Circulating cancer cells (CCC)
Circulating tumor cells (CTC) are tumor cells spread in blood and/or lymphatic vessels from solid tumors, thus including all types of tumor cells except those derived from leukemia and lymphoma. CTC may also circulate as aggregated tumor cells, which are defined as circulating tumor microemboli or “collective tumor cell migration (CTM)” (Fig. 1).
Tumor cells may circulate in blood spontaneously, i.e., because of their invasive capabilities or for other causes of cell spreading. Spontaneous circulation of tumor cells represents the early hallmark of the invasive behavior of a proportion of cancer cells and the first step of the process leading to the formation of metastases, which are known to account for 90 % of cancer-related morbidity and mortality. Nonspontaneous circulation of tumor cells may derive from iatrogenic invasive procedures (biopsy, surgical intervention, etc.), tumor compression, and tumor inflammation.
The process by which tumor cells spreading from solid tumors give rise to metastases includes the following steps (Fig. 2): tumor cell growth involving genetic and epigenetic changes and tumor-induced microenvironment reprogramming (Meseure et al. 2014), angiogenesis, tumor cell detachment, epithelial to mesenchymal transi-
tion (EMT), motility, intravasation, survival in vessels and embolization, collective tumor cell migration (CTM), possible extravasation, mesenchymal to epithelial transition (MET), formation of micrometastases, and growth of macrometastases. Epithelial–mesenchymal cell plasticity is thought to play a central role in cancer progression, generation of cancer stem cells (CSC), and metastasis formation (Ye and Weinberg 2015).
Fig. 1. CTC and CTM enriched by ISET and diagnosed by cytopathology. (a) CTC from a patient with mesothelioma (May–GrЂunwald–Giemsa staining, 100x). (b) CTM from a patient with breast cancer (May–GrЂunwald–Giemsa staining, 100x)
Fig. 2. Main steps leading to development of metastases. Growing tumor cells outstrip oxygen supply and activate angiogenesis. Invading tumor cells undergo the phenotype switch “epithelial to mesenchymal transition (EMT)”: they progressively lose epithelial antigens, acquire mesenchymal antigens, and motile propensities (like fibroblasts). After entering blood vessels (intravasation), circulating tumor cells (CTC) undergo apoptosis or circulate as isolated CTC. After extravasation to distant organs, CTC remain as dormant solitary cells or undergo limited proliferation (micrometastases). Unrestrained CTC proliferation gives rise to metastases, via phenotype reversion “mesenchymal to epithelial transition (MET)” and angiogenesis. Circulating tumor microemboli (CTM) represent “collective tumor cell migration” of tumor cells. They cannot extravasate, but arrest in capillaries and proliferate, rupturing the capillary walls and giving rise to metastases
Growing cells rapidly outstrip the supply of nutrients and oxygen and suffer from stress and hypoxia. Hypoxia-inducible factor (HIF), which mediates the transcriptional response to hypoxia, is a strong promoter of tumor growth and invasion and controls angiogenesis via two key angiogenic factors (VEGF-A and angiopoietin-2). Hypoxia determines cell necrosis and release of inflammatory mediators such as cytokines and chemokines which recruit, among other cells, leukocytes and macrophages. These, in turn, stimulate angiogenesis, extracellular matrix breakdown, and tumor cell motility (Noman et al. 2014). Local production of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor beta (TGF-beta) mediates the control of tumor cell survival/apoptosis balance and of E-cadherin downregulation leading to reduced cell adhesion and increased tumor cell invasiveness.
Hypoxia, acting through LOX induction and Snail activation, leads to E-cadherin repression, a crucial feature of the EMT. Furthermore, platelets may induce CTC to undergo EMT (Meseure et al. 2014; Ye and Weinberg 2015). During EMT, Twist may need to activate antiapoptotic programs in order to allow epithelial cells to convert to a mesenchymal fate while avoiding anoikis.
It is noteworthy that, in the journey of the tumor cells from the tumor tissue to the bloodstream and to the metastatic site, the cross talk of tumor cells with the microenvironment of the “primary” tissue, of blood, and of the metastatic tissue and the plasticity of tumor cells, i.e., their ability to shift from a differentiated to an undifferentiated phenotype and vice versa, are thought to play a central role in metastasis formation (Meseure et al. 2014; Ye and Weinberg 2015; Noman et al. 2014).
