Application of circulating tumor cells scope technique on circulating tumor cell research

Circulating tumor cells (CTCs) are becoming promising biomarkers in several cancers, such as colon, prostate, and breast carcinomas. Independent research groups have reported a correlation between CTC numbers and patient prognosis. Even more, the development of personalized medicine gives physicians impetus to utilize the advancement of molecular characterization of CTCs. This review introduces a new technique, CTCscope, and compares it with the current methods of CTCs detection, with particular emphasis on cancer research, and discusses the future application of this new method from bench to bed-side.


Introduction
Circulating tumor cells (CTCs) are becoming promising biomarkers in several cancers, such as colon, prostate, and breast carcinomas. Due to the lost expression of common epithelial markers by certain types of CTCs, such as EpCAM or the cytokeratin (CK), current positive selection strategies are usually with low sensitivity and efficiency [1,2]. For example, most breast tumour stem cells are estrogen receptor negative, which could not be detected by positive selection method [3] (Table 1).
In 2011, RNAscope or CTCscope was developed for detecting multiple tumour-specific marker mRNAs in tumor tissues [9,10], as well as CTCs from blood [11]. In brief, peripheral blood mononuclear cells (PBMC) including CTCs are enriched and then placed on the slides. After fixed by 10% formaldehyde solution, cells are hybridized with specifically designed target probes. The fluorescent signal is amplified by a series of nucleic acid hybridization and imaged by fluorescent microscopy for CTCscope analysis. The schematic of CTCscope assay procedure was showed in Figure 1. Compared to the current CTCs detection methods, CTCscope approach is more sensitive and allows single molecule detection in situ in individual cells by using a novel system of probe design and signal amplification. This ability makes CTCscope a powerful platform in routine clinical assays for CTCs detection.

Tumorgenesis research
In vivo study, Burd et al. used standard homologous recombination procedures to target firefly luciferase on the SV40 polyadenylation site to exon 1α of the endogenous p16 INK4a gene [12]. Then they applied RNA in situ hybridization (ISH) by using RNAscope 2.0 technology to detect this faithfully reports expression gene, which serves as a tumor suppressor and aging biomarker. Their work suggests that p16 INK4a activation is a characteristic of all emerging cancers and it could be set as a sensitive, unbiased reporter of neoplastic transformation. While in situ RNA study, Staudt et al. applied macrophage inflammatory protein-1a/CCL3-specific RNA target probe set, which targeted nucleotides 23 to 771 of the CCL cDNA sequence to detect CCL3 mRNA in tumor samples [13]. CCL3 is a chemokine, which is known to amplify inflammation. They found inhibiting NF-ҡB reversed the increase in CCL3 expression associated with LDL receptor-related protein (LRP1) gene silencing in macrophage-like cells. LRP1 is a type 1 transmembrane receptor, which mediates ligand endocytosis and impact cell migration. This phenomenon suggested to prevent LRP1 down regulation in myeloid cells might suppress monocyte recruitment to tumors and cancer angiogenesis. Step 1 Step 2 Step 3 Step anti-EpCAM antibodies conjugated with magnetic beads, for enriching CTCs in a magnetic field -MagSweeper; anti-EpCAM antibodies on microposts or columns of nanobeads -ELISPOT/Ephesia; anti-EpCAM antibodies conjugated to 3-μm beads to increase the size of CTCs before filtration -Cytometry. D: Schematic of the RNAscope assay procedure -CTCscope. In step 1, cells are fixed and permeabilized to allow for target probe access. In step 2, target RNA-specific oligonucleotide probes (Z) are hybridized in pairs (ZZ) to multiple RNA targets. In step 3, multiple signal amplification molecules are hybridized, each recognizing a specific target probe, and each unique label probe is conjugated to a different fluorophore or enzyme. In step 4, signals are detected using an epifluorescent microscope (for fluorescent label) (CTCscope image are reproduced from Payne et al. [4]) PBMC: peripheral blood mononuclear cells; CTC: circulating tumor cells; RBC: red blood cell.
fibronectin) expressed in CTCs which have undergone EMT [4]. They found that breast cancer cells could be identified by strong pan-CK staining, whereas the surrounding PBMCs showed minimal fluorescent signals ( Figure 1D). Also they found different EGFR mRNA expression levels consistent with the known EGFR protein expression status. The advance of application of fluorescence in situ hybridization technique is to detect the gene translocation. Tana et al. applied RNAscope formalin fixed paraffin embed (FFPE) assay in epithelioid hemangioendothelioma (EHE) tissue sections to detect the translocation (the chimeric WWTR1/CAMTA1 transcription factor), which would assist in the evaluation of this diagnostically challenging neoplasm as well as may represent a therapeutic target for EHE [14]. By multispectral imaging system, WWTR1/CAMTA1 fusion transcripts presented yellow punctate dots, while wild-type WWTR1 and CAMTA1 transcripts were stained with red or green punctate dots, respectively. Among these studies, it confirms that CTCscope provides additional prognostic and predictive information in therapy monitoring.

Cancer stem cell research
Currently, In situ study, by RNAscope technology, groups of cancer stem cell biomarker was confirmed. Barry et al. applied RNAscope for Olfm4, which marks crypt base columnar (CBCs) stem cells in intestinal tissue, and to determine if intestinal stem cell (ISC) numbers was reduced by YAP (a protein known as its powerful growth-inducing and oncogenic properties) expression [15]. And in their research, they found epithelial-specific expression of YAP suppressed intestinal cell renewal, which occurred through inhibition of the Wnt-singaling pathway. Ziskin et al. also applied multiplex fluorescent ISH and chromogenic nonisotopic ISH in 57 colorectal adenocarcinomas to detect 19 putative intestinal stem cell markers [16]. They found the G protein-coupled receptor (lgr5) and intestinal stem cells signature gene (Ascl2) was expressed on mainly colorectal cancers, which supported the hypothesis that the cancer cells were derived from lgr5 + /Ascl2 + crypt stem cells. Yan et al. applied fluorescence in situ hybridization (FISH) to detect lgr5 mRNA and genetic signature of panyellow fluorescent protein (YFP) expression within the Polycomb group protein (Bmi1) + clonally derived spheroids [17]. The result indicated that the Bmi1+ ISC lineage could generate Lgr5+ cells in vitro.

Other application
On several virus infection models, CTCscope became a popular research tool for detection certain mRNA expression. In order to confirm Hamster-adapted Sin Nombre Virus (HA-SNV) replication in pulmonary endothelial cells, Safronetz et al. applied monoclonal antibodies targeting the nucleoprotein (anti-SNV NP clone 5 F1/F7) nucleotides) and polyclonal rabbit anti-CD31 antibodies in a HA-SNV infection model [18].

Conclusions
This recently developed new technique, CTCscope, is designed for detection mRNA expression on CTCs. Due to its advantage of highly sensitivity, RNA ISH empower its high sensitivity and specificity to define low expression of commonly used biomarkers. However the future application of CTCscope from bench to bed-side seems both promising and challenged. Certain limitations of this approach may hamper its clinical use, e.g. the assay protocol which contains multiple steps is not easy to perform in a clinical lab, the changing of hybridization probes against the target mRNA may reduce the reliability of the detection, and the cut off value and clinical significance of CTC numbers need to be determined for different cancers.