Anti‐tumour immune response in GL261 glioblastoma generated by Temozolomide 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‐d TMZ administration at 60 mg/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 d, and the oscillatory behaviour (6.2 ± 1.5 d period from the TMZ administration day) was confirmed during response. Furthermore, IMS‐TMZ produced significant improvement in mice survival (22.5 ± 3.0 d 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 1 mo. These animals were considered “cured” and a GL261 re‐challenge experiment performed, with no tumour reappearance in five out of six 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 role in therapy response/escape in GL261 tumours under IMS‐TMZ therapy. This is associated to changes in the metabolomics pattern, oscillating every 6 d, in agreement with immune cycle length, which is being sampled by MRSI‐derived nosological images.

Unraveling response to temozolomide in preclinical GL261 glioblastoma with MRI/MRSI using radiomics and signal source extraction

“…Last but not least, our preliminary data show that timing is a relevant factor while evaluating tumour response to therapy 6,9 , and our time points were validated by MRSI, growth arrest and, in some cases, histopathological data. Since MRSI can provide earlier data and inform about tumour relapse before tumour volume changes 8 , this may be used in future studies to improve MRI information and refine prediction of whether a tumour is properly responding to therapy or will relapse in a near future.…”

“…In this sense, it is not surprising its misclassification with radiomics as well, since local tissue effects may have a clear impact on MRI features. In this respect, it is wise to mention here that MRSI features detected from treated GL261 mice have been found to oscillate along time 6,9 , with a ca. 6-day period.…”

“…41 also describe Alanine as a distinctive trait between untreated and treated mice. It is also worth mentioning that such metabolomic pattern changes have been also seen during transient responses to a variable set of therapeutic approaches: TMZ in standard or metronomic schedules 9 , CPA 6 , immunotherapy alone or in combination 42 , hinting that changes are not restricted to TMZ treatment.…”

“…Recent work 38 also described the value of GLRLM and GLSZM for evaluating response to therapy in GB, being able to distinguish pseudoprogression from true progression. Recent results 6 suggest that treated GL261 tumors responding to TMZ treatment present significantly higher immune system elements within the tumor region. The local effects of these cells against the GB mass may also cause local changes that could be spotted by MRI, although further work would be needed to establish a proper correlation between MRI features and immune system elements presence/action.…”

“…Data. The retrospective data analyzed in this study were acquired from 63 mice 6,8,9 , in which tumors were induced by stereotactical injection of GL261 GB cells. Each individual is identified by a unique code (CXXX, being XXX a sequential number).…”