1 Introduction
1.1 Biology of CTC clusters
1.2 CTC clusters categories
1.3 Relationship between CTCs and microorganisms
1.4 CTC clusters and metastasis
1.5 CTC clusters immunology
1.6 Comparison between single CTCs and CTC clusters in BC metastasis
Study | Tumor type | Metastatic potential | CTCs and CTC Cluster detection method | Study main findings | Reference |
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Cheung et al. (2016) | Breast cancer mouse model | The ex vivo colony formation increased by > 15-fold, and in vivo metastasis development increased by > 100-fold when tumor cells were aggregated into clusters | Multicolor lineage tracing | In a mouse model, multicolored tumor cell clusters were observed across all main phases of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases | [45] |
Donato et al. (2020) | Breast cancer mouse model | The average number of cells in hypoxic CTC clusters was larger than that of normoxic CTC clusters, with 5.3 cells per hypoxic CTC cluster and 2.82 cells per normoxic CTC cluster | Live imaging of HIF1a reporter | In a mouse model, Most CTC clusters are hypoxic; conversely, most single CTCs are normoxic | [55] |
Szczerba et al. (2019) | Patients with breast cancer and mouse models | Among breast CTCs, CTC-neutrophil clusters are the most effective subpopulation for metastasis formation, and a patient's bloodstream containing these cells is linked to a poor prognosis | Parsortix microfluidic device | - In breast cancer patients, those with 7.5 ml of peripheral blood containing at least one CTC-neutrophil cluster had a substantially worse progression-free survival than those with five or more CTCs in 7.5 ml of peripheral blood - Research revealed that mice administered CTCs from CTC–neutrophil clusters experienced a significantly lower survival period and overt metastasis than those injected with CTCs alone | [60] |
Aceto et al. (2014) | Breast cancer mouse model | CTC clusters have been estimated to be between 23 and 50 times more than that of single CTCs | Herringbone HB CTC-Chip | In a mouse model, clusters were demonstrated to make up around 50% of breast cancer metastases despite making up just 2–5% of total CTCs. Still, CTC clusters have been estimated to be between 23 and 50 times more than that of single CTCs and contribute to approximately half of all metastatic lesions in orthotopic breast cancer models | [20] |
Duda et al. (2010) | Mouse lung cancer cell line | The presence of fibroblasts in clusters makes cancer cells more viable in the bloodstream and at the secondary location | Whole-mount fluorescence microscopy | Using diphtheria toxin treatment 24 h after cell infusion to eliminate carcinoma-associated fibroblasts (CAFs), the number of metastases assessed two weeks after infusion did not change significantly. These findings demonstrate the ability of tumor-associated fibroblasts to stimulate lung metastasis. This advancement cannot occur when the CAFs are not directly associated with the cancer cells within the metastatic foci | [24] |
Liu et al. (2019) | Patients with breast cancer and mouse models | CTC clusters are more potent in facilitating metastasis formation than single CTCs, particularly in triple-negative patient-derived BC models (PDXs) | Cell search and fluorescence microscopy | -CTC clusters are more potent in facilitating metastasis formation than single CTCs, particularly in triple-negative patient-derived BC models (PDXs) -The breast cancer stem cell marker CD44 was highly expressed by aggregating tumor cells, encouraging carcinogenesis and polyclonal metastases - The interactions mediate tumour cluster aggregation between CD44 homophiles and, subsequently, CD44–PAK2. That will encourage the creation of new targeted techniques to prevent polyclonal metastasis and result in novel biomarker applications that predict prognosis | [22] |
Murlidhar et al. (2017) | Patients with surgically resectable (clinical stage I-III) lung cancer | The cells within CTC clusters can avoid cell death, given their prognostic relevance and capacity for metastasis. CTC clusters have been demonstrated to be linked to a poor prognosis | Microfluidic device | In early-stage lung cancer, (CTCs) can help predict an early relapse and assist in the early diagnosis of metastases. A notably greater quantity of CTCs in the (PV) blood has been discovered than in the Preoperative (Pe) blood. Gene ontology analysis enriched cell migration and immune-related pathways in CTC clusters, indicating a possible survival benefit of the clusters while in circulation | [106] |
Gkountela et al. (2019) | BC patients and mouse models | CTCs' ability to shape clusters has been connected to expanded metastatic potential | Whole-genome bisulfite sequencing (sc-WGBS) investigation | Demonstrated that clustering causes hypomethylation of Linking locations associated with stemness and prevalence regulatory, including SOX2, SIN3A, OCT4, and NANOG. Moreover, hypermethylation of Polycomb target genes is also shown to increase stemness and proliferation concurrently | [56] |
Paoletti et al. (2015) | Metastatic triple-negative breast cancer (TNBC) | Metastatic | CellSearch® | In correlational studies, CTC clusters are significant in metastatic triple-negative breast cancer (TNBC) and apoptosis | [107] |
