Clinical significance and heterogeneity of circulating tumor cells and clusters in breast cancer subtypes

Biological functions and heterogeneity of CTCS

Metastasis represents the most fatal manifestation of breast cancer and constitutes a primary determinant of patient mortality and poor prognosis (McGinnis et al., 2024). Cancer metastasis is a complex process encompassing the detachment of cancer cells from the primary site, their intravasation into the circulatory system, extravasation from the bloodstream, and subsequent colonization at distant metastatic sites (Liu et al., 2024). CTCs denote cancer cells that detach from the primary tumor and extravasate into the bloodstream (Fig. 1) (Jiang et al., 2025). CTCs are regarded as pivotal mediators of cancer metastasis (Eslami et al., 2022). Although originating from the primary tumor, these cells may undergo alterations in gene expression, molecular characteristics, and biological behavior upon entering the bloodstream (Jie, Zhang & Xu, 2017), thereby enhancing their adaptability and metastatic potentia (Zhan et al., 2023; Cohen et al., 2023).

The formation of CTCs encompasses two critical stages. The first stage, detachment, occurs when tumor cells undergo epithelial–mesenchymal transition (EMT), leading to the downregulation of adhesion molecules such as E-cadherin, thereby facilitating their release from the primary tumor into the bloodstream. The second stage, survival, necessitates that CTCs evade immune clearance, withstand oxidative stress, and resist shear forces within the bloodstream, thereby ensuring their viability and eventual establishment of distant metastases (Li et al., 2023). Accordingly, the survival capacity and metastatic potential of CTCs exert a direct influence on cancer progression and patient prognosis.

Recent studies show that CTCs are not a uniform cell type but a highly diverse population, varying significantly in shape, size, gene expression, and behavior (Pereira-Veiga et al., 2022). This heterogeneity not only affects the survival rates, metastatic potential, and drug resistance of CTCs, but is also closely associated with patient prognosis and the formulation of personalized treatment strategies. The heterogeneity of CTCs is primarily manifest in their morphological and molecular diversity (Gu, Wei & Lv, 2024), and such heterogeneity directly influences the metastatic process, patient prognosis, and the selection of therapeutic strategies.

Biological functions of CTC clusters and comparison with individual CTCs

CTC clusters typically arise through the aggregation of multiple tumor cells and the collective shedding from in situ carcinoma (Yang et al., 2024), In addition to tumor cells, these clusters may also contain red blood cells, lymphocytes, leukocytes, platelets, and cancer-associated fibroblasts (Pereira-Veiga et al., 2022). CTC clusters are classified into homotypic and heterotypic clusters based on the presence of non-neoplastic cells (Aceto, 2020). Homotypic clusters are defined as multicellular aggregates composed of two or more CTCs interconnected via cell–cell junctions, with their prevalence increasing as the disease progresses (Kurzeder et al., 2025). Heterotypic clusters comprise cellular assemblies that contain one or more CTCs in conjunction with non-malignant stromal cells (e.g., leukocytes, fibroblasts, endothelial cells) or immune cells, frequently incorporating platelets (Pereira-Veiga et al., 2022). Such heterotypic clusters further enhance immune surveillance evasion and vascular wall adhesion through synergistic interactions with blood components, thereby promoting metastatic niche formation (Heeke et al., 2019).

Although CTC clusters are considerably less abundant in circulation than individual CTCs, studies have demonstrated that their metastatic potential is markedly elevated—approximately 25 to 50 times greater than that of single CTCs. CTC clusters depend on intercellular adhesion molecules, including E-cadherin and N-cadherin, to maintain cohesion (Liu et al., 2021b). Morphologically, they exhibit greater structural complexity and demonstrate enhanced resistance to shear forces within the bloodstream (Gu, Wei & Lv, 2024). Furthermore, owing to intercellular cooperation, CTC clusters exhibit heightened survival capacity and enhanced immune evasion within circulation. Additionally, their three-dimensional structure and hypoxic microenvironment facilitate colonization at distant metastatic sites (Donato et al., 2020).

CTC clusters diverge from individual CTCs primarily in their cellular composition, prevalence, metastatic and invasive potential, survival capacity, and drug resistance. A comparative analysis of CTCs and CTC clusters is presented in Table 2.

