Research progress on the multi-omics and survival status of circulating tumor cells

Genomic research has played a crucial role in identifying genes that are specific or of particular significance in various types of cancer. And it has led to significant advancements in uncovering genetic mutations associated with cancer initiation and progression, encompassing mechanisms of pathogenesis, immune evasion, metastasis, proliferation, and resistance to apoptosis [16]. The genome of cancer is markedly unstable, which may be a major driver of cancer development [17, 18]. Certain gene mutations have been clinically validated to predict cancer prognosis, establish associations with chemotherapy or immunotherapy responsiveness, and facilitate the development of tailored treatment plans for individual variations [19‐26]. However, unveiling the "veil" of the cancer metastasis mechanism cannot be achieved solely through the exploration of the genome of solid cancer [27]. Profiling the genome of CTCs reveals unique implications distinct from those of the primary tumor. Distant metastasis from primary tumors involves a genetic mutation process, with most distant metastases acquiring driving mutations that are absent in primary tumors [28]. The process of hematogenous metastasis entails genetic changes in tumor cells. In the research of metastatic breast cancer, certain mutations with targeted potential, such as BRCA2 p.Q1931X and PTCH1 p.E1242X mutations, were present in CTCs and remain undetected in the corresponding tumor tissue [29]. Similarly, in pancreatic ductal adenocarcinoma, the KRAS mutations present in CTCs are inconsistent with those observed in the primary tumors, indicating genetic differences in their characteristics [30]. In expanded genomic profiling of CTCs in metastatic breast cancer patients, differences in the mutation statuses of ESR1 and ERBB2 between CTCs and their matched primary tumors were observed [31]. CTCs genome also holds potential significance for personalized therapy. On the one hand, the presence of genetic mutations in CTCs exhibits pronounced inter-patient heterogeneity. In genomic analysis of CTCs in colorectal cancer, there were notable disparities in the mutation statuses of KRAS, BRAF, and PIK3CA, as well as variations in the amplification status of epidermal growth factor receptor-1 (EGFR), among CTCs from different patients. Consequently, CTCs exhibit considerable heterogeneity from one patient to another, underscoring their potential as a foundation for personalized cancer treatment [32]. The genomic research findings on CTCs in non-small cell lung cancer similarly support this conclusion [33]. One the other hand, gene mutations in CTCs have a certain impact on treatment. In a study of metastatic breast cancer, disparities were identified in the CTC mutation status before and after treatment. Remarkably, a small quantity of CTCs carrying specific mutations could remain survival after treatment. These findings indicate that CTCs with certain gene mutations may develop resistance to drugs [34]. Gene mutations in CTCs can be used to study resistance mechanisms or to develop targeted therapeutics [35]. The utilization of targeted medications against drug-resistant mutations in cancer has demonstrated some effectiveness [36]. Consequently, genomic studies of circulating tumor cells (CTCs) offer substantial potential not only in understanding their heterogeneity but also in refining treatment strategies to tackle the challenging of cancer drug resistance.

