In tumors, nutrient availability and metabolism are known to be important modulators of growth signaling. However, it remains elusive whether cancer cells that are growing out in the metastatic niche rely on the same nutrients and metabolic pathways to activate growth signaling as cancer cells within the primary tumor. We discovered that breast cancer-derived lung metastases, but not the corresponding primary breast tumors, use the serine biosynthesis pathway to support mTORC1 growth signaling. Mechanistically, pyruvate uptake through Mct2 supported mTORC1 signaling by fueling serine biosynthesis-derived α-ketoglutarate production in breast cancer-derived lung metastases. Consequently, expression of the serine biosynthesis enzyme PHGDH was required for sensitivity to the mTORC1 inhibitor rapamycin in breast cancer-derived lung tumors but not in primary breast tumors. In summary, we provide in vivo evidence that the metabolic and nutrient requirements to activate growth signaling differ between the lung metastatic niche and the primary breast cancer site.

Metabolism is at the cornerstone of all cellular functions and mounting evidence of its deregulation in different diseases emphasizes the importance of a comprehensive understanding of metabolic regulation at the whole-organism level. Stable-isotope measurements are a powerful tool for probing cellular metabolism and, as a result, are increasingly used to study metabolism in in vivo settings. The additional complexity of in vivo metabolic measurements requires paying special attention to experimental design and data interpretation. Here, we review recent work where in vivo stable-isotope measurements have been used to address relevant biological questions within an in vivo context, summarize different experimental and data interpretation approaches and their limitations, and discuss future opportunities in the field. Metabolism: A Central Node for Cellular Processes Metabolism can be seen as the engine of the cell, providing energy, redox cofactors, and building blocks for cell maintenance, growth, and renewal, as well as playing a key role in modulating cell signaling [1,2]. To orchestrate all these functions, metabolism consists of a complex network of genes, enzymes, and metabolites, steadily modulated in response to different stimuli [3-5]. Defining the mechanisms at the basis of this regulation and understanding their physiological role is among the most important pursuits in biological and medical research [6], particularly since the realization that many pathologies are driven by metabolic deregulations [7]. An in-depth understanding of metabolic pathways in vivo and how these are deregulated in different diseases is thus fundamental for the discovery of new therapeutic targets and clinical biomarkers, enabling the development of more robust diagnosis approaches and personalized treatments, eventually improving the overall outcome for patients [8]. In this context, stable-isotope tracers (see Glossary) have become a standard for probing cellular metabolism [9] and their increasing implementation in in vivo settings has revolutionized our current understanding of mammalian metabolism in health and disease, unraveling novel regulatory principles at both the cellular and whole-organism level [10,11]. In this review, we summarize the latest advances in in vivo metabolic measurements using stable-isotope tracers, highlighting the importance of systems-level integrative approaches and careful experimental design, pointing out open challenges and opportunities for advancing the field, and outlining strategies and potential pitfalls when interpreting these measurements (Figure 1). Tracer-Based Methods for Measuring Cellular Metabolism In Vivo Many complementary methods exist to study metabolism in vivo and, consequently, the selection of a specific approach (or set of approaches) depends largely on the biological question being addressed. Nontracer-based methods, such as assessing bioenergetics by measuring dynamic changes in oxygen consumption [12] or evaluating changes in metabolite levels via metabolomics [13],...

Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs. Genetic, transcriptional and translational intra-tumor heterogeneity contributes to this dynamic process. Beyond this, metabolic intra-tumor heterogeneity has also been observed, yet its role for cancer progression remains largely elusive. Here, we discovered that intra-tumor heterogeneity in phosphoglycerate dehydrogenase (PHGDH) protein expression drives breast cancer cell dissemination and metastasis formation. Specifically, we observed intra-tumor heterogeneous PHGDH expression in primary breast tumors, with low PHGDH expression being indicative of metastasis in patients. In mice, Phgdh protein, but not mRNA, expression is low in circulating tumor cells and early metastatic lesions, leading to increased dissemination and metastasis formation. Mechanistically, low PHGDH protein expression induces an imbalance in glycolysis that can activate sialic acid synthesis. Consequently, cancer cells undergo a partial EMT and show increased p38 as well as SRC phosphorylation, which activate cellular programs of dissemination. In turn, inhibition of sialic acid synthesis through knock-out of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) counteracts the increased cancer cell dissemination and metastasis induced by low PHGDH expression. In conclusion, we find that heterogeneity in PHGDH protein expression promotes cancer cell dissemination and metastasis formation.