Fluorescence lifetime imaging microscopy (FLIM) of metabolic coenzymes NAD(P)H and FAD monitors cancer treatment response and heterogeneity in in vitro and in vivo models. FLIM offers the potential to inform cancer patient treatment in a clinical setting, but requires further preclinical validation as an accurate predictor of patient response. Tissue banks offer readily available tumor samples with follow-up patient data that could be used for FLIM validation, but the effects of fixation and processing on autofluorescence have not been well characterized. This study aims to determine if trends seen with treatment in live tumor samples are conserved in formalin-fixed, paraffin-embedded tumors. Previously, fluorescence lifetime images of FaDu xenografts were acquired in vivo in mice before fixation and embedding. Here, corresponding lifetime images of the fixed FaDu tumors were acquired and compared with the in vivo data. The results demonstrate that while NAD(P)H lifetime values are generally conserved between in vivo and fixed tumors, FAD lifetime values are not. Additionally, the cancer response trends seen in vivo are positively correlated for most FLIM components in fixed tumors, but they vary in magnitude. Further investigation is required to determine the cause of the discrepancies.
Engineered tissues offer great promise as engrafted therapies and in vitro models, but these tissues require a vascular network to retain viability at large scales. Significant efforts are focused on optimizing these in vitro vascular constructs, yet current evaluation methods require fixation and immunostaining. These destructive evaluation methods alter vascular network morphology, and cannot non-invasively monitor vascular assembly over time. Here, we demonstrate that autofluorescence multiphoton microscopy (MPM) can quantitatively assess the morphology of living 3D vascular networks without fixation, labels, or dyes. Autofluorescence MPM was used to non-invasively monitor the effect of culture conditions on 3D vascular network formation. Human embryonic stem (ES) cell-derived endothelial cells and primary human pericytes cultured in polyethylene glycol (PEG) hydrogels self-assembled into 3D vascular networks. Autofluorescence MPM of the metabolic co-enzyme NAD(P)H (excitation/emission wavelengths of 750 nm/400-460 nm) was used to quantify morphological parameters at day 6 of culture. Specifically, vessel diameter, vascular density, branch point density, and integration of endothelial cells into the network were quantified. Dynamic culture conditions (flow at 1μL/sec) led to vascular networks with higher mean vessel diameter compared to static culture (p<0.05). Standard immunohistochemistry found that vascular networks were positive for markers of endothelial cells, pericytes, and tight junctions. Scanning electron micrographs confirmed vessel lumen formation with pericytes wrapped around vessels. Dye transit of FITC-dextran through the network confirmed leaky endothelial barrier function. Our results demonstrate that autofluorescence MPM can non-invasively evaluate in vitro 3D vascular networks, and could be used for quality control of engineered tissues.
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