Biliary tract cancer (BTC) arises from biliary epithelial cells (BECs) and includes intrahepatic cholangiocarcinoma (IHCC), gallbladder cancer (GC), and extrahepatic cholangiocarcinoma (EHCC). Although frequent KRAS mutations and epigenetic changes at the INK4A/ARF locus have been identified, the molecular pathogenesis of BTC is unclear and the development of corresponding anticancer agents remains inadequate. We isolated epithelial cell adhesion molecule (EpCAM)-positive BECs from the mouse intrahepatic bile duct, gallbladder, and extrahepatic bile duct, and established organoids derived from these cells. Introduction of activated KRAS and homozygous deletion of Ink4a/Arf in the cells of each organoid type conferred the ability to form lethal metastatic adenocarcinoma with differentiated components and a pronounced desmoplastic reaction on cell transplantation into syngeneic mice, indicating that the manipulated cells correspond to BTC-initiating cells. The syngeneic mouse models recapitulate the pathological features of human IHCC, GC, and EHCC, and they should therefore prove useful for the investigation of BTC carcinogenesis and the development of new therapeutic strategies. Tumor cells isolated from primary tumors formed organoids in three-dimensional culture, and serial syngeneic transplantation of these cells revealed that their cancer stem cell properties were supported by organoid culture, but not by adherent culture. Adherent culture thus attenuated tumorigenic activity as well as the expression of both epithelial and stem cell markers, whereas the expression of epithelial-mesenchymal transition (EMT)-related transcription factor genes and mesenchymal cell markers was induced. Our data show that organoid culture is important for maintenance of epithelial cell characteristics, stemness, and tumorigenic activity of BTC-initiating cells.

Cancer stem cells (CSCs) drive tumor initiation and metastasis in several types of human cancer. However, the contribution of ovarian CSCs to peritoneal metastasis remains unresolved. The cell adhesion molecule CD44 has been identified as a major marker for CSCs in solid tumors, including epithelial ovarian cancer. CD44 exists as a standard form (CD44s) and also as numerous variant isoforms (CD44v) generated by alternative mRNA splicing. Here we show that disseminated ovarian tumors in the pelvic peritoneum contain highly enriched CD44v6-positive cancer cells, which drive tumor metastasis and are responsible for tumor resistance to chemotherapy. Clinically, an increased number of CD44v6-positive cancer cells in primary tumors was associated with a shortened overall survival in stage III-IV ovarian cancer patients. Furthermore, a subpopulation of CD44v6-positive cancer cells manifested the ability to initiate tumor metastasis in the pelvic peritoneum in an in vivo mouse model, suggesting that CD44v6-positive cells show the potential to serve as metastasis-initiating cells. Thus, the peritoneal disseminated metastasis of epithelial ovarian cancer is initiated by the CD44v6-positive subpopulation, and CD44v6 expression is a biomarker for the clinical outcome of advanced ovarian cancer patients. Given that a distinct subpopulation of CD44v6-positive cancer cells plays a critical role in peritoneal metastasis, definitive treatment should target this subpopulation of CD44v6-positive cells in epithelial ovarian cancer.

CD44 is a widely expressed polymorphic adhesion molecule that has pleiotropic functions in development and tumor progression. Its mRNA undergoes alternative splicing to generate multiple variant (CD44v) isoforms, although the function of each CD44v isoform is not fully elucidated. Here, we show that CD44v plays an important role in the induction of vimentin expression upon transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT). Among multiple CD44v isoforms expressed in NUGC3 gastric cancer cells, CD44v8-10 and CD44v3,8-10 are involved in the acquisition of migratory and invasive properties associated with TGF-β1-induced EMT, and only CD44v3,8-10 induces the transcription of vimentin mediated by the EMT transcription factor Slug. In primary tumor specimens obtained from patients with gastric cancer, CD44-containing variant exon 9 (CD44v9) expression and EMT features [E-cadherin(-)vimentin(+)] were significantly correlated, and EMT features in the cells expressing CD44v9 were associated with tumor invasion depth, lymph node metastasis, and pStage, which indicate invasive and metastatic properties, and poor prognosis. These results indicate that certain CD44v isoforms promote tumor cell motility and metastasis in gastric cancer in association with EMT features, and CD44v3,8-10 may contribute to these clinical characteristics.

