Periodic Reporting for period 1 - TargMacropin (Targeting macropinocytosis in glioblastoma to achieve precision medicine)
Período documentado: 2022-09-01 hasta 2025-02-28
Macropinocytosis is a non-specific endocytic process that allows cancer cells to uptake extracellular fluids into macropinosomes which are large intracellular vesicles. In the harsh tumor microenvironment, macropinocytosis has been reported to be a critical and unique cellular route of entry for nutrients (such as glutamine, or adenosine 5′-triphosphate). In addition to their large size and the ability to internalize high molecular weight dextran such as tetramethylrhodamine-Dextran (TMR-Dextran), macropinosomes are also characterized by their unique sensitivity to amiloride (such as 5-(Nethyl-N-isopropyl) amiloride (EIPA)) and its derivatives, which is the most effective and selective method currently used to pharmacologically inhibit micropinocytosis.
The objectives of this study are to investigate the molecular mechanisms underlying cancer cell addiction to macropinocytosis, with the ultimate goal to identify and validate targets for the therapeutic exploitation of this mechanism. To achieve this goal, we will combine various functional assays, metabolomic, transcriptomic, and proteomic analyses with a cutting-edge technique of laser capture microdissection together with mass-spectrometry-guided protein identification of the macropinosomes.
The objectives of this study are to investigate the molecular mechanisms underlying cancer cell addiction to macropinocytosis, with the ultimate goal to identify and validate targets for the therapeutic exploitation of this mechanism. To achieve this goal, we will combine various functional assays, metabolomic, transcriptomic, and proteomic analyses with a cutting-edge technique of laser capture microdissection together with mass-spectrometry-guided protein identification of the macropinosomes.
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.
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.
By identifying macropinosome components in GBM, our study will set up new methods for investigating macropinocytosis in different cellular models (not only in cancer cells but also in normal cells such as macrophages of dendritic cells). Indeed, our protocol describing laser capture microdissection combined with mass spectrometry will be submitted for publication and can serve the scientific community. Moreover, after validation, these new macropinocytosis-modulator can replace EIPA, amiloride known and used in the field as an inhibitor of macropinocytosis. Therefore, by identifying a new specific inhibitor of macropinocytosis, this study could provide a useful and suitable tool in the field.
Regarding GBM treatment, this project will pave the way for expanding our understanding of how GBM cells uptake nutrients and adapt/respond to stress and this dependency pathway could be exploited to design new therapeutic strategies.
The metabolomics analysis allowed a relative quantification of metabolites involved in several metabolic pathways (such as glycolysis, pentose phosphate pathway, tricarboxylic acid cycle…) under several microenvironmental stressors. By revealing the metabolic shift occurring upon various microenvironmental stress, our study will shed light on GBM metabolism under stress conditions and their metabolic vulnerabilities. Because the biological abnormalities driving GBM progression are not the same in every tumor, our study included several GBM subtypes allowing a unique opportunity to highlight a dependence on macropinocytosis and metabolic shift.
Regarding GBM treatment, this project will pave the way for expanding our understanding of how GBM cells uptake nutrients and adapt/respond to stress and this dependency pathway could be exploited to design new therapeutic strategies.
The metabolomics analysis allowed a relative quantification of metabolites involved in several metabolic pathways (such as glycolysis, pentose phosphate pathway, tricarboxylic acid cycle…) under several microenvironmental stressors. By revealing the metabolic shift occurring upon various microenvironmental stress, our study will shed light on GBM metabolism under stress conditions and their metabolic vulnerabilities. Because the biological abnormalities driving GBM progression are not the same in every tumor, our study included several GBM subtypes allowing a unique opportunity to highlight a dependence on macropinocytosis and metabolic shift.