New horizons in the identification of circulating tumor cells (CTCs): An emerging paradigm shift in cytosensors

https://doi.org/10.1016/j.bios.2022.114043Get rights and content

Highlights

  • The detailed overview on the principle, merits, and shortcomings of these state-of-art strategies based on a new feature in CTC preliminary identification are provided.

  • Some strategies based on combining two or more features for discriminating specific types of cancers are summarized.

  • The application challenges and the potential directions of current identification methods in clinical CTCs analysis are discussed.

Abstract

Circulating tumor cells (CTCs) are cancer cells that are shed from a primary tumor into the bloodstream and function as seeds for cancer metastasis at distant locations. Enrichment and identification methods of CTCs in the blood of patients plays an important role in diagnostic assessments and personalized treatments of cancer. However, the current traditional identification methods not only impact the viability of cells, but also cannot determine the type of cancer cells when the disease is unknown. Hence, new methods to identify CTCs are urgently needed. In this context, many advanced and safe technologies have emerged to distinguish between cancer cells and blood cells, and to distinguish specific types of cancer cells. In this review, at first we have briefly discussed recent advances in technologies related to the enrichment of CTCs, which lay a good foundation for the identification of CTCs. Next, we have summarized state-of-the-art technologies to confirm whether a given cell is indeed a tumor cell and determine the type of tumor cell. Finally, the challenges for application and potential directions of the current identification methods in clinical analysis of CTCs have been discussed.

Introduction

Cancer metastasis refers to the metastasis of tumor cells from primary tumors to distant organs through blood circulation, which directly leads to most cancer-related deaths. Tumor cells transported via the bloodstream are called circulating tumor cells (CTCs). Thomas Ashworth, an Australian physicist, in 1869, discovered CTCs in blood of a woman who died of metastatic cancer. It was not until 1889 that researchers discovered the critical functions of CTCs in tumor metastasis; CTCs can be termed as "seed" of the "seed and soil" theory (Ashworth, 1869; Friedlander et al., 2014). Proliferation and metastasis of tumors are complex processes. After epithelial-to-mesenchymal transition (EMT) process, tumor cells are transformed from a primary tumor and shed into the blood circulatory system, resulting in metastases. Following this process, a reverse EMT pathway (mesenchymal-to-epithelial transformation, MET) tends to occur, leading to tumor cell redifferentiation and the formation of micro-metastases (Fig. 1) (Esmaeilsabzali et al., 2013; Paterlini-Brechot and Benali, 2007).

CTCs play a role as central actors in the above-mentioned dynamic phases, and their concentration and abundant biological information have a significant impact on metastasis. Clinical trials have shown that early diagnosis, monitoring of treatment, prognosis, and recurrence can be predicted by quantitation of CTCs. The Food and Drug Administration (FDA) approved the CellSearch method in 2004 as a diagnostic tool for detection of CTCs to predict progression-free survival (PFS) and overall survival (OS) in patients with metastatic breast cancer. Furthermore, the FDA approved its use in patients with metastatic colorectal cancer and prostate cancer in 2007 and 2008, respectively. Several studies have also shown that the development and prognoses of cancer patients such as targeted treatment and drug resistance can be precisely defined by estimating the number of CTCs and carrying out extensive genetic analysis using cell-free DNA profiling. CTCs carry comprehensive genetic information related to primary and metastatic lesions, which is of great significance in the detection of cancer development, research on biological mechanisms, and guidance regarding treatment options. Therefore, it is important to develop techniques for the detection of CTCs.

For accurate detection of CTCs, a standardized procedure is necessary, which should include the following three steps: (1) an enrichment step is required to increase the concentration of CTCs by several log units; (2) after enrichment, the CTCs are likely to still contain significant quantities of contaminating blood cells, necessitating methods to distinguish cancer cells from hemocytes; a preliminary identification step is needed to verify if putative tumor cells are indeed authentic CTCs; (3) in early screening of an unknown cancer, the type of the cancer tumor cells remains unknown; therefore, a more accurate identification step is required to identify the CTC types. However, CTCs found in peripheral blood are rare, with the presence of only 1 CTC every 106 to 107 mononuclear cells. Another problem is that the biological and physical properties of CTCs are extremely heterogeneous. For example, the expression of epithelial cell adhesion molecule (EpCAM) might be downregulated during the EMT process (Liu et al., 2015), resulting in a very low probability of the most aggressive cancer cells being enriched (Bhagat et al., 2011; Pecot et al., 2011). These aspects present challenges to efficiently detect CTCs. Thus, the analysis of CTCs is validated by detecting one biomarker (EpCAM) using a sensitive enrichment method, and another biomarker (CK7,8,18,19, etc.) using a specific identification method. However, traditional identification methods impact the viability of cells, which affects the analysis of DNA or RNA at a single-cell level. Furthermore, the type of cancer cells cannot be determined when the cancer remains unidentified using traditional immunofluorescence identification methods.

The present state of CTCs identification has changed significantly due to a greater understanding of which features are altered in the biological processes within cancer cells. These new features were detected by various innovative approaches that have been developed in the past decade, which allowed distinguishing between tumor cells and blood cells, as well as distinguishing between various types of cancer cells. In this review, we first introduced the current status of CTCs enrichment technologies briefly because they are well-documented elsewhere (Lin et al., 2020; Nasiri et al., 2020). Next, we have provided a detailed overview of the principle, merits, and shortcomings of these state-of-art strategies based on a new feature in preliminary identification method of CTCs. Next, we summarize some strategies that combine two or more features to discriminate specific types of cancers. Finally, the challenges faced in the applications and potential directions of the current identification methods in clinical analysis of CTCs have been discussed.

Section snippets

Pre-enrichment of CTCs

To provide a good foundation for accurate identification of CTCs, an enrichment step is needed to increase the concentration of CTCs by several log units. Conventional isolation methods of CTCs can be divided into two categories: physical property-based technologies and protein expression-based technologies. In this section, we provide a brief overview of CTCs enrichment, focusing on technologies that have been developed recently.

Preliminary identification methods for discriminating tumor cells from hematocytes

After enrichment of CTCs using the above-mentioned enrichment tools, many leukocytes are still present in CTCs components due to an overlap of the characteristics of cancer cells and leukocytes. To increase the specificity of CTCs detection, some special features of cancer cells are chosen in subsequent characterization steps. These properties include high expression of telomerase activity, metabolic activities of mitochondria, and morphological and refractive index (RI) of tumor cells. Many

Accurate identification methods for discriminating specific types of cancers

In an identification method, tumor-specific markers-based systems are usually used to determine whether CTCs are authentic tumor cells. However, many tumor-associated biomarkers such as folate receptors and telomerase activity, are often shared by a variety of cancer cell types. Thus, the identification methods mentioned above may have off-target effects and may face difficulties in precise diagnosis and therapy. In recent years, many advanced strategies have been developed to accurately

Challenges and future outlook

Detection and quantitation of CTCs, as a minimally invasive and cost-effective approach, have proven to be valuable tools for the prognosis and prediction of cancer progression. Many methods and technologies have been developed for the enrichment and accurate identification of CTCs. However, enrichment and accurate methods for identification of CTCs are still in their early stages, and much effort is required to improve their characteristics. In this section, we discuss the current challenges

Conclusion

In this review, we have introduced in details the principle, merits, and shortcomings of the latest technologies used for preliminary and accurate identification of CTCs. We have also summarized the current challenges and future directions in this field. Although recently developed identification technologies have many significant characteristics such as negligible effect on cell quality, short reaction times, cost-effectiveness, and high accuracy compared to traditional methods, some

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81973099, 61874099).

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