Abstract Introduction Granzyme B (GZB), a serine protease secreted by cytotoxic T lymphocytes and natural killer cells, plays a critical role in immune defense by inducing apoptosis in target cells. Monitoring GZB production, e.g., during T cell activation, provides a direct functional readout of cytotoxic potential, distinguishing it from surface markers CD69 and CD25 that indicate activation status. Current methods for measuring GZB protein levels (flow cytometry and ELISA) involve multi-step, lengthy protocols. We developed a bioluminescent GZB activity assay for simplified, rapid GZB analysis to facilitate the incorporation of this functional indicator into experimental workflows. Methods The assay reaction consists of a peptide-luciferin prosubstrate and luciferase. Proteolytic cleavage by active GZB releases luciferin, which is oxidized by the luciferase enzyme. The resulting light signal is directly proportional to GZB activity. Results We used the assay to monitor GZB production during T cell activation. T cells were isolated from peripheral blood mononuclear cells and stimulated under various media conditions. Samples were collected during the activation phase on Days 1-3; culture medium and cell lysates were analyzed. GZB activity increased over time and varied by activation condition. On day 1, signals from cell lysates were ∼2 to 10-fold higher than medium background, increasing to ∼30 to 550-fold by day 3. Signals from non-activated cell lysates were not above background. GZB detected in culture medium also increased, reaching ∼11 to 106-fold above background on day 3. These results were confirmed by measuring intracellular GZB levels by flow cytometry and ELISA. GZB activity also aligned with CD69 and CD25 expression, as well as IFN-γ and TNF-α cytokine secretion. Conclusion This bioluminescent GZB activity assay provides a rapid and simple method for monitoring GZB, especially for studies requiring higher-throughput sample analysis. Funding Source Promega Corporation Topic Categories Technological Innovations in Immunology (TECH)

Abstract Defining cellular state requires understanding how multiple metabolic features change together, yet most assays capture only single parameters. We developed an integrated bioluminescent profiling strategy that measures coordinated metabolic and cell-health indicators from the same low-input sample, providing a practical way to resolve early pathway activity and cellular condition across immune and cancer systems. The approach uses a suite of luminescent assays to quantify ATP, NAD, total NADP(H), metabolic activity, and nutrient utilization including glucose consumption, lactate secretion, and malate accumulation. Together, these features report on glycolytic engagement, mitochondrial contribution, and redox balance, generating compact multiparametric profiles not achievable with isolated assays. In primary T cells, the integrated profiles distinguished early glycolytic, NAD-rich states linked to rapid expansion from more oxidative states associated with memory-biased phenotypes. These early metabolic patterns emerged within the first 72 hours of activation and aligned with later differences in proliferation and T cell-subset composition, demonstrating that early metabolic signatures capture functional trajectories beyond initial activation markers. In cancer-cell models, the same measurements resolved nutrient-dependent shifts in metabolic balance and stress adaptation, illustrating how environmental composition shapes pathway use and overall cellular fitness. The workflow uses standard luminescent instrumentation, minimal material, and is adaptable to additional metabolic markers as the platform evolves. By capturing coordinated metabolic and cell-health features within a scalable framework, this approach provides a practical way to define cellular state and relate early metabolic patterns to later functional behavior across diverse experimental contexts. Citation Format: Kayla Sylvester, Anthony C. Lauer, Gediminas Vidugiris, Donna Leippe, Jolanta Vidugiriene. Integrated metabolic and cell-health profiling as a framework for defining cellular state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3271.

Abstract Research scientists measure fatty acid oxidation (FAO) to assess how efficiently cells and tissues convert fats into energy and how this process shifts under different physiological or pathological conditions. Because FAO is central to mitochondrial energy metabolism, its measurement reveals insights into metabolic flexibility, mitochondrial health, and substrate preference. Altered FAO is implicated in many diseases including diabetes, obesity, heart failure, and cancer, making it an important marker for both mechanistic studies and drug screening. Additionally, FAO measurements help evaluate the effects of exercise, diet, or pharmacological interventions on energy balance and cellular metabolism. Radiolabeled fatty acids and oxygen consumption assays are the most common methods for measuring fatty acid oxidation (FAO), but these approaches can be labor-intensive and technically demanding. To simplify FAO measurement, we developed a bioluminescent assay based on a fatty acid-linked pro-luciferin substrate. The substrate readily enters cells, where FAO enzymes remove the fatty acid moiety, releasing a modified luciferin precursor. Addition of a detection reagent converts this intermediate to luciferin, generating luminescence proportional to FAO activity. The signal scales with cell number and incubation time and is compatible with both cancer cell lines and primary cells. With just 20,000 cells and a 1-hour incubation, the assay generates strong signal-to-background ratios - about 200 in HEK293 cells and 100 in primary human hepatocytes. The assay is sensitive to inhibition by etomoxir, confirming dependence on carnitine palmitoyltransferase (CPT1) activity, a key regulatory step in mitochondrial FAO. After media removal, all steps are performed in an add-and-read format suitable for 96- or 384-well plates, enabling convenient, high-throughput quantification of FAO. AI was used to help construct this abstract. Citation Format: Maggie Bach, Michael P. Valley, Hui Wang, Xavier Aguilar-Enriquez, Wenhui Zhou, Jolanta Vidugiriene. A bioluminescent assay for detection of fatty acid oxidation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 475.

Manufacturing chimeric antigen receptor (CAR) T cells with a stem cell memory phenotype can enhance their persistence in patients. Histone deacetylase inhibitors (HDACis) targeting class I HDACs promote chromatin remodeling in virally manufactured CAR T cells, leading to the activation of the Wnt pathway, and ultimately improve CAR T cell persistence. However, it is unknown whether the persistence of CRISPR-engineered CAR T cells can also be enhanced through such HDACi-mediated epigenetic modulation. CAR T cells engineered using CRISPR-Cas9 to integrate a CAR construct into the T cell receptor alpha constant (TRAC) were treated with various class- or isoform-selective HDACis, and phenotypic, functional, and metabolic changes were assessed in vitro and in a GD2+ neuroblastoma xenograft model. Compared to untreated GD2 TRAC-CAR T cells, chidamide and mocetinostat increased the stem cell memory marker expression (CD62L+/CCR7+/IL-7Rα+). Chidamide induced a fragmented mitochondrial morphology and, in vivo, enhanced the persistence and naive phenotype of the GD2 TRAC-CAR T cells without upregulating exhaustion markers (PD-1/LAG3). These findings suggest that in the management of treatment-resistant pediatric solid tumors, utilizing chidamide during the manufacturing of CRISPR-engineered CAR T cells may enhance their persistence while retaining a naive phenotype.

Chimeric antigen receptor (CAR) T cell therapy for solid tumours is challenging because of the immunosuppressive tumour microenvironment and a complex manufacturing process. Cellular manufacturing protocols directly impact CAR T cell yield, phenotype and metabolism, which correlates with in vivo potency and persistence. Although metabolic fitness is a critical quality attribute, how T cell metabolic requirements vary throughout the manufacturing process remains unexplored. Here we use optical metabolic imaging (OMI), a non-invasive, label-free method to evaluate single-cell metabolism. Using OMI, we identified the impacts of media composition on CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrate that OMI parameters can indicate cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. In a CRISPR-edited anti-GD2 CAR T cell model, OMI measurements allow accurate prediction of an oxidative metabolic phenotype that yields higher in vivo potency against neuroblastoma. Our data support OMI as a robust, sensitive analytical tool to optimize manufacturing conditions and monitor cell metabolism for increased CAR T cell yield and metabolic fitness. Monitoring T cell metabolism during chimeric antigen receptor T manufacturing using optical metabolic imaging enables fine-tuning of manufacturing conditions to increase T cell yield and fitness.

Advancing our understanding of T cell activation and its regulatory pathways is pivotal for immunology research and therapeutic development. This study evaluates T cell health and functional status under diverse activation conditions, illustrating key molecular mechanisms that govern immune responses. We focus on critical pathways, including mTOR, AMPK, and the metabolic interplay between glycolysis and oxidative phosphorylation. Using selective inhibitors, we identify critical immune regulatory modulators within these pathways. Our findings reveal a strong correlation between T cell metabolic state and cytokine secretion, with media composition identified as a significant factor influencing T cell function. Our methodology employs a panel of bioluminescent Lumit and Metabolite-Glo assays, enabling sensitive quantification of cytokines and metabolites. This method enables monitoring with minimal sample handling, preserving cell integrity and improving reproducibility. Its scalability and adaptability support analysis across diverse activation conditions and high-throughput screening applications. Furthermore, we show correlation to cell surface markers using traditional methods. Our findings and methods provide a robust platform for detailed exploration of signaling, metabolic, and immune dynamics during T cell activation, offering new insights for targeted immunomodulation strategies. Immune Response Regulation: Molecular Mechanisms (IRM)

