CD26, a multifunctional transmembrane glycoprotein, is expressed in various cancers and functions as dipeptidyl peptidase 4 (DPP4). We investigated whether CD26 expression is associated with hepatocellular carcinoma (HCC) progression and whether DPP4 inhibitors exert antitumor effects against HCC.
CD26 expression was examined in 41 surgically resected HCC specimens. The effects of DPP4 inhibitors on HCC were examined by using HCC cell lines (Huh-7 and Li-7), xenograft tumors in nude mice, and a nonalcoholic steatohepatitis-related HCC mouse model.
CD26 expression in HCC specimens was associated with increased serum DPP4 activity, as well as a more advanced stage, less tumor immunity, and poorer prognosis in HCC patients. The HCC cell lines and xenograft tumors exhibited CD26 expression and DPP4 activity. The DPP4 inhibitors did not exhibit antitumor effects in vitro, but natural killer (NK) and/or T-cell tumor accumulation suppressed growth of xenograft tumor and HCC in vivo. The antitumor effects of DPP4 inhibitors were abolished by the depletion of NK cells or the neutralization of CXCR3, a chemokine receptor on NK cells. EZ-TAXIScan, an optical horizontal chemotaxis apparatus, identified enhanced NK and T-cell chemotaxis by DPP4 inhibitors ex vivo in the presence of Huh-7 cells and the chemokine CXCL10, which binds to CXCR3. The DPP4 inhibitors prevented the biologically active form of CXCL10 from being truncated by Huh-7 cell DPP4 activity. DPP4 inhibitors also suppressed tumor angiogenesis.
These results provide a rationale for verifying whether DPP4 inhibitors clinically inhibit the progression of HCC or augment the antitumor effects of molecular-targeting drugs or immunotherapies against HCC.

Macrophages migrate and invade into damaged muscle rapidly and are important for muscle repair and subsequent regeneration. The exact cellular and biological events that cause macrophage migration toward injured muscle are not completely understood. In this study, the effect of macrophage differentiation on the chemotactic capability to invade local damaged muscle was investigated using an in vitro model of muscle injury. We used C2C12 cell myoblasts and J774 cell macrophages, and the \"killed-C2C12\" cells were combined with live C2C12 cells as a partially damaged muscle model. The cultured J774 cells, with or without lipopolysaccharide (LPS), were treated with Ly294002 (Ly), which is an inhibitor of phosphoinositide 3-kinase (PI3K). In order to evaluate the polarization effect of LPS stimulation on J774 cells, expression of cell surface Toll-like receptor 4 (TLR4), CD11c and CCR2, and expression of F-actin intensity, were analyzed by flow cytometry. The real-time horizontal chemotaxis assay of J774 cells was tested using the TAXIScan device. The expressions of TLR4, CD11c, and F-actin intensity in LPS-treated cells were significantly higher than those in Ctrl cells. In LPS-treated cells, the chemotactic activity toward damaged muscle cells completely disappeared. Moreover, the reduced chemotaxis depended far more on directionality than velocity. However, Ly treatment reversed the reduced chemotactic activity of the LPS-treated cells. In addition, cell-adhesion and F-actin intensity, but not CCR2 expression, in LPS-treated cells, was significantly reduced by Ly treatment. Taken together, our results suggest that the PI3K/Akt activation state drives migration behavior towards damaged muscle cells.

Migration of cancer cell correlates with distant metastasis and local invasion, which are good targets for cancer treatment. An optically accessible device \"TAXIScan\" was developed, which provides considerably more information regarding the cellular dynamics and less quantity of samples than do the existing methods. Here, we report the establishment of a system to analyze the nature of pancreatic cancer cells using TAXIScan and we evaluated lysophosphatidic acid (LPA)-elicited pancreatic cell migration.
Pancreatic cancer cell lines, BxPC3, PANC-1, AsPC1, and MIAPaCa-2, were analyzed for adhesion as well as migration towards LPA by TAXIScan using parameters such as velocity and directionality or for the number of migrated cells by the Boyden chamber methods. To confirm that the migration was initiated by LPA, the expression of LPA receptors and activation of intracellular signal transductions were examined by quantitative reverse transcriptase polymerase reaction and western blotting.
Scaffold coating was necessary for the adhesion of pancreatic cancer cells, and collagen I and Matrigel were found to be good scaffolds. BxPC3 and PANC-1 cells clearly migrated towards the concentration gradient formed by injecting 1 μL LPA, which was abrogated by pre-treatment with LPA inhibitor, Ki16425 (IC50 for the directionality ≈ 1.86 μM). The LPA dependent migration was further confirmed by mRNA and protein expression of LPA receptors as well as phosphorylation of signaling molecules. LPA1 mRNA was highest among the 6 receptors, and LPA1, LPA2 and LPA3 proteins were detected in BxPC3 and PANC-1 cells. Phosphorylation of Akt (Thr308 and Ser473) and p42/44MAPK in BxPC3 and PANC-1 cells was observed after LPA stimulation, which was clearly inhibited by pre-treatment with a compound Ki16425.
We established a novel pancreatic cancer cell migration assay system using TAXIScan. This assay device provides multiple information on migrating cells simultaneously, such as their morphology, directionality, and velocity, with a small volume of sample and can be a powerful tool for analyzing the nature of cancer cells and for identifying new factors that affect cell functions.

To compare the responses of individual neutrophils to chemoattractants, migration pathway data were obtained using TAXIScan, an optically accessible/horizontal apparatus in which a concentration gradient is established reproducibly for a given stimulus. The observed linear-mode trajectory pattern of neutrophils toward N-formyl-methionyl-leucyl-phenylalanine (fMLP) or Interleukin (IL)-8/CXCL8 was distinguished from random migration patterns toward leukotriene (LT) B4 or platelet activating factor (PAF). The median values of velocity and directionality calculated for individual cells toward fMLP and IL-8 were both relatively similar and high, whereas the values toward LTB4 and PAF were widely dispersed over a lower range of directionality and from low to high ranges of velocity. The different patterns between the groups may be explained by unique morphology with single polarity toward fMLP and IL-8, and unstable morphology with multiple polarities toward LTB4 and PAF. Unique morphologies toward fMLP and IL-8 were not affected by coexisting LTB4 or PAF. On the other hand, the addition of suboptimum concentrations of fMLP or IL-8 to LTB4 or PAF induced a nearly maximum chemotactic response in most cells. These data suggest that exogenous formyl peptides and endogenous chemokines augment neutrophil accumulation at inflammation sites, whereas lipid mediators may play a role in supporting activation of the inflammatory cells for recruitment.

To identify potential regulators of normal human melanocyte behaviour, we have developed an in vitro human melanocyte migration assay, using the optically accessible, real-time cell motility assay device TAXIScan. Coating of the glass surface with an extracellular matrix that served as scaffolding molecule was essential to demonstrate efficient melanocyte migration. Among several chemokines tested, stromal cell-derived factor (SDF)-1α/CXCL12 was the most effective driver of human normal skin melanocytes. Incubation of melanocytes with α-melanocyte-stimulating hormone (MSH) before the assay specifically enhanced CXCR4 expression and consequently chemotaxis towards SDF-1α/CXCL12. These results suggest that α-MSH acts on melanocytes to produce melanin as well as stimulates the cells to migrate to the site where they work through CXCR4 up-regulation, which is a new dynamic mode of action of α-MSH on melanocyte physiology.