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Circulating cancer cells (CCCs) are closely related to the process of distant metastasis. In early step of the metastasis cascade, CCCs must evade the detachment-induced cell death (anoikis) for their survival. Here, we examined whether Na + /K + -ATPase α3-isoform (α3NaK) in CCCs contributes to avoidance of anoikis. In CCCs isolated from gastric cancer patients, α3NaK was predominantly localized in the plasma membrane (PM), but it moved to the cytoplasm when the CCCs were attached to culture dishes. The CCCs showed significant expression of integrin α5 but not fibronectin, one of components of the extracellular matrix (ECM). In human gastric cancer MKN45 cells, digoxin (20 and 50 nM), a cardiac glycoside, significantly inhibited the enzyme activity and translocation (from cytoplasm to PM) of α3NaK, while they had no significant effect on ubiquitous Na + /K + -ATPase α1-isoform (α1NaK) in the PM. The translocation of α3NaK required the loss of ECM components from the cells. Additionally, digoxin significantly enhanced caspase 3/7 activity, as well as the expression of cleaved caspase 3, while reducing the viability of detached (floating) cells. In the MKN45 xenograft mouse model, intraperitoneal administration of digoxin (2 mg/kg/day) significantly decreased the number of CCCs and suppressed their liver metastasis. Our results suggest that α3NaK plays an essential role in the survival of CCCs in gastric cancer, and that digoxin enhances anoikis in detached (metastatic) gastric cancer cells by inhibiting the α3NaK translocation from cytoplasm to PM, thereby reducing CCCs. Targeting α3NaK may be a promising therapeutic strategy against CCC survival. Subject terms: Metastasis, Gastric cancer, Apoptosis

Circulating tumor cells (CTCs) hold significant promise for cancer diagnosis, prognosis, and treatment monitoring. We previously developed a technique for a single‐cell filtering device known as the microcavity array (MCA), specifically designed for the efficient recovery of CTCs from whole blood samples. Efficient enrichment and release of cells from the MCA remains challenging because of cell adhesion that occurs on the MCA surface during the enrichment phase. This study investigated the effects of surface modification with 2‐methacryloyloxyethyl phosphorylcholine (MPC) on the recovery efficiency of cancer cell lines from MCA. Scanning electron microscope (SEM) demonstrated reduced cell‐substrate interactions, leading to improved recovery efficiency. Comparative analyses showed that the MCA method provided superior recovery efficiency and reduced processing time compared to traditional methods such as density gradient centrifugation (DGC), while maintaining cell viability and proliferative capacity. CTCs were successfully detected in patients with gastric cancer, and short‐term cultures were achieved even when fewer than 20 CTCs per milliliter of blood were isolated. These findings emphasize the importance of surface modification for enhancing CTC isolation and the need for optimized culture conditions. The optimized MCA method offers a promising approach for rapid CTC recovery and potential integration with automated systems. Practical application : The Microcavity array (MCA) is a device specifically designed for efficient recovery of CTCs from whole blood. However cell adhesion on the MCA surface can limit release efficiency. This study demonstrated that surface modification with MPC signigicantly reduces cell‐substrate adhesion, improving recovery efficiency while maintaining cell viability and proliferative capacity. Compared to traditional density gradient centrifugation, the MPC‐modified MCA offers shorter processing time and better performance. CTCs were successfully detected in gastric cancer, and short‐term cultures were achieved even when fewer than 20 CTCs per mL of blood were isolated. The method supports downstearm applications such as cancer cell characterization and treatment monitoring. With potential for integration into automated system, the optimized MCA provides a practical, scalable solution for clinical liquid biopsy and personalized oncology.

Atezolizumab plus bevacizumab and lenvatinib are standard treatments for unresectable hepatocellular carcinoma; however, tumor markers such as alpha-fetoprotein and des-gamma-carboxy prothrombin have a limited ability to reflect treatment responses. Circulating tumor cells are non-invasive biomarkers associated with cancer stemness and treatment resistance. We assessed circulating tumor cell subsets expressing cancer stem cell markers (CD90, epithelial cell adhesion molecule, CD133, vimentin) using multiparametric flow cytometry at early and maximal response phases in patients receiving atezolizumab plus bevacizumab or lenvatinib. Early decreases in CD90-positive circulating tumor cells after therapy initiation were associated with tumor shrinkage and longer progression-free survival in both groups, as well as prolonged overall survival in the atezolizumab plus bevacizumab group. At maximal response, changes in CD90-positive circulating tumor cells reflected tumor burden more accurately than alpha-fetoprotein or des-gamma-carboxy prothrombin. These findings highlight the potential of CD90-positive circulating tumor cells to become dynamic biomarkers in systemic therapy for unresectable hepatocellular carcinoma.