The importance of the blood-brain barrier in preventing effective pharmacotherapy of glioblastoma has been controversial. The controversy stems from the fact that vascular endothelial cell tight junctions are disrupted in the tumor, allowing some systemic drug delivery. P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) efflux drugs from brain capillary endothelial cells into the blood. We tested the hypothesis that although the tight junctions are “leaky” in the core of glioblastomas, active efflux limits drug delivery to tumor-infiltrated normal brain and consequently, treatment efficacy. Malignant gliomas were induced by oncogene transfer into wild-type (WT) mice or mice deficient for Pgp and BCRP (KO). Glioma-bearing mice were orally dosed with dasatinib, a kinase inhibitor and dual BCRP/PgP substrate that is being tested in clinical trials. KO mice treated with dasatinib survived over twice as long as WT mice. Microdissection of the tumor core, invasive rim, and normal brain revealed 2-3 fold enhancement in dasatinib brain concentrations in KO mice relative to WT. Analysis of signaling demonstrated that poor drug delivery correlated with the lack of inhibition of a dasatinib target, especially in normal brain. A majority of human glioma xenograft lines tested expressed BCRP or PgP and were sensitized to dasatinib by a dual BCRP/Pgp inhibitor, illustrating a second barrier to drug delivery intrinsic to the tumor itself. These data demonstrate that active efflux is a relevant obstacle to treating glioblastoma and provide a plausible mechanistic basis for the clinical failure of numerous drugs that are BCRP/Pgp substrates.

Cancer vaccines have overall had a record of failure as an adjuvant therapy for malignancies that are treated with alkylating chemotherapy, and the contribution of standard treatment to that failure remains unclear. Vaccines aim to harness the proliferative potential of the immune system by expanding a small number of tumor-specific lymphocytes into a large number of anti-tumor effectors. Clinical trials are often conducted after treatment with alkylating chemotherapy, given either as standard therapy or for immunomodulatory effect. There is mounting evidence for synergy between chemotherapy and adoptive immunotherapy or vaccination against self-antigens; however, the impact of chemotherapy on lymphocytes primed against tumor neo-antigens remains poorly defined. We report here that clinically relevant dosages of standard alkylating chemotherapies such as temozolomide and cyclophosphamide significantly inhibit the proliferative abilities of lymphocytes in mice. This proliferative impairment was long lasting and led to quantitative and qualitative defects in B and T cell responses to neo-antigen vaccines. High affinity responder lymphocytes receiving the strongest proliferative signals from vaccines experienced the greatest DNA damage responses, skewing the response toward lower affinity responders with inferior functional characteristics. Together these defects lead to inferior efficacy and overall survival in murine tumor models treated by neo-antigen vaccines. These results suggest that clinical protocols for cancer vaccines should be designed to avoid exposing responder lymphocytes to alkylating chemotherapy.

Purpose: Atmospheric oxygen (∼20% O 2 ) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines.Experimental Design: GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O 2 . Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by Western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on MHC class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8 + T cells.Results: Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O 2 lysates. Antigen-specific CTL activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O 2 versus 20% O 2 through a mechanism that involved enhanced cross-presentation of exogenous antigen on MHC I. Vaccination with 5% O 2 GL261 cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O 2 lysate vaccines predominantly evoked an antibody response.Conclusions: Tissue culture oxygen functions as an "immunologic switch" by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen.