Molecular-based companion diagnostic tests are being used with increasing frequency to predict their clinical response to various drugs, particularly for molecularly targeted drugs. However, invasive procedures are typically required to obtain tissues for this analysis. Circulating tumour cells (CTCs) are novel biomarkers that can be used for the prediction of disease progression and are also important surrogate sources of cancer cells. Because current CTC detection strategies mainly depend on epithelial cell-surface markers, the presence of heterogeneous populations of CTCs with epithelial and/or mesenchymal characteristics may pose obstacles to the detection of CTCs.
Methods
We developed a new approach to capture live CTCs among millions of peripheral blood leukocytes using a green fluorescent protein (GFP)-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401, TelomeScan).
Results
Our biological capturing system can image epithelial and mesenchymal tumour cells with telomerase activities as GFP-positive cells. After sorting, direct sequencing or mutation-specific PCR can precisely detect different mutations in KRAS, BRAF and KIT genes in epithelial, mesenchymal or epithelial–mesenchymal transition-induced CTCs, and in clinical blood samples from patients with colorectal cancer.
Conclusions
This fluorescence virus-guided viable CTC capturing method provides a non-invasive alternative to tissue biopsy or surgical resection of primary tumours for companion diagnostics.
<p>Supplementary Movie S3 - MOV file 6227K, Time-lapse imaging of treatment dynamics of FUCCI-expressing dormant tumor spheres treated with OBP-301 or conventional therapy in three-dimensional culture. Tumor spheres were treated with mock (upper left), OBP-301 (upper right), cisplatin (lower left) or radiation (lower right). Images were acquired every 20 min. The playback speed is 30,240 x real time. Total imaging time = 7days. The cells in G0/ G1, S, or G2/M phases appear red, yellow, or green, respectively</p>
A 69-year-old man underwent endoscopic submucosal dissection (ESD) for early gastric cancer (EGC) at the lesser curvature in the angle of stomach. Histological examination revealed tub1, pM, ly0, v0, pLM(-), pVM(-), and the resection was considered curative. The scar after ESD was followed by esophagogastroduodenoscopy (EGD) and biopsy. Twenty months later, EGD showed an ulcerative lesion in the vicinity of the ESD scar, and histological examination of the biopsy specimen showed adenocarcinoma. A distal gastrectomy with lymph node dissection was then performed. Postoperative pathology showed tub1, pM, pN0, ly0, v0, and Stage 1A. Skip lesions were seen in the specimen resected by ESD, and the histological review confirmed so-called "dysplasia-like atypia" (DLA) between the lesions. It has been reported recently that in DLA, the dysplasia-like change involves only the bases of the pits, without upper pit or surface epithelium involvement, and it is said that the rate of DLA is higher in gastric cancer patients. We speculated that a precancerous lesion close to the resected cancer developed into a local recurrence.
Abstract Currently available methods for detection of tumors in vivo such as X-ray, computed tomography, and ultrasonography are noninvasive and have been well studied; the images, however, are not specific for tumors. Direct optical imaging of tumor cells in vivo that can clearly distinguish them from surrounding normal tissues may be clinically useful. Here, we describe a new approach to visualizing tumors whose fluorescence can be detected using tumor-specific replication-competent adenovirus (OBP-301, Telomelysin) in combination with Ad-GFP, a replication-deficient adenovirus expressing green fluorescent protein (GFP). Human telomerase reverse transcriptase is the catalytic subunit of telomerase, which is highly active in cancer cells but quiescent in most normal somatic cells. We constructed an adenovirus 5 vector in which the human telomerase reverse transcriptase promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site and showed that OBP-301 replicated efficiently in human cancer cells, but not in normal cells such as human fibroblasts. When the human lung and colon cancer cell lines were infected with Ad-GFP at a low multiplicity of infection, GFP expression could not be detected under a fluorescence microscope; in the presence of OBP-301, however, Ad-GFP replicated in these tumor cells and showed strong green signals. In contrast, coinfection with OBP-301 and Ad-GFP did not show any signals in normal cells such as fibroblasts and vascular endothelial cells. We also found that established subcutaneous tumors could be visualized after intratumoral injection of OBP-301 and Ad-GFP. A549 human lung tumors and SW620 human colon tumors transplanted into BALB/c nu/nu mice were intratumorally injected with 8 × 105 plaque-forming units of Ad-GFP in combination with 8 × 106 plaque-forming units of OBP-301. Within 3 days of treatment, the fluorescence of the expressed GFP became visible by a three-chip color cooled charged-coupled device camera in these tumors, whereas intratumoral injection of Ad-GFP alone could not induce GFP fluorescence. Moreover, intrathoracic administration of Ad-GFP and OBP-301 could visualize disseminated A549 tumor nodules in mice after intrathoracic implantation. Our results indicate that intratumoral or intrathoracic injection of Ad-GFP in combination with OBP-301 might be a useful diagnostic method that provides a foundation for future clinical application.
Abstract Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancer. However, the role of inflammatory microenvironment in the EMT-related tumor progression remains unclear. To explore the relationship between inflammatory microenvironment and EMT, a live imaging system for EMT is a promising strategy on the in vitro and in vivo experiments. In this study, we developed a fluorescence-guided live cell imaging system for the assessment of spatiotemporal dynamics of EMT, and investigated the potential of inflammatory microenvironment for the induction of EMT phenotype in human colorectal cancer. Methods: Two human colorectal cancer cell lines, HCT116 and RKO, were stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. Both cell lines were treated with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or co-cultured with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis and q-PCR. EMT-induced HCT116 and RKO cells were treated with anti-inflammatory agents, aspirin (1mM) and salicylic acid (1 mM), for the suppression of EMT. Results: Inflammatory cytokines (IL-1β and TNF-α) induced red fluorescence intensity and morphological change like mesenchymal phenotype in HCT116 and RKO cells. Removal of inflammatory cytokines attenuated red fluorescence intensity and morphological change in both cells. Inflammatory cytokines also induced the migration and invasion properties in association with EMT-related markers. Moreover, co-culture with LPS-stimulated inflammatory macrophages also induced red fluorescence intensity and morphological change as well as inflammatory cytokines. Anti-inflammatory agents significantly suppressed the fluorescence-related EMT phenotype under inflammatory microenvironment. Conclusions: These results suggest that inflammatory microenvironment has a great potential for the induction of EMT process during colorectal cancer progression. This unique fluorescence-guided EMT imaging system is useful method for the exploration of inflammation-mediated tumor progression. Citation Format: Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Takeshi Imamura, Toshiyoshi Fujiwara. Fluorescence-guided spatiotemporal dynamics of epithelial-mesenchymal transition under inflammatory microenvironment during colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5809. doi:10.1158/1538-7445.AM2017-5809
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