Yamauchi Akira, Yamamura Masahiro, Katase Naoki, Itadani Masumi, Okada Naoko, Kobiki Kayoko, Nakamura Masafumi, Yamaguchi Yoshiyuki, Kuribayashi Futoshi
Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192, Japan.
Department of Clinical Oncology, Kawasaki Medical School, Okayama, Japan.
BMC Cancer. 2017 Mar 31;17(1):234. doi: 10.1186/s12885-017-3218-4.
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 (IC 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. LPA mRNA was highest among the 6 receptors, and LPA, LPA and LPA 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.
癌细胞迁移与远处转移和局部侵袭相关，是癌症治疗的良好靶点。研发了一种光学可及的装置“TAXIScan”，与现有方法相比，它能提供更多关于细胞动力学的信息且所需样本量更少。在此，我们报告使用TAXIScan建立了一个分析胰腺癌细胞特性的系统，并评估了溶血磷脂酸（LPA）诱导的胰腺细胞迁移。
使用TAXIScan通过诸如速度和方向性等参数分析胰腺癌细胞系BxPC3、PANC-1、AsPC1和MIAPaCa-2对LPA的黏附及迁移情况，或通过博伊登室法分析迁移细胞的数量。为证实迁移由LPA引发，通过定量逆转录聚合酶反应和蛋白质印迹法检测LPA受体的表达及细胞内信号转导的激活情况。
支架包被对胰腺癌细胞的黏附是必要的，发现I型胶原和基质胶是良好的支架。BxPC3和PANC-1细胞明显向注射1 μL LPA形成的浓度梯度迁移，用LPA抑制剂Ki16425预处理可消除这种迁移（方向性的IC≈1.86 μM）。LPA受体的mRNA和蛋白表达以及信号分子的磷酸化进一步证实了LPA依赖性迁移。LPA mRNA在6种受体中表达最高，在BxPC3和PANC-1细胞中检测到LPA、LPA和LPA蛋白。LPA刺激后，在BxPC3和PANC-1细胞中观察到Akt（Thr308和Ser473）和p42/44MAPK的磷酸化，用化合物Ki16425预处理可明显抑制这种磷酸化。
我们使用TAXIScan建立了一种新型的胰腺癌细胞迁移检测系统。该检测装置能同时提供关于迁移细胞的多种信息，如形态、方向性和速度，所需样本量小，可成为分析癌细胞特性及鉴定影响细胞功能新因子的有力工具。
