It is generally known that the tissue specimens for cancer diagnosis are typically collected through a surgical biopsy to carry out diagnostic tests for the treatment and monitoring of cancer treatments and to assess the effectiveness of traditional therapies. The specimens are then stained using an immunohistochemistry procedure, which enables the pathologist to see how the patient's treatment has progressed. However, the drawbacks of using tissue samples are either difficulties or limitations that arise during surgery for the patient and the surgeon due to the several steps needed and the patient's state of mind. Additionally, it might not be feasible to get new tissue samples for follow-up treatment, particularly for tumors that affect internal organs. Using blood samples from cancer patients has become one of the emerging methods for cancer diagnosis and monitoring that has gained interest over the past ten years. These blood samples are referred to Liquid Biopsy (LB).

LB as defined by the National Cancer Institute of the United States (1), covers various biological samples including blood, urine, or other bodily fluids to be used to detect cancer cells or small pieces of genetic material (DNA/RNA) or other molecules that cancer cells release into body fluids. Therefore, it is very likely that we will be able to detect traces of cancer. One benefit of using LB in clinical work is that just one blood draw is required, which makes it simple for doctors to evaluate cancer progression and their patients' responses to treatment. We can repeatedly draw blood and/or ascetic fluid from the peritoneal cavity, or collect urine for the duration of drug activities to monitor treatment without disturbing the patient's daily life. One area of interest that is currently being widely studied is the detection of cancer cells in the bloodstream, known as Circulating Tumor Cells or CTCs for cancer surveillance and/or diagnosis.

CTCs are cancer cells that have detached from the primary tumor of the organ where the cancer originated and enter the bloodstream before traveling to other organs to grow into a secondary tumor (Figure 1). Normally, CTC cells have distinctive physical features when viewed under light microscopy; the cell size is larger than the white blood cells (PBMC) as shown in Figure 2. In addition, the size of the nucleus is larger than normal cells. To obtain an accurate CTC identification, immunofluorescence staining is therefore preferable.

Figure 1. Stages of CTCs metastasis and liquid biopsy sample collection.

The spread of cancer cells from the primary tumor into the blood vessels by changing their appearance to emerge as single cells (1) through the expression of the TWIST1 gene, for example. Tumor enters the EMT state before penetrating through the blood vessel wall into the bloodstream (2). Cancer cells can migrate far from the initial tumor and generate new tumors when they settle and grow in a distant section of the body (3). CTCs will fall out of the blood vessel and be ready to grow into a secondary tumor. During the migration and spread of cancerous cells in the bloodstream, we can draw blood as a liquid biopsy to collect CTCS (4) and isolate them for counting and/or additional biological molecular studies.

Immunofluorescence staining is performed to determine the cytokeratin (CK) protein expression within CTCs and/or Epithelial Cell Adhesion Molecule (EpCAM) on the surface of CTCs while WBCs express a protein called CD45 on their cell surface. We can use these proteins to differentiate CTCs from WBCs as seen in Figure 3. In addition to the expression of both CK and EpCAM, CTCs also have another distinguishing feature that they are the outcome of the phenomenon called “Epithelial-Mesenchymal Transition” or EMT. It is the biological process by which cancer cells change their appearance from being bound together as solid tumors in primary cells to single cells capable of escaping from the tumor into blood vessels at any time.

Clinical utilization of CTCs can be performed throughout the patient's treatments either chemotherapy or radiotherapy (2, 3). The response of the cancer to the drug/radiation used in therapy enables the doctor to plan the treatment, for example, adjusting the size or type of medicine as appropriate for each patient. The data from the study of CTCs for use in cancer treatment will focus on 1) the number of CTCs that can be discovered from patient blood samples and 2) the molecular profiling of isolated CTCs.

Figure 2. Size comparison between cultured cancer cells (bile duct cancer) and white blood cells.

Cultured cancer cells (black arrows) have very large cell size compared to the size of white blood cells (PBMCs) (white arrows).

CTCs enumeration for cancer diagnosis and treatment

Current techniques for detecting CTCs include various mechanisms for separating CTCs from the blood of patients with various types of cancer are shown in Table 1. However, it is well known that the number of CTCs in the bloodstream is very small, averaging 1-10 cells per 1 ml of blood. The chance of detecting CTCs depends on the factors used to separate them. This is because CTCs naturally have various cellular characteristics, including a larger size than blood cells, the appearance of a large nucleus that occupies almost all of the cytoplasm, and the expression of proteins in the cytoplasm and/or cell membrane especially protein expression found in the event of EMT, which is a distinctive feature of the transformation from a cancerous tumor mass to a single cell.

