Unravelling the Complexity of Post-surgical Glioblastoma Invasion: Insights into Tumour-Neuronal Cellular Interactions and Microtubule Dynamics

Background:
Glioblastoma (GBM) is an aggressive malignant tumour that arises in the astrocytic cells of the brain and is characterised by infiltrative growth and resistance to treatment. Whilst surgical resection represents the primary treatment option for GBM, the presence of residual infiltrative GBM cells left behind contributes to post-surgical tumour recurrence. Recent studies have revealed how GBM cells establish synapses with healthy c neurons, the functionality of such synapses, and the formation of an intricate network of GBM cells and healthy brain tissue (1). Recent research also highlights the role of cellular crosstalk and microtubule/ tumour microtube dynamics in GBM invasion (2). Understanding the intricate crosstalk between GBM cells and neurons, as well as the dynamics of microtubules within the tumour microenvironment through laboratory models, is essential for developing targeted therapies which may block or delay GBM recurrence, thereby improving patient outcomes.

Importance of the study:
As GBM is an exemplar of cancer invasion, the project will be broadly applicable to infiltrative disease including other somatic cancers. Specifically, the study will elucidate converging underlying mechanisms associated with GBM invasion, a critical phenotype that contributes to its infiltrative spread, recurrence, and resistance to treatment. Establishing functionality vs. the cellular state of infiltrative GBM cells in direct physical contact with healthy brain cells, can help us understand the post-surgical brain response to infiltrative GBM.

By unravelling the complex interplay between GBM cells and the surrounding microenvironment, particularly the interactions with healthy neurons and the dynamics of microtubules, the study may open new avenues for molecular targeted therapeutics.

Hypothesis:
Glioblastoma invasion is characterised by aberrant tumour-neuronal interactions and dysregulated microtubule dynamics.

Workplan
Objective 1: Measuring cellular functionality using electrophysiology.
The cellular-cross talk between GBM cells and neurons will be assessed by electrophysiological methods such as whole-cell patch clamping (including automated). The co-cultures will be generated from fluorescently tagged primary patient-derived cell lines isolated from GBM infiltrative margin regions. Through a multidisciplinary approach, including electrophysiological and metabolic analyses, we seek to elucidate the intricate interplay between tumour cells and neurons within the tumour microenvironment. To further study the synapses between GBM cells and neurons, the functionality of synaptic inputs will be monitored via measurements of intracellular calcium and potassium, as well as adenosine triphosphate (ATP) and glutamate.

Objective 2: Testing the cellular GBM state and its role in invasiveness.
Studies show tumour microtubes as a predominant site for interaction with neurons in the brain (2). To understand this interaction, the microtubules in the healthy neuron must be characterised and compared with GBM tumour microtubes to understand their respective role in crosstalk leading to tumour invasion. As microtubules are composed of tubulin, the BioID proteomics tool can be used to map protein-protein interactions . This will further enable us to compare the GBM interactome vs. neuronal interactome. This will be validated using microscopical and biochemical methods.

Objective 3: Proteomic profiling of invasive GBM
To further understand the cellular state of co-cultured cells (GBM and neurons) relative to GBM cells alone, the key proteins involved in the crosstalk need to be elucidated. This comparative analysis will allow us to identify proteins th"