E-cadherin, an epithelial cell-specific cell adhesion molecule, has both promoting and suppressing effects on tumor invasion and metastasis. It is often downregulated during cancer progression through gene deletion/mutation, transcriptional repression, or epigenetic silencing. We describe a novel regulatory switch to induce stimulus-dependent downregulation of mRNA encoding E-cadherin (CDH1 mRNA) in KRAS-mutated cancer cells. The regulatory switch consists of ZEB1 and oncogenic K-Ras, does not target the promoter region of CDH1, and requires an external cue to temporally downregulate E-cadherin expression. Its repressive effect is maintained as long as the external stimulus continues and is attenuated with cessation of the stimulus. Contextual external cues that turn this regulatory switch on include activation of protein kinase C or fibroblast growth factor signaling. The mode of action is distinct from that of EPCAM repression by ZEB1, which does not require an external cue. Thus, KRAS-mutated cancer cells acquire a novel mode of regulating E-cadherin expression depending on ZEB1, which could contribute to phenotypic plasticity of cancer cells during malignant progression.

Bone-related events caused by breast cancer bone metastasis substantially compromise the survival and quality of life of patients. Because triple-negative breast cancer (TNBC) lacks hormone receptors and Her2-targeted therapeutic options, progress in the treatment of TNBC bone metastasis has been very slow. Intercellular adhesion molecule 1 (ICAM1) is highly expressed in various cancers and plays an important role in tumorigenesis and metastasis. However, the effect and mechanism of ICAM1 in TNBC bone metastasis are still unknown. We found that ICAM1 was highly expressed in TNBC and correlated with prognosis in TNBC patients. Cell lines with high expression of ICAM1 exhibited enhanced bone metastasis in tumor-bearing mice, and silencing ICAM1 expression significantly inhibited bone metastasis in mice. ICAM1 interacted with integrins to activate the epithelial-to-mesenchymal transition program through TGF-β/SMAD signaling, ultimately enhancing cell invasiveness. Therefore, the findings of the present study provide a strong rationale for the application of ICAM1-targeted therapy in TNBC patients with bone metastasis.

Detection of rare tumor cells circulating in the blood (CTCs) presents technical challenges. CellSearch, the only approved system for clinical use, fails to capture epithelial cell adhesion molecule-negative CTCs such as malignant pleural mesothelioma (MPM). We have developed a novel microfluidic device (CTC-chip) in which any Ab to capture CTCs is conjugated. The CTC-chip was coated with an Ab against podoplanin that is abundantly expressed on MPM. Circulating tumor cell-detection performance was evaluated in experimental models in which MPM cells were spiked in blood sampled from a healthy volunteer and in clinical samples drawn from MPM patients. The CTC-chip showed superior CTC-detection performance over CellSearch in experimental models (sensitivity, 63.3%-64.5% vs 0%-1.1%; P < .001) and in clinical samples (CTC-positivity, 68.8% vs 6.3%; P < .001). A receiver operating characteristic (ROC) analysis showed that the CTC test provided a significant diagnostic performance in discrimination of unresectable disease from resectable disease (area under the ROC curve, 0.851; P = .003). The higher CTC count (≥2 cells/mL) was significantly associated with a poor prognosis (P = .030). The novel CTC-chip enabled sensitive detection of CTCs, which provided significant diagnostic and prognostic information in MPM.

Methods for the enumeration and molecular characterization of circulating tumor cells (CTC) have been actively investigated. However, such methods are still technically challenging. We have developed a novel epithelial cell adhesion molecule independent CTC enumeration system integrated with a sorting system using a microfluidics chip. We compared the number of CTC detected using our system with those detected using the CellSearch system in 46 patients with various cancers. We also evaluated epidermal growth factor receptor (EGFR) and PIK3CA mutations of captured CTC in a study of 4 lung cancer and 4 breast cancer patients. The percentage of samples with detected CTC was significantly higher with our system (65.2%) than with CellSearch (28.3%). The number of detected CTC per patient using our system was statistically higher than that using CellSearch (median 5, 0; P = 0.000172, Wilcoxon test). In the mutation analysis study, the number of detected CTC per patient was low (median for lung, 4.5; median for breast, 5.5); however, it was easy to detect EGFR and PIK3CA mutations in the CTC of 2 lung and 1 breast cancer patient, respectively, using a commercially available kit. Our system is more sensitive than CellSearch in CTC enumeration of various cancers and is also capable of detecting EGFR and PIK3CA mutations in the CTC of lung and breast cancer patients, respectively.

