The Anti-Tumor Effects of Adipose Tissue Mesenchymal Stem Cell Transduced with HSV-Tk Gene on U-87-Driven Brain Tumor.

Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 μM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues

Bystander effect of transduced AT-MSC-Tk in U-87 cells.

<p>U-87 cells were co-cultured in each well with the indicated AT-MSC cells at the same proportion. GCV 25 and GCV 50 indicate 25 μM and 50 μM of GCV, respectively; CONTROL indicates co-culture of U-87 and AT-MSC-GFP without GCV; GFP and TK-GFP indicate AT-MSC-GFP and AT-MSC-GFP-Tk cells, respectively. (A) Cell images were acquired, using an inverted microscope 8 days after GCV addition. (B) AT-MSC-Tk + GCV (25 and 50 μM) on the 15th day of culture. Bar = 50 μm.</p

Co-localization of tumor and AT-MSC-Tk by immunohistochemistry.

<p>U-87 tumor and AT-MSC were labeled with human nuclei monoclonal antibody conjugated to FITC (green) and AT-MSC-Tk-GFP labeled with anti-GFP antibody conjugated to Alexa Fluor 594 (red). Therefore, AT-MSC double labeled is in yellow. (A-C) U-87 + AT-MSC-Tk + GCV (200 X); (A) anti-Hu; (B) Anti-GFP; (C) Merge; (D-F) U-87 + AT-MSC-Tk no GCV (100 X); (D) anti-Hu; (E) Anti-GFP; (F) Merge;(G-I) Brain slices from the contralateral hemisphere (200 X); (G) anti-Hu; (H) Anti-GFP; (I) Merge of G and I under visible light. (J-L) human brain tissue (100 X); (J) anti-Hu; (K) Anti-GFP; (L) Merge; (M) Digital amplification of the square area of the Fig 4C; (N) Digital amplification of the square area of the Fig 4F. White arrows indicate areas where tumor cells died by treatment, supposedly. Bar = 50 μm.</p

AT-MSC characterizations and Transduction efficiency.

<p>In freshly isolated cells major two subpopulations were present: (A) Hematopoietic (P1—CD45 positive) and non-hematopoietic cells (events outside P1, CD45 negative). Non-hematopoietic cells were analyzed in (B) and (C). Pre-adipocytes were detected in B (P2—CD146 negative and CD34 positive) and in C (P2—CD31 negative and CD34 positive). MSC were detected in B (P5—CD146 positive, CD34 negative). Endothelial progenitors were detected in C (P5—CD31 positive, CD34 positive). (D—F) After seeding, AT-MSC from monolayer was still negative for CD45 and positive for CD34 and CD146. (G-I) AT-MSC were also positive for CD105, CD73 and CD90, which are all mesenchymal surface markers in vitro. (J—L) AT-MSC are multipotent for adipogenic (Oil Red O staining), osteogenic (Alizarin staining) and chondrogenic (Pellet culture, safranin staining) lineages, respectively. (M) Transduction efficiency in AT-MSC of the lentivirus containing Tk-GFP is 80%.</p