Table 1.

Studies comparing the biological and pathological characteristics of CTCs and CTC clusters

Study	Tumor type	Metastatic potential	CTCs and CTC Cluster detection method	Study main findings	Reference
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]