Radiotherapy is an indispensable part of the standard care for glioblastoma (GBM) patients; however, despite initial responses to radiotherapy, GBMs invariably recur. A proposed strategy for improving GBM radiotherapy involves combining both radiotherapy and therapy targeting tumor-associated macrophages (TAMs). However, lack of an established mechanism by which TAM-targeted therapy improves GBM radiotherapy has posed a barrier to clinical translation. Thus, there is an urgent need to establish mechanisms by which TAM-targeted therapy alters radiotherapy. The main objective of this project is to determine the cytotoxic mechanisms and anti-tumor efficacy of integrating TAM targeting within clinically relevant radiotherapy regimens. For these studies, therapeutic targeting of TAMs will be accomplished by repurposing the FDA-approved agent ferumoxytol. The main hypothesis is that ferumoxytol will reduce immunosuppressive, tumor-promoting TAMs. This reduction in TAMs is expected to increase glioma cell sensitivity to radiotherapy by both disrupting TAM-glioma cell heterotypic survival signaling and increasing radiation-induced anti-tumor immune responses. This hypothesis will be evaluated using radiotherapy regimens that are similar to clinical standards of care for two types of patients: those with newly diagnosed GBM (Aim 1); and those with recurrent GBM (Aim 2). This will include completion of the following aims: Aim 1) determine cytotoxic mechanisms and efficacy of combining ferumoxytol with conventionally fractionated radiotherapy; and Aim 2) evaluate ferumoxytol’s ability to augment hypofractionated radiotherapy. Aim 1 will evaluate the combination of ferumoxytol with radiotherapy in both in vitro coculture models and syngeneic rodent models. Aim 2 will evaluate the combination of ferumoxytol and hypofractionated radiotherapy in a translationally-relevant canine companion study. For both aims, the delivery and retention of ferumoxytol within tumor regions will be verified non-invasively using magnetic resonance imaging (MRI). The investigators believe the proposed research is innovative because it repurposes an established glioma imaging agent (ferumoxytol), for theragnostic TAM targeting. If ferumoxytol does not prove as effective as expected for enhancing radiotherapy, these studies will shift focus to other promising TAM-targeted therapeutics being developed by the investigative team. Upon completion of the proposed studies, this project will have established the therapeutic efficacy and cytotoxic mechanisms of combining TAM-targeted therapy with radiotherapy. This contribution is expected to be significant for two reasons: it will provide knowledge regarding the role of TAMs in modulating glioma cell resistance to radiotherapy; and it will lead to development of more effective radiotherapeutic strategies. Given the prevalence of radiotherapy for treating cancer, development of this therapeutic approach has the potential to improve outcomes ...