Project Summary Most studies investigate whole cancer cell populations and rarely address how single cells or sub-populations cooperate to promote their survival and spread. This is especially true in the context of metabolism, where tumors harbor distinct sub-populations of glycolytic and oxidative cells forged in part by microenvironmental pressures. How this metabolic heterogeneity drives tumor invasion and metastasis is an unexplored area and requires approaches that can isolate these specific cancer cell sub-populations. This multi-PI application in lung adenocarcinoma attempts to address this by determining how metabolically heterogeneous cancer cell sub- populations function and cooperate to drive invasion and metastasis. We build upon our published image-guided genomics technology (SaGA) to extract living and phenotypically defined cell sub-populations within collectively invading packs. We used SaGA to deconstruct the lung cancer collective invasion pack, which is comprised of hierarchical groups of invasive leader and proliferative follower cells invading as a cohesive unit. Our published and preliminary data show that follower cells consume twice as much glucose as leaders, and rely on glycolysis and the oxidative pentose phosphate pathway (PPP) to maintain their proliferative state. By simply disrupting the glucose transporter, GLUT1, followers become invasive and take on a leader-like phenotype. In contrast, leaders rely on oxidative phosphorylation (OXPHOS) via pyruvate dehydrogenase (PDH) activity to drive invasion, where disrupting PDH creates a more follower-like phenotype. These data lead to our overarching hypothesis that metabolic heterogeneity sustained by differential GLUT1 and PDH-driven metabolism facilitates lung cancer metastasis by maintaining distinct phenotypes in the collective invasion pack. We propose that this metabolic heterogeneity warrants a co-targeting therapeutic approach that disrupts both metabolic populations to inhibit metastasis. Thus, the objective of this proposal is to 1) elucidate the molecular basis and mechanistic underpinnings of how metabolic heterogeneity drives collective invasion and 2) test if co-targeting different metabolic sub-populations limits metastasis. We propose that these studies will directly impact our understanding of how metabolic heterogeneity sustains cooperativity to promote collective invasion/metastasis.