[Adeno-associated virus-mediated CD40 ligand transfer into human lung cancer cells].
OBJECTIVE
To investigate the transduction efficiency of serotype 1, 2, 5, 6, 7, 8, 9, 10 recombinant adeno-associated viruses (rAAV) in human lung cancer cell line A549 cells and compare the transduction efficiency of conventional AAV vectors with that of self-complementary AAV (scAAV) vectors. Furthermore, the capacity of A549 cells expressing transgenic CD40L to stimulate dendritic cells (DCs) was evaluated.


METHODS
Lung cancer A549 cells were infected with 1 x 10(4) particules per cell of AAV encoding the green fluorescent protein (GFP) or human CD40L driven by CMV promotor, and transgene expression was analyzed by flow cytometry and fluorescence microscopy. Stimulation of isolated human dendritic cells by CD40L-expressing tumor cells was quantified by measuring secreted interleukin-12 with immunoassay.


RESULTS
Serotype AAV2/5 transduced A549 cells much more efficiently than serotypes AAV2/1, AAV2/2, AAV2/6, AAV2/7, AAV2/8, AAV2/9 and AAV2/10. The transduction efficiency of scAAV2/5 was significantly higher than that of conventional AAV2/5. Furthermore, pre-treatment with carboplatin substantially increased AAV-mediated transgene expression. The scAAV2/5 vectors encoding human CD40L was used to generate CD40L. A549 cells transduce by these vectors were co-cultured with immature human DCs. As a consequence, interleukin-12 was released and measured in the culture supernatant. Specificity of immunostimulatory effect of CD40L was confirmed by blocking with a monoclonal antibody binding to human CD40L.


CONCLUSIONS
scAAV2/5 transduce lung adenocarcinoma A549 cell efficiently, and co-administration of chemotherapeutic agent carboplatin further enhances its transduction efficiency. It is confirmed that lung cancer cells infected with a CD40L-encoding scAAV2/5 construct can activate human DCs to secrete interleukin-12. Our findings provided a basis for future immunotherapeutic approaches including intratumoral transfer of stimulating factors.
Wu
Jian-qing
JQ
Department of Geriatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing, China. jianqingwu@gmail.com
Zhao
Wei-hong
WH
Yin
Kai-sheng
KS
Cheng
Yun-lin
YL
D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D023201:CD40 Ligand / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016190:Carboplatin / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D000229:Dependovirus / Q000145:classification / Q000235:genetics*; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods; D018664:Interleukin-12 / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012703:Serotyping; D014162:Transfection
D000970:Antineoplastic Agents; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins; D018664:Interleukin-12; D016190:Carboplatin