Immune escape of Circulating Tumor Cells mediated by tumor derived molecules

1. Molecules produced by tumor cells

1.1 Membrane proteins

NK cells are important cytotoxic cells in the immune system, as they can kill tumor cells by releasing cytotoxic granules containing perforin and granzymes and this function can be regulated by activating receptors on the cell surface such as NKG2D (Natural killer group2, member D), DNAM-1(DNAX accessory molecule-1), and NCR (Natural Cytotoxicity Receptors, NKp30, NKp44, NKp46). CTCs can inhibit the function of NK cells by abnormally expressing many membrane proteins.

1.1.1 Survivin

Survivin, a major inhibitor of apoptosis protein, is expressed in normal tissues, but its expression is upregulated by adipokines such as leptin or cytokines in tumor cells including CTCs (Nicolazzo et al, 2017). Jiang et al. (2007) reported that the expression of survivin in breast cancer cells was activated by the leptin/STAT3 signaling pathway. Survivin can block the cytotoxicity of NK cells by inhibiting the formation of the death-inducing signaling complex (DISC), and the activation of caspase-6 to resist granzyme B (Végran et al, 2013).

1.1.2 Major histocompatibility complex (MHC)

Major histocompatibility complex (MHC) class I molecules participate in immune activation and cytotoxic killing of the abnormal cells. Down-regulation or loss of expression is an important strategy for CTCs to escape the immune effector cells (Yoshihama et al, 2016). The mechanism of MHC class I gene transactivation is not very clear. Since an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein NLRC5 is a critical regulator for the transcriptional activation of MHC class I genes and other genes involved in the MHC class I antigen presentation pathway, its mutation may lead to the down-regulation or loss of MHC I (Vijayan et al, 2019). CTCs also can use platelet-derived MHC class I as a pseudonormal phenotype to escape NK cell immunosurveillance (Placke et al, 2012).

1.1.3 NK-cell receptors and ligands

NKG2D serves as a major activating receptor for NK cells. NKG2D ligand (NKG2DL), which is often expressed in tumor cells, can activate NK cells by binding to NKG2D receptor, and activated NK cells can then kill tumor cells. Tumor cells themselves can also express NKG2D receptor and compete with NK cells for binding to NKG2DL, thereby indirectly inhibiting the activation of NK cells (Tang et al, 2016). Tumor cells also can reduce the expression of NKG2DL at levels of transcription, translation and post-translation and thereby escape recognition by NK cells (Duan et al, 2019).

1.1.4 TRF2

Upregulation of a telomere stability protein TRF2 in cancer cells can facilitate the recruitment of Myeloid-derived suppressor cells (MDSCs) via the TLR2/MyD88/IL-6/STAT3 pathway, leading to the inhibition of NK cells (Cherfils-Vicini et al, 2019). TRF2 was also found to be highly expressed in CTCs (Long et al, 2016); therefore it can be inferred CTCs can escape from the immune surveillance in the same way.

1.1.5 Programmed death ligand 1 (PD-L1) and CD47

PD-L1 on the surface of CTCs provides a "don't find me" signal to the immune system. It can induce the apoptosis of activated T-cells via specific binding with the receptor molecule of PD-1 on T-cells. CD47 is a ubiquitously expressed glycoprotein, and plays a critical role in self-recognition. CD47 on the surface of CTCs can interact with signal regulatory protein-alpha (SIRPα) expressed on myeloid cells, and deliver an anti-phagocytic-"don't eat me"-signal (Liu et al, 2017). Silencing of CD47 and PD-L1 could significantly inhibit the growth of tumors and increased anti-tumor T cell and NK cell response (Lian et al, 2019).

1.1.6 Fas/Fas ligand (CD95/CD95L)

Fas receptor is a death receptor localized on the surface of various cells, which triggers a signal transduction pathway leading to apoptosis with its ligand FasL. The Fas/Fas ligand (CD95/CD95L) system utilized by antigen-specific T cells can play important roles in lymphocyte-mediated tumor regression in vivo (Abrams, 2005). Tumor cells can express non-functional Fas or non/low-expressed Fas, thus they can avoid the apoptosis mediated by FasL on the surface of T cells.

Up-regulation of CD95 in peripheral T-helper cells or CD95L in CTCs can induce the apoptosis of T cells via the CD95/CD95L (ligand) pathway, and lead to tumor cell escape (Gruber et al, 2013).

