To better understand their molecular composition and the macropinocytosis activity, the identification of macropinosome components is critical. Here, we used laser capture microdissection combined with mass spectrometry (collaboration with Dr. Frédéric Saltel at the BaRITOn Bordeaux Research in Translational Oncology) in two GBM patient-derived differentiated cells (GDCs), Ge518 and Ge269. Our results revealed 11 proteins involved in several mechanisms included glucose homeostasis, calcium regulation, actin modulation, axon growth and guidance, metabolism, and cargo receptor, in common between both Ge518 and Ge269 macropinosomes. Next, we will use a pharmacologic and/or a genetic gain or loss-of-function approach to modulate the expression of the identified molecules (expression plasmid, shRNAs and/or CRISPR-Cas9 KO technologies). As a readout, we will evaluate macropinocytosis activity using TMR-Dextran uptake and/or the self-quenched albumin (DQ-BSA; Life Technologies). We will also evaluate whether the modulation of their expression affects cell viability/apoptosis/necrosis (using the RealTime-Glo assay kit).
Tumor cells addicted to macropinocytosis are usually found in nutrient and oxygen depleted regions. As shown by several studies, macropinocytosis modulates cancer cell metabolism through nutrient internalization (glutamine, albumin). and is likely a result of tumor cell adaptation to this harsh tumor microenvironment. To measure the concentration of various metabolites in several GBM patient-derived stem cells (GSCs), we used high resolution LC-MS (collaboration with the Metabolomics Unit of the University of Lausanne, Switzerland). The metabolite profiling revealed several metabolic pathways dysregulated under the microenvironmental stressors present in the tumor.
Moreover, to investigate whether nutrient deprivation/hypoxia, oxidative stress and drug treatments induce an enhanced micropinocytosis, we measured TMR-Dextran uptake and cell viability under various forms of stress in a panel of GSCs. Our results revealed a greater increase of macropinocytosis activity in non-addicted cell lines compared to the addicted ones when glutamine and glucose are deprived in the media. In parallel, we also measured in these models the Warburg effect as the ratio of the oxygen consumption rate (OCR) to the extracellular acidification (ECAR) by using a Seahorse XF analyser. In nutrient-deprived conditions, our data showed a shift towards a quiescent metabolism when GSCs are exposed to these conditions regardless their basal energetic, glycolytic or aerobic metabolic status.
Finally, to identify the downstream signalling that induces or regulates macropinocytosis, we have established a collaboration with the team of Dr. Jean Armengaud (CEA, DMTS UMR0496, Bagnols-sur-Cèze), expert in next-generation proteomics, proteogenomics, and metaproteomics, to combine our metabolomics and next-generation proteomics analyses in our GBM patient-derived models under the different conditions. In addition, we will ask whether the progression of macropinocytosis-addicted tumors can be halted or slowed down in vivo by knocking down the different identified drivers of macropinocytosis or by targeting their regulatory pathway.
