Glioblastoma 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 Findings
Using 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.
Our 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.
Maria Castro and colleagues use cell line and transgenic mouse approaches to study the mechanisms underlying the immune response to glioblastoma multiforme.
Every year, more than 175,000 people develop a primary brain tumor (a cancer that starts in the brain rather than spreading in from elsewhere). Like all cancers, brain tumors develop when a cell acquires genetic changes that allow it to grow uncontrollably and that change other aspects of its behavior, including the proteins it makes. There are many different types of cells in the brain and, as a result, there are many different types of brain tumors. However, one in five primary brain tumors is glioblastoma multiforme (GBM; also known as grade 4 astrocytoma), a particularly aggressive cancer. With GBM, the average time from diagnosis to death is one year and only one person in 20 survives for five years after a diagnosis of GBM. Symptoms of GBM include headaches, seizures, and changes in memory, mood, or mental capacity. Treatments for GBM, which include surgery, radiotherapy, and chemotherapy, do not “cure” the tumor but they can ease these symptoms.
Why Was This Study Done?
Better treatments for GBM are badly needed, and one avenue that is being explored is immunotherapy—a treatment in which the immune system is used to fight the cancer. Because many tumors make unusual proteins, the immune system can sometimes be encouraged to recognize tumor cells as foreign invaders and kill them. Unfortunately, attempts to induce a clinically useful anti-GBM immune response have been unsuccessful, partly because the brain contains very few dendritic cells, a type of immune system cell that kick-starts effective immune responses by presenting foreign proteins to other immune system cells. Another barrier to immunotherapy for GBM is immune evasion by the tumor. Many tumors develop ways to avoid the immune response as they grow. For example, they sometimes reduce the expression of proteins that the immune system might recognize as foreign. In this study, the researchers test a new combined treatment strategy for GBM in which dendritic cells are encouraged to enter the brain and tumor cells are killed to release proteins capable of stimulating an effective antitumor immune response.
What Did the Researchers Do and Find?
The researchers first established brain tumors in mice. Then, they injected harmless viruses carrying the genes for Fms-like tyrosine kinase 3 ligand (Ftl3L; a protein that attracts dendritic cells) and for thymidine kinase (TK; cells expressing TK are killed by a drug called gancyclovir) into the tumor. Expression of both Flt3L and TK (but not of either protein alone) plus gancyclovir treatment shrank the tumors and greatly improved the survival of the mice. The researchers show that their strategy increased the migration of dendritic cells into the tumor provided they expressed an immune system protein called Toll-like receptor 2 (TLR2). TLR2 expression on the dendritic cells was also needed for an effective anti-tumor immune response and for tumor regression. TLR2 normally activates dendritic cells by binding to specific proteins on invading pathogens, so what was TLR2 binding to in the mouse tumors? The researchers reveal that TLR2 was responding to high-mobility-group box 1 (HMGB1), a protein released by the dying tumor cells by showing that treatment of the tumor-bearing mice with the HMGB1 inhibitor glycyrrhizin blocked the therapeutic effect of Flt3L/TK expression. Finally, the researchers report that other tumor cell types release HMGB1 when they are killed and that the Flt3L/TK expression strategy can also kill other tumors growing in mouse brains.
What Do These Findings Mean?
Results obtained in mouse models of human diseases do not always lead to effective treatments for human patients. Nevertheless, the findings of this study provide new insights into how an effective immune response against brain tumors might be brought about. Most importantly, they show that an effective strategy might need to both attract dendritic cells into the brain tumor and to kill tumor cells, so they release proteins that can activate the dendritic cells. That is, the authors suggest it's important to combine immunotherapies with tumor-killing strategies to provide effective treatments for primary and metastatic brain tumors
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000010.
The US National Cancer Institute provides information about brain tumors for patients and health professionals and about the the immune system and how it can be harnessed to fight cancer (in English and Spanish)
Cancer Research UK provides information on all aspects of brain tumors for patients and their caregivers
MedlinePlus provides links to further information about brain cancer, (including some links to information in Spanish)
The American Brain Tumor Association provides brain tumor resources and information
The National Brain Tumor Society provides educational and support services regarding brain tumors