Year: 2026
Type: conference-abstract
Abstract: Background CAR T cell therapies have revolutionized treatment of blood cancers, though barriers remain in achieving durable remissions in solid tumors like neuroblastoma. Many studies have shown that T cells undergo extreme metabolic changes with activation and that naïve, exhausted, and memory-like T cells have distinct metabolic features that impact fitness, subtype, and function. In vivo multiphoton autofluorescence microscopy provides label-free, single-cell imaging of metabolic changes quantified via the metabolic coenzymes NAD(P)H and FAD, which are autofluorescent molecules endogenous to all cells. We aim to study metabolic changes of CAR T cells in vivo to improve assessment of product efficacy.Methods Human neuroblastoma cells (GD2 + CHLA-20) were implanted into the flanks of NSG mice to generate xenograft tumors. Once tumors were established, human GD2 CAR T cells were administered intravenously. Third generation GD2 CAR T cell constructs included an appended T2A mCherry fluorescent reporter to allow for in vivo identification and tracking of CAR+ T cells. CAR T cells with varying co-stimulatory domains and TCR knockout were compared to monitor metabolic fitness, persistence, and tumor response. Tumors and lymphoid tissues were imaged at several timepoints following CAR T administration, either fresh ex vivo or in vivo under anesthesia. Multiphoton imaging was performed to collect NAD(P)H, FAD, and mCherry signal through a 40X objective. Fluorescence lifetime data were collected using time correlated single photon counting electronics. Tissues were also harvested and analyzed via flow cytometry to corroborate intravital imaging findings.Results GD2 + CAR T cells circulate in lymphoid tissues and infilitrate neuroblastoma tumors at day 3 post injection – with CAR T cells persisting until at least day 18 (figure 1A). CAR T cells exhibit different metabolic phenotypes including changes in NAD(P)H and FAD protein binding and redox ratio over time. Quantified single-cell mCherry+ CAR+ T cell metabolic changes over time show decreased free fraction of NAD(P)H and increased NAD(P)H mean lifetime – both suggesting a more oxidative metabolic shift over time (figure 1B). CAR T cell size and morphology, often indicative of proliferation and cytotoxicity, also change over time with the largest change on day 7 (figure 1B).Conclusions These results show that autofluorescence in vivo imaging enables single-cell quantification of GD2+ CAR T cell metabolic changes across time and CAR product. With continued efforts, this imaging platform may be leveraged to help develop more effective CAR T cell therapy for solid tumors, including neuroblastoma.Ethics Approval All animal work was approved by the University of Wisconsin Institutional Animal Care and Use Committees.Abstract 191 Figure 1In vivo metabolic imaging of human GD2+ mCherry+ CAR T cells against neuroblastoma. (A) Metabolic images of CHLA-20 tumors (top) and inguinal lymph nodes (bottom) show mCherry+ CAR+ T cells (red) infiltrating tumors (blue and green) and blood vessels. Scale bar 25 μm; (B) Quantified single-cell CAR T cell metabolic changes over time
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225145488