Date Published: January 13, 2009
Publisher: Public Library of Science
Author(s): James F Curtin, Naiyou Liu, Marianela Candolfi, Weidong Xiong, Hikmat Assi, Kader Yagiz, Matthew R Edwards, Kathrin S Michelsen, Kurt M Kroeger, Chunyan Liu, A. K. M. Ghulam Muhammad, Mary C Clark, Moshe Arditi, Begonya Comin-Anduix, Antoni Ribas, Pedro R Lowenstein, Maria G Castro, Robert Weil
Abstract: BackgroundGlioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs.Methods and FindingsUsing a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4+ and CD8+ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1.ConclusionsOur data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.
Partial Text: The systemic immune system can be ignorant of antigens derived from tumors growing within the confines of the brain parenchyma  owing to the paucity of professional antigen presenting cells (APCs) and lymphatic drainage. Added to the brain’s immune privilege , and tumor immune escape , these factors pose clinically significant obstacles for successful immunotherapy. The most aggressive intracranial primary brain tumor is glioblastoma multiforme (GBM), which is associated with 5% survival rates 5-y postdiagnosis, despite advances in surgery, chemotherapy, and radiotherapy . Novel experimental therapies that harness the power of the patient’s immune system are being pursued to improve therapeutic efficacy [5,6]. We postulated that the lack of dendritic cells (DCs) within the brain parenchyma is central to the failure of glioma cell killing approaches to stimulate a strong adaptive antiglioma immune response. To this effect, we developed a combined gene therapeutic approach aimed at engineering the tumor microenvironment to induce the migration into the tumor mass of APCs, combined with tumor cell death to release tumor antigens. Our approach consists of expressing Fms-like tyrosine kinase 3 ligand (Flt3L), which induces DC infiltration into the brain parenchyma , in combination with the conditional cytotoxic gene thymidine kinase (TK) .
This paper elucidates the mechanisms that mediate an antibrain tumor immune response directly from within the tumor microenvironment. Utilizing syngeneic brain tumor models we demonstrate that it is possible to stimulate a systemic antitumor immune response by modifying the brain tumor microenvironment through the recruitment of DCs to the tumor milieu in combination with tumor cell killing. Further, we demonstrate an essential link that has so far been difficult to determine. Namely, it has been known for some time that stimulation of the innate immune system is necessary to achieve activation of the systemic adaptive immune response. However, how this is achieved in tumors, in the absence of exogenous activators of TLR signaling has remained elusive. Clinically, tumor immunization paradigms have attempted to provide innate immune stimulation through the administration of vaccines with known TLR activators, such as CpG [64,65].