However, CTCs still retain some genetic features of primary cells. These characteristics of CTCs were revealed by Semaan and colleagues (4), who found that CTCs from blood samples of pancreatic cancer patients exhibited characteristics of four subtypes: epithelial, mesenchymal cells, a group of cells that have EMT phenotype, and a group of cells that share the characteristics of stem-like cells. These characteristics will promote the proliferation and drug resistance of cancer cells. Another benefit of counting the number of CTCs is that it can help doctors in the diagnosis and monitoring of cancer treatment. According to Jiang and colleagues (5), they found that the higher the number of CTCs discovered in cholangiocarcinoma patients, the wider the spread of cancer to lymph nodes and other distant organs, as well as being associated with the cancer stage. The study additionally discovered that there is a good chance that the patient's overall survival time may be predicted based on the number of CTCs.

Molecular characteristics of CTCs for treatment

As mentioned above, CTCs need to transform to escape from the primary tumor and become individual cells ready to spread to other organs. Naturally, there will be altered gene expression to adjust itself to be able to travel in the bloodstream. The ways to achieve this are by upregulating, downregulating, or mutating genes in CTCs. For example, a study of the spread of bile duct cancer due to changes in TWIST1 protein expression accelerated the EMT process (6). Another study by Khales and colleagues (7) found that the TWIST1 gene, which is that controls the activity of other genes, promotes EMT. TWIST1 decreases the expression of E-cadherin protein and increases the expression of vimentin protein. Both proteins control the detachment of CTCs. Moreover, TWIST1 gene expression reduced the death of esophageal squamous cell carcinoma (ESCC) cancer cells through upregulating Bcl-2 gene expression and reducing Bax protein expression, but increasing ABCG2 gene expression and ABCC4 which controls the entry and exit of many types of molecules. This may lead to drug resistance in another way as well.

Uses of CTCs in clinical applications

Early Diagnosis: For people who have no history of cancer, CTCs can be used as an indicator of cancer risk. In case cancer cells are identified in his/her bloodstream, the doctor who reviews the blood test data may propose additional testing for cancer biomarkers to be sure. In this sense, if it becomes apparent that the person undergoing the checkup has cancer, it will be feasible to begin treatment sooner, boosting the chances of cure.

Prognosis: As you have learned earlier, the number of CTCs is associated with severity and patient survival time, indicating a poor prognosis. In this instance, we may find a large number of CTCs in the blood of a cancer patient, increasing the likelihood that the patient will be in a late stage and/or have a shorter survival period.

Disease follow-up: The doctor will be able to evaluate the effectiveness of the treatment after the patient has undergone surgery (8), or after they have received chemotherapy and/or radiotherapy for a while. By collecting the patient's blood and comparing the quantity of CTCs before and after treatment, the success of treatment can be assessed more easily and quickly. Moreover, the number of CTCs can also be used to track disease recurrence.

Personalized medical treatment: Information on gene and protein expression of CTCs obtained from patient samples will be used to plan treatment for more efficiency suitable for individual patients. It may reduce the risk of unwanted side effects and shorten the time of treatment due to the selection of more targeted chemotherapy drugs.

In conclusion, the detection of CTCs in cancer patients' blood can help doctors quickly evaluate the chemical and/or radiological treatment or monitor cancer recurrence after surgery. In other words, it is expected to be the beginning point for personalized targeted therapy to give targeted treatment and achieve positive responses from patients on a case-by-case basis, improving the patient's chances of survival for a longer period.

Figure 3. Immunofluorescence staining of cultured cholangiocarcinoma cells and white blood cells

Cytokeratin in cultured cholangiocarcinoma cells are immunostained by antiobody pan-cytokeratin conjugated to AF680 (Santa Cruz Biotechnology, USA) visble in yellow (white arrows), while white blood cells are inmmunostained with antibody CD45 conjugated to PE (ImmunoTools, Germany) visble in red (red arrows). The nucleus are counterstained with DAPI (blue). Image is taken by an Axio Imager Z2 Epi-Fluorescence microscope (Zeiss GmbH, Jena, Germany).

References

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