1. Volume 19, Issue 12, Pages 2249-2257 (December 2011)
The Development of a Novel Cancer Immunotherapeutic Platform Using Tumor- targeting Mesenchymal Stem Cells and a Protein Vaccine 
Hon-Jian Wei, Alexander TH Wu, Chung-Huei Hsu, Yi-Ping Lin, Wen-Fang Cheng, Ching-Hua Su, Wen-Ta Chiu, Jacqueline Whang- Peng, Frank L Douglas, Win-Ping Deng 
Molecular Therapy 
Volume 19, Issue 12, Pages (December 2011)
DOI: /mt
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
In vitro immunological analyses of mice vaccinated with protein vaccine. (a) Splenocyte proliferation assay in response to E7 peptide in vitro. Splenocytes from mice vaccinated with PE(ΔIII)-E7-KDEL3 showed increased proliferation in response to E7 peptides containing a major histocompatibility complex (MHC) class II epitope. (b) Dot blot analysis of E7-specific antibodies purified from mice vaccinated with PE(ΔIII)-E7-KDEL3. (c) E7-specific antibodies mediated complement-dependent cytotoxicity. After incubation with the coculture of NG4TL4-TK and KP-hMSC cells, the sera from mice vaccinated with PE(ΔIII)-E7-KDEL3 induced more cell lysis than that from naive mice. Error bars represent SD among three independent experiments. *P < 0.05, **P < hMSCs, human mesenchymal stem cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
In vivo imaging of mice-bearing tumor with protein vaccine/mesenchymal stem cells (MSCs) combined treatment. (a) Illustration of experimental design. (b) Representative planar γ-camera images from the subcutaneous tumor model. Images were taken at 7 and 21 after subcutaneous (s.c.) injection of 5 × 104 NG4TL4-TK cells in the right flanks. (c) Representative planar γ-camera images of lung-metastasis model. Images were collected at 7 and 14 days after intravenous injection of 1 × 105 NG4TL4-TK cells. There are four groups in both models. Control group, tumor bearing with no treatment (PE(ΔIII)-E7-KDEL3) group, tumor bearing with protein vaccine treatment only; KP-hMSCs, tumor bearing with MSCs treatment only; KP-hMSCs+PE(ΔIII)-E7-KDEL3 group, tumor bearing with combined treatment. In the subcutaneous tumor model, mice with combined treatment showed a significantly lower level of signal intensity from the tumor than other groups at day 21. Similar to the subcutaneous model, signal intensity from the tumor in the lungs was significantly weaker in the combined treatment group. Dotted circle represents the position of the lungs.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Assessments of mice-bearing tumor with protein vaccine/mesenchymal stem cell (MSC) combined treatment. (a) The volumes of subcutaneous tumors from different treatment groups. The tumors in combined-treatment group were significantly smaller than that in other groups at day 21, and slightly increased at day 28. Even after 34 days, the signal intensity of planar γ-camera imaging remains weak in combined-treatment group (n = 6). (b) Representative macro-morphological images of pulmonary nodules from different treatment groups. The least number of tumor nodules were observed in the combined-treatment group. (c) Quantification of pulmonary nodules from control and treatment groups. A significant decrease was observed in the number of pulmonary nodules in the combined-treatment group when compared with other groups (n = 5). Error bars indicate SD. *P < 0.05, compared with control group.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Ex vivo validation of mesenchymal stem cells (MSCs) targeting and vaccine/MSCs combined treatment in pulmonary metastatic tumor. (a) The presence of HSV1-tk gene in the lung tissues from different treatment groups was detected using reverse transcription (RT)-PCR. There were five different groups namely, without any treatment (lane 1), vaccine only treatment (lane 2), MSCs only treatment (lane 3), the combined treatment (lane 4), and positive control NG4TL4-TK cells (lane 5). HSV1-tk gene was used as an indicator for that the pulmonary tumors were developed by NG4TL4-TK cells, and could be detected in all groups. (b) RT-PCR detection of HPV-16 E6/E7 expression in lung tissues from the mice received different treatments. The group arrangement is the same as in a except lane 5, which represents a positive control, MSCs only. HPV-16 E6/E7 gene in the MSC was detected in MSCs only treatment group (lane 3) and MSCs control (lane 5). GAPDH was used as an internal standard. M represents DNA ladder. (c) Hematoxylin and eosin (H&E) stained parafilm sections of the lung tissues from different treatment groups. Pulmonary alveoli were appeared more intact in the combined-treatment group than in other groups. Blue arrow indicated tumor nodule formation.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Evaluation of tumor inhibition by the protein vaccine/mesenchymal stem cells (MSCs) combined treatment. (a) Western blots probed with anti-E7-antibody from sera of the mice received the combined treatment. A negative correlation between the level of anti-E7 antibody and the tumor volume was observed. (b) TUNEL analysis of tumor sections obtained from the combined treatment and MSCs only treatment groups. TUNEL staining was shown in green and propidium iodide (PI) in red. The sample from the combined treatment contained more TUNEL-positive cells than the MSCs only group. (c) Quantification of TUNEL analysis (n = 6). Error bars represent SD among three independent experiments. *P < 0.05, compared with KP-hMSCs group.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Proposed mechanism underlying the antitumor effect mediated by the vaccine/mesenchymal stem cell (MSC) combined treatment. Diagrammatic representation of antitumor mechanisms mediated by KP-hMSCs+PE(ΔIII)-E7-KDEL3 combined treatment. Antibody-mediated tumor immunity have been shown could be achieved by complement dependent cytotoxicity and antibody dependent cell-mediated cytotoxicity (NK cells). Hence, we proposed two possible scenarios that KP-hMSCs facilitated antitumor effect of PE(ΔIII)-E7-KDEL3. First, injected KP-hMSCs upon targeting and interacting with tumor cells, integrated into tumor-associated stroma by differentiating into vascular endothelial cells and stroma fibroblasts. The E7-antibody recongnized antigen and inhibited the tumor via destroying tumor-associated and antiangiogenic effect. Second, KP-hMSCs and the tumor cells underwent cellular fusion so that the hybrid progenies also expressed E7 antigen. Thus, antitumor effect was then achieved by anti-E7 antibodies from the immunization leading to the reduced tumor burden and metastasis.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions