulation display apoptotic features, such as condensed or fragmented nuclei, due to the stressful vascular that promotes rapid clearance of most CTCs, often within minutes to hours via and anoikis. Standard exclusion criteria for confirming CTCs include CD45 negativity to eliminate leukocytes and the absence of other hematopoietic markers, ensuring analysis focuses on non-blood cells. Viability assessment incorporates dyes like or propidium iodide, which penetrate compromised membranes to identify dead or dying cells and quantify live CTC fractions. As of 2025, tools have emerged for automated morphological scoring, leveraging image analysis to quantify CTC size, shape irregularity, and nuclear features with enhanced precision and reduced manual variability. These morphological evaluations are often supported by molecular markers for robust CTC confirmation.

Molecular Profiling

Molecular profiling of circulating tumor cells (CTCs) involves advanced techniques to interrogate their genetic, transcriptomic, and proteomic landscapes at the single-cell level, providing insights into tumor heterogeneity, , and therapeutic vulnerabilities. These analyses reveal how CTCs, as components of biopsies, can noninvasively capture dynamic molecular changes that may not be evident in biopsies. For instance, often begins after morphological confirmation of CTC identity to ensure targeted interrogation of viable tumor cells. Key techniques include single-cell RNA sequencing (scRNA-seq), which enables high-resolution transcriptomic analysis of individual CTCs to uncover patterns associated with and . Seminal studies have demonstrated scRNA-seq's utility in identifying gene expression in pancreatic CTCs, highlighting their mesenchymal features. (FISH) is widely used for detecting specific amplifications, such as HER2 and in and CTCs, allowing real-time assessment of targetable alterations. Whole-genome sequencing (WGS) of single CTCs detects copy number variations and mutations with high fidelity, as shown in low-pass WGS approaches that accurately profile CTCs from blood and non-blood body fluids. Proteomic profiling via , including , characterizes surface and intracellular proteins in low-abundance CTCs, identifying subgroups based on multiplexed markers. A prominent aspect of CTC molecular profiling is intratumoral heterogeneity, exemplified by variable PIK3CA mutations in breast cancer CTCs, where single-cell analysis reveals discordant statuses across cells from the same patient, indicating evolutionary divergence. Epithelial-mesenchymal transition (EMT) signatures, including elevated TWIST1 and ZEB1 expression, are frequently observed in CTCs, promoting survival in circulation and correlating with stem-like properties. Drug resistance markers, such as efflux pumps (e.g., ABC transporters), are enriched in CTCs, contributing to chemotherapy evasion and underscoring their role in acquired resistance. These profiles often correlate with primary tumor evolution, as CTC mutations and expression patterns recapitulate metastatic progression and diverge from the originating lesion over time. As a liquid biopsy modality, CTC molecular holds potential for real-time monitoring of tumor dynamics, enabling personalized therapy adjustments without invasive procedures. However, challenges persist due to low CTC yields, necessitating whole-genome amplification that can introduce biases and artifacts in downstream analyses. Recent 2025 advances in have enhanced sensitivity for CTCs, paving the way for integrated spatial approaches to map heterogeneity , though adaptation to rare CTCs remains technically demanding. Recent 2025 studies integrating scRNA-seq with have further elucidated CTC heterogeneity in , identifying novel resistance mechanisms as of October 2025.

