MRI monitoring of monocytes to detect immune stimulating treatment response in brain tumor

Abstract

Background: Glioblastoma (GBM) is an aggressive brain cancer with a poor prognosis. The use of immune therapies to treat GBM has become a promising avenue of research. It was shown that amphotericin B (Amp B) can stimulate the innate immune system and suppress the growth of brain tumor initiating cells (BTICs). However, it is not feasible to use histopathology to determine immune activation in patients. We developed an MRI technique that can rapidly detect a therapeutic response in animals treated with drugs that stimulate innate immunity. Ultra-small iron oxide nanoparticles (USPIOs) are MRI contrast agents that have been widely used for cell tracking. We hypothesized that the increased monocyte infiltration into brain tumors due to Amp B can be detected using USPIO-MRI, providing an indicator of early drug response.

Methods: We implanted human BTICs into severe combined immunodeficient mice and allowed the tumor to establish before treating the animals with either Amp B or vehicle and then imaged them using MRI with USPIO (ferumoxytol) contrast.

Results: After 7 days of treatment, there was a significantly decreased T2* in the tumor of Amp B but not vehicle animals, suggesting that USPIO is carried into the tumor by monocytes. We validated our MRI results with histopathology and confirmed that Amp B-treated animals had significantly higher levels of macrophage/microglia that were colocalized with iron staining in their brain tumor compared with vehicle mice.

Conclusion: USPIO-MRI is a promising method of rapidly assessing the efficacy of anticancer drugs that stimulate innate immunity.

Keywords: drug response; glioblastoma; innate immunity; iron oxide; MRI.

Fig. 1.

Changes on the T₂* map 24 hours after USPIO injection. There was no significant T₂* darkening in the tumor of vehicle animals, but significant darkening in the tumor can be seen in Amp B–treated animals. The white region of interest demarcates the region of interest used for the tumor and the contralateral brain.

Fig. 2.

Quantification of tumor volume and T₂* changes in vehicle- and Amp B–treated animals. (A) There was no significant difference in the tumor volume between vehicle- and Amp B–treated animals; this is likely because Amp B treatment occurred for only 7 days in mice with large, established tumors. (B) T₂* measured in the brain contralateral to the tumor. There were no significant changes in contralateral brain T₂* in the vehicle- and Amp B–treated animals. (C) T₂* measured in the tumor. Amp–B treated animals showed a significant reduction in tumor T₂* compared with tumor T₂* of the vehicle group. *P<.05, Student’s t-test.

Fig. 3.

T1w rapid acquisition with relaxation enhancement (RARE) sequence (TR = 500ms, nominal TE = 7ms, RARE factor = 4) before and after Gd (Magnevist), showing that vehicle animals had similar degrees of Gd enhancement compared with Amp B–treated animals. Black arrows denote the location of the tumor.

Fig. 4.

Amp B‒induced decrease in T₂* post ferumoxytol contrast is not associated with a leaky BBB. There was a significant decline in T₂* post ferumoxytol in the tumor of Amp B–treated animals (C), but not on the contralateral side (A). Fluorescence imaging with FITC revealed similar leakiness of the vasculature in the tumor (D) compared with the contralateral healthy brain (B). Scale bars represent 100 μm. *P<.05, paired t-test.

Fig. 5.

(A–D) Immunohistochemistry shows that there is significantly more Iba1 staining in the Amp B animals (B and D) compared with vehicle-treated controls (A and C). (E) Quantification of Iba1 staining showed that there are significantly more Iba1-positive cells (normalized to tumor area) in the Amp B–treated animals compared with vehicle. Scale bars represent 100 μm. *P<.05, Student’s t-test.

Fig. 6.

Histological staining in Amp B tumor for (A, C) macrophage/microglia (Iba1), and (B, D) DAB-enhanced Perl’s (iron) staining on the adjacent paraffin slide. This shows that there are macrophages and microglia (brown cells) and that there is iron accumulation in the tumor (small brown spots). Scale = 100 μm. MRI data for this animal is shown in Fig. 2.