Metronomic treatment in immunocompetent preclinical GL261 glioblastoma: effects of cyclophosphamide and temozolomide

Abstract

Glioblastoma (GBM) causes poor survival in patients even when applying aggressive treatment. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment, but resistance always ensues. In previous years, efforts have focused on new therapeutic regimens with conventional drugs to activate immune responses that may enhance tumor regression and prevent regrowth, for example the "metronomic" approaches. In metronomic scheduling studies, cyclophosphamide (CPA) in GL261 GBM growing subcutaneously in C57BL/6 mice was shown not only to activate antitumor CD8 T-cell response, but also to induce long-term specific T-cell tumor memory. Accordingly, we have evaluated whether metronomic CPA or TMZ administration could increase survival in orthotopic GL261 in C57BL/6 mice, an immunocompetent model. Longitudinal in vivo studies with CPA (140 mg/kg) or TMZ (range 140-240 mg/kg) metronomic administration (every 6 days) were performed in tumor-bearing mice. Tumor evolution was monitored at 7 T with MRI (T -weighted, diffusion-weighted imaging) and MRSI-based nosological images of response to therapy. Obtained results demonstrated that both treatments resulted in increased survival (38.6 ± 21.0 days, n = 30) compared with control (19.4 ± 2.4 days, n = 18). Best results were obtained with 140 mg/kg TMZ (treated, 44.9 ± 29.0 days, n = 12, versus control, 19.3 ± 2.3 days, n = 12), achieving a longer survival rate than previous group work using three cycles of TMZ therapy at 60 mg/kg (33.9 ± 11.7 days, n = 38). Additional interesting findings were, first, clear edema appearance during chemotherapeutic treatment, second, the ability to apply the semi-supervised source analysis previously developed in our group for non-invasive TMZ therapy response monitoring to detect CPA-induced response, and third, the necropsy findings in mice cured from GBM after high TMZ cumulative dosage (980-1400 mg/kg), which demonstrated lymphoma incidence. In summary, every 6 day administration schedule of TMZ or CPA improves survival in orthotopic GL261 GBM with respect to controls or non-metronomic therapy, in partial agreement with previous work on subcutaneous GL261.

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

“…The start of MRSI explorations was conditioned by the measured tumour volume. According to our experience and data described by our group, tumour volumes below 10 to 20 mm 3 are not properly segmented by MRSI, and this was the cut‐off point to start MRSI acquisitions. An example can be seen in Figure S2b.…”

“…Our surrogate biomarker for therapy response, coded in nosological images, was developed with TMZ‐treated cases and one of the fundamental questions then was whether it would be useful with other therapeutic agents. Indeed, it was also proven robust to detect tumour response in cyclophosphamide (CPA)‐treated mice, suggesting that the changes sampled are not restricted to TMZ, but rather being related to local changes during different treatments. Those would be probably related to immune system action against tumours, which could be an extremely valuable biomarker in tuning therapy administration to obtain maximal effectiveness and improve outcome.…”

“…In addition to the beneficial effects related to immune system, the IMS administration schedule also allows us to reduce the cumulative amount of administered TMZ and avoid consequent development of other tumours due to the mutagenic effects of TMZ, such as lymphomas . Moreover, if a translational protocol reaches the clinical pipeline, the reduction of TMZ dosage should also decrease undesired side effects in patients.…”

“…30 Tumour volume evolution of vehicle and TMZ-treated mice is shown in Figure S1b. As described before, 31 TMZ treatment with the IMS administration has a positive impact in the survival of GL261 tumour-bearing mice ( Figure S1d). Thus, the average survival rate for control mice was 22.5 ± 3.0 d whereas the TMZ-treated animals survived significantly more, 135.8 ± 78.2 d (P < .05 according to log-rank test), being 61.5% of TMZ-treated mice alive at 150 d. p.i.…”

“…In addition to the beneficial effects related to immune system, the IMS administration schedule also allows us to reduce the cumulative amount of administered TMZ and avoid consequent development of other tumours due to the mutagenic effects of TMZ, such as lymphomas. 31 Moreover, if a translational protocol reaches the clinical pipeline, the reduction of TMZ dosage should also decrease undesired side effects in patients. The curative effect of the IMS-TMZ protocol alone in 38% of the overall investigated mice indicates that similar protocols, avoiding damage to the amplification phase of the immune cycle, could be of relevance to patients…”