Circulating Tumor Cell
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A circulating tumor cell (CTC) is a cell that has shed into the
vasculature The blood circulatory system is a system of organs that includes the heart, blood vessels, and blood which is circulated throughout the entire body of a human or other vertebrate. It includes the cardiovascular system, or vascular system, tha ...
or
lymphatics The lymphatic vessels (or lymph vessels or lymphatics) are thin-walled vessels (tubes), structured like blood vessels, that carry lymph. As part of the lymphatic system, lymph vessels are complementary to the cardiovascular system. Lymph vessel ...
from a primary
tumor A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...
and is carried around the body in the
blood circulation The blood circulatory system is a system of organs that includes the heart, blood vessels, and blood which is circulated throughout the entire body of a human or other vertebrate. It includes the cardiovascular system, or vascular system, tha ...
. CTCs can
extravasate __NOTOC__ Extravasation is the leakage of a fluid out of its container into the surrounding area, especially blood or blood cells from vessels. In the case of inflammation, it refers to the movement of white blood cells from the capillaries to th ...
and become ''seeds'' for the subsequent growth of additional tumors (
metastases Metastasis is a pathogenic agent's spread from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, then ...
) in distant organs, a mechanism that is responsible for the vast majority of cancer-related deaths. The detection and analysis of CTCs can assist early patient prognoses and determine appropriate tailored treatments. Currently, there is one FDA-approved method for CTC detection, CellSearch, which is used to diagnose
breast The breast is one of two prominences located on the upper ventral region of a primate's torso. Both females and males develop breasts from the same embryological tissues. In females, it serves as the mammary gland, which produces and sec ...
,
colorectal The large intestine, also known as the large bowel, is the last part of the gastrointestinal tract and of the digestive system in tetrapods. Water is absorbed here and the remaining waste material is stored in the rectum as feces before being ...
and
prostate The prostate is both an Male accessory gland, accessory gland of the male reproductive system and a muscle-driven mechanical switch between urination and ejaculation. It is found only in some mammals. It differs between species anatomically, ...
cancer. The detection of CTCs, or
liquid biopsy A liquid biopsy, also known as fluid biopsy or fluid phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. Like traditional biopsy, this type of technique is mainly used as a diagnostic and monitoring tool for ...
, presents several advantages over traditional tissue biopsies. They are non-invasive, can be used repeatedly, and provide more useful information on metastatic risk, disease progression, and treatment effectiveness. For example, analysis of blood samples from cancer patients has found a propensity for increased CTC detection as the disease progresses. Blood tests are easy and safe to perform and multiple samples can be taken over time. By contrast, analysis of solid tumors necessitates invasive procedures that might limit patient compliance. The ability to monitor the disease progression over time could facilitate appropriate modification to a patient's therapy, potentially improving their prognosis and quality of life. The important aspect of the ability to prognose the future progression of the disease is elimination (at least temporarily) of the need for a surgery when the repeated CTC counts are low and not increasing; the obvious benefits of avoiding the surgery include avoiding the risk related to the innate tumor-genicity of cancer surgeries. To this end, technologies with the requisite sensitivity and reproducibility to detect CTCs in patients with metastatic disease have recently been developed. On the other hand, CTCs are very rare, often present as only a few cells per milliliter of blood, which makes their detection challenging. In addition, they often express a variety of markers which vary from patient to patient, which makes it difficult to develop techniques with high
sensitivity and specificity ''Sensitivity'' and ''specificity'' mathematically describe the accuracy of a test which reports the presence or absence of a condition. Individuals for which the condition is satisfied are considered "positive" and those for which it is not are ...
.


