Diffuse Midline Glioma (DMG) is a rare form of brain tumour which commonly occurs within the midline structures of the brain. This disease occurs mostly in children, with 20-30 children across Australia and New Zealand children diagnosed each year. Of these, over 90% succumb within two years post-diagnosis. Presently, no curative treatments exist with the current standard of therapeutic modality being palliative radiation therapy. Further to this, DMG is known to exhibit multiple mechanisms for radiation resistance which reduces the efficacy of this mode of treatment for most patients.
In order to better understand the ways in which this cancer responds and builds resistance to radiation, we utilised single cell proteomics (SCP) to elucidate whether radiation response and subsequent resistance is driven by a heterogeneous mechanism within a cellular population. Patient-derived, treatment-naïve DMG cells were subjected to two different doses of radiation (5 Gy and 10 Gy) alongside a non-irradiated control, and single cell isolation was performed using the CellenONE single cell sorter (Cellenion) 72 hours post-radiation. A total of 40 single cells from each condition were then lysed and digested in the CellenONE, followed by LC-MS/MS analysis on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher) via data-independent acquisition. A global analysis of the cells suggests that proteome shifts between conditions in response to radiation are not dose-dependent, however non-linear dimension reduction via UMAP and additional bioinformatic interrogation clearly indicates subpopulations of cells which exhibit differential expression of key response proteins, particularly those belonging to phosphorylation and ubiquitination signalling cascades, and apoptotic pathways. This study highlights that SCP is a rapidly emerging technology which shows great promise in understanding complex disease models through interrogating cellular heterogeneity.