Mu et al. (2015) | Metastatic Breast cancer (Stage III-IV) | Metastatic | CellSearch® | Patients with breast cancer phases III and IV had a lower PFS when their baseline numbers of both single CTCs and CTC clusters (classified as C2 CTCs) were high | [35] |
Jansson et al. (2016) | Metastatic Breast cancer (Stage III-IV) | Metastatic | CellSearch® | In an observational study, follow-up (FU) samples of patients having CTCs and CTC clusters following systemic irradiation had the worst prognosis in terms of PFS and OS compared to those without such cells | [36] |
2 Methods for CTC cluster detection and isolation
2.1 Label-free methods
2.2 Affinity-based methods
2.3 Predictive value of CTC cluster counts in monitoring therapy response
2.3.1 CTC clusters in primary BC
2.3.2 CTC clusters in MBC
Study Name | Study TYPE | Number of patients | Treatment GROUPS | Primary endpoint results | Breast cancer subtype | CTC cluster detection method | Statistical significance of results |
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Jansson et al. [36] | A prospective observational study (cohort study) | Fifty-two patients newly diagnosed with a first metastatic incident were scheduled to begin first-line therapy in the metastatic context | They did not have different treatment groups but followed the patients who received first-line therapy in the metastatic context according to their BC subtype (hormone receptor-positive, HER2-positive, or triple-negative) | The CTC clusters detected in the blood at 1–3 months and 6 months were linked to a shorter PFS. At 1–3 months, CTC clusters were also linked to shorter OS. Still, at 6 months, OS could not be assessed because every patient in the group that had clusters died before patients in the group without clusters | They followed the patients according to their BC subtype (hormone receptor-positive, HER2-positive, or triple-negative). They found that CTC clusters were less commonly detected at baseline and between 1 and 3 months in patients with hormone receptor-positive tumors than in HER2-positive and triple-negative BC patients. Still, at 6 months, there was no significant variance | Used CellSearch analysis to detect apoptotic CTC, CTC clusters, and WBC-CTCs. They defined CTC clusters as two or more CTCs close to each other within the same image field | The CTC clusters in the blood at 1–3 and 6 months were linked to shorter PFS. At 1–3 months, CTC clusters were also related to shorter OS. Patients with clusters had a greater risk of cancer progression and mortality compared to those who had no CTC clusters at any stage during the trial duration |
Wang et al. [37] | Cohort study | Before beginning a new therapy, there were one hundred twenty-eight female patients with metastatic BC | They did not have different treatment groups but followed the research types and received various therapies according to their physician's discretion | Baseline CTC clusters significantly correlated with reduced PFS outcome. Additionally, the longitudinal analysis found that CTC clusters had more predictive value than CTC enumeration alone. Also, changes in both CTCs and CTC clusters from baseline to the first follow-up predicted patient survival | NA | Used the CellSearch System to detect CTCs and CTC clusters. They defined CTC clusters as two or more CTCs close to each other within the same image field | Baseline CTC clusters significantly correlated with reduced PFS outcome. CTC clusters had more excellent predictive value than CTC enumeration alone. Changes in both CTCs and CTC clusters significantly predicted patient survival. CTC cluster size significantly correlated with patient outcomes [115] |
Larsson et al. [38] | A prospective observational study (cohort study) | One hundred fifty-six patients with recently diagnosed metastatic BC | They did not have different treatment groups but followed the patients who received systemic therapy according to their physician's discretion | Patients with CTC clusters at baseline had poorer OS and PFS rates than those without CTC clusters. The hazard ratio of both PFS and OS increased with the presence of CTC clusters during systemic therapy | NA | Used CellSearch technology to identify CTCs and CTC clusters. They defined CTC clusters as two or more CTCs close to each other within the same image field | The patients with CTC clusters at baseline had worse OS and PFS. PFS and OS hazard ratios increased when CTC clusters were present during therapy. Moreover, mortality was 11 times higher in patients with high CTC counts (CTCs 5) and CTC clusters compared to individuals without these characteristics |
Costa et al. [133] | Cohort study | Fifty-four female metastatic BC patients | They did not have different treatment groups but followed the patients who received various therapies according to their physician's discretion and their BC subtype (HR + HER2-, HR + HER2 + , HR-HER2 + , or triple-negative) | At baseline, the CTC cluster showed a higher risk of disease progression and death. The PFS, OS, and survival time were also shorter. Furthermore, it was discovered that patients with a CTC cluster of four or more cells had a higher likelihood of disease progression than those with a CTC cluster of 2–3 cells (which, compared to patients without a CTC cluster, had a greater risk of disease progression) | They followed the patients according to their BC subtype (HR + HER2-, HR + HER2 + , HR-HER2 + , or triple-negative). They found that patients with the HR + HER2 subtype were more frequently found to have CTC clusters | Used the CellSearch System to isolate and count CTCs and CTC clusters. They defined CTC clusters as two or more CTCs close to each other within the same image field | At baseline, the CTC-cluster had a higher chance of disease progression and death, along with shorter PFS, OS, and survival time. Patients with a 4 + cell CTC-cluster had a greater probability of disease progression. The continuous existence of CTC clusters in the circulatory blood was related to shorter OS and a greater mortality risk |