Types

CTCs that originate from
carcinomas Carcinoma is a malignancy that develops from epithelial cells. Specifically, a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that arises from cells originating in the endodermal, mesoder ...
(cancers of epithelial origin, which are the most prevalent) can be classified according to the expression of epithelial markers, as well as their size and whether they are apoptotic. In general, CTCs are
anoikis Anoikis is a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix (ECM). Usually cells stay close to the tissue to which they belong since the communication between proxima ...
-resistant, which means that they can survive in the bloodstream without attaching to a substrate. # Traditional CTCs are characterised by an intact, viable nucleus; the expression of
EpCAM Epithelial cell adhesion molecule (EpCAM), also known as CD326 among other names, is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell–cell adhesion in epithelia. EpCAM is also involved in cell signaling, migration, prol ...
and
cytokeratins Cytokeratins are keratin proteins found in the intracytoplasmic cytoskeleton of epithelial tissue. They are an important component of intermediate filaments, which help cells resist mechanical stress. Expression of these cytokeratins within epi ...
, which demonstrate epithelial origin; the absence of CD45, indicating the cell is not of hematopoietic origin; and their larger
size Size in general is the Magnitude (mathematics), magnitude or dimensions of a thing. More specifically, ''geometrical size'' (or ''spatial size'') can refer to linear dimensions (length, width, height, diameter, perimeter), area, or volume ...
, irregular shape or subcellular morphology. # Cytokeratin-negative CTCs are characterised by the lack of EpCAM or cytokeratins, which may indicate an undifferentiated phenotype (circulating
cancer stem cells Cancer stem cells (CSCs) are cancer cells (found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sampl ...
) or the acquisition of a mesenchymal phenotype (known as epithelial-mesenchymal transition or EMT). These populations of CTCs may be the most resistant and most prone to metastasis. They are also more difficult to isolate because they express neither cytokeratins nor CD45. Otherwise, their morphology, gene expression and genomics are similar to those of other cancer cells. # Apoptotic CTCs are traditional CTCs that are undergoing
apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
(programmed cell death). These may be used to monitor treatment response, as done experimentally by the Epic Sciences method, which identifies nuclear fragmentation or cytoplasmic blebbing associated with apoptosis. Measuring the ratio of traditional CTC to apoptotic CTCs—from baseline to therapy—provides clues to treatment efficacy in targeting and killing cancer cells. # Small CTCs are cytokeratin-positive and CD45-negative, but with sizes and shapes similar to white blood cells. Importantly, small CTCs have cancer-specific biomarkers that identify them as CTCs. Small CTCs have been implicated in progressive disease and differentiation into small cell carcinomas, which often require a different therapeutic course.


CTC clusters

CTC clusters are two or more individual CTCs bound together. The CTC cluster may contain traditional, small or CK- CTCs. These clusters have cancer-specific biomarkers that identify them as CTCs. Several studies have reported that the presence of these clusters is associated with increased metastatic risk and poor prognosis. For example, one study involving prostate cancer showed an eight-fold longer mean survival rate for patients with only single CTCs versus those with CTC clusters, while other studies have shown similar correlations for colon cancer. In addition, enumerating CTC clusters can provide useful prognostic information for patients with already elevated CTC levels. However, one study has reported that contrary to existing consensus, at least a discrete population of these clusters are non-malignant, and derive instead from the tumor endothelium. These circulating tumor-endothelial clusters also show epithelial-mesenchymal markers but do not mirror the genetics of the primary tumor. Previously it was assumed that CTC clusters could not pass through narrow vessels, such as capillaries, due to their overall size. However, it has been shown that CTC clusters can "unwind" through "selective cleavage of intercellular adhesions" to traverse these constrictions single-file, then reverse the process once clear. This behavior could be a factor in why CTC clusters have such a significant metastatic potential.