Clinical Applications

Prognostic and Diagnostic Value

Circulating tumor cells (CTCs) serve as a key prognostic in various cancers, with elevated counts at baseline associated with poorer clinical outcomes. In , a threshold of ≥5 CTCs per 7.5 of is linked to significantly reduced overall survival (median 16.0 months versus 36.3 months for <5 CTCs) and . Similarly, in metastatic hormone-sensitive , baseline CTC counts of ≥5 per 7.5 correlate with worse overall survival (27.9 months) compared to 0 CTCs (not reached at 78 months follow-up), with a of 3.22. These associations hold independently of other factors like nodal status, establishing CTC as a strong predictor of progression and mortality. CTC clusters, multicellular aggregates detected in circulation, further amplify prognostic risk by enhancing metastatic potential. In and cancers, clusters exhibit 20- to 100-fold greater ability to form metastases than single CTCs, and their presence in is tied to inferior overall and beyond single CTC counts. The FDA-cleared CellSearch system, approved in 2004 for enumerating CTCs of epithelial origin, uses a ≥5 CTCs per 7.5 mL threshold to predict shorter progression-free and overall survival in patients with metastatic , , and colorectal cancers. Meta-analyses reinforce this, with a pooled analysis of 2,436 metastatic cancer patients confirming ≥5 CTCs per 7.5 mL as an independent prognostic factor for recurrence and death, tripling the risk. Diagnostically, CTCs enable non-invasive assessment of and early detection, often outperforming traditional in for disseminated disease. In early-stage (stages I-IIIA), CTC detection rates range from 20% to 26.7%, correlating with tumor aggressiveness and aiding in identifying patients at risk for before clinical symptoms arise. For , CTC positivity in localized disease exceeds 50% and predicts outcomes with an area under the curve of 0.811, supporting its role in refining staging accuracy. In , CTCs distinguish from other conditions with 75% and 96.3% specificity ( 0.867), providing a liquid alternative to invasive procedures. Thresholds for prognostic significance vary by cancer type, reflecting differences in tumor and patterns. For instance, ≥1 CTC per 7.5 mL in early yields a hazard ratio of 2.55 for death, escalating to 5.20 at ≥5 CTCs, while in non-small cell , ≥5 CTCs per mL predicts a fourfold higher mortality risk. Dynamic baseline monitoring of CTC levels can predict recurrence up to several months in advance; in post-surgery, persistent CTC detection signals relapse risk 7-9 weeks prior to confirmation. Molecular markers on CTCs, such as EpCAM expression, can enhance these predictions when integrated with .

Therapeutic Monitoring and Targeting

Circulating tumor cells (CTCs) serve as dynamic biomarkers for assessing treatment efficacy in various cancers, with serial enumeration revealing responses to therapies such as . A decline in CTC counts following administration often indicates a positive therapeutic response, while an increase or persistence signals emerging resistance, as observed in where pre-treatment CTC levels exceeding 5 per 7.5 mL blood correlate with poorer outcomes and reduced . In , elevated CTCs expressing plastin 3 or 1 (ALDH1) post-treatment predict chemoresistance and shorter survival. This monitoring approach enables real-time adjustments to treatment regimens, distinguishing it from baseline prognostic assessments by focusing on longitudinal changes during active therapy. CTCs also play a critical role in detecting (MRD) after primary treatment, identifying aggressive subclones that may lead to relapse before clinical detection. In , persistent CTCs post-therapy have been linked to recurrence within 4-11 months, providing an early window for intervention. Combining CTC analysis with (ctDNA) in liquid biopsies enhances monitoring, as seen in where CTC counts and ctDNA mutations track immune inhibitor efficacy, with reductions indicating sustained responses. Recent 2025 studies in non-small cell demonstrate that PD-L1-expressing CTCs, alongside ctDNA, predict outcomes, supporting personalized dosing adjustments based on molecular resistance markers like ESR1 mutations. Targeting CTCs directly addresses their metastatic potential through therapies exploiting specific vulnerabilities. Anti-epidermal growth factor receptor () inhibitors target EpCAM- and HER2-positive CTCs, reducing survival and dissemination in preclinical models of and cancers. Cluster-disrupting agents, such as inhibitors like , prevent E-cadherin-mediated CTC aggregation, which enhances metastatic efficiency; a phase I (NCT03928210) in advanced evaluates 's ability to lower CTC clusters and risk. In 2025 trials for , CTC-derived models guide personalized dosing by testing drug sensitivity, with successful cultures from over 400 CTCs informing cisplatin responsiveness in head and neck . Challenges in viable CTC isolation limit their utility for ex vivo drug screening, primarily due to their rarity (often fewer than 10 per mL blood) and heterogeneity, which complicates culture establishment and clonal expansion. Short-term cultures are achievable for drug susceptibility testing, but long-term viability remains inconsistent across cancer types, often requiring high CTC yields and optimized media to avoid adaptation artifacts. Advances like microfluidic chips (e.g., MyCTC) enable and screening from liquid biopsies, yet low success rates underscore the need for refined protocols to support personalized therapeutic decisions.

History and Advances

Historical Milestones

The concept of circulating tumor cells (CTCs) dates back to 1869, when physician Thomas Ashworth first observed cells resembling those from a in the blood of a who died from metastatic cancer, suggesting that such cells could disseminate through the bloodstream to form distant metastases. This pioneering microscopic examination laid the groundwork for understanding CTCs as potential metastatic precursors, though the observation remained anecdotal for decades. In the 1950s, research advanced through animal models demonstrating via circulating cells; for instance, experiments injecting tumor cells into animal bloodstreams showed that clusters of these cells had enhanced metastatic potential compared to single cells. By the , isolation techniques improved with the introduction of immunohistochemical methods, enabling more reliable detection of CTCs based on specific markers like cytokeratins, which correlated their presence with metastatic disease progression. The 1990s marked a shift toward molecular detection, with the development of EpCAM-based methods, such as immunobead-PCR using anti-EpCAM antibodies, allowing sensitive enrichment and identification of epithelial CTCs in samples. This era transitioned from purely morphological assessments to targeted capture strategies, improving specificity. A major milestone occurred in 2004 when the CellSearch system, an automated platform combining EpCAM immunomagnetic capture with fluorescent enumeration of CTCs (defined as EpCAM+, CK8/18/19+, CD45-), received FDA clearance for prognostic use in patients. In the 2010s, discoveries included the identification of CTC clusters as highly metastatic entities; for example, a 2014 study demonstrated that CTC clusters, originating from oligoclonal primary tumor cell groups, exhibited up to 100-fold greater metastatic potential than single CTCs due to enhanced survival and colonization abilities. Additionally, the first genomic sequencing of CTCs from patients in 2011 revealed actionable mutations, such as AR amplifications, enabling insights into tumor and resistance mechanisms without invasive biopsies. These advances underscored the from morphological to molecular and genomic of CTCs, facilitating their role in precision oncology.

Recent Developments and Challenges

Recent advancements in circulating tumor cell (CTC) research have leveraged (AI) and (ML) to improve detection accuracy and efficiency. Platforms developed since 2023 integrate algorithms for automated CTC identification from microfluidic devices and imaging data, enabling analysis of CTC morphology, clonal diversity, and epithelial-mesenchymal transition () markers with enhanced sensitivity. For instance, AI-driven clustering has facilitated real-time profiling of CTC heterogeneity, supporting predictive modeling for therapeutic responses in cancers such as and . CTC-derived organoids have emerged as powerful tools for disease modeling and personalized medicine, with notable progress in recent studies. These three-dimensional cultures, generated from isolated CTCs, recapitulate tumor heterogeneity and metastatic potential, with success rates for long-term establishment around 35% as reported in 2025 reviews; earlier work in 2021 using nanoemulsion enhancements achieved 75% success in short-term ex vivo CTC cultures lasting over 23 days in breast cancer samples. Applications include drug sensitivity screening, such as evaluating Survivin inhibitors in colorectal cancer organoids, and investigating EMT mechanisms in breast and renal cancers. Multi-omics integration has advanced CTC characterization by combining , transcriptomics, and to uncover survival mechanisms and therapeutic targets. Single-cell RNA sequencing of CTCs has revealed distinct mutation profiles, such as and variants, differing from primary tumors, while transcriptomic analyses highlight metastasis-related genes like BIRC5 in . These approaches enable comprehensive profiling of CTC heterogeneity, informing precision therapies despite challenges in low-yield and . In 2025, imaging techniques have progressed through deep multiplexed marker profiling and , allowing non-invasive, real-time CTC detection in preclinical models at sensitivities of 1 CTC per 10^6 cells. Hybrid liquid biopsies combining CTCs with (ctDNA) have shown promise in gastrointestinal cancers, where integrated analyses predict and treatment responses, such as ctDNA clearance correlating with improved survival in . Progress in mesenchymal CTC capture has been marked by polymeric microfluidic devices like the CTC-Chip, which target markers to enrich EMT-phenotype cells, increasing yield in samples. Despite these innovations, significant challenges persist in CTC research. Standardization remains elusive due to variability in CTC definitions (e.g., CD45-/cytokeratin+ criteria) and enrichment methods, complicating inter-laboratory comparisons and clinical validation. Low CTC viability, with half-lives of seconds to minutes under circulatory stress, limits ex vivo culture success to under 1% for metastatic modeling. Regulatory hurdles, including scalability of technologies like leukapheresis and FDA requirements for explainable AI in diagnostics, delay widespread adoption. Looking ahead, CTC-targeted nanotherapies hold potential for inhibition through biomimetic nanoparticles that disrupt CTC-neutrophil interactions, integrating photothermal and immunotherapeutic agents for synergistic effects. Ethical concerns in CTC-based early detection, particularly with / integration, include data risks under HIPAA/GDPR, algorithmic biases exacerbating healthcare disparities, and the need for transparent models to build clinical .