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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

BMC Cancer 1/2017

Evaluation of pancreatic cancer cell migration with multiple parameters in vitro by using an optical real-time cell mobility assay device

Zeitschrift:
BMC Cancer > Ausgabe 1/2017
Autoren:
Akira Yamauchi, Masahiro Yamamura, Naoki Katase, Masumi Itadani, Naoko Okada, Kayoko Kobiki, Masafumi Nakamura, Yoshiyuki Yamaguchi, Futoshi Kuribayashi
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12885-017-3218-4) contains supplementary material, which is available to authorized users.

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.
Zusatzmaterial
Additional file 1: Table S1. Primers used for the quantitative RT-PCR. Total 6 pairs of primers for LPA receptors (LPA1, LPA2, LPA3, LPA4, LPA 5, and LPA6) were used for this study, based on the information reported previously (27). (DOCX 14 kb)
12885_2017_3218_MOESM1_ESM.docx
Additional file 2: Table S2. Targets for PathScan RTK signaling array. The phosphorylation of 39 different molecules in BxPC3 and PANC-1 cells was evaluated using the PathScan array. Details are described in Methods section. (DOCX 14 kb)
12885_2017_3218_MOESM2_ESM.docx
Additional file 7: The modified Boyden chamber assay. A) Schematic diagram (sagittal section) of one well of the modified Boyden chamber assay (Transwell). Cells in the chemotaxis buffer are located in the upper chamber and the chemoattractant the chemotaxis buffer is added to the lower chamber. B) Schematic diagram of the membrane part of the modified Boyden chamber. The membrane separates the upper and the lower chamber. The matrix is coated on the lower side of the membrane. C) Photographs of the lower side of the membrane after the assay. Cells are stained with the staining solution accompanied with the assay kit. Magnification: 400×. (TIFF 98113 kb)
12885_2017_3218_MOESM7_ESM.tif
Additional file 8: The TAXIScan assay. A) Schematic diagram (sagittal section) of one channel of the TAXIScan chamber. The chamber is filled with the chemotaxis buffer (light brown color). Cells are located on the one side of the micro-channel and the chemoattractant (red color) is placed on the other side of the micro-channel. B) Schematic diagram (sagittal section) of the micro-channel. The chemoattractant is defused in the micro-channel, which forms the stable concentration gradient. Cells on the matrix-coated coverslip migrates towards the gradient of the chemoattractant in the micro-channel. C) Photograph of cells migrating towards the chemoattractant. The image is taken from underneath of the TAXIScan chamber. (TIFF 98112 kb)
12885_2017_3218_MOESM8_ESM.tif
Literatur
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