Investigating the potential of a novel TLR3 agonist, BO-112, to enhance the therapeutic effects of radiotherapy

Abstract

Radiotherapy (RT) is commonly used in the treatment of those diagnosed with cancer, with over 60% of patients receiving it at some point during their treatment plan. Whilst the most common recognised mechanism of action of RT is its ability to cause cell death via DNA damage, it can also result in both local and systemic immunity. However, this is rarely seen clinically, and what is more prevalent is its ability to result in immune suppression. Thus, research has focused on combining immunotherapies with RT to promote RT-induced immune stimulation and reduce immune supression. Utilising toll-like receptor 3 (TLR3) agonists with RT has shown success both in vitro and in vivo cancer models showing increased tumour control through changing the contexture of the tumour microenvironment from immune suppressive to immune stimulatory. Therefore, TLR3 agonists combined with RT could enhance the therapeutic effect of RT which is critical in cancers like bladder cancer where survival rates are poor and there is an urgent need for effective treatment options to enhance both survival and quality of life. BO-112 is a nanoplexed form of the well-studied TLR3 agonist poly I:C. It has been shown to cause apoptosis of cancer cells as well as increase tumour control and antitumour immunity in vivo. Moreover, when combined with RT it has been shown to enhance tumour control though inducing local antitumour immunity, as well as showing it can induce systemic immunity using a dual tumour model. In this report, the ability of BO-112 to increase cell death alone and with RT in MBT2 and MB49 murine bladder cancer cell lines was investigated using various cell death/survival assays in vitro. Development of an orthotopic model of bladder cancer which more closely represented what is observed clinically was also investigated. BO-112 with RT was shown to decrease colony formation and cell viability as analysed via WST-1 assay, BO-112 with RT increased caspase dependent cell death which increased over time, as analysed via annexin V/propidium iodide staining, suggesting apoptotic cell death. This combination therapy also resulted in cleavage of caspase 3 in MB49 cells and cleaved PARP in both cell lines. Combination therapy resulted in the release of HMGB1 and HSP90 from MBT2 cells, but not MB49 cells. MB49 cells were significantly more treatment resistant than the MBT2 cell line. With BO-112 previously being shown to cause the release of IFNβ, mimicry of this using exogenous IFNβ resulted in decreased colony formation in both cell lines and deletion of IRF3 from MB49 cells resulted in a loss of the additive effect BO-112 had to RT. Intramural injections of MB49 cells into the bladder resulted in consistent and rapid tumour growth, with mice tolerating the procedure well. Taken together, these results show that BO-112 has an additive effect to RT as it increases cell death in mouse bladder cancer cells. Further investigations are required to verify this combination’s ability to increase cell death in vitro, and to elucidate its ability to increase tumour control in vivo.

Date of Award	6 Jan 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Timothy Illidge (Supervisor) & Jamie Honeychurch (Supervisor)