Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo

Abstract

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrowderived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.Intravital microscopy has provided unprecedented molecular, cellular, anatomical and functional insight into tumor biology and response to treatment 1 . This technique captures fluorescence from molecules that are injected into a host or expressed by cells 2,3 . Additionally, intrinsic signals such as second harmonic generation (SHG) emanating from collagen can be imaged using multiphoton microscopy 4,5 . Traditional fluorophores are prone to photobleaching, compromising the ability to image the same region repeatedly, and have relatively narrow excitation and broad emission spectra. Also, several excitation wavelengths may be required to excite all fluorophores and intrinsic signals, and overlapping emissions may obscure the delineation between multiple probes. Quantum dots, colloidal semiconductor nanocrystals 6 , have the potential to overcome these limitations: they are photostable, tunable to a desired narrow emission spectrum, relatively insensitive to the wavelength of excitation Correspondence should be addressed to R.K.J. (E-mail: jain@steele.mgh.harvard.edu). Note: Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. light, and are especially bright fluorophores 7 . Recent studies exploit these optical properties for imaging of cells 8 or whole tumors 9 . The ability of quantum dots to show crucial information at the length scale between these two extremes has yet to be established 10 . Here, we present studies that highlight the synergy of quantum dots and multiphoton intravital microscopy for tumor pathophysiology studies: differentiating tumor vessels from both perivascular cells and matrix, assaying the ability of microparticles to access the tumor, and monitoring the trafficking of precursor cells. NIH Public Access RESULTS Customizing quantum dot emissionBecause quantum dot emissions are tunable by both size and chemical composition 6 , we prepared ...

In vivo quantum dot labeling of mammalian stem and progenitor cells

“…Quantum dots (QDs) are a recently developed class of highly stable, nanometer-sized inorganic fluorophores (Gao et al, 2004;Medintz et al, 2005;Michalet et al, 2005;Stroh et al, 2005). QDs have significant advantages compared with traditional organic fluorophores, including size-tunable emission, increased quantum efficiency, broader absorption spectra, narrower emission spectra (i.e., smaller full-width half-maximum), and remarkable resistance to photo bleaching (Bruchez et al, 1998;Chan and Nie, 1998;Han et al, 2001;Rieger et al, 2005).…”

Real-Time Intravital Imaging of RGD−Quantum Dot Binding to Luminal Endothelium in Mouse Tumor Neovasculature

“…Only recently have intravital techniques begun to emerge as a vital tool for studying the behavior of nanomaterials in vivo. [23][24][25][26] Still, no studies have explored nanoparticle binding in the tumor neovasculature. This critical step has regulatory and design implications for imaging and therapeutic nanoparticle targeting in tumor neovasculature.…”

“…In general, commercially avail able Angiosense remained in the vasculature at approximately constant levels for at least 2-3 h postinjection, while qdots cleared from the vasculature (via reticuloendothelial system, or RES, uptake 33 ) in a linear fashion within 1-1.5 h ( Figure S1 in Supporting Information). Many studies report extravasation of nanostructures (and larger particles 18,23,34 ) from tumor vessels in animal models, 1,2,4,5,23 but the molecular size cut-offs in such vessels have been reported to vary significantly between different xenograft models reported 18 and even between different regions of the same vessel of a given tumor. 23 Our results suggest that this cutoff size variability occurs even in the low nanoscale range.…”

“…Many studies report extravasation of nanostructures (and larger particles 18,23,34 ) from tumor vessels in animal models, 1,2,4,5,23 but the molecular size cut-offs in such vessels have been reported to vary significantly between different xenograft models reported 18 and even between different regions of the same vessel of a given tumor. 23 Our results suggest that this cutoff size variability occurs even in the low nanoscale range. We found that although qdots do not extravasate in the SKOV-3 ear or flank xenografts, they clearly and rapidly extravasate when exposed to the ears of mice inoculated with certain other tumor cell lines (e.g., LS174T).…”

Recent Advances in Nanobiotechnology and High-Throughput Molecular Techniques for Systems Biomedicine

“…QDs have been applied to in vitro live cell imaging, in vivo imaging in animals and medical diagnostics (Cheki et al, 2013;Michalet et al, 2005;Stroh et al, 2005).…”