Circulating tumor cell
A circulating tumor cell is a cancer cell from a primary tumor that has shed into the blood of the circulatory system, or the lymph of the lymphatic system. The circulatory system is a highly toxic environment for CTCs, and they must make significant adaptations to survive. If CTCs successfully adapt to the physical and biological threats of the circulatory system, they will be carried around the body to other organs where they may leave the circulation. When CTCs exit circulation they become the seeds for the subsequent growth of secondary tumors. This is known as metastasis, which is responsible for most 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, colorectal and prostate cancer.
The detection of CTCs, or liquid biopsy, 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 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.
Types
CTCs that originate from carcinomas 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-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 and cytokeratins, which demonstrate epithelial origin; the absence of CD45, indicating the cell is not of hematopoietic origin; and their larger size, irregular shape or subcellular morphology.
- Cytokeratin-negative CTCs are characterised by the lack of EpCAM or cytokeratins, which may indicate an undifferentiated phenotype or the acquisition of a mesenchymal phenotype. 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. 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
There are two types of circulating tumor cell cluster, one that consists of cancer cells only is termed homotypic. A CTC cluster that also incorporates other cells including white blood cells, fibroblasts, endothelial cells, and platelets, is termed heterotypic. Heterotypic clusters are also known as microemboli. It is suggested that these microemboli might enhance metastatic potential.
The cancer exodus hypothesis suggests that CTC clusters remain intact throughout the metastatic process, rather than dissociating into single cells, which was previously assumed. According to this hypothesis, the clusters enter the bloodstream, travel as a cohesive unit, and exit circulation at distant metastatic sites without breaking apart. This allows the clusters to retain their multicellularity, enhancing their metastatic efficiency. The hypothesis posits that the survival advantage provided by intercellular support within clusters increases their metastatic potential compared to single CTCs.
CTC clusters exhibit distinct gene expression profiles, which confer resistance to certain cancer therapies, making them more resilient than individual tumor cells. Their ability to remain multicellular throughout metastasis, may explain their superior survival and metastatic potential.
Research on CTC clusters and their role in metastasis continues to evolve, with the cancer exodus hypothesis offering a new perspective on how these clusters contribute to cancer progression. Detecting and analyzing CTC clusters provides critical prognostic information and could help guide therapeutic decisions for cancer patients.
Surviving Circulation
For most cancers, tumor cells must enter the bloodstream and survive as circulating tumor cells before metastasizing. However, the circulatory system is a highly hostile environment, and less than 0.01% of CTCs will survive in circulation and go on to metastasize. Cancer cells in circulation face several major threats, including fluid shear stress, deformation, anoikis, and immune 'system attack' from cytotoxic T cells and natural killer cells. To establish metastases, circulating tumor cells must adapt to withstand or evade these threats. These challenges, along with the best-understood mechanisms cancer cells use to overcome them, are described in more detail below.Fluid Shear Stress
Fluid shear stress is the mechanical force exerted by a moving fluid on a solid object, which, in this case, refers to the force of fluid in the bloodstream on the tumor cell. Exposure to such stresses can rapidly damage or kill many detached tumor cells, so circulating tumor cells must adapt to survive FSS in order to metastasize.One way CTCs adapt to FSS is by creating a physical barrier between themselves and the fluid. To form this physical barrier, tumor cells may circulate in clusters, which distribute forces across multiple cells, reducing the stress on any individual cell. The clusters may consist solely of tumor cells or include various other cell types including macrophages, red blood cells, and fibroblasts. Additionally, CTCs often recruit platelets to form a shield that absorbs the fluid shear forces.
CTCs also exhibit cytoskeletal adaptations in response to FSS. The findings across studies are inconsistent: some studies suggest that CTCs increase cytoskeletal stiffness to withstand deformation, while others suggest they become more fluid to accommodate the mechanical stress. However, it is clear that CTCs' cytoskeletal responses are vital to their survival under FSS.
Deformation
Circulating tumor cells are larger than most capillaries, so the cells are forced into a very different shape as they attempt to squeeze through these narrow vessels. When the cell is forced into a different shape, it is called deformation. Deformation puts significant strain on the plasma membrane, cytoskeleton, and nucleus, and this strain can easily rupture the cell.The nucleus is a large, stiff, and vital organelle, so it is often the limiting factor in how much the cell can deform. The nuclear matrix is a cross-linking layer of proteins that gives structure to the membrane of the nucleus. This matrix is made up of lamins, either type A/C or type B. Lamin A/C is stiffer than lamin B. When a cell nucleus has more lamin B than lamin A/C, it can better withstand the compressive forces of deformation. However, this also makes the cell more prone to damage from fluid shear stress.
Additionally, deformation leads to the activation of many signaling pathways within the cell to help the cytoskeleton adapt to the compression. The best understood of these pathways is the RhoA-ROCK pathway. When the cell membrane is stretched, mechanosensitive channels will open, flooding the cell with calcium ions. These calcium ions activate a number of proteins that eventually activate RhoA, a GTPase. RhoA activates ROCK, a protein kinase, which activates myosin light chain and deactivates myosin light chain phosphatase. This allows myosin to bind to actin and contract the cytoskeleton, causing the whole cell to constrict.