1.1.7 Human leukocyte antigen-G (HLA-G)

In many types of cancers, HLA-G is found to express in seven different forms, four are membrane-bound (HLA-G1 to -G4) and three are soluble (HLA-G5 to -G7 ). The soluble molecules secreted via extracellular vesicles (EVs) were found to be highly related to the detection of stem cell-like circulating tumor cells (König et al, 2016). HLA-G is considered to be a crucial molecule in immune escape (Garziera et al, 2014). It can interact with inhibitory receptors such as immunoglobulin-like transcript 2 (ILT2) and immunoglobulin-like transcript 4 (ILT4) and the killer cell immunoglobulin-like receptor [KIR2DL4/p49 (CD158d)], on the immune effector cells, such as NK cells, T cells, B cells, endothelial, macrophages, monocytes and DCs, inhibiting their cytotoxicity,, proliferation/differentiation, and suppressing the cytokine production or inducing the apoptosis of immune cells, stimulating the generation of Tregs, one type of tumor-infiltrating lymphocytes, which play an important role in immunologic escape of tumor cells, and expanding the MDSCs (Zhang et al, 2018).

HLA-G polymorphism is believed to be related to cancer development, but the association between it and the immune escape of tumor cells remains unknown.

1.2. Cytokines

Cancer cells can produce many kinds of cytokines to escape from the immune surveillance. Transforming growth factor (TGF)-β is one of the major immunosuppressive cytokines found in many cancer cells (Xiang et al, 2009; Yamada et al, 2016). TGF-β can suppress T-cell proliferation, differentiation and the activity of NK cells (Czernek et al, 2017).

Interleukins are another kinds of cytokines. Interleukin-8 (IL-8) may enhance the resistance of colorectal cancer cells to anoikis by activating AKT, TOPK and ERK pathways (Xiao et al,2 015). Gastric cancer mesenchymal stem cells derived IL-8 can induce immune escape by elevating PD-L1 expression in gastric cancer cells via a STAT3/mTOR-c-Myc signal axis (Sun et al, 2018).

IL-33 is important for the immune recognition, and is a strong inducer of MHC-I and antigen processing in metastatic cancers, down-regulation of IL-33 ultimately decreases functionality of MHC-I and reduces immune-surveillance (Saranchova et al, 2016).

1.3. MicroRNAs (miRNAs)

MiRNAs are another kinds of key molecules in regulating genes related to cancer progression and immune evasion (Pucci et al, 2018; Grange et al, 2019). In liver cancer miR-544 promoted the immune escape of liver cancer cells by downregulating NCR1 via targeting Runt-related transcription factor 3 (RUNX3) (Pan et al, 2018). In breast cancer MiR-519a-3p not only can inhibit several tumor suppressor genes, but also can inhibit TRAIL- and FasL-induced apoptosis by directly targeting the proapoptotic TNFRSF10B (TRAIL-R2) and CASP8 (caspase-8) mRNAs. Moreover, miR-519a-3p can decrease NK cell-mediated killing of breast cancer cells by downregulating tumor cell ligands for the NK cell-activating receptor NKG2D (Breunig et al, 2017). miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype (Ortega et al,2015). It could  increase the levels of IL-10 and prostaglandin E2, so as to suppress antitumor immunity mediated by T-cells in in colorectal cancer (Nosho et al,2016). miR-10b could express in circulating tumor cells (Gasch et al,2015), and downregulated the ligand of NK cell receptor NKG2D MICB, leading to immune escape(Tsukerman et al.2012).

2. Transportation of tumor cells derived molecules

Extracellular Vesicles coming from both normal and tumor cells play a key role in the physiological and pathological process, and are considered to be efficient messengers (Grange et al, 2019). Those from tumor cells display features of their originating cells and carry multiple immunoinhibitory signals such as proteins, DNAs, metabolites and microRNAs, to force recipient cells towards a pro-tumorigenic phenotype by modulating the expression of immunoregulatory genes (Navarro-Tableros et al, 2018). They could promote the formation of MDSCs (Umansky et al.2017), promote the production of prostaglandin E2 (PGE2), IL-6, and tumor growth factor β (TGF-β) from MDSCs to disable anti-tumor immune effector cells, or induce apoptosis in CD8+ T cells and enhance CD4+ T regulatory cell suppressor activity; they could repress NK activity by interacting with NKG2D or downregulating NKG2D (Tucci et al, 2018). Czystowska-Kuzmicz et al (2019) recently reported that small extracellular vesicles containing arginase-1 could suppress CD4+ and CD8+ T-cell responses in ovarian carcinoma. Estrogen receptor-binding fragment-associated antigen 9 (EBAG9) was reported to be a cancer biomarker and EVs from EBAG9-overexpressing prostate cancer cells could facilitate immune escape of tumors by inhibiting T-cell cytotoxicity and modulating immune-related gene expression in T cells (Miyazaki et al, 2018).

3. Conclusions

Escape from the immune surveillance for CTCs is a complicated process, besides the role of tumor derived molecules, epithelial-mesenchymal transition (EMT) can promote the survival of CTCs, tumor microenvironment (TME) composed of a variety of cells and molecules, also can influence this process. Study on the relationship between them will be helpful for further understanding the immune escape mechanisms.

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