IntroductionT cell metabolism is a key determinant of immune function and therapeutic efficacy, yet current expansion protocols often neglect how culture conditions influence metabolic programming. We employed a modular, low-input bioluminescent assay platform to profile how media, activation strength, and metabolic perturbation define metabolic trajectories that persist through early expansion and influence downstream outcomes.MethodsA multifactorial experimental design was used to evaluate early T-cell activation across media (ICXF, TexMACS, RPMI+FBS) and activators (TransAct, Dynabeads, ImmunoCult). Low-input bioluminescent assays were used to quantify metabolic cofactors (ATP, NAD+, NADP(H)), reducing capacity, and nutrient usage (glucose, lactate, malate). Conditions that yield metabolically distinct phenotypes were selected for deeper analysis of proliferation, cytokine secretion, cytotoxicity, and flow cytometric profiling. To validate and functionally confirm these phenotypes, pathway-specific metabolic inhibitors were introduced in follow-up experiments.ResultsBy measuring intracellular ATP, NAD+, NADP(H), reducing capacity, and nutrient flux, we identified media- and activation-specific metabolic states that emerged upon T-cell activation and persisted through early expansion. ICXF with TransAct promoted a glycolytic, NAD-rich phenotype associated with rapid expansion. In contrast, TexMACS with ImmunoCult supported oxidative metabolism, enriched for TSCM-like cells, and enhanced cytotoxicity despite slower growth. Early lactate levels strongly predicted downstream expansion (r = 0.68, p < 0.0001), highlighting glycolytic activity as a key determinant of proliferative potential. Functional validation with pathway-specific inhibitors revealed media-dependent vulnerabilities, highlighting distinct metabolic wiring.ConclusionThis approach enables predictive, multiplexed metabolic profiling using minimal sample input and offers a scalable strategy to optimize T-cell manufacturing for memory enrichment and cytotoxic potency.

Advancing our understanding of T cell activation and its regulatory pathways is critical for developing effective cancer immunotherapies. This study characterizes T cell health and functional dynamics under diverse activation conditions, highlighting key molecular mechanisms that influence immune responses within the tumor microenvironment. We investigate mTOR and AMPK signaling pathways, as well as the metabolic balance between glycolysis and oxidative phosphorylation, which are integral to T cell function in immunotherapeutic contexts. Using selective inhibitors, we identify modulators that regulate cytokine secretion and T cell metabolic states, revealing media composition as a significant variable affecting immune function. Our methodology leverages a suite of bioluminescent Lumit and Metabolite-Glo assays for sensitive quantification of cytokines and metabolites, enabling precise, non-invasive monitoring while preserving cell integrity. This approach is scalable and adaptable for analyzing diverse activation conditions and high-throughput screening applications. Additionally, our findings correlate these metabolic and functional profiles with cell surface markers assessed by traditional methods, providing a comprehensive framework for immune monitoring. These insights underscore the importance of metabolic and signaling interplay in T cell activation, offering a robust platform for studying immune dynamics and uncovering novel immunomodulatory targets. Our work supports the development of precision strategies to modulate T cell activity and optimize immune responses in cancer therapy. Kim Haupt, Kayla Sylvester, Tony Lauer, Natasha Karassina, Jolanta Vidugiriene. Metabolic and functional dynamics of T cell activation: bioluminescent assays for immune monitoring and modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4855.