Frequency

The detection of CTCs may have important prognostic and therapeutic implications but because their numbers can be very small, these cells are not easily detected. It is estimated that among the cells that have detached from the primary tumor, only 0.01% can form metastases. Circulating tumor cells are found in frequencies on the order of 1-10 CTC per mL of whole blood in patients with metastatic disease. For comparison, a mL of blood contains a few million white blood cells and a billion red blood cells. This low frequency, associated to difficulty of identifying cancerous cells, means that a key component of understanding CTCs biological properties require technologies and approaches capable of isolating 1 CTC per mL of blood, either by enrichment, or better yet with enrichment-free assays that identify all CTC subtypes in sufficiently high definition to satisfy diagnostic pathology image-quantity requirements in patients with a variety of cancer types. To date CTCs have been detected in several epithelial cancers (breast, prostate, lung, and colon) and clinical evidences indicate that patients with metastatic lesions are more likely to have CTCs isolated. CTCs are usually (in 2011) captured from the vasculature by using specific antibodies able to recognize specific tumoral marker (usually
EpCAM Epithelial cell adhesion molecule (EpCAM), also known as CD326 among other names, is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell–cell adhesion in epithelia. EpCAM is also involved in cell signaling, migration, prol ...
); however this approach is biased by the need for a sufficient expression of the selected protein on the cell surface, event necessary for the enrichment step. Moreover, since EpCAM and other proteins (e.g.
cytokeratin Cytokeratins are keratin proteins found in the intracytoplasmic cytoskeleton of epithelial tissue. They are an important component of intermediate filaments, which help cells resist mechanical stress. Expression of these cytokeratins within epi ...
s) are not expressed in some tumors and can be down regulated during the epithelial to mesenchymal transition ( EMT), new enrichment strategies are required. First evidence indicates that CTC markers applied in human medicine are conserved in other species. Five of the more common markers including CK19 are also useful to detect CTC in the blood of dogs with malignant mammary tumors. Newer approaches are able to identify more cells out 7.5 ml of blood, like IsofFux or Maintrac. In very rare cases, CTCs are present in large enough quantities to be visible on routine
blood smear A blood smear, peripheral blood smear or blood film is a thin layer of blood smeared on a glass microscope slide and then stained in such a way as to allow the various blood cells to be examined microscopically. Blood smears are examined in the ...
examination. This is referred to as
carcinocythemia Carcinocythemia, also known as carcinoma cell leukemia, is a condition in which cells from malignant tumours of non-hematopoietic origin are visible on the peripheral blood smear. It is an extremely rare condition, with 33 cases identified in t ...
or carcinoma cell leukemia and is associated with a poor prognosis.


Detection methods

To date, a variety of research methods have been developed to isolate and enumerate CTCs. The only U.S.
Food and Drug Administration The United States Food and Drug Administration (FDA or US FDA) is a List of United States federal agencies, federal agency of the United States Department of Health and Human Services, Department of Health and Human Services. The FDA is respon ...
(FDA) cleared methodology for enumeration of CTC in whole blood is the CellSearch system. Extensive clinical testing done using this method shows that presence of CTCs is a strong prognostic factor for
overall survival Survival rate is a part of survival analysis. It is the proportion of people in a study or treatment group still alive at a given period of time after diagnosis. It is a method of describing prognosis in certain disease conditions, and can be use ...
in patients with metastatic breast, colorectal or prostate cancer. CTCs are pivotal to understanding the biology of metastasis and promise potential as a biomarker to noninvasively evaluate tumor progression and response to treatment. However, isolation and characterization of CTCs represent a major technological challenge, since CTCs make up a minute number of the total cells in circulating blood, 1–10 CTCs per mL of whole blood compared to a few million white blood cells and a billion red blood cells. Therefore, the major challenge for CTC researchers is the prevailing difficulty of CTC purification that allows the molecular characterization of CTCs. Several methods have been developed to isolate CTCs in the peripheral blood and essentially fall into two categories: biological methods and physical methods, as well as hybrid methods that combine both strategies. Techniques may also be classified based on whether they select CTCs for isolation (positive selection) or whether they exclude all blood cells (negative selection).


Biological methods

Biological methods isolate cells based on highly specific antigen binding, most commonly by monoclonal antibodies for positive selection. Antibodies against tumor specific biomarkers including
EpCAM Epithelial cell adhesion molecule (EpCAM), also known as CD326 among other names, is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell–cell adhesion in epithelia. EpCAM is also involved in cell signaling, migration, prol ...
,
HER2 Receptor tyrosine-protein kinase erbB-2 is a protein that in humans is encoded by the ''ERBB2'' gene. ERBB is abbreviated from erythroblastic oncogene B, a gene originally isolated from the avian genome. The human protein is also frequently refer ...
and PSA have been used. The most common technique is magnetic nanoparticle-based separation (immunomagnetic assay) as used in CellSearch or MACS. Other techniques under research include microfluidic separation and combination of immunomagnetic assay and microfluidic separation. As the development of microfabrication technology, microscale magnetic structures are implemented to provide better control of the magnetic field and assist the CTCs detection. Oncolytic viruses such as
vaccinia ''Vaccinia virus'' (VACV or VV) is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, which encodes approximately 250 genes. The dimensions of the ...
viruses are developed to detect and identify CTCs. Alternative methods exist which use
engineered proteins Engineering is the use of scientific principles to design and build machines, structures, and other items, including bridges, tunnels, roads, vehicles, and buildings. The discipline of engineering encompasses a broad range of more speciali ...
instead of antibodies, such as the malaria VAR2CSA protein, which binds to oncofetal
chondroitin sulfate Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating sugars ( N-acetylgalactosamine and glucuronic acid). It is usually found attached to proteins as part of a proteoglycan. A chondroitin chain can have ov ...
on the surface of CTCs. CTCs may also be retrieved directly from the blood by a modified
Seldinger technique The Seldinger technique, also known as Seldinger wire technique, is a medical procedure to obtain safe access to blood vessels and other hollow organs. It is named after Sven Ivar Seldinger (1921–1998), a Swedish radiologist who introduced th ...
, as developed by GILUPI GmbH. An antibody coated metal wire is inserted into a peripheral vein and stays there for a defined period (30 min). During this time, CTCs from the blood can bind to the antibodies (currently anti-EpCAM). After the incubation time, the wire is removed, washed and the native CTCs, isolated from the blood of the patient, can be further analysed. Molecular genetics as well as immunofluorescent staining and several other methods are possible. Advantage of this method is the higher blood volume that can be analysed for CTCs (approx. 750 ml in 30 min compared to 7.5 ml of a drawn blood sample).


CellSearch method

CellSearch is the only FDA-approved platform for CTC isolation. This method is based on the use of iron nanoparticles coated with a polymer layer carrying biotin analogues and conjugated with antibodies against EpCAM for the capture of CTCs. Isolation is coupled to an analyzer to take images of isolated cells upon their staining with specific fluorescent antibody conjugates. Blood is sampled in an
EDTA Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2sub>2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes ev ...
tube with an added preservative. Upon arrival in the lab, 7.5mL of blood is centrifuged and placed in a preparation system. This system first enriches the tumor cells immunomagnetically by means of ferrofluid nanoparticles and a magnet. Subsequently, recovered cells are permeabilized and stained with a nuclear stain, a fluorescent antibody conjugate against
CD45 Protein tyrosine phosphatase, receptor type, C also known as PTPRC is an enzyme that, in humans, is encoded by the ''PTPRC'' gene. PTPRC is also known as CD45 antigen (CD stands for cluster of differentiation), which was originally called leuko ...
(leukocyte marker) and
cytokeratin Cytokeratins are keratin proteins found in the intracytoplasmic cytoskeleton of epithelial tissue. They are an important component of intermediate filaments, which help cells resist mechanical stress. Expression of these cytokeratins within epi ...
s 8, 18 and 19 (epithelial markers). The sample is then scanned on an analyzer which takes images of the nuclear, cytokeratin, and CD45 stains. To be considered a CTC a cell must contain a nucleus, be positive for cytoplasmic expression of cytokeratin as well as negative for the expression of CD45 marker, and have a diameter larger than 5 µm. If the total number of tumor cells found to meet the criteria cited above is 5 or more, a blood sample is positive. In studies done on prostate, breast and colon cancer patients, median survival of metastatic patients with positive samples is about half the median survival of metastatic patients with negative samples. This system is characterized by a recovery capacity of 93% and a detection limit of one CTC per 7.5 mL of whole blood. For specific cancer types, alternative methods such as IsoFlux have shown greater sensitivity.


