Circulating tumor cells (CTCs) originate from solid tumors, and represent an early step in blood-borne metastasis. They are a minor fraction of blood cells, as there is usually less than 10 CTCs in 1 ml of blood, thus almost 1 million lower number compared to leukocytes.
To avoid apoptosis connected with lack of attachment, or to escape immune system surveillance, circulating tumor cells reshape their integrin expression profile and upregulate certain cell surface proteins, such as CD47 (IAP) or CD106 (VCAM-1). Unlike leukocytes, CTCs do not express CD45. Typical markers of CTCs are CD326 (EpCAM), CD325 (N-cadherin), CD44 (HCAM), and cytokeratins.
The later ones represent not only a tool for detection of CTCs, but often also for identification of the original tumor (details were presented in our older blog Cytokeratins - markers in tumor diagnostics). As these two goals differ in the requirements on anti-cytokeratin antibodies, there are two types of antibodies, that are being used. As CTCs are very heterogeneous population, for their detection it is better to use pan-reactive antibodies, detecting multiple cytokeratins simultaneously, or even combinations of these pan-reactive antibodies. On the other hand, when trying to identify a particular source of circulating tumour cells, specific combinations of anti-cytokereatin antibodies are needed.
Fig. 1: Flow cytometry surface staining pattern of MCF-7 cell line suspension stained using anti-human CD326 (323/A3) PE antibody (10 μl reagent per million cells in 100 μl of cell suspension). Separation of human MCF-7 cells (red-filled) from SP2 cells (black-dashed) in flow cytometry analysis (surface staining) stained using anti-human CD326 (323/A3) PE antibody (10 μl reagent per million cells in 100 μl of cell suspension).
This is not a simple task, as after leaving the primary tumor, these cells usually change also their cytokeratin profile and down-regulate expression of these antigens, thus they can often express only some of the original cytokeratins. CTC markers help to identify and isolate these very rare cells, hence they can be further characterized by genetic methods, to improve the tumor diagnostics and prediction. To detect those CTCs, that have lost their original epithelial phenotype, or that are of mesenchymal origin, vimentin is being used as a marker. Epithelial-mesenchymal transition, a dedifferentiation process favoring cell migration and associated with gaining stem-like properties, can be monitored by combination of antibodies specific for cytokeratins (pan-cytokeratin) and vimentin.
Fig. 2: Flow cytometry analysis (intracellular staining) of cytokeratin expression in HeLa cells and in peripheral blood cells using anti-cytokeratin antibody (C-11) Alexa Fluor® 488.
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Trop-2 is thought to be associated with the epithelial phenotype of cancer cells and many studies have reported that epithelial markers positively correlate with its expression, whereas mesenchymal markers typically exert negative correlation.
Within the programmed cell death pathway, the interaction between PD-1 and PD-L1 and L2 particularly affects T-lymphocytes, in which they cause a phenomenon called functional exhausting, by attenuating TCR-mediated T-cell proliferation by negative costimulation control of activation of T-lymphocytes during the presentation of antigens by dendritic cells.