Fengshu Zhao

Using ABCG2-molecule-expressing side population cells to identify cancer stem-like cells in a human ovarian cell line

CSCs (cancer stem cells) are a small subset of cells within a tumour that possesses the characteristics of stem cells and are considered to be responsible for resistance to chemoradiation. Identification of CSCs through stem cell characteristics might have relevant clinical implications. In this study, SP (side population) cells were sorted from a human ovarian cancer cell line by FACS to determine whether cancer stem cell‐like SP cells were present. A very small fraction of SP cells (2.6%) was detected in A2780 cells. SP cells possessed the following characteristics: highly proliferative activity, marked ability for self‐renewal in soft agar and culture medium, high expression of ABCG2, drug resistance to vinblastine in vitro, and strong tumourigenic potential in Balb/c nude mice. It is concluded that there exists in the A2780 cell line a small number of SP cells with high expression of ABCG2. The cells have the characteristics of cancer stem‐like cells, and identification and cloning of such human SP cells can help in improving therapeutic approaches to ovarian cancer in patients.

Enhancing therapy of B16F10 melanoma efficacy through tumor vaccine expressing GPI-anchored IL-21 and secreting GM-CSF in mouse model

In the present study, we developed the tumor vaccine expressing IL-21 in the GPI-anchored form together with secreting GM-CSFs and investigated its antitumor efficacy in C57BL/6 mouse model. The fusion genes containing IL-21 and the GPI anchor signal sequence were acquired by overlaping PCR, inserted into the downstream of two multi-clone sites in recombinant plasmid pRSC/GM-CSFs to form pRSC/IL-21-gpi-GM-CSFs that was transfected into the B16F10 cells. The tumor cell vaccine B16F10/IL-21-gpi-GM-CSFs was identified by reverse transcription PCR, IFA and FCM, respectively. The results showed that the pRSC/IL-21-gpi-GM-CSFs had no cell cycle and proliferative state impact on the B16F10 cells after transfected, and that the tumor vaccine B16F10/IL-21-gpi-GM-CSFs increased the cytotoxicities of NK cells and CD8(+)CTL, enhanced the level of serum IFN-gamma, augmented therapy of tumor effect and prolonged survival time in the tumor-bearing mice immunized with the tumor vaccine B16F10/IL-21-gpi-GM-CSFs. The data that we presented here provided a rationale and practical platform for clinical testing of enhancing cell therapy of B16F10 melanoma efficacy by modified tumor vaccine expressing GPI-anchored IL-21 and secreting GM-CSF.

Identifying tumor stem-like cells in mouse melanoma cell lines by analyzing the characteristics of side population cells

Increasingly more evidence shows that TSCs possess the characteristics of stem‐like cells. However, a link between stem cells and TSCs remains to be shown. We have sorted SP cells and non‐SP cells from the B16F10 cell lines by FACS, and then studied their cellular biological characteristics by using a SFCM culture method, proliferative assay in vitro, clone formation assays in soft agar and normal media, tumorigenic assays in C57BL/6 mice, and resistance to chemotherapy assay in vitro, the quantitative detecting expression of ABCG2 and their CD phenotype analysis by a FCM. We detected 0.96% SP cells in the B16F10 cells and found that they had obvious differences in characteristics from non‐SP cells. They possessed a marked capacity for self‐renewal in soft agar and culture medium, strong tumorigenic potential in C57BL/6 mice, apparent resistance to vinblastin in vitro, upregulated ABCG2 expression, and a high expression of CD44+CD133+CD24+ phenotypes. We conclude that there were a few of SP cells that had the characteristics of tumor stem‐like cells which may provide a useful tool and a readily accessible source for further study when specific TSCs markers are unknown.

Antitumor efficacy induced by human ovarian cancer cells secreting IL-21 alone or combination with GM-CSF cytokines in nude mice model.

The ovarian cancer cells (SKOV3) secreting IL-21 alone or combination with GM-CSF cytokines was developed and its antitumor effect was evaluated in the nude mice. The gene of IL-21 was amplified from plasmid pRSC-IL-21 by PCR, cloned into the plasmid pRSC-GM-CSF, and the plasmid pRSC-GM-CSF-IL21 was constructed. The plasmids of pRSC-GM-CSF, pRSC-IL21, pRSC-GM-CSF-IL21 and pRSC were respectively transfected into the SKOV3 cells and antitumor efficacy induced by the SKOV3 cells secreting IL-21 or combination with GM-CSF was evaluated by surveying the tumor growth and the nude mices survival. The results indicated that the secreted IL-21 and GM-CSF were functional because the culture supernatant of SKOV3 cells transfected with the plasmid pRSC-GM-CSF-IL21 enhanced NK cytotoxicity in vitro. The expressions of MIC A/B, NKG2D and ICAM-1 molecules on the SKOV3 cells were up-regulated. The level of IFN-gamma and TNF-alpha, the NK cytotoxicity and the antitumor efficacy were significantly increased in the null mice inoculated with the SKOV3 cells secreting both IL-21 and GM-CSF in comparison with the nude mice inoculated with the other different SKOV3 cells. We concluded that the SKOV3 cells genetically engineered to secrete biologically active IL-21 and GM-CSF elicited antitumor immunity effectively through enhancing NK cytotoxicity, promoting the expressions of MIC A/B , ICAM-1 and NKG2D molecules as well as elevating level of IFN-gamma and TNF-alpha in the nude mice model.

