Role of circulating tumor cells in future diagnosis and therapy of cancer

Introduction

Cancer is a collective term for uncontrolled malignant tumor growth taking place in any tissue of the body. More than 100 types of cancers are known till date, some of them being more common in specific genders such as in case of women; breast cancer is of the most common whereas in men, prostate cancer is quite common. Other types of cancer like lung, colon, blood, lymph are found in both men and women. Surgery, radiotherapy, chemotherapy are the established treatments for cancer which also constitute significant side effects. However, there is still a long way to go to constitute 100% efficacious results because of heterogeneity and resistant of tumor cells to available therapies of cancer. Each of the subtypes responds differently to the treatment and makes it difficult to attain a replete cytogenic response. Cancers are also known for attaining complex diversity which makes it difficult for clinicians to choose the treatment procedure.  Some prevalent mutations or the ones attained during the course of treatment may also result resistance to the therapy. In such cases, continuation of the same treatment only worsens the condition, therefore, there is a need of extremely specific and targeted therapy which can help the survival of patients in such situations. It is increasingly becoming a prerequisite to take a "fingerprint" of a given tumor and then proceed with a "tailor-made" treatment. Circulating tumor cells (CTCs) can provide us with the required information and pave a new avenue in future cancer therapies.

Mechanism of Cancer Development

Most cancer remains asymptomatic at early stages and start showing up signs only in later stages of development. It is difficult to treat the patient in advanced stages of cancer, because the tumor spreads itself in various tissues of the body which is referred as invasion and metastasis of cancer.  The actual trigger which initiates this process remains obscure. However, CTC-based technologies may predict the pathway of metastasis. A malignant tumor cell has many cell cycle pathways abnormally regulated. Initially, the epithelial cells of a primary tumor infest nearby blood or lymphatic vessels and circulate in them as shown in [Figure 1]. Of the many altered pathways in these cells, one of them is the production of a protein called matrix metalloproteinase (MMP). Upon metastasis of a tumor cell, it breaks from the main tumor and enters the extracellular space which is mainly made up of collagenous fibers. The tumor cells secrete MMP, which breaks collagen fibers as well as the basement membrane surrounding the blood and lymph vessels. The tumor cells now gain direct access to the epithelial membrane of the vessels and squeeze into them through the tight junctions.  Once into the bloodstream, they can easily transport to other tissues of the body and invade them. An aggressive tumor cell can attach itself to the endothelial membrane of the vessel and create a "pore" through which it escapes out and invade the nearby tissue. Other less aggressive tumor cells can use this pore to enter the same tissue and establish a new tumor. Malignant tumors also initiate angiogenesis to enhance blood supply around the tumor and support its growth and development. All this time, when several changes are taking place during the course of tumor growth, an important phenomenon is the shedding of cells from the primary tumor in the bloodstream as CTCs. These cells carry tremendous information about the presence of tumor, its growth stage and mutations that it harbors. Due to this vital data, they have enormous applications in the detection, staging and treatment guidance of solid tumor malignancies. In this review, we have discussed about their significance, isolation, enrichment techniques and the advancements in the field of molecular biology of CTCs in major types of cancers including breast, prostate, colorectal, and lung cancer.

Circulating Tumor Cells

CTCs are described as cells shed by a primary tumor into vasculature and they keep circulating in the blood stream of cancer patients. Scientists have tried to decipher their nature and significance. CTCs are known to be circulating in the body fluids before they metastasize to various parts of the body even in primary stages of the disease.  However, they are not easily identified, as they are present in a very small numbers. It is estimated that a teaspoon of blood might contain just about 5-50 CTCs. CTCs first exuviate from the primary tumor and remain in the blood stream for a while till the time it wedges itself in a new tissue as shown in [Figure 1]. Some CTCs can adhere to the wall of capillaries and bunk to enter a new tissue. While in the blood stream, they might even clog capillaries due to their big size.  Many CTCs can be shed from a given tumor in different locations. A given tumor may vary in nature at different locations, that is, it may display heterogeneity. CTCs released from different locations of a tumor may exhibit discrepancies of a given tumor. Thus, CTCs can contribute to a potpourri of heterogeneous cells disgorged from the same tumor.