Most hepatocellular carcinomas (HCC) develop as a result of chronic liver inflammation. We have shown that the oncoprotein gankyrin is critical for inflammation-induced tumorigenesis in the colon. Although the in vitro function of gankyrin is well known, its role in vivo remains to be elucidated. We investigated the effect of gankyrin in the tumor microenvironment of mice with liver parenchymal cell-specific gankyrin ablation (Alb-Cre;gankyrinf/f ) and gankyrin deletion both in liver parenchymal and non-parenchymal cells (Mx1-Cre;gankyrinf/f ). Gankyrin upregulates vascular endothelial growth factor expression in tumor cells. Gankyrin binds to Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1), mainly expressed in liver non-parenchymal cells, resulting in phosphorylation and activation of signal transducer and activator of transcription 3 (STAT3). Gankyrin deficiency in non-parenchymal cells, but not in parenchymal cells, reduced STAT3 activity, interleukin (IL)-6 production, and cancer stem cell marker (Bmi1 and epithelial cell adhesion molecule [EpCAM]) expression, leading to attenuated tumorigenic potential. Chronic inflammation enhances gankyrin expression in the human liver. Gankyrin expression in the tumor microenvironment is negatively correlated with progression-free survival in patients undergoing sorafenib treatment for HCC. Thus, gankyrin appears to play a critical oncogenic function in tumor microenvironment and may be a potential target for developing therapeutic and preventive strategies against HCC.

Liquid biopsy of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) is gaining attention as a method for real-time monitoring in cancer patients. Conventional methods based upon epithelial cell adhesion molecule (EpCAM) expression have a risk of missing the most aggressive CTC subpopulations due to epithelial-mesenchymal transition and may, thus, underestimate the total number of actual CTC present in the bloodstream. Techniques utilizing a label-free inertial microfluidics approach (LFIMA) enable efficient capture of CTC without the need for EpCAM expression. In this study, we optimized a method for analyzing genetic alterations using next-generation sequencing (NGS) of extracted ctDNA and CTC enriched using an LFIMA as a first-phase examination of 30 patients with head and neck cancer, esophageal cancer, gastric cancer and colorectal cancer (CRC). Seven patients with advanced CRC were enrolled in the second-phase examination to monitor the emergence of alterations occurring during treatment with epidermal growth factor receptor (EGFR)-specific antibodies. Using LFIMA, we effectively captured CTC (median number of CTC, 14.5 cells/mL) from several types of cancer and detected missense mutations via NGS of CTC and ctDNA. We also detected time-dependent genetic alterations that appeared during anti-EGFR therapy in CTC and ctDNA from CRC patients. The results of NGS analyses indicated that alterations in the genomic profile revealed by the liquid biopsy could be expanded by using a combination of assays with CTC and ctDNA. The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (ID: UMIN000014095).

We aimed to isolate circulating tumor cells (CTCs) using a microfluidic technique with a novel lateral magnetophoretic microseparator. Prostate cancer-specific gene expressions were evaluated using mRNA from the isolated CTCs. A CTC-based multigene model was then developed for identifying advanced prostate cancer. Peripheral blood samples were obtained from five healthy donors and patients with localized prostate cancer (26 cases), metastatic hormone-sensitive prostate cancer (mHSPC, 10 cases), and metastatic castration-resistant prostate cancer (mCRPC, 28 cases). CTC recovery rate and purity (enriched CTCs/total cells) were evaluated according to cancer stage. The areas under the curves of the six gene expressions were used to evaluate whether multigene models could identify mHSPC or mCRPC. The number of CTCs and their purity increased at more advanced cancer stages. In mHSPC/mCRPC cases, the specimens had an average of 27.5 CTCs/mL blood, which was 4.2 × higher than the isolation rate for localized disease. The CTC purity increased from 2.1% for localized disease to 3.8% for mHSPC and 6.7% for mCRPC, with increased CTC expression of the genes encoding prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and cytokeratin 19 (KRT19). All disease stages exhibited expression of the genes encoding androgen receptor (AR) and epithelial cell adhesion molecule (EpCAM), although expression of the AR-V7 variant was relatively rare. Relative to each gene alone, the multigene model had better accuracy for predicting advanced prostate cancer. Our lateral magnetophoretic microseparator can be used for identifying prostate cancer biomarkers. In addition, CTC-based genetic signatures may guide the early diagnosis of advanced prostate cancer.