Parsortix® method

This automated method uses size filtration to enrich larger and less compressible circulating tumor cells from other blood components. The Parsorti®x system can take in blood samples ranging from 1 mL to 40 mL. A disposable microfluidic cassette with a 6.5 micron gap allows the vast majority of red blood cells and white blood cells to pass through, while larger rare cells, including circulating tumor cells and fetal cells get caught. Trapped cells can either be automatically stained with antibodies for identification or can be released out of the cassette for subsequent analysis. These released / harvested cells are alive and can be analyzed by downstream cellular and molecular techniques, as well as cultured. The filtration cassette captures a plethora of different cancer cell types. In May 2022, the Parsortix® PC1 system was cleared by the FDA as a medical device for the capture and harvest of circulating tumor cells (CTCs) from metastatic breast cancer patient blood for subsequent analysis. In addition to the IVD application, the PC1 may be used with the MBC-01 Metastatic Breast Cancer Kit for use in research studies or Lab Developed Tests (LDTs) that have been created and validated in a clinical laboratory.


Epic Sciences method

This method involves technology to separate nucleated cells from red blood cells, which lack a nucleus. All nucleated cells, including normal white blood cells and CTCs, are exposed to fluorescent-tagged antibodies specific for cancer biomarkers. In addition, Epic's imaging system captures pictures of all the cells on the slide (approximately 3 million), records the precise coordinates of each cell, and analyzes each cell for 90 different parameters, including the fluorescence intensity of the four fluorescent markers and 86 different morphological parameters. Epic can also use
FISH Fish are aquatic, craniate, gill-bearing animals that lack limbs with digits. Included in this definition are the living hagfish, lampreys, and cartilaginous and bony fish as well as various extinct related groups. Approximately 95% of li ...
and other staining techniques to look for abnormalities such as duplications, deletions, and rearrangements. The imaging and analysis technology also allows for the coordinates of every cell on a slide to be known so that a single cell can be retrieved from the slide for analysis using next-generation sequencing. A hematopathology-trained algorithm incorporates numerous morphology measurements as well as expression from cytokeratin and CD45. The algorithm then proposes candidate CTCs that a trained reader confirms. Cells of interest are analyzed for relevant phenotypic and genotypic markers, with regional white blood cells included as negative controls. Epic's molecular assays measure protein expression and also interrogate genomic abnormalities in CTCs for more than 20 different cancer types.


Maintrac

Maintrac is a diagnostic blood test platform applying microscopic ''in vitro'' diagnostic methods to identify rare cells in body fluids and their molecular characteristics. It is based on positive selection using EpCAM-specific antibodies. Maintrac uses an approach based on microscopic identification of circulating tumor cells. To prevent damage and loss of the cells during the process, Maintrac uses just two steps towards the identification. In contrast to many other methods, maintrac does not purify the cells or enrich them, but identifies them within the context of the other blood compounds. To obtain vital cells and to reduce stress of those cells, blood cells are prepared by only one centrifugation step and erythrocyte lysis. Like CellSearch, maintrac uses an EpCAM antibody. It is, however, not used for enrichment but rather as a fluorescent marker to identify those cells. Together with the nuclear staining with propidium iodide the maintrac method can distinguish between dead and living cells. Only vital, propidium excluding EpCAM positive cells are counted as potential tumor cells. Only living cells can grow into tumors, therefore dying EpCAM positive cells can do no harm. The suspension is analysed by fluorescence microscopy, which automatically counts the events. Simultaneous event galleries are recorded to verify whether the software found a true living cell and to differentiate between skin epithelial cells for example. Close validation of the method showed that additional antibodies of cytokeratins or CD45 did not have any advantage. Unlike other methods maintrac does not use the single cell count as a prognostic marker, rather Maintrac utilizes the dynamics of the cell count. Rising tumor cell numbers are an important factor that tumor activity is ongoing. Decreasing cell counts are a sign for a successful therapy. Therefore, maintrac can be used to verify the success of a chemotherapy and to supervise the treatment during hormone or maintenance therapy Maintrac has been used experimentally to monitor cancer recurrence. Studies using Maintrac have shown that EpCAM positive cells can be found in the blood in patient without cancer. Inflammatory conditions like Crohn's disease also show increased levels of EpCAM-positive cells. Patients with severe skin burns can also carry EpCAM positive cells in the blood. Therefore, the use of EpCAM-positive cells as a tool for early diagnosis is not optimal.


