{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T18:20:47Z","timestamp":1790619647806,"version":"4.1.0"},"reference-count":58,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:00:00Z","timestamp":1787788800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"McCabe Fund Advisory Committee, the University of Pennsylvania School of Medicine"},{"id":[{"id":"https:\/\/ror.org\/00b30xv10","id-type":"ROR","asserted-by":"publisher"}]},{"name":"NIH","award":["R01CA191207"],"award-info":[{"award-number":["R01CA191207"]}]},{"name":"NIH","award":["R01-CA278837"],"award-info":[{"award-number":["R01-CA278837"]}]},{"name":"NIH","award":["R01CA266285"],"award-info":[{"award-number":["R01CA266285"]}]},{"name":"NIH","award":["R01CA301356-01A1"],"award-info":[{"award-number":["R01CA301356-01A1"]}]},{"name":"Thomas B. McCabe and Jeannette E. Laws McCabe Fund Pilot Award"},{"name":"Harrison Scholarship, the University of Pennsylvania School of Medicine"},{"id":[{"id":"https:\/\/ror.org\/00b30xv10","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Bruder Family Breast Cancer Immunotherapy Research Funds"},{"name":"Roy & Diana Vagelos Program in the Molecular Life Sciences"},{"name":"EXON20-IRC"},{"name":"Ludwig Research Foundation Princeton Branch"},{"DOI":"10.13039\/100000050","name":"NHLBI","doi-asserted-by":"crossref","award":["R01 HL165792"],"award-info":[{"award-number":["R01 HL165792"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000062","name":"NIDDK","doi-asserted-by":"crossref","award":["R01 DK098656"],"award-info":[{"award-number":["R01 DK098656"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NCI","award":["R01CA277037"],"award-info":[{"award-number":["R01CA277037"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Metabolites"],"abstract":"<jats:p>Background\/Objectives: Triple-negative breast cancer (TNBC) exhibits substantial metabolic heterogeneity and plasticity, contributing to variable therapeutic responses. We investigated whether optical redox imaging (ORI) could characterize metabolic phenotypes, monitor responses to metabolic perturbation, and relate these responses to functional outcomes in TNBC cells. Methods: Four TNBC cell lines were treated with the lactate dehydrogenase A inhibitor FX11 or the glutaminase inhibitor CB-839, alone or in combination with paclitaxel. Intensity-based label-free ORI was performed and followed by imaging of fluorescent probes to assess mitochondrial membrane potential (MMP), re-active oxygen species (ROS), and cell number in the same dishes. Seahorse assays were used to evaluate mitochondrial respiration and glycolytic flux. Results: TNBC cell lines exhibited distinct basal redox phenotypes and differential responses to acute glycolytic and glutaminolytic perturbation. HCC1806 cells showed the strongest acute ORI responses to both FX11 and CB-839. Acute FX11 treatment induced a rapid reductive shift accompanied by ROS accumulation and loss of MMP, whereas CB-839 produced a more modest early reductive response without detectable ROS accumulation or MMP loss. Prolonged treatment revealed distinct temporal redox trajectories in HCC1806 cells: FX11-treated cells evolved from an acute reductive response toward a more oxidized state, while CB-839-treated cells transitioned from an early reductive shift to a sustained oxidized redox state accompanied by marked reductions in OCR and ECAR. Functionally, in two representative models (HCC1806 and MDA-MB-231), CB-839 reduced cell numbers and enhanced the anti-proliferative effect of paclitaxel, whereas FX11 had no significant effect on cell number despite inducing pronounced acute redox perturbations. Conclusions: Integrating ORI with metabolic flux assays and imaging-based functional measurements enables characterization of multiple dimensions of metabolic behavior, including basal phenotype, pathway-specific responsiveness, temporal redox responses, and treatment-associated outcomes. These findings support intensity-based wide-field ORI as a practical and accessible tool for probing metabolic heterogeneity and characterizing context-dependent metabolic responses in TNBC cells.<\/jats:p>","DOI":"10.3390\/metabo16090613","type":"journal-article","created":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T08:02:27Z","timestamp":1787817747000},"page":"613","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Live-Cell Optical Redox Imaging Reveals Metabolic Heterogeneity and Context-Dependent Responses to Metabolic Perturbation in TNBC Cells"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5127-2629","authenticated-orcid":false,"given":"He N.","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jack","family":"Kollmar","sequence":"additional","affiliation":[{"name":"Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7202-0906","authenticated-orcid":false,"given":"Allison","family":"Podsednik","sequence":"additional","affiliation":[{"name":"Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mihiar","family":"Wannousse","sequence":"additional","affiliation":[{"name":"Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Shestov","sequence":"additional","affiliation":[{"name":"Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roddy S.","family":"O\u2019Connor","sequence":"additional","affiliation":[{"name":"Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph A.","family":"Baur","sequence":"additional","affiliation":[{"name":"Department of Physiology, Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Tchou","sequence":"additional","affiliation":[{"name":"Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rong","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9447-7982","authenticated-orcid":false,"given":"Lin Z.","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1938","DOI":"10.1056\/NEJMra1001389","article-title":"Triple-negative breast cancer","volume":"363","author":"Foulkes","year":"2010","journal-title":"N. 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