M2-Receptor Stimulation in Blood-Brain-Tumour Barrier: Implications in Glioblastoma Therapy

Glioblastoma (GBM) is an aggressive brain tumour, with high drug resistance and recurrence caused by the cancer stem cells' capability of self-repairing their DNA following standard therapy. Another limitation in the development of new GBM therapies is the difficulty in achieving drug penetration across the Blood-Brain Barrier (BBB), which daily regulates brain homeostasis and function by limiting molecular access to the brain parenchyma. This selective permeability is maintained by endothelial cells (ECs), which possess tight junctions (on the lateral membrane) and efflux pumps (on the apical surface). However, our published studies on GBM cells showed that the M2 muscarinic receptor (M2R) stimulation with cholinergic dualsteric agonist Iper-8-Naphthalimide (N8) significantly reduces cell proliferation and survival. Furthermore, co-treatment with N8 and conventional chemotherapy drugs increases the cytotoxicity in GBM stem cells by downregulating the expression of efflux pumps. Therefore, the present work aims to assess the effect of N8 on the BBB. Interestingly, we recently developed an auto-fluorescent form of N8 highly useful for elucidating its mechanism of action and tracking its passage across the BBB. Data were obtained using a human in vitro BBB model, including the three cell types required to maintain barrier properties - endothelial cells, pericytes, and astrocytes – or with astrocytes replaced by GBM cells to extend the model to the Blood–Brain Tumour Barrier (BBTB). Interestingly, N8 demonstrated the ability to cross slowly the barrier, and preserve BBB integrity without causing any toxic effects on junction integrity or efflux pump functionality. In this context, N8 appears to be a promising candidate as an adjuvant to current standard glioblastoma therapies due to its effect on GBM, which enhances the retention of chemotherapy drugs without toxicity on the BBB.