Transcriptomic research serves as a vital bridge connecting the genome to the proteome. The analysis of the transcriptome is indispensable for understanding the intricacies of the genome, unveiling the molecular composition of tumor cells, and addressing the progression of diseases. Together, the genome and transcriptome provide a comprehensive perspective on individual cancer patients, significantly influencing clinical decisions [37]. Notably, the gene expression of CTCs does not align perfectly with that of the primary tumor. In certain breast cancer patients, markers such as EGFR, epidermal growth factor receptor-2 (HER-2), and estrogen receptor (ER) may exhibit a negative status in the primary tumor, but CTCs can still manifest positive expression [38‐40]. It has been proven to have certain prognostic significance in breast cancer and bladder cancer [41, 42]. In CTCs with specific phenotypes, there are specific pathway activation that can induce CTC proliferation [38]. The gene expression profiles discussed above carry significant implications for therapeutic strategies, such as targeted therapies. Within CTCs, there exist numerous promising targets awaiting exploration. The incorporation of this data is anticipated to enhance the precision and efficacy of cancer treatment [43]. Through transcriptome sequencing, a wealth of metastasis-related information can be obtained from CTCs, enriching genes associated with enhanced tumor cell proliferation and increased invasive capacity. These are essential components in exploring the mechanism of tumor metastasis [44‐46]. Among them, there are numerous potential therapeutic targets, various pro-cancer pathways or more invasive CTC subpopulations [47‐51]. Given the substantial heterogeneity observed among tumor cells, the need for single-cell transcriptome studies focusing on CTCs is evident. Single-cell transcriptomics has proven its worth in investigating CTC heterogeneity [52], making it a promising avenue for identifying precise diagnostic and prognostic markers, as well as actionable therapeutic targets for personalized cancer treatments [53]. And single-cell analysis can provide unique insights into the mechanisms of metastatic colonization by CTCs and precision medicine for cancers [54, 55]. Rapidly developing single-cell isolation technologies include the MagSweeper device [56], laser capture microdissection [57], NanoVelcro-LMD technology [58] and manual cell picking through micromanipulators [59]. Single-cell technology now allows comprehensive analysis of CTCs to reveal aspects of metastasis and resistance mechanisms in cancer therapy [52]. Moreover, existing sequencing technologies such as Hydro-Seq [60], Smart-seq [61], and Smart-seq2 [62] gave a strong basis to the feasibility of individual CTCs for RNA-seq analysis, allowing contamination-free high-throughput and sensitive analysis of the transcriptome of CTCs. Substantial progress has been achieved in revealing the heterogeneity of CTCs using single-cell transcriptomics. By comparing the single-cell RNA-sequencing results of CTCs from different vascular sites, including the hepatic vein, peripheral artery, peripheral vein and portal vein, in patients with hepatocellular carcinoma, Sun YF et al. found there was significant heterogeneity in CTCs between different vascular sites and different patients, involving differences in gene expression related to immunity, oxidative phosphorylation/metabolism, G protein-coupled receptor signaling, cell cycle, EMT, tumor cell-platelet microaggregates, and immune-suppressive chemokines [63, 64]. In the study of CTCs in breast cancer, CTCs in the blood from patients at different time stages (including active and dormant phases) as well as from mouse models show differential expression of cytokinesis and mitotic genes such as Ki67 [65]. Therefore, transcriptome sequencing for CTCs needs to be more systematic, taking into account time and space. Single-cell RNA sequencing (scRNA-seq) for CTCs is also an important source of information for metastatic targets and drug resistance mechanisms. The results of scRNA-seq on individual CTCs isolated from patients with pancreatic cancer, breast cancer, and prostate cancer have revealed that extracellular matrix (ECM) protein genes are highly expressed in human CTCs [66]. Considering the role of ECM lectins in cancer metastasis [67, 68], and the high expression of ECM gene family members in CTCs from several cancer types,  their upregulation may play a crucial role in the generation of CTCs from primary tumors. ScRNA-seq analysis of prostate cancer CTCs by David T. Miyamoto has shown that CTCs exhibit various alterations in androgen receptors (AR), including AR splice variants and point mutations, which are associated with clinical resistance to anti-androgen therapy [46]. In pancreatic ductal adenocarcinoma, scRNA-seq analysis has revealed that the expression of apoptosis inhibitor protein family member BIRC5 (survivin) is higher in CTCs compared to the primary tumor. This may be closely related to the survival mechanisms of CTCs in the bloodstream. Therefore, BIRC5 may be a significant target for reducing metastasis [69].

At present, research on CTCs primarily focuses on genomics and transcriptomics. However, various modifications of proteins are necessary during the translation process, and transcriptomic data cannot fully reflect the qualitative and quantitative changes in protein expression during tumor development [70, 71]. Moreover, transcriptome and protein expression are not completely correlated [72‐74]. Typically, single-cell proteomics analysis methods offer higher precision compared to current scRNA-seq methods. Proteomics technology enables high-throughput and rapid analysis of various proteins expressed in tumors, making it possible to identify numerous protein markers with diagnostic value [75, 76]. These proteins may provide new targets for tumor treatment and effective tumor biomarkers for early diagnosis. However, the limited number of CTCs in peripheral blood, the complexity of protein types within single cells, low protein content, and non-amplifiability pose challenges to CTC proteomics research. Mass spectrometry-based single-cell proteomics research on CTCs has not been reported yet. Nevertheless, some studies have identified some of the proteins present on CTCs by specific means, such as targeting assays. Single-cell resolution western blotting have identified glyceraldehyde-3-phosphate dehydrogenase, β-microtubulin, pankeratin, extracellular regulated protein kinases, epithelial cell adhesion molecule (EpCAM), ER, eIF6E and other proteins expressed in CTCs derived from breast cancer patients [77]. Reza KK et al. successfully detected the heterogeneity of melanoma CTCs and changes in biomarkers after treatment by a surface-enhanced Raman spectroscopy-based simple microfluidic device [78]. The studies described above were based on targeted detection of known proteins and could not detect the possible presence of unknown proteins in CTCs, which may have implications for metastatic drivers, tumor markers, and therapeutic targets. Single-cell proteomics based on mass spectrometry is rapidly advancing, allowing for the identification of biomarkers and the uncovering of heterogeneity within CTCs [79‐83]. Therefore, proteomic studies of CTCs are very promising.

