Clin Exp Otorhinolaryngol.  2013 Sep;6(3):176-183.

Selective Delivery of a Therapeutic Gene for Treatment of Head and Neck Squamous Cell Carcinoma Using Human Neural Stem Cells

• Kwon SK
• Kim SU
• Song JJ
• Cho CG
• Park SW

Abstract

OBJECTIVES
Based on studies of the extensive tropism of neural stem cells (NSCs) toward malignant brain tumor, we hypothesized that NSCs could also target head and neck squamous cell carcinoma (HNSCC) and could be used as a cellular therapeutic delivery system.
METHODS
To apply this strategy to the treatment of HNSCC, we used a human NSC line expressing cytosine deaminase (HB1.F3-CD), an enzyme that converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), an anticancer agent. HB1. F3-CD in combination with 5-FC were cocultured with the HNSCC (SNU-1041) to examine the cytotoxicity on target tumor cells in vitro. For in vivo studies, an HNSCC mouse model was created by subcutaneous implantation of human HNSCC cells into athymic nude mice. HB1.F3-CD cells were injected into mice using tumoral, peritumoral, or intravenous injections, followed by systemic 5-FC administration.
RESULTS
In vitro, the HB1.F3-CD cells significantly inhibited the growth of an HNSCC cell line in the presence of the 5-FC. Independent of the method of injection, the HB1.F3-CD cells migrated to the HNSCC tumor, causing a significant reduction in tumor volume. In comparison to 5-FU administration, HB1.F3-CD cell injection followed by 5-FC administration reduced systemic toxicity, but achieved the same level of therapeutic efficacy.
CONCLUSION
Transplantation of human NSCs that express the suicide enzyme cytosine deaminase combined with systemic administration of the prodrug 5-FC may be an effective regimen for the treatment of HNSCC.

Keyword

Head and neck neoplasms; Neural stem cells; Tropism; Cytosine deaminase; Molecular targeted therapy

MeSH Terms

Animals
Brain Neoplasms
Carcinoma, Squamous Cell
Cell Line
Cytosine Deaminase
Flucytosine
Fluorouracil
Head
Head and Neck Neoplasms
Humans
Injections, Intravenous
Mice
Mice, Nude
Molecular Targeted Therapy
Neck
Neural Stem Cells
Suicide
Transplants
Tropism
Tumor Burden
Cytosine Deaminase
Flucytosine
Fluorouracil

Figure

• 

  Fig. 1 Analysis of cytosine deaminase (CD) expression by reverse transcription PCR. The HB1.F3-CD (F3-CD) line was derived from HB1.F3 (F3) cells by retroviral transduction with the gene for Escherichia coli CD. The CD transcript was expressed in F3-CD cells, but not in F3 cells.

• 

  Fig. 2 Selective cytotoxic effects on SNU-1041 tumor cells in vitro by HB1.F3-CD (F3-CD)/5-fluorocytosine (5-FC) regimen. (A) No significant decrease in proliferation was caused by four days treatment with 5-FC (2,500 µg/mL) in HB1.F3 (F3) cells or SNU-1041 cells. F3-CD cells and SNU-1041 cells co-cultured with F3-CD cells exhibited significant inhibition of proliferation by same dose of 5-FC. (B) MTT assay showed that SNU-1041 and F3 cells proliferated without significant changes from controls by high concentrations of 5-FC, confirming the low toxicity of the prodrug. F3-CD cells and SNU-1041 cells cocultured with F3-CD cells exhibited significant inhibition of proliferation. Inhibition of proliferation increased as 5-FC concentration in the culture media increased. *P<0.05, Mann-Whitney U-test.

• 

  Fig. 3 Tumor homing capabilities of HB1.F3-CD (F3-CD) cells (Prussian blue staining, ×40). F3-CDs were labeled with Feridex and injected into the tail vein (A), subcutaneously 1.5 cm from the tumor injection site (B), or at the tumor center (C) on 7 day after tumor cell inoculation. Mice were sacrificed on day 14, and Prussian blue staining was performed. F3-CDs were found primarily at the tumor border (white arrowheads). F3-CDs were not observed in tissues from brain (D), heart (E), kidney (F), liver (H), or spleen (I). F3-CDs were observed in lung tissue (G, black arrowhead) at a very low frequency.

• 

  Fig. 4 Antitumor effects of therapeutic HB1.F3-CD (F3-CD) cells in the presence of 5-fluorocytosine (5-FC) in vivo. (A) Timeline for experiment. (B) Representative mice from control (group 4) and 5-FC treated (group 3) groups. (C) The F3-CD/5-FC regimen caused an inhibitory effect on tumor growth in all experimental animals, irrespective of stem cell injection method. Significant decreases in tumor growth occurred in groups 1, 2, and 3 in comparison to the control group (group 4). In comparison with the conventional treatment group (group 5), there was no significant difference in tumor volume. s.c., subcutaneous injection; PBS, phosphate-buffered saline; 5-FU, 5-fluorouracil; i.v., intravenous injection. *P<0.05. **P<0.01, Mann-Whitney U-test.

• 

  Fig. 5 Side effects of treatment. (A) Differences in mouse size in representative animals indicate the low toxicity of 5-fluorocytosine (5-FC) administration (upper) in comparison with the serious weight loss caused by 5-fluorouracil (5-FU) treatment (down). (B) There was no significant difference in weight between animals treated with 5-FC and animals treated with phosphate-buffered saline. Animals treated with 5-fluorouracil showed significant weight loss compared with animals treated with 5-FC. PBS, phosphate-buffered saline. *P<0.01, Mann-Whitney U-test.

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