Augmenting therapy of ovarian cancer efficacy by secreting IL-21 human umbilical cord blood stem cells in nude mice.

In the present study, CD34+ human umbilical cord blood stem cells (UCBSCs) were engineered to express interleukin-21 (IL-21) and then were transplanted into A2780 ovarian cancer xenograft-bearing Balb/c nude mice. The therapeutic efficacy of this procedure on ovarian cancer was evaluated. The findings from the study indicated that UCBSCs did not form gross or histological teratomas until up to 70 days postinjection. The CD34+ UCBSC-IL-21 therapy showed a consistent effect in the ovarian cancer of the treated mice, delaying the tumor appearance, reducing the tumor sizes, and extending life expectancy. The efficacy was attributable to keeping CD34+ UCBSC-IL-21 in the neoplastic tissues for more than 21 days. The secreted IL-21 not only increased the quantity of CD11a+ and CD56+ NK cells but also increased NK cell cytotoxicities to YAC-1 cells and A2780 cells, respectively. The efficacy was also associated with enhancing the levels of IFN-γ, IL-4, and TNF-α in the mice as well as the high expressions of the NKG2D and MIC A/B molecules in the tumor tissues. This study suggested that transferring CD34+ UCBSC-IL-21 into the nude mice was safe and feasible in ovarian cancer therapy, and that the method would be a promising new strategy for clinical treatment of ovarian cancer.

Investigation of immunogenic effect of the BCG priming and Ag85A- GM-CSF boosting in Balb/c mice model.

Mycobacterium tuberculosis (M. tuberculosis) has once again become a major public health threat owing to the combined effects of a worldwide anti-tuberculosis drug resistance and the emergence of the human immunodeficiency virus pandemic. The Bacille Calmette-Guérin (BCG)-based vaccine has displayed inconsistent efficacy in different trials although it continues to be used to prevent tuberculosis in many countries. In current work, we have developed DNA vaccines expressing M. tuberculosis antigen 85A (Ag85A) and cytokine granulocyte macrophage colony stimulating factor (GM-CSF) and aimed to investigate the immune effect in mice based on BCG priming and DNA vaccine boosting and immune protection against M. tuberculosis challenge. Our results showed that the activity of cytotoxic T lymphocyte and spleen cell proliferative responses to Ag85A and IFN-gamma level as well as the specific antibody titer against Ag85A were significantly increased in mice immunized with prime-boost strategy in comparison with the mice immunized with BCG or DNA vaccine expressing Ag85A and GM-CSF alone. Meanwhile, the immune strategy of BCG-prime and DNA vaccine boost induced mice to generate efficient immune protection against M. tuberculosis challenge. Our data demonstrate that BCG-prime and DNA vaccine expressing Ag85A and GM-CSF boost provides a rational strategy for further development of DNA vaccine against M. tuberculosis infection.

Comparison of Immune Responses Induced in Mice by Vaccination with DNA Vaccine Constructs Expressing Mycobacterial Antigen 85A and Interleukin-21 and Bacillus Galmette-Guérin

In this paper, we addressed the immune adjuvant effects of interleukin(IL)-21 on DNA vaccine constructs expressing mycobacterium tuberculosis (TB) Ag85A and compared immune responses induced in mice inoculated DNA vaccine constructs expressing Ag85A and IL-21 with mice inoculated DNA vaccine constructs expressing Ag85A alone or Bacillus Galmette-Guérin(BCG.). In this experiment, the gene of IL-21 was firstly amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned into the plasmid pRSC, forming recombinant plasmid pRSC-IL21. Then, the gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned into the recombinant pRSC-IL21 again, finally forming co-expression DNA vaccine constructs pRSC-IL21-Ag85A. It was identified by the analysis of endonuclease digestion, DNA sequencing, the IL-21 and Ag85A expression in SP2/0 cells. Mice were i.m. immunized with BCG, DNA vaccine constructs pRSC-Ag85A or pRSC-IL21-Ag85A respectively, and the immune responses induced in mice was compared with other vaccines. The results showed that the DNA vaccine constructs pRSC-IL21-Ag85A was successfully constructed since the Ag85A and IL-21 was correctly expressed in SP2/0 cells respectively, and it elicited stronger immune responses in Balb/c mice than that of mice immunized with pRSC-Ag85A and the efficiency was as BCG did. We concluded that the IL-21 was a promising immune adjunctive modality to enhance immunigenicity of DNA vaccine containing Ag85A and the study provided the possibility of further development of immune accessory effect of IL-21 on DNA vaccine against TB.