Clinical detection of CTCs and CTC clusters

The detection of CTCs typically comprises two primary stages: enrichment and separation, followed by identification and analysis (Fabisiewicz & Grzybowska, 2017). Conventional methodologies include immunomagnetic bead capture and physical separation techniques. Immunomagnetic capture employs antibodies conjugated to magnetic beads that recognize surface markers such as EpCAM (e.g., the CellSearch system) (He et al., 2024), However, due to tumor cell heterogeneity and EMT, EpCAM expression may be downregulated, thereby compromising detection sensitivity (Hwang et al., 2024). In contrast, physical separation methods, such as the ISET and Ficoll techniques, facilitate CTC enrichment independent of specific surface markers (Stamatakis et al., 2021; Wang et al., 2024), thus avoiding marker-related detection loss associated with EMT. Nonetheless, these approaches often require specialized equipment and may compromise cellular integrity, potentially interfering with downstream molecular analyses (Paoletti et al., 2019).

Beyond physical and immunoaffinity-based methods, molecular biology techniques—such as PCR, single-cell sequencing, and fluorescence in situ hybridization (FISH) (Li, Wu & Bai, 2018; Ma et al., 2024; Smilkou et al., 2024)—are utilized to investigate gene mutations (e.g., HER2, PIK3CA) and gene expression profiles in CTCs (Gasch et al., 2016), These technologies enable high-sensitivity molecular typing and dynamic monitoring of drug resistance, thereby informing targeted therapeutic decisions (Table 3).

Current detection platforms for CTC clusters largely mirror those used for single CTCs. However, owing to their distinct biological characteristics, CTC clusters offer greater utility in clinical prediction and monitoring of cancer progression. For instance, in patients with stage IV breast cancer, the presence of more than five CTC clusters per 7.5 mL of blood is frequently correlated with aggressive disease phenotypes, suggesting that CTC cluster detection may serve as a prognostic indicator of disease progression (Zhang et al., 2024).

Nonetheless, the detection of CTC clusters is impeded by several challenges. One such challenge is detection sensitivity—owing to high heterogeneity and loss of EpCAM expression during EMT, traditional immuno-enrichment techniques often fail to recover CTC clusters effectively (Hwang et al., 2024). Another limitation lies in current enrichment techniques: due to their larger size and increased rigidity, CTC clusters are poorly captured by single-cell enrichment methods such as CellSearch. Optimization strategies have included physical enrichment methods such as microfluidic sorting and filtration-based separation, which selectively isolate CTC clusters based on size and mechanical properties (Mishra et al., 2025). Emerging approaches incorporating machine learning, such as Smart-Seq2 integration, have further improved detection specificity (Pastuszak et al., 2024).

Despite recent advances, the effective separation and detection of CTC clusters remain challenging. Future investigations may focus on developing multi-dimensional detection models that integrate CTC clusters with other biomarkers (e.g., cfDNA, exosomes) to enhance the diagnostic accuracy of breast cancer (Khandare et al., 2024). Additionally, the incorporation of artificial intelligence technologies to optimize automated detection platforms, alongside clinical validation, represents a critical avenue for future investigation.

The frequency and quantity of CTCs and CTC clusters vary among breast cancer subtypes

Current research suggests that the number of CTCs and CTC clusters varies substantially among breast cancer subtypes and is strongly correlated with their respective invasiveness and metastatic potential. A clinical trial comparing HER2-positive and luminal A patients has demonstrated elevated CTC counts in the HER2-positive cohort, potentially attributable to HER2 gene amplification and the activation of signaling pathways that facilitate EMT. HER2 overexpression may enhance tumor cell proliferation, invasiveness, and endothelial translocation, consequently resulting in an increased release of CTCs into the circulation (Zhang et al., 2021).

In luminal A-type breast cancer, CTC and CTC cluster counts are generally limited, which may underlie its reduced invasiveness and metastatic capacity. In contrast, luminal B-type breast cancer exhibits markedly increased levels of CTCs and CTC clusters (Smerage et al., 2014). supporting the hypothesis that luminal A tumors demonstrate lower metastatic aggressiveness compared to luminal B subtypes (Chai et al., 2023).

Notably, although TNBC is recognized for its pronounced invasiveness and metastatic behavior, several studies have paradoxically reported lower CTC counts in this subtype (Munzone et al., 2012), a trend corroborated by additional clinical reports (Costa et al., 2020). This paradox may be explained by the higher invasiveness of TNBC-derived CTCs or their increased propensity to form clusters. Given that CTC clusters exhibit significantly greater metastatic potential than individual CTCs, this may account for the elevated metastatic propensity of TNBC despite lower overall CTC counts.

