Patient-derived organotypic tissue cultures as a platform to evaluate metabolic reprogramming in breast cancer patients.

Abstract

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 non-cancer (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 ERlow/PR-/HER2- tumor. They all exhibited tissue outgrowth in one 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 (GNG), 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 GNG products were preferentially diverted to support purine nucleotide synthesis. When blocking this novel process with an inhibitor of phosphoenolpyruvate carboxykinase (3-mercaptopicolinic acid or 3-MPA), metastatic, ERlow/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.