Brain microsurgery in glioblastoma mouse models for local administration of Temozolomide-loaded hydrogels

Glioblastoma is the most common and aggressive malignant tumor of the central nervous system in adults. It can occur at any age, but 70% of patients are diagnosed between the ages of 45 and 70. These tumors show a high proliferation rate with diffuse infiltration of adjacent brain tissue and a rapidly progressive course (around 2-3 months).Tumors are usually located in the cerebral hemispheres, but can be found throughout the central nervous system. The first choice treatment is usually surgical, both to confirm the diagnosis through a biopsy, and to remove the tumor mass as extensively as possible. Unfortunately, a complete resection is very infrequent, as cancer cells usually infiltrate the surrounding brain. Therefore, the goal of surgery is only to obtain a histological diagnosis, decrease the symptoms due to the increase in intracranial pressure and prolong survival. Surgery is usually followed by radiation therapy and chemotherapy but there is a treatment gap of 2 to 3 weeks between tumor resection and subsequent therapies. The post-surgical therapeutic standard currently consists of a chemo-radiotherapy association with Temozolomide (TMZ) for the entire duration of radiotherapy, followed by adjuvant TMZ. However, tumor recurrences due to residual infiltrative cells at the resection margin are extremely common. This study aims at developing a mouse model of glioblastoma recurrences by a surgical protocol of partial tumor removal in mouse brains, for subsequent on-site treatment with thermogel, a “smart” material loaded with TMZ. For this purpose, the U87MG human glioblastoma cell line was chosen for the development of a mouse orthotopic model in the striatum, a subcortical region of the brain. Once tumors of sufficient size were obtained, a microsurgery protocol with craniotomy was optimized for the partial removal of the tumor mass in order to study the phenomenon of recurrence. The cavity thus obtained was filled with thermogel containing TMZ. The effect due to this treatment was confirmed by two different types of analysis: histological and bioluminescent. The histological analysis allowed us to verify the correct inoculation region of the tumor cells and to verify their growth. Furthermore, by measuring the tumor present in the brain slices removed from treated and untreated mice, we were able to measure the area of each tumor with a specific software, verifying the effectiveness of the treatment. To confirm obtained data, we carried out another type of analysis, using the IVIS In Vivo Imaging System, usingU87MG cells stably expressing the firefly enzyme luciferase from Luciola Italica (Red-FLuc). By recording the bioluminescence emitted by the tumor cells inoculated inside the brain of the mice, following an adequate stimulus, we confirmed the effect of the treatment of the thermogel containing the chemotherapeutic.