To date, no large-scale, systematic clinical studies have quantitatively compared CTC and CTC cluster levels across the four major molecular breast cancer subtypes. Future research should incorporate multi-center clinical trials and leverage advanced techniques—such as single-cell sequencing and circulating biomarker profiling—to elucidate the quantitative characteristics and clinical relevance of CTCs and CTC clusters in different breast cancer subtypes. Furthermore, investigating whether TNBC exhibits a greater tendency to form CTC clusters, and the extent to which this contributes to its metastatic aggressiveness, represents an important avenue for future investigation.

Biological functions of CTCs and CTC clusters in different subtypes

Significant differences in metastatic potential exist across various breast cancer subtypes, and the biological functions of CTCs and CTC clusters may play a crucial role in these variations. In the highly metastatic TNBC subtype, CTCs and CTC clusters exhibit elevated Notch1 signaling pathway activity, which substantially promotes EMT, thereby enhancing their motility and invasiveness and facilitating distant metastasis (Boral et al., 2017). Additionally, CTCs in TNBC patients display elevated PD-L1 expression, which interacts with the PD-1 receptor on T cells, effectively suppressing T cell–mediated anti-tumor immunity and permitting immune evasion (Vardas et al., 2023). Moreover, studies indicate that CAIX—a target gene of hypoxia-inducible factor 1α (HIF1α)—is markedly upregulated in TNBC and HER2-positive breast cancer cell lines, playing a critical role in promoting CTC survival, immune evasion, and drug resistance(Twomey & Zhang, 2023). Regarding CTC cluster formation, desialylation of CTCs in TNBC promotes intercellular adhesion by exposing galactose residues—for example, via galectin-3–mediated aggregation—thereby enhancing cluster formation and chemotherapy resistance(Glinsky et al., 2003; Gvozdenovic & Aceto, 2023). Simultaneously, elevated expression of epidermal growth factor receptor (EGFR) effectively promotes CTC cluster formation and enhances their stability (Liu et al., 2021a). Notably, CTCs detected in TNBC patients often exhibit a TLR4+/pSTAT3+ phenotype, with Toll-like receptor 4 (TLR4) and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) playing pivotal roles in tumor cell proliferation, invasion, and migration (Papadaki et al., 2022). Collectively, these factors contribute to the increased invasiveness and drug resistance of CTCs and CTC clusters in TNBC, thereby accelerating distant metastasis.

HER2-positive breast cancer represents one of the most metastatic subtypes, with CTCs displaying higher Ki67 expression—indicative of enhanced proliferative activity and survival capacity (Boral et al., 2017). Regarding cell migration, HER2-positive CTCs may demonstrate increased migratory and invasive capabilities during the EMT process. Moreover, activation of the HER2 signaling pathway may promote EMT and further enhance CTC survival and migration by stimulating downstream pathways such as PI3K/AKT and MAPK (Bulfoni et al., 2016). Additionally, overexpression of the HER2 receptor is common in both HER2-positive CTCs and CTC clusters, which not only supports cell proliferation and survival but may also enhance intercellular adhesion, thereby improving the ability of CTC clusters to resist shear forces and evade immune surveillance in circulation (Nasr & Lynch, 2023).

Luminal-type breast cancer is classified into luminal A and luminal B subtypes based on hormone receptor expression and cellular characteristics. Studies have demonstrated that CTCs in luminal-type breast cancer predominantly exhibit an epithelial phenotype, with both CTCs and CTC clusters displaying relatively lower invasiveness (Bulfoni et al., 2016). In luminal A breast cancer, CTCs demonstrate lower PD-L1 expression (Papadaki et al., 2021). Combined with relatively weak estrogen receptor (ER) signaling, this may result in a shortened survival time of CTCs in the bloodstream, thereby reducing their metastatic potential (Koch et al., 2020). In contrast, in luminal B breast cancer, the CTC count and the formation rate of CTC clusters are generally higher than in luminal A–type, potentially contributing to its greater metastatic potential (Holler et al., 2023).

Overall, these studies reveal significant differences in the invasiveness, survival mechanisms, and immune evasion capabilities of CTCs and CTC clusters across various breast cancer subtypes, providing novel insights into their metastatic characteristics. However, research on the biomarkers and immune evasion mechanisms of CTCs and CTC clusters across the four subtypes remains limited. Future studies should further investigate the mechanisms underlying CTC cluster formation and their roles in metastasis and drug resistance, as well as evaluate the clinical utility of CTC-related biomarkers in precision medicine for breast cancer, thereby enhancing our understanding of the relationships among different breast cancer subtypes.

