A Novel Liquid Biopsy Strategy to Detect Small Amounts of Cancer Cells Using Cancer-Specific Replication Adenoviruses

Abstract

Circulating tumor cells (CTCs) are a promising source of clinical and biological cancer information and can be a material for liquid biopsy. However, detecting and capturing these cells remains a challenge. Various biological factors (e.g., cell surface proteins, cell size, deformability, or dielectrophoresis) have been applied to detect CTCs. Cancer cells dramatically change their characteristics during tumorigenesis and metastasis. Hence, defining a cell as malignant using such a parameter is difficult. Moreover, immortality is an essential characteristic of cancer cells. Telomerase elongates telomeres and plays a critical role in cellular immortality and is specifically activated in cancer cells. Thus, the activation of telomerase can be a good fingerprint for cancer cells. Telomerase cannot be recognized by antibodies in living cells because it is a nuclear enzyme. Therefore, telomerase-specific replication adenovirus, which expresses the green fluorescent protein, has been applied to detect CTCs. This review explores the overview of this novel technology and its application in gynecological cancers.

Keywords: adenovirus; circulating tumor cells (CTCs); telomerase.

Figure 1

Structures of telomerase-specific replicating adenoviruses. (A) Representative transcription factor binding sites on the human telomerase reverse transcriptase (hTERT) promoter are shown. The sites on the promoter are not to exact scale. +1 indicates the transcription start site. ATG indicates the translation starting codon. (B) In OBP-301, the hTERT extended core promoter drives the expression of E1A and E1B genes linked with internal ribosome entry site (IRES). OBP-401 is a green fluorescent protein (GFP)-expressing variant, in which the cytomegalovirus promoter drives the expression of GFP inserted in the E3 region. In OBP-1101, the adenoviral fibers were replaced with type 35 fibers to enable infection of coxsackievirus–adenovirus receptor-negative cells. The miR142-3p responsive elements were inserted into the 3′ untranslated regions (UTR) of the E1 and GFP genes to attenuate non-specific GFP expression in blood cells. ITR, internal terminal repeat; pA, bovine growth hormone polyadenylation signal; AP1, Activator protein 1; SP1, Specificity protein 1; ER, estrogen receptor; MZF-2, myeloid Zinc-Finger Protein 2; NF-κB, nuclear factor kappa B; c-Myc, c-myelocytomatosis oncogene product; E2F, E2 promoter binding factor; AP-2, Activating enhancer-binding protein-2; Ets, erythroblast transformation specific proteins; GFP, green fluorescent protein; Ad35, adenovirus type 35.

Figure 2

Representative pictures of CTCs detected by telomerase-specific replicating adenoviruses; (A) OBP-401 was infected with the blood samples from a patient with cervical cancer (top row) and a healthy volunteer (bottom row). GFP(+)/CD45(−) cells were recognized as CTCs, and GFP(+)/CD45(+) cells were false-positive blood cells. (B) OBP-1101 was infected with the blood samples from patients with cervical cancer. As with OBP-401, CTCs were GFP(+)/CD45(−) and false-positive cells were GFP(+)/CD45(+). The observed CTCs were negative for the epithelial markers EpCAM and cytokeratin.