Treatment of experimental intracranial glioblastoma (GBM) with adenoviruses expressing TK and Flt3L induce intratumoral infiltration of phagocytic immature plasmacytoid dendritic cells
Authors: Marianela Candolfi, Gwendalyn King, Akm Muhammad, James Curtin, Chunyan Liu, Kurt Kroeger, David Foulad, Jonathan Lerner, Pedro Lowenstein, Maria Castro
Journal: Cancer Research (2008)

2856  We previously demonstrated that adenoviral vectors (Ads) expressing TK (Ad-TK) and Flt3L (Ad-Flt3L) increase the survival of rats bearing large intracranial GBM. This approach combines the cytotoxic effect of TK that kills glioma cells in the presence of ganciclovir (GCV), and the immunostimulatory effect of Flt3L, which attracts antigen presenting cells (APCs) to the brain tumor mass. Here, we characterized the APCs that infiltrate the tumor mass after Ad-TK/GCV+Ad-Flt3L treatment. We implanted CNS1 tumors in the striatum of Lewis rats and 10 days later treated them intratumorally with Ad-TK/GCV+Ad-Flt3L. We extracted immune cells from the intracranial tumors 5 and 12 days after the treatment using Percoll gradient, and characterized them by flow cytometry. We found that this immunotherapy induces active recruitment of plasmacytoid dendritic cells (pDCs) into the tumor mass and increases the infiltration of macrophages when compared to controls. To study the ability of intratumoral pDCs to uptake tumor antigen we injected fluorescent beads (1μm FluoSpheres) intratumorally 4 days after Ad-TK/GCV+Ad-Flt3L treatment. We found that the beads were rapidly taken up by intratumoral APCs 24 h after administration. APCs containing beads were detected 1, 4 and 7 days after delivery in the tumor mass and approaching or entering tumor blood vessels. Four days after bead delivery, APCs containing beads were detected in the draining cervical limph nodes. Intratumoral immune cells containing beads were positively labeled for CD68, a marker for macrophages/activated microglia, and CD45R, a marker for pDCs and B cells.To specifically characterize the intratumoral pDCs, immune cells from the tumor were collected by Percoll gradient 5 days after the treatment and pDCs were purified by flow cytometry (CD3-, CD4+, CD45R+). We studied the expression of activation markers (CD86, MHCII), their ability to release IFN-α and their phagocytic activity in vitro. We found that intratumoral pDCs recruited by Ad-TK/GCV+Ad-Flt3L do not express CD86 or MHCII and are capable of phagocytosis of beads in vitro. Also, while intratumoral pDCs do not release IFN-α basally, they can be activate in vitro after 24 h incubation with the TLR-9 agonist ODN Cpg2216 (50 µg/ml), releasing IFN-α and upregulating MHCII expression. Our results suggest that APCs infiltrating intracranial GBMs after Ad-TK/GCV+Ad-Flt3L treatment are macrophages and immature phagocytic pDCs and that these cells can phagocytose tumor Ag and migrate to the draining limph node. This mechanism could elicit immune-mediate tumor regression in our model after treatment with Ad-TK/GCV+Ad-Flt3L.  Support: NIH/NINDS R01 NS44556.01, R21-NSO54143.01; UO1 NS052465.01; RO3 TW006273-01 (MGC).; RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, R21 NS047298-01 (PRL); F32 NS058156.01 (MC)