Project Summary/Abstract Living cells require a constant supply of nutrients that provide energy and building blocks to support their vital activities and growth. Fluctuations in nutrient availability are inevitable. Thus, to survive, cells need to adapt to these changes by rewiring their metabolism. Studying this metabolic adaptation in lower organisms has revolutionized our understanding of biological systems. For example, the discovery of how prokaryotes respond to changes in the accessibility to lactose and glucose as a carbon source led to the concept of gene regulation after the identification of the lac operon. In eukaryotes the evolution of subcellular organelles provided an optimal environment for biochemical reactions to proceed. Moreover, this system allowed the eukaryotic cell to evolve additional strategies to acquire nutrients besides passive diffusion or transport across the cell membrane. Through the endo- lysosomal compartment, cells can scavenge nutrients from extracellular macromolecules, which provides them with metabolic flexibility to survive various states of nutrient availability by balancing the composition of their microenvironment with their nutrient demands. Nutrient acquisition strategies are fully exploited by malignant cells to survive the harsh tumor microenvironment. Pancreatic cancer, a lethal malignancy, is a paradigm of metabolic adaptation. Hypo- vascularization of pancreatic ductal adenocarcinoma (PDAC) limits the delivery of free nutrients and oxygen to cancer cells. To overcome nutrient scarcity, cancer and stromal cells rely on scavenging nutrients from intra- and extracellular macromolecules via autophagy and macropinocytosis, respectively. Both pathways converge on the lysosome, a cellular organelle that degrades macromolecules to recycle their nutrient content. Despite their essential role in cancer, studying lysosomes in highly heterogenous tumors in vivo is challenging because of the lack of tools that allow the functional profiling of lysosomal content during tumorigenesis at a cell-type-specific resolution. In this proposal, I will describe our novel approach to develop an innovative technology that allows the rapid capturing of lysosomes from specific cell types in the tumor to profile their metabolite, lipid and protein contents to understand how lysosomes in malignant and stromal cells mediate metabolic adaptation. We will also design a modular mouse model system that will allow the selective interrogation of the lysosomal response to major metabolic stressors that exist in the tumor microenvironment. Our innovative approaches combined with functional characterization of the lysosomal components using genetic tools will result in an unprecedent subcellular and cell-type-specific understanding of tumor metabolism. We believe that our work has the potential to revolutionize our understanding of metabolic adaptation in mammalian systems, and to identify vulnerabilities that can be exploited as ...