BMC Cancer 2006, 6:66 http://www.biomedcentral.com/1471-2407/6/66 TFuigmuorre v2olume analysis after treatment in BALB/c nude mice Tumor volume analysis after treatment in BALB/c nude mice. Implanted tumors were generated by injecting MCF-7 cells in the flank in BALB/c nude mice. When subcuta- neous tumors reached approximately 7–8 mm in diameter, rAAV/TRE/HSVtk/Tet-On virus (2 × 1011 particles/ml) or mock-infection (DMEM) were performed direct intratumor injection to human breast cancer model on day 0, followed by a second injection on day 7. GCV treatment (100 mg/kg) was started on day 0 and continued for another 14 days after induced by Dox for one week. Tumor size was measured every 3 days. Tumor volume values (mm3) are expressed as means ± SD. The tumors of the rAAV/tk/On+Dox+GCV group demonstrated significant tumor growth suppression after GCV treatment compared with mock (P < 0.05 on day 30 after infection). in a regulated manner in implanted human breast cancer infected intratumorally with rAAV. Histological analysis of human breast cancer in BALB/c nude mice BALB/c nude mice bearing human breast cancer with dif- ferent treatments were given sodium pentobarbital (50 mg/kg, i.p.) anesthesia to excise the subcutaneous tumors. In the hematoxylin and eosin stained sections, few accom- panying infiltrating cells were seen in control groups. On the other hand, remarkable levels of infiltration of lym- phocytes were observed throughout the tumors that had been injected with Dox followed by GCV treatment, and breast cancer cells were severely damaged (Fig. 3). TFiugmuorre s3lices stained with hematoxylin and eosin Tumor slices stained with hematoxylin and eosin. Subcutaneous tumors were excised from BALB/c nude mice with different treatments under sodium pentobarbital (50 mg/kg, i.p.) anesthesia. Tumors were rinsed twice in normal saline (NS) then fixed in 4% polyparaformaldehyde for 12 hours, embedded in paraffin, sliced and sections stained with hematoxylin and eosin for light microscopic observation (× 400 magnification). A: DMEM group; B: GCV+Dox group; C: GCV+Virus group; D: Dox+GCV+Virus group. HSVtk gene expression in tumor tissue and rAAV-infected MCF-7 cells In order to evaluate the efficiency of gene transfer in vivo, we used RT-PCR to detect the expression of HSVtk gene. A 420 bp fragment of HSVtk gene was observed, which was consistent with predicted results (Fig. 4). We detected high level of expression of HSVtk in tumor tissues of GCV + Dox+ rAAV treated group. The faintest band could be detected in the rAAV+GCV group and no band in other two groups (the DMEM group and the Dox+GCV group) could be seen (Fig. 5). After induction of Dox, the HSVtk expression level was enhanced by approximately fourfold in tumors subjected to treatment with GCV and viruses. This result was agreement with the growth of breast cancer in mice. As expected, without HSVtk expression, the growth of parental MCF-7 cells was not affected by GCV and tumors continued to grow until the mice were killed because of large tumor loads. A similar result was obtained in rAAV-infected MCF-7 cells. Discussion We constructed rAAV vector, which contained Tet-On reg- ulatory sequences and the HSVtk gene, in an effort to develop a novel treatment for breast cancer and other solid tumors. Our results demonstrate that this vector is Page 5 of 8 (page number not for citation purposes)