Glucose metabolism is fundamental to the function and survival of both cancer cells and immune cells within the tumor microenvironment. Cancer cells often exhibit altered metabolic pathways to meet the elevated energy and anabolic demands of rapid proliferation, including increased glucose uptake for enhanced glycolysis and glycogen storage. This can deplete glucose availability for infiltrating immune cells, restricting their ability to execute an effective anti-tumor response and promoting an immunosuppressive environment. Understanding glucose metabolism in cancer cells and T cells is important for elucidating the role of nutrient competition in immunotherapy effectiveness. To investigate these dynamics, we developed sensitive bioluminescence assays capable of measuring multiple aspects of glucose metabolism in both cancer and immune cells, including glycogen synthesis and utilization. Glycogen serves as a glucose reservoir that can rapidly respond to glucose supply and demand to maintain cellular functions under fluctuating nutrient conditions. Changes in glycogen levels (both increases and decreases) were observed in cancer cell lines in response to medium composition (e.g., glucose concentration) and drug treatments. Glycogen was also measured in T cells upon activation, providing a useful method for studying requirements for glycogen synthesis in these cells and its relevance for T cell function. The increased sensitivity of the glycogen assay required low cell numbers and was amenable to reduced volumes, in 384-well plates, further facilitating these studies. In addition to glycogen synthesis and degradation, other glucose metabolic pathways, including glycolysis, oxidative phosphorylation, and the pentose phosphate pathway, were analyzed. We used assays to measure intermediates and products in these pathways, including lactate, pyruvate and malate, as well as assays for measuring dehydrogenase enzyme activity. Dehydrogenases representing all three pathways exhibited increased activity upon T cell activation, indicating enhanced engagement of these pathways. These assays should provide a valuable method for studying the impact of limited glucose on T cell activation and suggesting approaches to address it. To better understand the role of glucose availability and implications of nutrient competition in the tumor microenvironment for anti-tumor immune responses, we used sensitive bioluminescent assays to investigate metabolic profiles of cancer and immune cells. The assays can facilitate identification of potential approaches and targets to eliminate limited access to glucose and other nutrients as a barrier to efficient immunotherapies. Donna Marie Leippe, Natasha Karassina, Mike Valley, Kayla Sylvester, Anthony Lauer, Jolanta Vidugiriene. Investigating glucose utilization in cancer and immune cells using bioluminescent metabolite and enzyme assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4178.

The regulation of reactive oxygen species (ROS) such as superoxide (SO) and nitric oxide (NO) is crucial in biology, influencing metabolism and signaling pathways. Imbalances in these species lead to oxidative stress and various diseases. Traditional methods for measuring SO and NO face challenges in terms of sensitivity and specificity, particularly in complex biological matrices. This report introduces bioluminescent probes that leverage the intrinsic sensitivity of bioluminescence for direct and selective detection of SO and NO. These probes release analogs of d-luciferin upon reaction with their target ROS. Following addition of luciferase, luminescence is generated proportional to the amount of accumulated luciferin, allowing for quantitation of SO or NO. Both probes exhibit high specificity, confirmed through cell-free assays and cell-based studies in macrophages, demonstrating their utility in measuring cellular SO and NO production. These assays offer a robust, high-throughput platform for studying ROS, providing direct insights into oxidative stress-related mechanisms.

Obesity and environmental toxins are risk factors for breast cancer; however, there is limited knowledge on how these risk factors interact to promote breast cancer. Acrylamide, a probable carcinogen and obesogen, is a by-product in foods prevalent in the obesity-inducing Western diet. Acrylamide is metabolized by cytochrome P450 2E1 (CYP2E1) to the genotoxic epoxide, glycidamide, and is associated with an increased risk for breast cancer. To investigate how acrylamide and obesity interact to increase breast cancer risk, female mice were fed a low-fat (LFD) or high-fat diet (HFD) and control water or water supplemented with acrylamide at levels similar to the average daily exposure in humans. While HFD significantly enhanced weight gain in mice, the addition of acrylamide did not significantly alter body weights compared to respective controls. Mammary epithelial cells from obese, acrylamide-treated mice had increased DNA strand breaks and oxidative DNA damage compared to all other groups. In vitro, glycidamide-treated COMMA-D cells showed significantly increased DNA strand breaks, while acrylamide-treated cells demonstrated significantly higher levels of intracellular reactive oxygen species. The knockdown of CYP2E1 rescued the acrylamide-induced oxidative stress. These studies suggest that long-term acrylamide exposure through foods common in the Western diet may enhance DNA damage and the CYP2E1-induced generation of oxidative stress in mammary epithelial cells, potentially enhancing obesity-induced breast cancer risk.