Metabolomics is the analytical research of small molecule metabolites, similar to other "histological" techniques, which can provide critical information about the status of cancers [84]. The metabolic status of cancers is available to differentiate cancer cell subtypes [85], identify cancer biomarkers and drivers of tumorigenesis and can also provide advice for targeted therapies [86, 87]. Tumor cells can adapt to the complex microenvironment and promote immune escape by altering metabolic patterns or reprogramming multiple metabolic pathways [88]. Thus, CTCs in the circulation should have a unique and suitable metabolic pattern. Due to the trace presence of CTCs in the blood, the identification of CTC metabolites poses a formidable challenge. Studies on the metabolomics of CTCs are currently limited to the validation of the presence of known metabolic pathways in CTCs, such as through gene expression assays or performing in vitro cultures of CTC lineage cells to gain insight into the metabolism of CTCs [89‐91]. The in-depth exploration of CTCs metabolomics remains a significant gap in current research. The limited presence of CTCs in the bloodstream poses a formidable challenge for the non-targeted identification of their metabolites. On one front, it is focused on the intracellular metabolites of CTCs. Currently, single-cell metabolomics based on mass spectrometry is rapidly advancing. Takayuki Kawai integrated capillary electrophoresis with mass spectrometry and identified 40 metabolites from single HeLa cell [92]. Shuting Xu and colleagues developed a multidimensional organic mass cytometry platform based on chip-nanoelectrospray ionization mass cytometry system, capable of providing 100 metabolic parameters at single-cell resolution [93]. The use of magnetic bead separation combined with downstream laser desorption/ionization mass spectrometry has been developed and holds potential for identifying metabolic products in CTCs [94]. However, the metabolic products secreted by CTCs and the influence of the blood microenvironment on CTCs are factors that cannot be ignored in the study of CTC metabolism. This includes factors such as blood flow shear stress, oxygen levels, nutrients, and the impact of immune cells on CTCs. Simulating the microenvironment in which CTCs exist in the blood is essential. Currently, relevant research on simulating the blood environment has made some progress, including the simulation and real-time monitoring of hemodynamics, which involves shear stress, blood oxygen transport, and content [95‐99], as well as red blood cell simulation [100]. The nutritional gradients in the blood are also a critical factor to consider. Moreover, in blood simulations, it may be necessary to extract and culture white blood cells from patients’ blood for blood simulation [101]. In summary, metabolomics is rapidly advancing, and despite facing numerous challenges in the field of CTCs metabolomics research, with continuous technological progress, the study of CTCs metabolism is gradually becoming feasible and holds tremendous potential in addressing the challenges of cancer metastasis.

CTC clusters are rare multicellular populations that can be detected in the early stages of cancers such as pancreatic cancer [10], breast cancer [11] and small cell lung cancer [12]. This implied that the transmission of CTC clusters is an early event in the cancer progression. Cohort studies across different cancer types have consistently shown that patients with CTC clusters tend to have significantly shorter survival compared to those without CTC clusters. Analyzing CTC clusters not only provides valuable prognostic information beyond individual CTC counts but also reveals a correlation between the size of CTC clusters and patient outcomes. Larger CTC clusters are associated with a higher risk of death [102‐108]. The existence of CTC clusters is a significant factor that impacts the prognosis of cancer patients, making it a highly valuable area of research. The analysis of CTC clusters was also based on their enrichment and separation, and many CTC isolation techniques were incompatible with CTC cluster isolation. In recent years, techniques for CTC clusters have developed rapidly. The two-stage sequential microfluidic chip invented by Sam H. Au et al. could successively capture CTC clusters with less cell damage based on the larger size and asymmetry of CTC clusters [109]. The ScreenCell Cyto-Cl filter could separate individual CTCs and CTC clusters in a single blood sample [110], and the NanoVelcro chip based on EpCAM enrichment with densely packed silicon nanowires could capture CTC clusters with low EpCAM expression [111].

