Anti-tumour immune response in GL261 glioblastoma generated by Temozolamide Immune-Enhancing Metronomic Schedule monitored with MRSI-based nosological images

Abstract

Glioblastomas (GB) are brain tumours with poor prognosis even after aggressive therapy. Improvements in both therapeutic and follow-up strategies are urgently needed. In previous work we described an oscillatory pattern of response to Temozolomide (TMZ) using a standard administration protocol, detected through MRSI-based machine learning approaches. In the present work, we have introduced the Immune-Enhancing Metronomic Schedule (IMS) with an every 6-days TMZ administration at 60mg/kg, and investigated the consistence of such oscillatory behaviour. A total of n=17 GL261 GB tumour-bearing C57BL/6j mice were studied with MRI/MRSI every 2 days, and the oscillatory behaviour (6.2±1.5 days period from the TMZ administration day) was confirmed during response. Furthermore, IMS-TMZ produced significant improvement in mice survival (22.5±3.0 days for controls vs 135.8±78.2 for TMZ-treated), outperforming standard TMZ treatment. Histopathological correlation was investigated in selected tumour samples (n=6) analyzing control and responding fields. Significant differences were found for CD3+ cells (lymphocytes, 3.3±2.5 vs 4.8±2.9 respectively) and Iba-1 immunostained area (microglia/macrophages, 16.8±9.7% and 21.9±11.4% respectively). Unexpectedly, during IMS-TMZ treatment, tumours from some mice (n=6) fully regressed and remained undetectable without further treatment for one month. These animals were considered “cured” and a GL261 re-challenge experiment performed, with no tumour reappearance in 5 out of 6 cases. Heterogeneous therapy response outcomes were detected in tumour-bearing mice, and a selected group was investigated (n=3 non-responders, n=6 relapsing tumours, n=3 controls). PD-L1 content was found ca. 3-fold increased in the relapsing group when comparing with control and non-responding groups, suggesting that increased lymphocyte inhibition could be associated to IMS-TMZ failure. Overall, data suggest that host immune response has a relevant paper in therapy response/escape in GL261 tumours under IMS-TMZ therapy. This is associated to changes in the metabolomics pattern, oscillating every 6 days, in agreement with immune cycle length, which is being sampled by MRSI-derived nosological images