Despite consistent efforts, researchers are yet to gather its caboodle. Of the known properties, one of them is that they undergo epithelial to mesenchymal transition (EMT) as shown in [Figure 2]. This results in change with respect to epithelial markers and other cellular properties.  An epithelial cell starts behaving like a mesenchymal cell and can detach itself from the parent tissue and become a free flowing entity. CTCs use this property to invade blood and lymph capillaries and swim freely in them. Not all CTCs undergo complete EMT; some of them undergo just partial changes or partial EMT. CTCs undergone complete EMT can revert their phenotype by undergoing mesenchymal to epithelial transition (MET) out of which some can contribute to micro or macro-metastasis leading to cancer progression.  When a tumor cell undergoes reversion by MET, they regain properties of cell adhesion. These cells first adhere to the wall of capillaries and then evade from them to nearby tissues. Since they can now behave as epithelial cells again; they adhere to the target site and start dividing and re-dividing giving rise to a new tumor. However, EMT transition can also lead to a perplexed situation as there is a lot of diversity in the morphological transformations.

Significance of CTCs

One of the most axiomatic implications of CTCs is that they are minimally invasive indicators. Detection of CTCs can reveal mint of information rather than just the presence of a tumor. They can help us to realize the concept of tailor-made medicine. Analysis of CTCs can save a patient from worsening the condition with unsuitable medications. Furthermore, the earlier they are detected, faster and better treatment options can be made available to the patient. It provides the basis of understanding mutations and genotypic changes of malignant cells and hence provides the best suitable targeted therapy. CTCs are multifunctional biomarkers and enable us to assess the patient serially along the treatment journey. They are potentially an alternative to invasive biopsies for detection, characterization and monitoring of non-hematological cancers.  Although as of now it is not clear whether CTCs are the cause of metastasis, they still hold the potential for being a cause for disease progression. Metastasis is better known to be caused by cancer stem cells (CSCs), which are highly motile, self-renewing cancer initiators. They also have increased resistance to apoptosis as well as to certain treatment drugs. CTCs with such properties can be metastatic in nature. CTCs after undergoing EMT can also make non-CSC type cells to behave like CSCs. In addition, it is yet to be clarified whether cells with metastatic potential have increased motility and aggressive nature of CTCs as compared to non-metastatic tumor cells. On the whole, CTCs give us biological insights of the disease condition, progression, and treatment prediction. Reports indicate that patients with fewer numbers of CTCs survive longer than the patients which have more number of CTCs. Another important implication of CTCs is that they can form the constitutional basis of tumor staging. The types and quantity of CTCs can form prima facie of the degree and type of cancer. They can be periodically used to keep a check on disease progression. In some cases, they have even been able to identify the drug targets by analyzing the enumerated CTCs and its phenotype. They can even help in the selection of secondary treatment options while the patient has failed to respond to first line treatments. One such example is the detection of human epidermal growth factor receptor 2 (HER2)-positive CTCs in HER2-negative breast cancers. Thus, it gives us hints and specks about quiescent population that may be present in the tumor and be the cause of drug resistance or relapse of the disease. Since, CTCs hold such critical information about a tumor and its characteristics; they can definitely form the pedestal of patient-specific treatments. The great enigma about cancer can adjudicate with the help of information retrieved from CTCs analysis.