Relationship between CTCs and CTC clusters and prognosis of breast cancer patients

It is currently recognized that the quantity of CTCs and CTC clusters is closely associated with OS and DFS in patients with breast cancer. Numerous studies have demonstrated that elevated CTC counts are correlated with poorer prognostic outcomes, and the presence of CTC clusters further exacerbates the risk of disease progression and metastasis. Notably, CTC clusters possess greater predictive value for recurrence risk compared to individual CTCs in breast cancer patients. Existing evidence indicates that the metastatic efficiency of CTC clusters is approximately 50 times higher than that of solitary CTCs (Schuster et al., 2021), Moreover, reductions in CTC counts observed during treatment have been associated with enhanced therapeutic responses and improved survival outcomes, whereas persistent or increasing numbers of CTC clusters may reflect therapeutic resistance and indicate a heightened likelihood of tumor recurrence (Costa et al., 2020), Collectively, these findings underscore the critical role of CTC clusters in the clinical assessment of OS and DFS, as well as in prognostic evaluation.

Differences in the number of CTCs and CTC clusters between different subtypes of breast cancer

In clinical investigations, significant heterogeneity has been observed in both the detection rate and quantity of CTCs and CTC clusters across the molecular subtypes of breast cancer. Specifically, patients with luminal A tumors exhibit a CTC detection rate of approximately 60%, with the majority demonstrating low CTC counts (median: two cells per 7.5 mL of blood) (Zhou et al., 2022). CTC clusters are exceptionally rare in this subtype (median cluster count: 4) (Sayed et al., 2024). In contrast, patients with luminal B subtype demonstrate a CTC detection rate approaching 90% (Xu et al., 2018), though with a similar CTC count (median: 2 cells/7.5 mL) and cluster frequency to luminal A (Reduzzi et al., 2021). Notably, HER2-positive patients exhibit near-universal CTC detection (≈97%) with a median CTC count of 4 cells/7.5 mL (Xu et al., 2018), while CTC clusters are detected in 45% of cases (median: 0; range: 0–8) (Reduzzi et al., 2021). Of particular interest, TNBC patients universally present detectable CTCs (median: 2.5 cells/7.5 mL) along with the highest median cluster count (approximately 5) (Xu et al., 2018) (Table 4). These findings collectively suggest that CTC clusters occur more frequently in luminal subtypes and TNBC compared to HER2-positive disease, whereas HER2-positive tumors are associated with both higher CTC counts and detection rates than luminal subtypes. Critically, CTC enumeration (including cluster quantification) across all four subtypes demonstrates significant correlation with adverse clinical outcomes (Aceto et al., 2014; Gkountela et al., 2019).

The value of CTCs and CTC clusters in the treatment of different subtypes of breast cancer

Studies have demonstrated that the detection of HER2-positive CTCs is associated with reduced DFS in patients, suggesting that CTCs may serve as early indicators of resistance to anti-HER2 therapies (Masuda et al., 2016). In HER2-positive breast cancer, the continued expression of HER2 in CTCs may indicate a favorable response to trastuzumab treatment; in contrast, the presence of HER2-negative CTCs may reflect increased therapeutic resistance (Zhang et al., 2024).

In patients with TNBC, the detection of PD-L1-positive CTCs has been positively correlated with the therapeutic efficacy of PD-1/PD-L1 immune checkpoint inhibitors, such as atezolizumab, indicating that PD-L1-positive CTCs may function as predictive biomarkers for identifying likely responders (Vardas et al., 2023). Similarly, in luminal-type breast cancer, mutations in the estrogen receptor 1 (ESR1) gene detected in CTCs have been strongly associated with endocrine resistance. These mutations induce conformational changes in the estrogen receptor (ER), allowing for ligand-independent activation of ER signaling, thereby diminishing the effectiveness of anti-estrogen therapies such as tamoxifen or letrozole (Fridrichova, Kalinkova & Ciernikova, 2022). As such, ESR1-mutant CTCs may serve as predictive markers of endocrine therapy resistance and could inform early treatment adaptation to agents such as fulvestrant or cyclin-dependent kinase 4/6 (CDK4/6) inhibitors (Brett et al., 2021).

Additionally, gene expression analyses (GEA) of CTCs have revealed dynamic changes in ER status during treatment in some patients with luminal-type breast cancer. Specifically, a phenotypic shift from ER-positive to ER-negative CTCs has been observed, which may signify emerging resistance to endocrine therapy and necessitate timely modifications to the therapeutic regimen (Jakabova et al., 2017).

In conclusion, CTCs and CTC clusters expressing distinct biomarkers across breast cancer subtypes possess significant potential for prognostic evaluation and personalized treatment decision-making. Further large-scale prospective studies are warranted to validate the clinical utility of CTCs and CTC clusters in the context of precision medicine for breast cancer.