Tumor‐targeted fluorescence labeling systems for cancer diagnosis and treatment

Abstract Conventional imaging techniques are available for clinical identification of tumor sites. However, detecting metastatic tumor cells that are spreading from primary tumor sites using conventional imaging techniques remains difficult. In contrast, fluorescence‐based labeling systems are useful tools for detecting tumor cells at the single‐cell level in cancer research. The ability to detect fluorescent‐labeled tumor cells enables investigations of the biodistribution of tumor cells for the diagnosis and treatment of cancer. For example, the presence of fluorescent tumor cells in the peripheral blood of cancer patients is a predictive biomarker for early diagnosis of distant metastasis. The elimination of fluorescent tumor cells without damaging normal tissues is ideal for minimally invasive treatment of cancer. To capture fluorescent tumor cells within normal tissues, however, tumor‐specific activated target molecules are needed. This review focuses on recent advances in tumor‐targeted fluorescence labeling systems, in which indirect reporter labeling using tumor‐specific promoters is applied to fluorescence labeling of tumor cells for the diagnosis and treatment of cancer. Telomerase promoter‐dependent fluorescence labeling using replication‐competent viral vectors produces fluorescent proteins that can be used to detect and eliminate telomerase‐positive tumor cells. Tissue‐specific promoter‐dependent fluorescence labeling enables identification of specific tumor cells. Vimentin promoter‐dependent fluorescence labeling is a useful tool for identifying tumor cells that undergo epithelial–mesenchymal transition (EMT). The evaluation of tumor cells undergoing EMT is important for accurately assessing metastatic potential. Thus, tumor‐targeted fluorescence labeling systems represent novel platforms that enable the capture of tumor cells for the diagnosis and treatment of cancer.


| INTRODUC TI ON
Tumor cells exhibit characteristics that increase the potential for malignancy, such as unlimited cell proliferation, chemoresistance, migratory and invasive abilities, and metastasis, which can lead to poor prognosis in cancer patients. Recent advances in antitumor techniques such as surgery, chemotherapy, radiotherapy, and immunotherapy have improved the clinical outcome of cancer patients.
However, advanced cancers are often refractory to antitumor therapies and exhibit tumor recurrence, distant metastasis, and poor prognosis. Therefore, early diagnosis of cancer is one of the most prominent strategies to cure cancer patients.

| TUMOR-TARG E TEDFLUORE SCEN CE L AB ELINGSYS TEMS
Indirect reporter labeling is one of the leading tumor-targeted fluorescence labeling systems that contribute to the diagnosis and treatment of tumors ( Figure 1). Indirect reporter labeling induces the expression of genes encoding fluorescent proteins in tumor cells using tumor-specific gene promoters, which is useful for the diagno-

| Telomerasepromoter-dependent fluorescence labeling
Telomerase is an enzyme that elongates the length of telomeres by repeated TTAGGG nucleotide sequences at the ends of a chromosome. 21 Most tumor cells exhibit high telomerase activity in association with unlimited cell proliferation, whereas most types of normal somatic cells exhibit no telomerase activity. Telomerase is a ribonucleoprotein complex consisting of two different subunits, a catalytic subunit (hTERT and telomerase-associated protein 1) 22,23 and an RNA subunit (human telomerase RNA component). 24 As hTERT expression is highly associated with telomerase activity in  Table 1). As miR-142-3p is ubiquitously expressed in various types of hematopoietic cells, including monocytes, 33,34 OBP-1101-mediated E1 and GFP expression is attenuated in miR-142-3p-positive hematopoietic cells, 32 leading to an improvement in tumor-specific fluorescent labeling. Although Ad5 can bind to CAR on the surface of target cells, 35 Ad35 binds with greater affinity to CD46, which is expressed on a variety of tumor cells more ubiquitously than CAR.
OBP-1101 with Ad35 fibers can infect CAR-negative tumor cells by binding to CD46, 32 leading to an improvement in viral infectivity.
Togo et al. 36 reported that the OBP-1101-based fluorescence labeling system is a promising tool for detecting highly malignant CTCs expressing the mesenchymal marker vimentin in non-small-cell lung cancer patients. OBP-1101 thus appears to be superior to OBP-401 in terms of inducing tumor-specific GFP expression.
Herpes simplex virus type-1 can be also utilized as a tool to induce tumor-specific GFP expression using the hTERT promoter.
Zhang et al. 37 developed a telomerase-dependent replicative herpes simplex virus, oHSV1-hTERT-GFP, in which the endogenous ICP4 promoter is replaced with the hTERT gene promoter to drive the expression of ICP4 for tumor-specific viral replication ( Figure 2 and Table 1). oHSV1-hTERT-GFP contains the GFP expression cassette in the ICP34.5 region, thus enabling visualization of tumor cells as GFP-positive cells. The oHSV-hTERT-GFP-based GFP induction system is useful for detecting CTCs in the peripheral blood of cancer patients. 38 Human telomerase reverse transcriptase promoter-driven replicative viruses have emerged as not only novel diagnostic tools but also novel antitumor methods for cancer treatment.  (Figure 3 and Table 1). The fiber F I G U R E 2 Fluorescence labeling systems using telomerase promoterdependent replicative viruses expressing fluorescent proteins. Ad5, adenovirus type 5; Ad35, adenovirus type 35; CMV, cytomegalovirus; GFP, green fluorescent protein; hTERT, human telomerase reverse transcriptase; IRES, internal ribosome entry site; ITR, inverted terminal repeat; miR, microRNA of AdSur-SYE contains the SYENFSA ligand, which is sequence identified to bind with greater affinity to human pancreatic cancer cells. 51 As AdSur-SYE harbors the EGFP expression cassette in the E3 region, the AdSur-SYE-based EGFP induction system is highly effective for targeting pancreatic cancer cells. In addition, Li et al. 52 developed a survivin promoter-specific replicative adenovirus, CRAd5/11-Sp-eGFP, which expresses EGFP and chimeric Ad5/11 fiber consisting of an Ad5 tail and an Ad11 shaft and knob ( Figure 3 and Table 1

| Carcinoembryonicantigenpromoterdependent fluorescence labeling
Pancreatic ductal adenocarcinoma is a highly lethal disease, with a 5- year survival rate of less than 10%. 59 Tools enabling early diagnosis of PDAC are needed to improve the clinical outcome of PDAC patients.
Carcinoembryonic antigen is the standard serum tumor marker for assessing a variety of gastrointestinal cancers, including pancreatic cancer. 60 The secretion of CEA to the extracellular environment is highly associated with CEA promoter activity in PDAC cells. 61 To target CEA-positive PDAC cells, Xu et al. 61

| Alpha-fetoproteinpromoter-dependent fluorescence labeling
Hepatocellular carcinoma is the most common type of liver cancer. 62 Alpha-fetoprotein is widely used as serum tumor marker for the diagnosis of HCC. 63 Alpha-fetoprotein expression is highly associated with clinical stage, early recurrence, and poor prognosis in HCC patients. 64 For the fluorescence labeling of AFP-positive HCC cells, Yoon et al. 65 developed an AFP promoter-specific nonreplicative adenovirus carrying the GFP gene (Ad/Ha2bm-GFP) ( Table 1)

| IrreversibleEMT-dependent fluorescence labeling
For the fluorescence labeling of tumor cells undergoing EMT,

CO N FLI C T SO FI NTE R E S T
Dr. Imamura Takeshi is an editorial board member of Cancer Science.
The other authors have no potential conflicts of interest to disclose.