Epithelial cancer cells have very low survival rates in circulation. Animal studies, in which tumor cells are directly introduced into the systemic circulation, have established that approximately 1/40 CTC give rise to micrometastases and only approximately 0.01 % proliferate into macrometastasis (Luzzi et al. 1998). The fate of intravasated tumor cells includes a rapid phase of intravascular cancer cell disappearance related to sheer forces, detection by immune system, and “anoikis.” Many cancer cell types with increased metastatic potential are resistant to anoikis and to elimination by the immune system compared with the parental cells, a tumor cell behavior related to the expression of apoptosis inhibitors and CTC chaperoning by endothelial cells and/or platelets.
Metastatic inefficiency is principally determined by CTC susceptibility to apoptosis, failure of solitary cells extravasated in distant organs to initiate growth, and failure of early micrometastases in distant organs to stimulate angiogenesis and continue growth into macromatastases.
Both solitary cells in organs, defined as disseminated tumor cells (DTC), and micrometastases may remain in “dormancy” for years (Sosa et al. 2014). The immune system and angiogenesis have been shown to play a role in tumor cell dormancy, as well as extracellular and stromal microenvironments, autophagy, tumor cell epigenetics, and heterogeneity (Sosa et al. 2014). Finally, it has been suggested that any factor that tips the balance between proliferation and apoptosis may result in tumor progression or regression.
The mechanisms involved in the preferential choice of a target organ for metastatic tumor cell proliferation (“seed and soil” theory) are still not completely understood, but include the close interaction between tumor cells (the “seeds”) and the microenvironment of the “soil” (Langley and Fidler 2011). Organ-specific attractant molecules (chemokines) can stimulate migrating tumor cells to invade the walls of blood vessels and enter specific organs. Tumor–endothelial interaction, appropriate adhesion molecules expressed by endothelial cells in distant organs, and local growth factors can drive metastatic tumor cell proliferation. Once the target organ is reached, mesenchymal-like CTC may need to reverse to epithelial-like tumor cells via MET in order to regain the ability to proliferate.
Tumor cells can also invade as multicellular aggregates or clusters, a process known as “collective tumor cell migration.” Multicellular aggregates of tumor cells, also called circulating tumor microemboli (CTM), are thought to have potential advantages for survival, proliferation, and establishment of micrometastatic lesions in distant organs. Actually, it has been shown that CTM may bring their own soil and give rise to metastasis without extravasation, by proliferating within the vasculature (Fig. 2). Thus, it is generally accepted that the presence of CTM in blood is a marker of highly metastatic potential (Paterlini-Breґchot 2014).
Convergent results have led to the present knowledge that invasion can be early and sometimes clinically dormant (Sosa et al. 2014). Tumor cell dissemination may precede evident primary tumor outgrowth by many years (Sosa et al. 2014; Kohn and Liotta 1995). The capacity to metastasize may be preordained by the spectrum of mutations acquired early in tumorigenesis, which means that some cancers start out “on the wrong foot.” In fact, it has been demonstrated that cancer cells in the primary tumor may harbor a gene expression signature matching that observed in the metastatic colony and that this signature can help to predict whether the tumor will remain localized or not (Soundararajan et al. 2015).
CTC detection and characterization
The challenge of CTC/CTM detection is related to the requirement of high sensitivity combined with high specificity. Since invasion can start very early during tumor development, identification and counting of CTC when they are very rare (few CTC/CTM per 10 ml of blood, which means few CTC/CTM mixed with approximately 50–100 million leukocytes and 50 billion erythrocytes) could alert the oncologist about a developing tumor invasion process (Paterlini-Breґchot 2014).
Specificity is also an absolute requirement in this field. In fact, a wrong identification of circulating epithelial non-tumor cells as “tumor cells” is expected to generate wrong clinical and therapeutical choices with bad impact on cancer patients’ survival.
Indirect methods to detect CTC do not provide a diagnostic identification of CTC (Paterlini-Breґchot 2014) as they target epithelial cells and/or use organ-specific markers which identify cells from organs but do not demonstrate their tumorous nature. These include immune-mediated methods and RT-PCR (reverse transcriptase polymerase chain reaction) methods. Since antigens or transcripts completely specific for CTC are not known (i.e., antigens or transcripts expressed by all tumor cells from a solid tumor type and not expressed by leukocytes nor by other circulating non-tumor cells), epithelial-specific or organ-specific antigens have been used to identify CTC (for instance, EpCAM, BerEP4, cytokeratins).