Physical methods

Physical methods are often filter-based, enabling the capture of CTCs by
size Size in general is the Magnitude (mathematics), magnitude or dimensions of a thing. More specifically, ''geometrical size'' (or ''spatial size'') can refer to linear dimensions (length, width, height, diameter, perimeter), area, or volume ...
rather than by specific
epitope An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The epitope is the specific piece of the antigen to which an antibody binds. The p ...
s. ScreenCell is a filtration based device that allows sensitive and specific isolation of CTCs from human whole blood in a few minutes. Peripheral blood is drawn and processed within 4 hours with a ScreenCell isolation device to capture CTCs. The captured cells are ready for cell culture or for direct characterization using ViewRNA in situ hybridization assay. The Parsortix method separates CTCs based on their size and deformability.


Hybrid methods

Hybrid methods combine physical separation (by gradients, magnetic fields, etc.) with antibody-mediated cell retrieval. An example of this is a sensitive double gradient centrifugation and magnetic cell sorting detection and enumeration method which has been used to detect circulating epithelial cancer cells in breast cancer patients by negative selection. The principle of negative selection is based on the retrieval of all
blood cells A blood cell, also called a hematopoietic cell, hemocyte, or hematocyte, is a cell produced through hematopoiesis and found mainly in the blood. Major types of blood cells include red blood cells (erythrocytes), white blood cells (leukocytes), a ...
by using a panel of antibodies as well as traditional gradient centrifugation with
Ficoll Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll radii range from 2-7 nm. It is prepared by reaction of the polysaccharide with epichlorohydrin. Ficoll is a regi ...
. A similar method known as ISET Test has been employed to detect circulating prostate cancer cells and another technique known as RosetteStep has been used to isolate CTCs from
small-cell lung cancer Small-cell carcinoma is a type of highly malignant cancer that most commonly arises within the lung, although it can occasionally arise in other body sites, such as the cervix, prostate, and gastrointestinal tract. Compared to non-small cell c ...
patients. Similarly, researchers at Massachusetts General Hospital have developed a negative selection method which employs inertial focusing on a microfluidic device. The technique, called CTC-iChip, first removes cells too small to be CTCs, such as red blood cells, and then uses magnetic particles to remove white blood cells.


