Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment.
The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF) self-quenched macromolecular probe (PGC-Cy5.5) has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation) and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 microm) showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total) and GFP-negative (19% total) populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFP-negative cells (2.5-3.0%) had seven times higher NIRF intensity than the majority of GFP-positive or GFP-negative cells (372 and 55 AU, respectively). Highly NIRF-positive, FP-negative cells were CD45- and MAC3-positive. Our results indicate that: 1) intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2) tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.
Bogdanov
Alexei A
AA
Center for Molecular Imaging Research, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA.
Lin
Charles P
CP
Simonova
Maria
M
Matuszewski
Lars
L
Weissleder
Ralph
R
D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis; D002232:Carbocyanines / Q000494:pharmacology; D002403:Cathepsins / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008853:Microscopy; D018613:Microscopy, Confocal; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000378:metabolism*; D017785:Photons; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D000943:Antigens, Differentiation; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011506:Proteins; D012333:RNA, Messenger; C036763:monocyte-macrophage differentiation antigen; D049452:Green Fluorescent Proteins; D017493:Leukocyte Common Antigens; D002403:Cathepsins