Cancer cell-oriented migration of mesenchymal stem cells engineered with an anticancer gene (PTEN): an imaging demonstration

Abstract

Background: Mesenchymal stem cells (MSCs) have been considered to hold great potential as ideal carriers for the delivery of anticancer agents since the discovery of their tumor tropism. This study was performed to demonstrate the effects of phosphatase and tensin homolog (PTEN) engineering on MSCs' capacity for cancer cell-oriented migration.

Methods: MSCs were engineered with a PTEN-bearing plasmid and the expression was confirmed with Western blotting. A human glioma cell line (DBTRG) was used as the target cell; DBTRG cell-oriented migration of MSCs was monitored with a micro speed photographic system.

Results: The expression of transfected PTEN in MSCs was identified by immunoblotting analysis and confirmed with cell viability assessment of target cells. The DBTRG cell-oriented migration of PTEN-engineered MSCs was demonstrated by a real-time dynamic monitoring system, and a phagocytosis-like action of MSCs was also observed.

Conclusion: MSCs maintained their capacity for cancer cell-directed migration after they were engineered with anticancer genes. This study provides the first direct evidence of MSCs' tropism post-anticancer gene engineering.

Keywords: cancer; gene therapy; mesenchymal stem cells; phosphatase and tensin homolog.

Figure 1

PTEN expression and identification on PTEN-engineered MSCs. Notes: (A) The images of MSCs 24 hours after mock transfection of control plasmid (pDsRed1-N1) (A1 and A2) or the same plasmid inserted with PTEN (A3 and A4). The left panel presents brightfield images and the right panel shows red fluorescence images. (B) The immunoblotting results with anti-PTEN antibody (B1) and anti-RFP antibody (B2). Cells were harvested 2 days after electrotransfection with control plasmid or PTEN-bearing plasmid. The protein size markers are shown on the left. Abbreviations: PTEN, phosphatase and tensin homolog; MSCs, mesenchymal stem cells; RFP, red fluorescent protein.

Figure 2

ELISA analysis of PTEN in MSC culture media. Notes: Conditioned media from control MSCs, MSCs with mock transfection, and MSCs with PTEN-RFP transfection were collected 2 days after transfection. Mean ± SEM for four independent experiments. Abbreviations: ELISA, enzyme-linked immunosorbent assay; PTEN, phosphatase and tensin homolog; MSCs, mesenchymal stem cells; RFP, red fluorescent protein; SEM, standard error of the mean; OD, optical density.

Figure 3

DBTRG cell viability of indirect cocultures. Notes: DBTRG cells were plated into 24-well plates (1×10⁴/well) on day 0. The culture media were replaced with conditioned media from native MSC and MSC^PTEN on day 1. Cell viability assessment was performed on day 3 with a LIVE/DEAD Viability/Cytotoxicity Assay Kit (Life Technologies, Carlsbad, CA, USA). (A) The images under different culture conditions. Medium type and percentage are indicated on the left of the graph. Bar size, 30 μm. (B) The summary of cell viability of the indirect cocultures. Mean ± SEM for three independent experiments. *P<0.05 and **P<0.01 versus control. Abbreviations: DBTRG, human glioma cell line; PTEN, phosphatase and tensin homolog; MSC, mesenchymal stem cell; SEM, standard error of the mean.

Figure 4

Imaging demonstration of MSC’s migration toward DBTRG cells. Notes: DBTRG cells were plated into 6-well plates (5×10⁴ cells/well) on day 0. The MSCs which were preengineered with PTEN by electroporation were seeded into the DBTRG culture on day 1. The round cells are DBTRG and the spindle cells are MSCs. The real-time imaging capture commenced on day 1 with a LEICA DMIRE2 microscope (Leica Microsystems, Wetzlar, Germany) under the same culture conditions, ie, 37°C and 5% CO₂ atmosphere. The capturing frequency was three pictures per hour. Both (A and B) are static pictures which were intercepted at the beginning and the end of the third hour, respectively, from the video record. The red boxes show the migration of a mesenchymal stem cell toward DBTRG cells and the blue boxes display the phagocytosis-like action of a mesenchymal stem cell. Abbreviations: DBTRG, human glioma cell line; PTEN, phosphatase and tensin homolog; MSCs, mesenchymal stem cells.