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Background/Aim: Trastuzumab is the only clinically approved targeted therapy for HER2 gene-amplified gastric cancer at present. However, the clinical significance of multi-targeting tyrosine kinase inhibitors (TKIs) in HER2-positive gastric cancer remains unclear. Materials and Methods: We examined the anti-tumor activity of lapatinib and afatinib, that are reversible and irreversible TKIs, in HER2 gene-amplified trastuzumab-sensitive and - resistant gastric cancer cells (GLM-1 and GLM-1HerR2) in vitro and in vivo. Results: Afatinib inhibited the growth of GLM-1 and GLM-1HerR2 cells in vitro more efficiently than lapatinib by inducing G1 cell-cycle arrest and apoptosis. Preclinical studies in mice revealed that afatinib inhibited growth of intraperitoneal GLM-1 and subcutaneous GLM-1HerR2 tumor more strongly than lapatinib. Afatinib was more effective than lapatinib in blocking PI3K/Akt and MAPK signaling in both GLM-1 and GLM-1HerR2 cells. Conclusion: Afatinib could be a potential new molecular-targeted therapy for trastuzumab-sensitive and trastuzumab-resistant HER2 gene-amplified gastric cancers.

Androgen deprivation therapy is given to suppress prostate cancer growth; however, some cells continue to grow hormone-independently as castration-resistant prostate cancer (CRPC). Sulfated glycosaminoglycans promote ligand binding to receptors as co-receptors, but their role in CRPC remains unknown. Using the human prostate cancer cell line C4-2, which can proliferate in hormone-dependent and hormone-independent conditions, we found that epidermal growth factor (EGF)-activated EGFR–ERK1/2 signaling via 3- O -sulfated heparan sulfate (HS) produced by HS 3- O -sulfotransferase 1 (HS3ST1) is activated in C4-2 cells under hormone depletion. Knockdown of HS3ST1 in C4-2 cells suppressed hormone-independent growth, and inhibited both EGF binding to the cell surface and activation of EGFR–ERK1/2 signaling. Gefitinib, an EGFR inhibitor, significantly suppressed C4-2 cell proliferation and growth of a xenografted C4-2 tumor in castrated mouse. Collectively, our study has revealed a mechanism by which cancer cells switch to hormone-independent growth and identified the key regulator as 3- O -sulfated HS.

Numerous technologies exist to detect circulating tumor cells (CTCs), although reports on cytological detection of CTCs remain limited. We recently developed a cytology-based CTC detection device using glass slides and light microscopy. In this study, we automated this previously manual device to improve its efficiency and cost effectiveness for clinical applications. We conducted a pilot study using this device to compare CTCs in peripheral blood (PB) and draining venous blood (DVB) from patients with colorectal cancer (CRC). The cytology-based automated CTC detection platform consisted of a disposable filtration device with a three-dimensional (3D) metal filter and multichannel automated CTC enrichment device. This platform allowed rapid and gentle filtration of CTCs and their efficient transfer from the filter to glass slides for subsequent Papanicolaou (Pap) and immunocytochemical (ICC) staining. Cytological diagnosis of CTCs was performed by observing permanent glass slide specimens by light microscopy. The current pilot clinical study enrolled CRC patients (n = 26) with stage I-IV tumors, who underwent surgery. PB was collected before surgery, and DVB was obtained from the mesenteric vein immediately after resection. Based on the CTC morphology obtained from PB and DVB samples, we proposed the following cytological criteria for the diagnosis of CTCs: pan-cytokeratin-positive, atypical cells with malignant morphological features identified by Pap staining. The numbers of CTCs defined by these criteria were significantly higher in DVB than PB from CRC patients (p<0.01), and the number of CTCs in DVB was increased significantly with stage progression (p<0.05). These results suggest that DVB may be another potential source of CTCs other than PB for liquid biopsies including downstream analysis. This automated cytology-based CTC detection device therefore provides a unique and powerful tool to investigate the significance of CTCs in CRC patients in a clinical setting.

The ability to accurately detect and analyze rare cells in a cell population is critical not only for the study of disease progression but also for next flow cytometry systems in clinical application. Here, we report the development of a prototype device, the 'Rare cell sorter', for isolating and recovering single rare cells from whole blood samples. On this device, we utilized an open-channel microfluidic chip for rare cell isolation. And the advantage of open-channel allows us to recover the isolated rare cell directly from the chip. We set the circulating tumor cell (CTC) as a target cell. For the clinical experiment, CTCs were isolated from blood samples collected from patients with metastatic breast cancer and healthy volunteers. There was a significant difference in the number of CTCs between the patients with metastatic breast cancer and healthy volunteers. To evaluate the damage to cells during isolation and recovery, we performed an RNA integrity assay using RNA extracted from CTCs recovered from the chip and found that our process for single CTC isolation and recovery is mild enough for gene analysis of CTCs.

