Abstract
The complexity of the tumor microenvironment necessitates that cell behavior is studied in a broad, multi-scale context. Although tomographic and microscopy-based far and near infrared fluorescence (NIRF, >650 nm) imaging methods offer high resolution, sensitivity, and depth penetration, there has been a lack of optimized NIRF agents to label and track cells in their native environments at different scales. In this study we labeled mammalian leukocytes with VivoTag 680 (VT680), an amine reactive N-hydroxysuccinimide (NHS) ester of a (benz) indolium-derived far red fluorescent probe. We show that VT680 diffuses into leukocytes within minutes, covalently binds to cellular components, remains internalized for days in vitro and in vivo, and does not transfer fluorescence to adjacent cells. It is biocompatible, keeps cells fully functional, and fluoresces at high intensities. In a tumor model of cytotoxic T lymphocyte (CTL) immunotherapy, we track and quantify VT680-labeled cells longitudinally at the whole-body level with fluorescence-mediated molecular tomography (FMT), within tissues at single cell resolutions by multiphoton and confocal intravital microscopy, and ex vivo by flow cytometry. Thus, this approach is suitable to monitor cells at multiple resolutions in real time in their native environments by NIR-based fluorescence imaging.
Highlights
The growing appreciation that cellular and molecular mechanisms are controlled by the microenvironment in which they operate–in vitro models seldom recapitulate in vivo behavior–indicates a need to simultaneously monitor biological processes at various scales non-invasively
We evaluated whether VivoTag 680 (VT680), a near-infrared fluorochrome NHS ester, can be used as a multi-scopic cell tracker for in vivo optical imaging; we compared it to CFSE, a cell tracker currently available and in wide use at the microscopic level
Mean fluorescent intensity (MFI), as determined by flow cytometry (FCM) (Laser Ex: 635; Em filters: 685/LP, 695/40), showed that VT680 tagged cells efficiently; increasing doses corresponded to increasing MFI and reached 46104 with 300 mg/mL compared to,10 MFI for control unlabeled cells (Fig. 1A)
Summary
The growing appreciation that cellular and molecular mechanisms are controlled by the microenvironment in which they operate–in vitro models seldom recapitulate in vivo behavior–indicates a need to simultaneously monitor biological processes at various scales non-invasively. Optical imaging allows in vivo visualization of biology as it unfolds, with relatively little perturbation of the native environment, but multi-scopic imaging that integrates single-cell and whole-body information from the same animal is often not possible, partly because of inadequate reporter tags. While design of cell trackers in the far and near-infrared region of the light spectrum partly resolves these problems, cell trackers must be considered for multiphoton excitation, biocompatibility, and cellular retention if they are to report effectively on biology. Their optimization, remains a challenge for multi-scopic imaging
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