Study of immunotherapy of murine myeloma by an IL-21-based tumor vaccine in BALB/C mice.

The tumor cells can be recognized and eliminated by the power of the immune response has result in intense interest in the development of tumor vaccines transfected with plasmid DNA containing target genes, and the tumor vaccines are being evaluated as prophylactic and therapeutic vaccines for tumor. In current study, we designed a murine myeloma cell(SP2/0) vaccine containing mIL-21 plasmid DNA and to evaluate its anti-tumor efficacy and analyze the mechanism of anti-tumor efficacy. It was up-regulated obviously that the MHC-Ⅰmolecule was expressed on SP2/0-mIL-21 tumor cells surface and the significant tumor regression and prolonged survival were observed in BALB/c mice injected with the SP2/0-mIL-21 tumor vaccine. The four mice without tumors growth were rechallenged with SP2/0 cells on the opposite site of the back and there was only one with growth a small tumor after 30 days and others remained tumor free. The cytotoxic activities of NK, CTLs and the IFN-γ were significantly increased respectively in immunized mice. The expression of I-TAC in the tumor tissue was up-regulated and the tumor tissue were showed the tumor cells were apoptosis and a lots of infiltrating lymphocytes and phagocytes. We conclude that the autologous IL-21-producing tumor vaccine can induce strong cell-mediated immune response and it is a promising immune adjunctive modality to prevent or inhibit growth of SP2/0 cells in mice model.

Effect of downregulation of ZEB1 on vimentin expression, tumour migration and tumourigenicity of melanoma B16F10 cells and CSCs.

Zinc‐finger E‐box binding homeobox 1 (ZEB1) is a master regulator of epithelial‐mesenchymal transition (EMT) and has been implicated in primary epithelial cancer biological processes, such as invasion and metastasis. However, the role of ZEB1 in progression of melanoma and cancer stem cells (CSCs) remains obscure. In this study, the recombinant plasmids of t3 shRNAs targeting mouse ZEB1 were constructed and transfected into melanoma B16F10 cells. The stable transfected cells were selected and the characteristics of ZEB1 downregulated B16F10 cells was assessed. The tumourigenicity of CD44+CD133+CSCs isolated from B16F10 cells stably transfected with the ZEB1‐shRNA2 recombinant was also assessed. ZEB1‐shRNAs B16F10 showed a lower expression of ZEB1 and vimentin, weaker migration, invasiveness, colony forming, and proliferation, and a lower tumourigenicity than the control cells. The tumourigenicity of the ZEB1‐shRNA2 CD44+CD133+CSCs was also inhibited. In conclusion, ZEB1‐shRNA2‐mediated downregulation of ZEB1 expression in B16F10 cells and CSCs is involved in the inhibition of the EMT process. ZEB1 may be a potential target in melanoma targeted.

Investigation on the anti-tumor efficacy by expression of GPI-anchored mIL-21 on the surface of B16F10 cells in C57BL/6 mice

GPI-anchored membrane cytokines have been shown to play an important role in host immune response against tumor cells. In the present study, we constructed the tumor vaccine expressing mIL-21 in the GPI-anchored form and investigated its anti-tumor effect in C57BL/6 mice model. The fusion genes containing mIL-21 and the GPI anchor signal sequence was acquired by overlaping PCR, inserted into plasmid pcDNA3.1 to form the pcDNA3.1 mIL-21-GPI recombinant, which was transfected into the B16F10 cells, and the tumor vaccine based on B16F10 cells expressing the GPI-anchored membrane mIL-21 was generated. Through transfection, it was found that GPI-anchored membrane mIL-21 has no proliferate impact on B16F10 cells, but it was functional and reflected in inducing CD3-activated murine splenocytes proliferation response to B16F10 cells, improving the cytotoxicities of CTL and NK cells, increasing the numbers of splenocytes-producing IFN-gamma in mice, augmenting therapeutic effect of tumor and prolonging longevity effects in tumor-bearing mice injected with the inactivated GPI-anchored mIL-21 tumor vaccine. We concluded the expression of mIL-21 on the B16F10 cells surface in the GPI-anchored form was proved to be effective in activating immune responses against tumor cells, and our results provided a good foundation for further investigating the immunotherapy of tumor by GPI-mIL-21.