Paoletti et al. [134] | prospectively designed retrospective translational medicine study | They include 595 female patients with established BC and signs of metastatic cancer on clinical and/or radiographic assessments | They had two treatment groups: one that received eribulin mesylate (a chemotherapy drug) and one that received capecitabine (another chemotherapy drug) | They found that patients with doublets and clusters had statistically lower OS at baseline and the First Follow-up than those with couples only, clusters only, or no doublets or clusters. Also, CTC clusters were associated with a poorer prognosis, regardless of their detection time | NA | Used the CellSearch system to count the CTCs, classifying them as clusters when there are three or more and as doublets when there are two. They used the "revised" CellSearch CTC enumeration algorithm, which counts the number of CTCs in a couple or cluster and each CTC to determine the total CTC enumeration | Paoletti et al. found that patients with doublets and clusters had lower OS at baseline and the first follow-up. CTC clusters were linked to a worse prognosis, whether discovered at baseline or the first follow-up |
2.4 Prevention of CTC cluster formation in BC patients
Treatment | Description | Mechanism |
---|---|---|
ADGRG1 inhibitors [150] | ADGRG1 is a protein that actively facilitates the process of tumorigenesis, invasion/migration, and cell–cell adhesion in cells associated with triple-negative breast cancer | Inhibitors of ADGRG1 could prevent CTC cluster formation by blocking its role in cell–cell adhesion |
Estrogen receptor alpha (ER/ESR1) inhibitors [151] | Mutations in ER/ESR1 have been detected in 20–40% of metastatic breast cancers resistant to endocrine therapy. These mutations are linked to unfavorable outcomes | Inhibitors of ER/ESR1 could prevent CTC cluster formation by blocking its role in metastasis |
Chemotherapy drugs [152] | Chemotherapy is the prevailing approach for managing systemic therapy in triple-negative breast cancer (TNBC) patients | It can still effectively prevent CTC cluster formation by killing cancer cells and preventing their spread |
Customized drug screening [153] | A culture assay of CTC derived from patients can be a valuable tool for evaluating anticancer drugs to guide therapy for personalized treatment | This approach allows for evaluating drug response using patient-derived CTC cultures obtained from a liquid biopsy |
VEGF inhibitors [55] | The protein VEGF serves to activate the genesis of novel blood vessels | The growth of new blood vessels that provide tumors with oxygen and nutrients exists through inhibitors that target VEGF |
Pro-angiogenic treatment [55] | The pro-angiogenic treatment promotes the genesis of novel blood vessels | This treatment could improve blood flow to tumors, allowing for better chemotherapy drugs and oxygen delivery |
Epigen [154] | Clusters promoted the expression of the low-affinity EGFR ligand epigen, which promotes effective metastatic outgrowth and is exhibited in the highest levels in metastatic tumors | The metastatic outgrowth was reduced by 94% upon Epigen knockdown. Additionally, there was a decrease in the size of lung metastases, although the total number was not affected |
Plakoglobin [155] | Plakoglobin, a component of desmosomes and adherence junctions, was overexpressed 219-fold more in CTC clusters and was associated with lower distant metastasis-free survival (p = 0.008) | Eliminating intercellular contacts crucial for cluster formation was achieved through Plakoglobin knockdown, and the count of tumor-derived CTC clusters decreased in experimental mouse tumors |
Keratin-14 [45] | Desmosome and hemidesmosome adhesion complex genes, which control cell–matrix adhesion, cell–cell adhesion, and immune evasion, were abundant in keratin 14 cells | The mean number of metastases was seven times lower in keratin 14 knockdown tumors than in control tumors |
ICAM-1 [156] | ICAM-1 is a transmembrane glycoprotein crucial for melanoma cells' adhesion to the endothelial monolayer [124] | ICAM-1 expression is increased with an increase in tumor cell adhesion. Thus, treatment with specific anti-ICAM-1 antibodies decreases this effect |
Hemophilic CD44 [157] | CD44, a multifaceted transmembrane glycoprotein, facilitates the epithelial-mesenchymal transition process [126] | CD44-positive cells exhibited a heightened propensity for tumor formation in immunodeficient mice relative to their CD44-negative counterparts |
Na + /K + ATPase exists on the cell membrane; its expression is highly expressed in breast cancer cases | Ouabain treatment inhibits the expression of Na + /K + ATPase in mice, which inhibits distant tumor formation in multiple mouse metastasis models |