biostudies-literatureFan TWNIBIB NIH HHSNCI NIH HHSNational Institutes of HealthNIGMS NIH HHS108495https://www.ebi.ac.uk/biostudies/studies/S-EPMC12137166biostudies-literatureUnknown301(5)Patient-derived organotypic tissue cultures (PD-OTC) are unique models for probing cancer metabolism and therapeutic responses. They retain patient tissue architectures/microenvironments that are difficult to recapitulate while affording comparison of cancer (CA) versus matched noncancer (NC) tissue responses to treatments. We have developed a long-term culturing method for fresh and cryopreserved PD-OTC of breast cancer patients bearing invasive ductal carcinoma. Five PD-OTC came from patients with treatment-naïve primary ER⁺/PR⁺/HER2^- tumors while one came from a patient with neoadjuvant therapy for locally metastatic ER^low/PR^-/HER2^- tumor. They all exhibited tissue outgrowth in 1 month with some CA OTC harboring isolatable organoids and fibroblasts. We interrogated reprogrammed metabolism in CA versus paired NC OTC with dual ²H₇-glucose/¹³C₅,¹⁵N₂-Gln tracers coupled with stable isotope-resolved metabolomic analysis. We noted variable activation of glycolysis, cataplerotic/anaplerotic Krebs cycle including reductive carboxylation, the pentose phosphate pathway, riboneogenesis, gluconeogenesis, de novo and salvage synthesis of purine/pyrimidine nucleotides, and ADP-ribosylation in CA PD-OTC. Altered metabolic activities were in part accountable by expression changes in key enzymes measured by reverse phase protein array profiling. Notably, Gln-fueled gluconeogenesis products were preferentially diverted to support purine nucleotide synthesis. When blocking this novel process with an inhibitor of phosphoenolpyruvate carboxykinase (3-mercaptopicolinic acid), metastatic, ER^low/PR^-/HER2^- CA OTC displayed compromised cellularity, reduced outgrowth, and disrupted growth/survival-supporting metabolism but the matched NC OTC did not. Thus, our PD-OTC culturing method not only promoted understanding of actual patient's tumor metabolism to uncover viable metabolic targets but also enabled target testing and elucidation of therapeutic efficacy.The Journal of biological chemistryPatient-derived organotypic tissue cultures as a platform to evaluate metabolic reprogramming in breast cancer patients.PMC12137166P30 CA177558P30CA1775585P20GM121327R21EB032515P20 GM121327R21 EB032515Kaddah MMYLin PYan JGoncalves CFLHigashi RMIslam JMMWang XFan TWLane ANZhu CfalsePatient-derived organotypic tissue cultures as a platform to evaluate metabolic reprogramming in breast cancer patients.Patient-derived organotypic tissue cultures (PD-OTC) are unique models for probing cancer metabolism and therapeutic responses. They retain patient tissue architectures/microenvironments that are difficult to recapitulate while affording comparison of cancer (CA) versus matched noncancer (NC) tissue responses to treatments. We have developed a long-term culturing method for fresh and cryopreserved PD-OTC of breast cancer patients bearing invasive ductal carcinoma. Five PD-OTC came from patients with treatment-naïve primary ER⁺/PR⁺/HER2^- tumors while one came from a patient with neoadjuvant therapy for locally metastatic ER^low/PR^-/HER2^- tumor. They all exhibited tissue outgrowth in 1 month with some CA OTC harboring isolatable organoids and fibroblasts. We interrogated reprogrammed metabolism in CA versus paired NC OTC with dual ²H₇-glucose/¹³C₅,¹⁵N₂-Gln tracers coupled with stable isotope-resolved metabolomic analysis. We noted variable activation of glycolysis, cataplerotic/anaplerotic Krebs cycle including reductive carboxylation, the pentose phosphate pathway, riboneogenesis, gluconeogenesis, de novo and salvage synthesis of purine/pyrimidine nucleotides, and ADP-ribosylation in CA PD-OTC. Altered metabolic activities were in part accountable by expression changes in key enzymes measured by reverse phase protein array profiling. Notably, Gln-fueled gluconeogenesis products were preferentially diverted to support purine nucleotide synthesis. When blocking this novel process with an inhibitor of phosphoenolpyruvate carboxykinase (3-mercaptopicolinic acid), metastatic, ER^low/PR^-/HER2^- CA OTC displayed compromised cellularity, reduced outgrowth, and disrupted growth/survival-supporting metabolism but the matched NC OTC did not. Thus, our PD-OTC culturing method not only promoted understanding of actual patient's tumor metabolism to uncover viable metabolic targets but also enabled target testing and elucidation of therapeutic efficacy.2025-01-01T00:00:00Z2025 May2026-05-29T14:39:22.287Z2025-07-26T03:06:36.715ZS-EPMC121371664020994810.1016/j.jbc.2025.108495