DiR-labeled Embryonic Stem Cells for Targeted Imaging of in vivo Gastric Cancer Cells

Abstract

Embryonic stem (ES) cells have great potential in applications such as disease modeling, pharmacological screening and stem cell therapies. Up to date, there is no related report on the use of ES cells as tracking and contrast reagents of cancer cells in vivo. Herein we report that DiR-labeled murine ES cells can recognize and target gastric cancer cells in vivo. DiR-labeled murine ES (mES) cells (5×10(6)) were intravenously injected into gastric tumor-bearing mice. The biodistribution of DiR-labeled mES cells was monitored by IVIS imaging within 24 h. Major organs were harvested and analyzed by immunofluorescence staining and Western blotting. Chemotaxis assay was employed to investigate the chemotaxis of ES cells tracking cancer cells. Fluorescent imaging results showed that DiR-labeled mES cells targeted gastric cancer tissue in vivo as early as 10 min post-injection, reaching a peak at 2h post-injection. Immunofluorescence staining and Western blotting results showed gastric cancer tissues specifically expressed SSEA-1. In vitro migration tests confirmed that mES cells actively moved to test sites with different concentration of CXCL12 in a dose-dependent manner. In conclusion, DiR-labeled mES cells may be used for gastric cancer targeted imaging in vivo, and have great potential in applications such as identifying and imaging of early gastric cancer in near future.

Keywords: chemotaxis.; gastric cancer cells; migration; murine embryonic stem cells; target imaging.

Fig 1

Fluorescence microscope images of the morphology of GFP-SV129 mES cells.(A) GFP-SV129 mES cells exhibited typical large colonies and clearly boundaries with feeder cells. (B) mES cells expressed strong green fluorescence. (C) Magnification of mES cells cultured on feeder cells. (D) Feeder-free culture of mES cells grew in monolayer. (E) Feeder-free culture of mES cells exhibited green fluorescence. (F) Magnification of feeder-free culture of mES cells.

Fig 2

(A) DiR-labeled mES cells were subjected to fluorescent imaging at different exposure time, the ratio of DiR-labeled mES cells to unlabeled mES cells shows that the DiR-mES cells had strong fluorescent signals within 24 h. (B) The quantitative analysis of fluorescence of DiR(+) cells showed a linear relationship (r²=0.9939) between Fluc activity and cell numbers. (C) Trypan blue staining shows that mES cells had good cell viability after being labeled with DiR. (D) mES cells grew very well after being labeled with DiR 24 h.

Fig 3

In vivo fluorescence images of gastric cancer mouse models at 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 10 h, 24 h post-injection DiR-mES cells at lateral position. It shows that tumor tissues had strong fluorescence signals after injection DiR-mES cells as shown in blue circles. The tissue to background ratio (TBR) values show that DiR-mES cells accumulated in the tumor tissues and reached peak value at 2 h post-injection.

Fig 4

Optical imaging of DiR-mES cells in vivo/ex vivo. (A) Fluorescence imaging of gastric cancer mouse models at 6 h post-injection at lateral position; (B) Bioluminescence imaging of gastric cancer mouse models at 6 h post-injection at lateral position; (C) Combination image of fluorescence and bioluminescence imaging shows that DiR-mES cells could target the tumor tissues exactly. (D)Ex vivo fluorescent image of the dissected organs and tumors in the control group and test group at 24 h post-injection. (E)Radiant efficiency of the tissues in the test group and control group.

Fig 5

(A) Immunofluorescence analysis of the tumor tissues. (B) Western blot analysis of the tumor tissues.

Fig 6

Analysis of chemotaxis. (A) Expression of CXCR4 in isotype control(a) and mES cells (b) were determined by flow cytometry. (B) Secretion of CXCL12 in MFC cells was measured by enzyme-linked immunosorbent assay. (C) Cell and chemoattractant dose-response curves.