Isolation and Analysis of CTCs

In the recent years, CTCs have gained increasing importance because of their multi potential uses. Despite their long known discovery and spates in clinical oncology, no method has been devised to isolate or enumerate CTCs efficiently. Primarily, their quantity in blood circulation is the biggest hurdle in isolation of CTCs. Out of the several CTCs shed by the primary tumor only about 0.1% survives in the circulation and only about 0.01% is responsible for metastasis. It has been reported that CTCs are not continuously shed in the circulation. They are discontinuous and might not be present in homogenous condition. Thus, while isolating CTCs a single blood sample might fall insufficient or may give inaccurate results.  This is accompanied by further reduction in their numbers when they get clogged in capillaries due to their large size. They can also form clusters while flowing and some of them may even adhere to the walls of the capillaries, or some might be cloaked by the platelets. Further reduction in CTCs number takes place during batch processes which are followed for their enrichment. Simpler methods involve size based separation, collagen adhesion method or density-based separation. Other sophisticated ones rely on epithelial markers, immunomagnetic techniques, microchips, and nanotech approaches.
Density-based Ficoll-Hypaque method
Gertler et al2003 have used Ficoll-Hypaque density-based separation method to separate tumor cells from bone marrow and peripheral blood aspirations. It is based on differential migration of cells which takes places during centrifugation and gives a layered separation of cells types. The porous barrier is permeable to the red blood cells and other smaller components of blood. The buffy coat above this layer is of concern, as it contains the tumor cells along with leukocytes. This layer can be easily aspirated and analyzed further to determine the presence and quantification of CTCs.
mmunomagnetic (antibody based) method
This method exploits the presence of surface markers on tumor cells or hematopoietic cells. In this method, antibodies are coupled with magnetic particles and then used for positive or negative selection of CTCs. In positive selection, surface markers of CTCs are targeted, whereas, in negative selection, depletion of blood cells other than CTCs is achieved by targeting their surface markers as shown in [Figure 3]. Epithelial cell adhesion molecule (EpCAM) is one of the most widely tapped markers on tumor cells. CD45 in case of lymphocytes and glycophorin for erythrocytes are two commonly used markers in case of negative selection. MACS ^® has introduced microbeads which can be used in such negative selection.

Food and Drug Administration (FDA) has approved CellSearch ^® (by Janssen Diagnostics) which is by far the most efficacious system for extraction and enumeration of CTCs. The CTCs according to this system are defined by a characteristic round oval shape cells with nucleus which is stained by 4',6-diamidino-2-phenylindole stain. This procedure may be laborious and intensive but gives the best enrichment results as a comparison to other existing techniques. It makes use of antibodies like EpCAM attached to magnetic beads for binding to specific tumor cell surface receptors. These cells can be pulled out from the rest of cells under the influence of a magnetic field.  Some tumor cells might escape the antibodies as they undergo EMT transitions while some other tumor cells belonging to a smaller sub-population might also be ignored. Some CTCs remain undetected throughout this process.  Hence, although this method is being used currently for experimental purposes, there is yet lot of scope for improvisation in the quantitative as well as qualitative aspects of tumor cell detection.

Microfluidics method
As antibody-dependent cell sorting is not a completely reliable source. There are a lot of hurdles in accomplishing higher percent enrichment of cells from the whole blood. Hence, it is important to take into consideration other methods which rely on antibody-free systems. In this method, cell size-based sorting is accomplished using microfluidic technology. The microfluidic chamber is made up of special materials and is usually spiral or curvilinear. When whole blood is allowed to pass through this micro-chamber, inertial lift forces and drag forces help in sorting of the cells. These forces rely on differential sizes of cell in the sample. In case of CTCs, whole blood or leukocyte along with CTCs fraction can be used as a feed in input. As they pass through the microfluidic chamber, the forces will act on the cells and start separating them based on size. The CTCs incline more towards the inner wall (larger size) while other cells such as white blood cells and red blood cells will incline towards the outer side of the wall (smaller size).  They can be collected in separate fragments at the end of the tube, where it bifurcates into collecting chambers as shown in [Figure 4]. Recent advances have allowed the procedure to be carried out with minimal loss of cell types.

Size based separation method
As CTCs are usually bigger in size compared to other components, this characteristic is put to use. This method can even be used to detect the presence of a single tumor cell in a quantity of blood as little as 1 mL (shown in [Figure 5]). ISET ^® is one such established method which is used for such type of cell size based sorting. Specially designed filter are employed to allow blood components to percolate through them. CTCs being bigger in size will not be able to pass through the membrane and hence remain over it. They can be then collected from over the membrane filter and subjected to analysis.

