Chemotherapeutic agents enhance AAV2-mediated gene transfer into breast cancer cells promoting CD40 ligand-based immunotherapy

Abstract

Purpose: Supplementing conventional treatment with gene therapy to induce an immune response might be beneficial to cancer patients. In this study, we evaluated the efficiency of transduction of breast cancer cells with recombinant adeno-associated virus (rAAV) and effects of cytotoxic agents used in chemotherapy. Furthermore, the capacity of tumor cells expressing transgenic CD40 ligand (CD40L) to stimulate dendritic cells was measured.

Methods: Breast cancer cell lines were infected with a rAAV encoding the enhanced green fluorescent protein (EGFP) or murine CD40L and transgene expression was analyzed by flow cytometry. Stimulation of isolated human dendritic cells by CD40L-expressing tumor cells was quantified by measuring secreted interleukin 12.

Results: Infection with an EGFP-encoding rAAV resulted in variable fractions (14-93%, mean 42%) of transgene-expressing cells. Pre-incubation of MM 157, MM 231, and MCF7 cells with epirubicin or carboplatin substantially increased AAV-mediated transgene expression. rAAV/CD40L was used to generate CD40L-transgenic tumor cells, which specifically activated immature dendritic cells, as confirmed by blocking with an antibody binding to CD40L.

Conclusions: The efficiency of rAAV-mediated gene transfer into breast cancer cells is significantly higher than previously reported and can be further enhanced by co-administration of chemotherapeutic agents. We also confirmed that breast cancer cells can activate human dendritic cells after infection with a CD40L-encoding rAAV.

Fig. 1

Effects of cytotoxic substances on viral transduction with rAAV/EGFP. Breast cancer cells with low susceptibility to AAV transduction were infected with rAAV/EGFP at MOI 50 after treatment with carboplatin (hatched bars) or epirubicin (gray bars). In comparison with untreated cells (black bars), the fractions of transgene-positive cells, analyzed after 48 h by flow cytometry, were considerably increased. Means and standard deviations of three transduction experiments are shown

Fig. 2

Transgene expression after transduction with rAAV/mCD40L. HeLa and breast cancer cells were transduced with rAAV/mCD40L at MOI 150 and transgene expression was analyzed after 48 h by flow cytometry after fluorescence immunostaining with an antibody specific for murine CD40L. Means and standard deviations of three transduction experiments are shown

Fig. 3

Co-incubation with AAV/mCD40L-transduced breast cancer cells stimulates dendritic cells to secrete interleukin 12. a Immature dendritic cells were co-cultivated with AAV/mCD40L-transduced breast cancer and HeLa cells, and release of interleukin 12 into the supernatant was measured by ELISA. The human CD40L stably over-expressing HeLa cells (HeLa-hCD40L) were used as a positive control. b Production of IL-12 by human dendritic cells co-cultivated with AAV/mCD40L-transduced cells (empty bars) was blocked by pre-incubation with a monoclonal antibody binding specifically to the murine CD40L (hatched bars). The human CD40L stably over-expressing HeLa cells (HeLa-hCD40L) were used as a negative control not affected by the mouse-specific antibody. The low background secretion of IL-12 was determined with not infected parental cells (black bars) and cells transduced with the EGFP-encoding rAAV/EGFP (gray bars). Data are shown normalized on basis of IL-12 secretion shown in (a)