Research by Nicola Aceto et al. in breast cancer revealed that CTC clusters originate from the primary tumor and are held together by plakoglobin proteins, which are found at bridging granules and adhesion junctions. [112, 113]. In colorectal cancer research, CTC clusters were also considered to be directly released from the solid tumor [114]. The formation of CTC clusters in hepatocellular carcinoma has shown correlation with the activation of the Wnt/β-catenin signaling pathway [115]. Xia Liu et al. observed by intravital multiphoton microscopic imaging in human MDA-MD-231 cell-derived tumor model mouse vasculature that individual CTCs could gather in clusters in the bloodstream and demonstrated that this process is inseparable from the role of CD44 [116]. Additionally, in breast cancer, the appearance of CTC clusters is closely related to intratumor hypoxia [117]. In several studies, the formation and metastatic potential of CTC clusters were also found to be regulated by acetyl heparinase and intercellular cell adhesion molecule-1 (ICAM); among them, ICAM was also found to have the ability to mediate CTC transendothelialization [118, 119]. Also, in breast cancer, disseminated tumor cell clusters are mainly composed of keratin 14-expressing cells[120]. In terms of genetics, genetic analysis of CTC clusters in breast cancer showed that CTC clusters were significantly associated with cadherin 1 (CDH1) [121] and ESR1 Y537S and D538G mutations [122]. CDH1 is a gene located on chromosome 16 that encodes E-calmodulin, a member of the classical calmodulin family [123]. Taken together, formation of CTC clusters is regulated by various factors, including genetic mutations, the action of various adhesion factors, and the activation of signaling pathways. (Fig. 2). However, the current research on the molecular characteristics of CTC clusters is largely concentrated in breast cancer. The complexity of the formation mechanism of CTC clusters implies that current studies are relatively limited. The presence of CTC clusters has been confirmed in various cancers, highlighting the importance of focusing on the diversity and heterogeneity across different cancer types.

CTC cluster size will not hinder their capacity to reach metastatic sites via capillaries. [124]. In breast cancer, compared to single CTCs, binding sites for stemness- and proliferation-associated transcription factors, including OCT4, NANOG, SOX2, and SIN3A, are specifically hypomethylated in the methylation region of CTC cluster DNA [125], which may confer enhanced metastatic and proliferative potential to CTC clusters. Furthermore, combination and collaboration among CTCs within the clusters could provide CTC clusters with an advantage through blood flow stresses, such as loss of nest apoptosis, shear force and immune attack, and colonization of distant organs [126]. In the mouse breast cancer model, there is an increase in the intercellular adhesion and epithelial gene expression of CTC clusters. This leads to a decrease in the expression of the activation ligands for NK cells, conferring low sensitivity of CTC clusters to NK-mediated suppression [127].

Clinical follow-up studies on colorectal cancer and hepatocellular carcinoma suggest that chemotherapy cannot completely eliminate CTC clusters [128, 129], which leads to potential risks of recurrence and metastasis. The persistence of CTC clusters significantly increases the probability of metastasis. Therefore, treatment targeting CTC clusters is of crucial importance in reducing the occurrence of metastasis and improving prognosis. This can be approached from two perspectives: firstly, disassembling CTC clusters. As mentioned above, the formation of CTC clusters is regulated by various factors in some cancers, and targeting the facilitators of CTC cluster formation may help reduce their occurrence or facilitate their disassembly. One study showed that urokinase-type fibrinogen activators had an inhibitory effect on CTC clusters and had the potential to improve survival in a mouse model of lung metastasis [130]. Na/K-ATPase inhibitors, such as ouabain and digitoxin (DT), could dissociate CTC clusters by increasing the intracellular calcium concentration, resulting in the inhibition of cell–cell junctions, and have the ability to inhibit the spontaneous shedding of CTC clusters from cancerous lesions and reduce the ability of cancer metastatic seeding [125]. What is more, the anti-EGFR monoclonal antibody (clone LA1) blocking EGFR has been shown to effectively inhibit in vitro the CD44-mediated aggregation of triple-negative breast cancer cells, reducing metastasis [131]. The use of celecoxib can reduce the formation of CTC clusters by inhibiting COX-2 and downregulating E-cadherin protein expression within xenograft tumors [132]. On the other hand, during tumor cell aggregation, it induced a hypoxic environment, including mitochondrial autophagy mediated by hypoxia-inducible factor 1-alpha (Hif1α) and restriction of reactive oxygen species (ROS). This ultimately reduces dependence on glycolysis for ATP production. Disrupting these metabolic adaptations may have the potential to decrease the survival and metastatic capability of tumor cells [133]. This is limited to cell line, and further expansion is needed to delve into research involving CTC clusters sourced directly from patients. However, treatment targeting CTC clusters also requires the development of efficient and convenient clinical detection methods [134]. Additionally, translating research into clinical applications necessitates a deeper understanding of the molecular characteristics of CTC clusters in different cancers. Clusters of CTCs in different types of cancer may harbor specific cancer-associated molecular characteristics. For instance, this could potentially offer more comprehensive guidance for the treatment of CTC clusters [135]. Treating solely targeting the CTC clusters may not achieve a significant enough effect, while combining with other treatment methods such as chemotherapy may lead to more pronounced outcomes [136]. The therapeutic potential and clinical applicability of identified targets also require further assessment.