Other techniques
The FDA approved cell detection method has quite some limitations. Hence, a lot of attempts are being made to invent better techniques which are highly efficient low on cost, less labor intensive, and time savers too. Metacell ^® is another cell size-based sorting method which has been introduced lately. Microchips and micro slides are being designed to exploit various differential properties of cells. Lu et al have introduced a device which they refer to as Nan Velcro CTCs Chip. They claim that this device is much more efficient and reproducible as compared to CellSearch ^® kit. This kit is composed of a patterned silicon nanowire substrate which is overlaid with polydimethylsiloxane mixture. While another cell surface marker-based systems is a flow cytometry fluorescence-activated cell sorting. Another emerging technique is making use of dielectric constants of cells such as the DEPArray system. Ju et alhave described a method where they make use telomerase activity to isolate melanoma cells in peripheral blood. As telomerase activity is elevated in cancerous cells rather than normal cells, they made use of an adenoviral vector human telomerase reverse transcriptase to drive the expression of green fluorescent protein which can be used to isolate CTCs in this method. An interesting device called VeriFAST is an integrated system which can isolate cells as well as perform down streaming processes including staining with EpCAM and other antibodies to isolate CTCs. Many more such technological advancements have been reported by scientists all over the world. There are several newer assays are being introduced which are focused on marker free isolation such as chromatography, filtration, and dielectrophoresis for capturing CTCs from cancer patients. Few of them have been mentioned under specific cancer categories discussed ahead in this review.

Characterization and Molecular Profiling of CTCs

We have discussed various CTCs enrichment techniques which are being used for isolation of CTCs from metastatic cancer patients. However, none of them has achieved much of quantitative success. The results have shown a great amount of variation from 10% to 90% of isolated CTCs and hence, it is crucial to analyze the collected cells for their quantity as well as their exact phenotype. A numerical indication of collected CTCs may not be able to reveal the true picture of the type of cells isolated from cancer patients. Similarly, tumor cells can undergo a variety of changes and be present in heterogeneous subpopulations. Hence, a mere number of CTCs can lead to faulty conclusions. Therefore, there is a need for true characterization of these isolated CTCs cells to come to logical conclusions. Molecular profiling of these isolated cells will crystallize the picture, as it reveals the true nature of the isolated CTCs cells.

A fundamental process in EMT, down-regulates E-cadherin, which can be attained by many transcriptional factors. Most of the molecular markers that have been isolated for characterizing CTCs are EMT indicators. During EMT process, a metastatic cell goes through a lot of modifications at cellular and molecular levels and many genes undergo transcriptional alterations.  Some of these genes play a role in initiating the effect of EMT while others play a role in regulating and maintaining its transited state. The other factors like inflammatory cytokines and physical changes in the tumor microenvironment also play a role in EMT promotion. TWIST1 and TWIST2 genes are most strongly expressed genes in EMT process which are responsible for inducing transformation alone or in co-operation with other factors such as TGFβ, Wnt, Notch, etc. E-cadherin is one of the most important proteins for maintaining the epithelial nature of cells. Snail1 and Snail2 suppress the transcription of E-cadherin as well as Zeb1 and Zeb2 genes. This results into downregulation of E-cadherin, which leads to initiation of EMT process. Other gate keeper's genes of epithelial state, such as alpha and gamma catenins are also been down-regulated along with downregulation of E-cadherin in this process.

Induction of certain mesenchymal characters during EMT process requires upregulation of two extracellular matrix proteins, that is, vimentin and fibronectin in these cells which escape the barriers of local tissue and proceeded for invasion. Similarly, other genes such as N-cadherin, CD44, intergrin β6 are also implicated for proper migration of these cells. Even understanding the mutational changes, abnormal size, and characteristics of CTCs, scientists are still pondering over the fact that these cells are able to survive in an environment which is totally hostile for them. It is postulated that out of the several hundred CTCs shed by the tumor, only a few remain in the circulation. There are reports suggesting that CTCs bearing mutations, such as upregulation of CD47, help them in escaping attack by natural killer cells and macrophages. Similarly, downregulation of chaperone protein-calreticulin again helps them to dodge the immune system.  Schölch et al in their studies have referred this state as an "immune-evasive" to the period between EMT and MET in circulation. Thus, overall it seems that CTCs have very evolved mechanisms to maintain and express their invasive aggressive nature by surpassing the body's natural immune system.

CTCs in Breast Cancer Diagnosis and Treatment

 Acknowledgments

Authors wish to acknowledge Management of Jaslok Hospital and Research Centre, Mumbai for providing facilities to establish CSC laboratory in the Department of Molecular Medicine and Biology. This gave an excellent exposure to develop this important technology in this laboratory and thus to write such an important review on this topic.

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