Abstract The efficacy of T cell-mediated immunotherapy is improved by the proliferation of T cells with a stem cell memory phenotype (Tscm). These cells are capable of long-term persistence, self-renewal, multipotency, and robust engagement with cancer cells. This study focuses on refining T cell activation protocols to bolster the presence of Tscm within the therapeutic arsenal against cancer. Through a series of cell health and metabolic assays, we traced the activation-induced metabolic reprogramming that results from Tscm enrichment. Ex vivo activation was characterized by a rapid 2.5-3 fold increase in ATP production and a 2 fold shift in intracellular reducing potential, paralleling the metabolic demands of Tscm in the cancer microenvironment. Progression of activation intensified the metabolic flux through catabolic and anabolic pathways, with significant fluctuations in metabolites such as glucose, lactate, glutamine, glutamate, branched chain amino acids, and pyruvate. These metabolic alterations were accompanied by a 2 fold increase in the prevalence of Tscm cells, and demonstrated a dependency on media composition. Our findings reveal that metabolic profiling during T cell activation provides early and actionable insights into the enrichment of Tscm, and may serve as a strategic guideline for the enhancement of cancer immunotherapies. Citation Format: Anthony Lauer, Natasha Karassina, Kayla Sylvester, Jolanta Vidugiriene. Metabolic screening of T cells guides the enrichment of stem cell memory phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 17.

Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact T-cell phenotypes. CRISPR/Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant ( TRAC ) gene resulting in TRAC -CAR T cells with an enriched stem cell memory T-cell population, a process that could be further optimized through modifications to the media composition. In this study we generated anti-GD2 TRAC -CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine low media and then expanded in glucose/glutamine high media. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro and potency against human GD2+ xenograft neuroblastoma models in vivo . Compared to standard TRAC -CAR T cells, MP TRAC -CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity and reduced IFN-γ, IL-2, IP-10, IL-1β, IL-17, and TGFβ production at the end of manufacturing ex vivo , with increased central memory CAR T cells and better persistence observed in vivo . Metabolic priming with media during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo , which could lead to better responses against solid tumors in vivo .

Abstract Dehydrogenases play a pivotal role in cellular metabolism, acting as gatekeepers in key metabolic pathways. Their activity reflects the metabolic state of the cell, which is particularly relevant in the context of cancer and the immune response, where metabolic reprogramming is a hallmark of tumor progression and differentiation stages. Here, we present the development and application of novel bioluminescent dehydrogenase assays for direct measurement of metabolic activity in cell lysates. Our assays are highly sensitive, requiring as few as 100-500 cells, and have been successfully miniaturized to a 384-well plate format conducive to high-throughput screening. This technological advance allows for the precise monitoring of dehydrogenase activity across the pentose phosphate pathway, glycolysis, and the mitochondrial tricarboxylic acid (TCA) cycle. We demonstrate how shifts in dehydrogenase activity can serve as indicators of the metabolic status of cells, and their modulation during differentiation. Moreover, our bioluminescent assays provide a rapid screening platform for pathway-specific metabolic inhibitors, with potential applications in identifying new therapeutic targets within cancer metabolism. Our data reveal a dynamic landscape of metabolic activity during T cell maturation, underscoring the potential of dehydrogenase activity profiling in cancer immunobiology and the development of targeted cancer therapies. Citation Format: Kim Haupt, Natasha Karassina, Anthony Lauer, Kayla Sylvester, Jolanta Vidugiriene. Bioluminescent dehydrogenase assays: A novel approach for metabolic profiling and inhibitor screening in cancer biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 441.

Abstract The generation of T cells with a stem cell memory phenotype (T_SCM) holds significant promise for immunotherapy due to their longevity and potent antitumor activity. This study aimed to optimize T cell activation and expansion conditions to selectively enrich for the T_SCM population. Utilizing a suite of cell health and metabolism assays, we monitored T cell cultures to identify predictive markers of T_SCM enrichment. We observed that early activation steps correlated with a surge in ATP production and alterations in cell reducing potential, indicative of metabolic reprogramming. Subsequent activation phases were marked by an upregulation in both catabolic and anabolic pathways, as evidenced by the increased turnover of key metabolites including glucose, lactate, glutamine, glutamate, branched-chain amino acids (BCAA), and pyruvate. Notably, the rate of T cell activation influenced the composition of the resultant T cell populations, with the media composition further affecting these dynamics. Our findings suggest that metabolic assays, particularly those measuring changes in ATP levels and primary metabolite flux, can serve as effective early predictors of T_SCM enrichment. These insights may guide the development of more efficacious T cell-based immunotherapies by facilitating the targeted generation of T cells with a stem cell-like memory phenotype.