Circulating tumor cells (CTCs) in tumor draining vein blood (DB) are potential sources for liquid biopsy. However, the identification of CTCs in DB of breast cancer has not been attempted. In this study, we investigated the feasibility of CTC detection in DB of breast cancer patients using a newly developed filtration-based microfluidic CTC detection device. Samples of peripheral vein blood (PB) and DB drawn from the lateral thoracic vein of the resected breast tissue were collected during the perioperative period. We investigated 41 breast cancer patients who underwent breast surgery with axillary lymph node dissection. DB was successfully collected in 36 patients (87.8%), with a mean amount of 0.85 ml. CTCs were detected in 58.3% of PB samples and 80.6% of DB samples. DB had significant higher number of CTCs compared with PB ( p  < 0.001). CTCs were detected in 75.0% of DB samples and 50.0% of PB samples from patients achieving pathological complete response after neoadjuvant chemotherapy. These results suggest that abundant CTCs are released into the DB of breast cancer patients, indicating that CTCs in DB would be alternative sources for liquid biopsy and potential indicators for monitoring of treatment response and prognosis in breast cancer patients.

Background Malignant mesothelioma (MM) is a very aggressive tumor that develops from mesothelial cells, mainly due to asbestos exposure. MM is categorized into three major histological subtypes: epithelioid, sarcomatoid, and biphasic, with the biphasic subtype containing both epithelioid and sarcomatoid components. Patients with sarcomatoid mesothelioma usually show a poorer prognosis than those with epithelioid mesothelioma, but it is not clear how these morphological phenotypes are determined or changed during the oncogenic transformation of mesothelial cells. Methods We introduced the E6 and E7 genes of human papillomavirus type 16 and human telomerase reverse transcriptase gene in human peritoneal mesothelial cells and established three morphologically different types of immortalized mesothelial cell lines. Results HOMC-B1 cells exhibited epithelioid morphology, HOMC-A4 cells were fibroblast-like, spindle-shaped, and HOMC-D4 cells had an intermediate morphology, indicating that these three cell lines closely mimicked the histological subtypes of MM. Gene expression profiling revealed increased expression of NOD-like receptor signaling-related genes in HOMC-A4 cells. Notably, the combination treatment of HOMC-D4 cells with TGF-β and IL-1β induced a morphological change from intermediate to sarcomatoid morphology. Conclusions Our established cell lines are useful for elucidating the fundamental mechanisms of mesothelial cell transformation and mesothelial-to-mesenchymal transition.

Circulating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

Background: Circulating tumor cells (CTCs) have recently been developed as biomarkers. Several studies have reported on the clinical use of CTCs to assess drug resistance in various cancers. However, sequential and multiple CTC measurements during chemotherapy are relatively rare. We recently reported a transient increase in CTCs early after chemotherapy by sequentially detecting CTCs in a human pancreatic cancer xenograft model in nude mice. Method: In the present study, using a newly developed immunocytology and glass slide-based convenient CTC detection platform, we examined CTC numbers sequentially before, during, and after chemotherapy in the peripheral blood of 14 pancreatic cancer patients, pathological stage (pStage) I-IV, who underwent surgery with preoperative chemotherapy and GS (Gem/S-1) and GnP (Gem/nab-PTX). Results: Among patients with strongly or weakly elevated CTC counts (3–44/5 mL of blood) following GS treatment, four out of six pancreatic cancer patients were judged to have a partial response (PR), and two out of six were deemed to have stable disease (SD) as a clinical response based on the CT image. In contrast, in patients with GnP therapy, three out of four patients showed no CTC response, and these three patients were judged to have progressive disease (PD), while the remaining one patient was judged to have SD in terms of their clinical response. Conclusion: These results suggest that sequential CTC monitoring during preoperative chemotherapy in pancreatic cancer patients can be a helpful liquid biopsy diagnostic tool as a therapeutic marker to predict tumor chemosensitivity and chemoresistance in clinical settings. Further large-scale clinical studies are required to confirm and clarify this hypothesis.

Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)–labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma–xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression–dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are powerful molecular targeted therapeutic agents for lung cancer. We recently developed an original immunocytology and glass slide-based circulating tumor cell (CTC) detection platform for both CTC enumeration and EGFR mutation analysis with DNA extracted from CTCs. Methods: Using this platform, we conducted a pilot clinical study for CTC enumeration in peripheral blood (PB), pulmonary arterial blood (PA), and pulmonary venous blood (PV) from 33 patients with lung cancer (Stage I–III) who underwent surgery, followed by digital PCR-based EGFR mutation analysis of CTCs in PV from 12 patients. Results: The results showed that CTC levels were significantly higher in PV and PA than in PB (p < 0.05, p < 0.01. respectively), with a notably greater number of small and large CTC clusters (p < 0.01). Genetic analysis of EGFR mutations of CTCs from PV (n = 12) revealed six mutations, including three Exon19del and three L856R, in CTCs and eight EGFR mutations, including five Exon19del and three L856R, in lung tumor tissue. CTC mutation status matched that of tissue samples in nine patients, was unmatched in two patients, and controversial in one patient, indicating a sensitivity of 0.75 (6/8) and specificity of 1.0 (4/4) with some false-negative results for the mutation analysis of CTCs. Conclusions: This immunocytology-based CTC detection platform is a convenient method for detecting both CTC number and EGFR mutation status under microscopy, suggesting its potential as a liquid biopsy tool in the hospital for patients with lung cancer in some clinical settings.