CTC characterization

Some drugs are particularly effective against cancers which fit certain requirements. For example,
Herceptin Trastuzumab, sold under the brand name Herceptin among others, is a monoclonal antibody used to treat breast cancer and stomach cancer. It is specifically used for cancer that is HER2 receptor positive. It may be used by itself or together wi ...
is very effective in patients who are
Her2 Receptor tyrosine-protein kinase erbB-2 is a protein that in humans is encoded by the ''ERBB2'' gene. ERBB is abbreviated from erythroblastic oncogene B, a gene originally isolated from the avian genome. The human protein is also frequently refer ...
positive, but much less effective in patients who are Her2 negative. Once the primary tumor is removed, biopsy of the current state of the cancer through traditional tissue typing is not possible anymore. Often tissue sections of the primary tumor, removed years prior, are used to do the typing. Further characterization of CTC may help determining the current tumor phenotype. FISH assays have been performed on CTC as well as determination of
IGF-1R The insulin-like growth factor 1 (IGF-1) receptor is a protein found on the surface of human cells. It is a transmembrane receptor that is activated by a hormone called insulin-like growth factor 1 ( IGF-1) and by a related hormone called IGF-2. ...
, Her2,
Bcl-2 Bcl-2 (B-cell lymphoma 2), encoded in humans by the ''BCL2'' gene, is the founding member of the Bcl-2 family of regulator proteins that regulate cell death (apoptosis), by either inhibiting (anti-apoptotic) or inducing (pro-apoptotic) apoptosi ...
,
ERG The erg is a unit of energy equal to 10−7joules (100 nJ). It originated in the Centimetre–gram–second system of units (CGS). It has the symbol ''erg''. The erg is not an SI unit. Its name is derived from (), a Greek word meaning 'work' o ...
, PTEN, AR status using immunofluorescence. Single cell level qPCR can also be performed with the CTCs isolated from blood. The organ tropism of patient-derived CTC has been investigated in a mouse model. CTCs isolated from breast cancer patients and expanded ''in vitro'' showed they could generate bone, lung, ovary and brain metastases in mice, partially reflecting the secondary lesions as found in the corresponding patients. Remarkably, one CTC line—isolated long before the appearance of brain metastasis in patient—was highly competent to generate brain metastasis in mice. This was the first predictive case for brain metastasis and a proof of concept that intrinsic molecular features of metastatic precursors amongst CTCs could provide novel insights into the mechanisms of metastasis.


Cell morphology

Morphological appearance is judged by human operators and is therefore subject to large inter operator variation. Several CTC enumeration methods exist which use morphological appearance to identify CTC, which may also apply different morphological criteria. A recent study in prostate cancer showed that many different morphological definitions of circulating tumor cells have similar prognostic value, even though the absolute number of cells found in patients and normal donors varied by more than a decade between different morphological definitions.


History

CTCs were observed for the first time in 1869 in the blood of a man with metastatic cancer by Thomas Ashworth, who postulated that "cells identical with those of the cancer itself being seen in the blood may tend to throw some light upon the mode of origin of multiple tumours existing in the same person". A thorough comparison of the
morphology Morphology, from the Greek and meaning "study of shape", may refer to: Disciplines * Morphology (archaeology), study of the shapes or forms of artifacts * Morphology (astronomy), study of the shape of astronomical objects such as nebulae, galaxies ...
of the circulating cells to tumor cells from different lesions led Ashworth to conclude that "One thing is certain, that if they TCcame from an existing cancer structure, they must have passed through the greater part of the circulatory system to have arrived at the internal saphena vein of the sound leg". The importance of CTCs in modern cancer research began in the mid 1990s with the demonstration that CTCs exist early on in the course of the disease. Those results were made possible by exquisitely sensitive magnetic separation technology employing
ferrofluid Ferrofluid is a liquid that is attracted to the poles of a magnet. It is a colloidal liquid made of nanoscale ferromagnetic or ferrimagnetic particles suspended in a carrier fluid (usually an organic solvent or water). Each magnetic particle ...
s (colloidal magnetic nanoparticles) and high gradient magnetic separators invented by Paul Liberti and motivated by theoretical calculations by Liberti and Leon Terstappen that indicated very small tumors shedding cells at less than 1.0% per day should result in detectable cells in blood. A variety of other technologies have been applied to CTC enumeration and identification since that time. Modern cancer research has demonstrated that CTCs derive from clones in the primary tumor, validating Ashworth's remarks. The significant efforts put into understanding the CTCs biological properties have demonstrated the critical role circulating tumor cells play in the metastatic spread of carcinoma. Furthermore, highly sensitive, single-cell analysis demonstrated a high level of heterogeneity seen at the single cell level for both protein expression and protein localization and the CTCs reflected both the primary biopsy and the changes seen in the metastatic sites.


References

{{DEFAULTSORT:Circulating Tumor Cell Anatomical pathology Oncology