Glycogen is a large polymer of glucose that functions as an important means of storing energy and maintaining glucose homeostasis. Glycogen synthesis and degradation pathways are highly regulated and their dysregulation can contribute to disease. Glycogen storage diseases are a set of disorders that arise from improper glycogen metabolism. Glycogen storage disease II, known as Pompe disease, is caused by a genetic mutation that leads to increased glycogen storage in cells and tissues, resulting in progressive muscle atrophy and respiratory decline for patients. One approach for treating Pompe disease is to reduce glycogen levels by interfering with the glycogen synthesis pathway through glycogen synthase inhibitors. To facilitate the study of glycogen synthase inhibitors in biological samples, such as cultured cells, a high-throughput approach for measuring cellular glycogen was developed. A bioluminescent glycogen detection assay was automated and used to measure the glycogen content in cells grown in 384-well plates. The assay successfully quantified reduced glycogen stores in cells treated with a series of glycogen synthase 1 inhibitors, validating the utility of the assay for drug screening efforts and demonstrating its value for therapy development and glycogen metabolism research.

Chimeric antigen receptor (CAR) T cell therapy for solid tumors remains challenging due to the complex manufacturing process and the immunosuppressive tumor microenvironment. The manufacturing condition directly impacts CAR T cell yield, phenotype, and metabolism, which correlate with in vivo potency and persistence. Optical metabolic imaging (OMI) is a non-invasive, label-free method to evaluate single cell metabolism based on autofluorescent metabolic coenzymes NAD(P)H and FAD. Using OMI, we identified the dominating impacts of media composition over the selection of antibody stimulation and/or cytokines on anti-GD2 CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrated that OMI parameters were indicative of cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. Notably, OMI accurately predicted oxidative metabolic phenotype of virus-free CRISPR-edited anti-GD2 CAR T cells that correlated to higher in vivo potency against neuroblastoma. Our data supports OMI’s potential as a robust, sensitive analytical tool that enables dynamic and optimal manufacturing conditions for increased CAR T cell yield and metabolic fitness.One sentence summary Autofluorescence imaging informs manufacturing conditions that enhance yield and metabolic fitness of CAR T cells for neuroblastoma.### Competing Interest StatementKS receives honoraria for advisory board membership for Andson Biotech and Notch Therapeutics. CMC receives honoraria for advisory board membership for Bayer, Elephas Bioscience, Nektar Therapeutics, Novartis, and WiCell Research Institute. MCS, DLP, KS, DC declare two patents pending based on this work. No other conflicts of interest are reported.

Abstract Energy uptake and utilization in eukaryotic cells is a dynamic process regulated by a series of interacting metabolic networks. Interrogation of this complex network relies on rapid, sensitive approaches that do not require extensive sample handling and are easily adaptable to 96- and 384-well plates. Previously, using the Ultra-Glo luciferase reaction we developed a panel of bioluminescent assays that can be used to monitor numerous aspects of cellular metabolism and mitochondrial function including ATP production, glucose and amino acid metabolism, and the TCA cycle. Here, we extend the use of the luciferase reaction and report on the development of novel bioluminescence probes for studying two important metabolic cellular responses: fatty acid β-oxidation (FAO) and production of reactive oxygen and nitrogen species. For studying FAO, we developed a cell permeable probe with caged-luciferin attached to a fatty acid chain. The probe enters the cells and following FAO cycling, free luciferin is released and detected using Luciferin Detection Reagent. We validated the approach using known FAO activators and inhibitors and used it to monitor the changes in FAO during T-cell activation. For measuring reactive oxygen and nitrogen species, highly selective bioluminescence probes were developed. Upon reaction of the probes with their corresponding ROS target, they form the stable D-luciferin reporter molecule, causing luciferin to accumulate. Upon treatment with luciferase in the detection step, the generated light allows for quantification of the superoxide or nitric oxide formed. Both probes are suitable for in vitro and cell-based detection of ROS in an “add and read” format, providing for a simple workflow amenable to high throughput experimentation. Citation Format: Kim Haupt, Matt Larsen, Hui Wang, Natasha Karassina, Mike Valley, Wenhui Zhou, Jolanta Vidugiriene. Novel bioluminescence approaches for measuring fatty acid β-oxidation and production of reactive oxygen and nitrogen species. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4778.