SignificanceIncreased collagen linearization and deposition during tumorigenesis can impede immune cell infiltration and lead to tumor metastasis. Although melanoma is well studied in immunotherapy research, studies that quantify collagen changes during melanoma progression and treatment are lacking.AimWe aim to image in vivo collagen in preclinical melanoma models during immunotherapy and quantify the collagen phenotype in treated and control mice.ApproachSecond-harmonic generation imaging of collagen was performed in mouse melanoma tumors in vivo over a treatment time course. Animals were treated with a curative radiation and immunotherapy combination. Collagen morphology was quantified over time at an image and single-fiber level using CurveAlign and CT-FIRE software.ResultsIn immunotherapy-treated mice, collagen was reorganized toward a healthy phenotype, including shorter, wider, curlier collagen fibers, with modestly higher collagen density. Temporally, collagen fiber straightness and length changed late in treatment (days 9 and 12), while width and density changed early (day 6) compared with control mice. Single-fiber collagen features calculated in CT-FIRE were the most sensitive to the changes among treatment groups compared with bulk collagen features.ConclusionsQuantitative second-harmonic generation imaging can provide insight into collagen dynamics in vivo during immunotherapy, with key implications in improving immunotherapy response in melanoma and other cancers.
Intravital multiphoton microscopy of the metabolic co-enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) provides label-free imaging of metabolic changes in vivo. Since the metabolism of tumor and immune cells is associated with cancer progression, we aim to study metabolic changes during a triple-combination immunotherapy regimen that cures murine melanoma tumors. Our results demonstrate that intravital OMI can capture tumor and T cell autofluorescence intensity and lifetime changes during immunotherapy. Overall, this technology enables analysis of single cell metabolic changes in vivo to provide insight for immunotherapy development.
Current methods to assess immune cell function use labels that are limiting for time-course studies of immune cell behavior in tumors. Here, we use multiphoton imaging of NAD(P)H and FAD, co-enzymes of metabolism, in T cells and macrophages within the tumor microenvironment. T cells alter their metabolism in response to tumor-like pH, glucose, and lactic acid levels, while macrophages alter their metabolism during tumor-stimulated migration. These results indicate that multiphoton autofluorescence imaging is a powerful label-free method to monitor immune cell metabolism within single cells in the tumor microenvironment.
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