Dual Ribosome Profiling reveals metabolic limitations of cancer and stromal cells in thetumor microenvironment

Cancer cells, immune cells, and stromal cells within the tumor microenvironment (TME) collaboratively influence disease progression and therapeutic responses. The nutrient-limited conditions of the TME, particularly the scarcity of glucose, amino acids, and lipids, challenge cancer cell survival1-4. However, the metabolic constraints faced by immune and stromal cells in comparison to cancer cells, and how these limitations affect therapeutic outcomes, remain poorly understood. Here, we introduce Dual Ribosome Profiling (DualRP), a method that allows for simultaneous analysis of translation and identification of ribosome stalling, revealing amino acid shortages in different cell types within tumors. Using DualRP, we uncover that interactions between cancer cells and fibroblasts trigger an inflammatory response, mitigating amino acid limitations during glucose starvation. In immunocompetent mouse models, we observe that immune checkpoint blockade therapy induces serine and glycine restrictions specifically in T cells, but not in cancer cells. We further demonstrate that these amino acids are essential for optimal T cell function both in vitro and in vivo, highlighting their critical role in effective immunotherapy. Our findings show that therapeutic interventions create distinct metabolic demands across different tumor cell types, with nutrient availability significantly influencing the success of immunotherapy. DualRPs ability to explore cell type-specific metabolic vulnerabilities offers a promising tool for advancing our understanding of tumor biology and improving therapeutic strategies.