However, due to the lack of tumor specificity, epithelial-specific antibodies and transcripts have been proven to generate false-positive results through the biased detection of circulating non-tumor epithelial cells. Furthermore, epithelial-specific antibodies and transcripts can generate false-negative results since they cannot detect invasive circulating tumor cells which have lost their epithelial antigens due to the EMT process. Finally, CTM cannot be reliably detected by immune-mediated and RT-PCR approaches (as multiple cell labeling tends to dissociate tumor cell aggregates and RT-PCR methods destroy cell membranes). Thus, it appears that a reliable unbiased isolation and diagnostic identification of CTC and CTM cannot be based on the expression of epithelial-specific antigens or transcripts.
Accordingly, since the term circulating tumor cells (CTC) has been referred to circulating cells detected with methods using epithelial-specific antigens or transcripts, which have been demonstrated to generate false-positive and falsenegative results, the terms of circulating cancer cells (CCC) and circulating cancer microemboli (CCM) have been introduced in 2014 to indicate circulating cells and microemboli isolated from blood without antibody-dependent bias and diagnostically (i.e., virtually without false-positive and false-negative results) identified by cytopathology (Paterlini-Breґchot 2014).
Direct methods, in particular density gradient isolation and ISET (isolation by size of tumor cells), which do not rely on the use of antibodies, isolate all types of CTC from blood without introducing bias of selection, thus without losing the most invasive tumor cells which have lost epithelial antigens. When they are followed by cytopathological analysis, they provide the diagnostic identification of CCC (Paterlini-Breґchot 2014).
CTC molecular characterization has revealed genetic heterogeneity of CTC and may detect potentially theranostic genetic abnormalities, useful to select targeted therapies and/or to detect escape mutants. Genotyping of CTC can be performed by several approaches including FISH (fluorescence in situ hybridization), CGH (comparative genomic hybridization), and NGS (next-generation sequencing). Analyses of oncogene abnormalities (e.g., HER2, ALK, BRAF) can be performed by FISH or by quantitative PCR. Immunolabeling of cancer cells isolated without using antibodies is an interesting approach to identify mutated oncogenic proteins (e.g., ALK, BRAF) and to characterize their invasive potential, for instance, through the expression of HER-2, metalloproteinases, EGF-R, uPAR, and alpha-fetoprotein.
Detection of apoptotic cells (for instance, by TUNEL (TdT-uridine nick end labeling) analysis) may be relevant before and after anticancer therapy, in order to assess the proapoptotic effect of therapeutic programs. However, the method used to prepare the cells for analysis may induce apoptotic cell death in cells made fragile by blood storage, multiple manipulations, and magnetic particles.
CTC culture, although potentially useful for CTC characterization and drug sensitivity studies, has been shown to be difficult, inconsistent, and with low efficiency up to now (Paterlini-Breґchot 2014).
CTC characterization assays are expected to expand our knowledge of the invasion process and generate new data aimed at improving cancer patients’ diagnosis, follow-up, and treatment. However, it is noteworthy that addressing characterization studies only to a proportion of CTC isolated by antibody-dependent approaches is susceptible to generate biased results and false conclusions potentially leading to harmful clinical choices.
Clinical impact of CTC detection
Several studies have shown the potential of CTC/CTM detection and counting for cancer prognosis and follow-up. However, the clinical impact of CTC detection is not completely established because a substantial number of studies do not meet essential criteria for quality assurance, stressing the need for a gold standard assay based on a highly sensitive, unbiased isolation of
CTC and their diagnostic cytopathologic detection (i.e., a gold standard method for CCC detection) (Paterlini-Breґchot 2014). This approach is crucial to assess the clinical impact of CCC by performing large clinical trials focused on patients with different types of solid cancers at different clinical stages. These trials are expected to generate reliable results and provide guidelines to the clinical use of CCC. In this setting, it is noteworthy that CCC diagnosed by a direct cytopathological assay (ISET) have been demonstrated to detect lung cancer before CT scan leading to its early diagnosis and surgical eradication (Ilie et al. 2014).
Cell plasticity — Capacity of cells to adopt the biological properties (gene expression profile, phenotype, etc.) of other undifferentiated (stem) or differentiated types of cells.
Escape mutants — Mutated forms of a microorganism or tumor cell which escape the attack of immune system or selected therapy.
Liquid biopsy — Detection of circulating tumor cells and/or cell-free molecules (DNA, RNA, miRNA) shed in blood by the primary tumor and/or metastases.
Theranostic — A form of diagnostic testing employed for selecting targeted therapy.
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