{
  "abstract": "Background While immune checkpoint inhibitors have shown success in a subset of melanoma patients, 1 their efficacy requires T cell infiltration and migration.2 Targeting T cell metabolism can enhance tumor infiltration.3 However, current studies on T cell migration and metabolism rely on bulk measurements, obscuring single-cell heterogeneity and real-time metabolic changes.3 Therefore, there is a need to understand T cell metabolic changes during migration at the single-cell level. Two-photon autofluorescence lifetime imaging, or optical metabolic imaging (OMI), of the metabolic co-enzyme NAD(P)H can track real-time metabolic changes in T cells during migration. The free and protein-bound conformations of NAD(P)H produce different autofluorescence lifetimes, which provide non-invasive and label-free readouts of metabolic changes in single cells. The adult zebrafish scale provides an optically accessible model of T cell migration including confinement during migration through tissue,4 and zebrafish models of melanoma provide insight into the tumor microenvironment.5 Here, we test the feasibility of monitoring single-cell metabolism and migration in zebrafish using OMI with two metabolic inhibitors: sodium cyanide (NaCN), which inhibits mitochondrial Complex IV, increasing glycolysis, and 2-Deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis.Methods Adult Ick: GFP zebrafish were anesthetized and then scales were plucked and placed in a dish containing PBS. For the NaCN experiment, NaCN was added after acquiring the first image prior to treatment. For the 2-DG experiment, scales in the treatment group were placed in a dish containing 2-DG for 90 minutes prior to imaging. NAD(P)H lifetimes were collected through a 40X objective using time-correlated single photon counting electronics. T cell migration was analyzed with TrackMate.Results We demonstrated that OMI can monitor single-cell metabolism changes during T cell migration in a zebrafish scale model. The proportion of free NAD(P)H (α1) in zebrafish scale T cells increased with NaCN treatment ( figure 1A) and decreased with 2-DG treatment (figure 1B), as expected, suggesting that our model is sensitive to metabolic perturbations. 2-DG treatment also affects T cell morphology, possibly related to the changes in T cell migratory pattern under planar confinement in the zebrafish epidermis (figure 1C-D). Furthermore, migratory parameters will be correlated to metabolic parameters to elucidate the relationship between metabolism and migration on a single-cell level (figure 2).Conclusions OMI of the zebrafish scale model can track real-time metabolism non-invasively on the single cell level. Future work in zebrafish models of melanoma may be used to develop immunotherapies that target distinct subgroups of T cells.Acknowledgements This work is supported by the NIH (R01 CA278051 and R35 GM118027).References Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science 2018;359(6382):1350-5.Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, Sher X, Liu XQ, Lu H, Nebozhyn M, Zhang C. Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science 2018;362(6411):eaar3593.Simula L, Fumagalli M, Vimeux L, Rajnpreht I, Icard P, Birsen G, An D, Pendino F, Rouault A, Bercovici N, Damotte D. Mitochondrial metabolism sustains CD8+ T cell migration for an efficient infiltration into solid tumors. Nature Communications 2024;15(1):2203.Robertson TF, Schrope J, Zwick Z, Rindy J, Horn A, Hou Y, Huttenlocher A. Live imaging in zebrafish reveals tissue-specific strategies for amoeboid migration. Development 2025;152(8):dev204351.Frantz WT, Ceol CJ. From tank to treatment: modeling melanoma in zebrafish. Cells 2020;9(5):1289.Abstract 759 Figure 1OMI captures metabolic and morphological changes in zebrafish scale T cells in response to metabolic perturbations from NaCN and 2-DG. (A) T cell NAD(P)H α1 in NaCN was recorded for 5 minutes. (B) T cell NAD(P)H α1 and (C) circularity in PBS and 2-DG were compared. (D) T cell circularity was recorded for 12 minutes and statistical differences are relative to the 1-min time pointAbstract 759 Figure 2Proof-of-concept schematic illustrating OMI and single cell tracking to relate migration and metabolism at a single-cell level. (A) T cell mean velocity was recorded for 10 min in PBS. (B) NAD(P)H α1 of the same T cells in (A) were tracked for 10 min. (C) Correlation of mean velocity and NAD(P)H α1 in the same T cells in (A-B) where each point is one T cell at one time point",
  "authors": [
    {
      "affiliations": [
        "University of Wisconsin-Madison, Madison, WI, USA",
        "Morgridge Institute for Research, Madison, WI, USA"
      ],
      "name": "Angela Hsu"
    },
    {
      "affiliations": [
        "Morgridge Institute for Research, Madison, WI, USA"
      ],
      "name": "Kayvan Samimi"
    },
    {
      "affiliations": [
        "University of Wisconsin-Madison, Madison, WI, USA"
      ],
      "name": "Tanner Robertson"
    },
    {
      "affiliations": [
        "University of Wisconsin-Madison, Madison, WI, USA"
      ],
      "name": "Anna Huttenlocher"
    },
    {
      "affiliations": [
        "Morgridge Institute for Research, Madison, WI, USA"
      ],
      "name": "Melissa C Skala"
    }
  ],
  "title": "759 Label-free imaging reveals in situ metabolic changes in zebrafish scale T cells during migration",
  "uid": "037f887a-eab6-5bd5-9b6f-ce6d4a1112fd"
}