Chemically conjugated cyanine dyes have proved to be useful for measuring blood flow and cardiac output, as well as imaging tumors (2
). The chemical structures of water-soluble pentamethine and heptamethine cyanine dyes have recently been modified to increase their chemical stability, photostability, and quantum yield (1
). IR-783 and MHI-148 are two such new dyes, modified with a rigid cyclohexenyl substitution in the polymethine linker. The present study describes for the first time that these NIR dyes can be actively taken up and accumulated by cancer cells but not by normal cells. The salient features of these newly discovered dual imaging and targeting NIR dyes are: (1
) Detecting cancer cells and cancer metastases directly without the requirement of chemical conjugation. (2
) Detecting many other tumor types and tumor cell populations under cell culture and in vivo
conditions. The cancer-specific uptake and retention of these dyes is likely to be mediated by OATPs since the transport of these dyes into cancer cells can be antagonized by BSP, an OATP competitive inhibitor (38
) Serving as potential carriers for drug payloads or radioactive agents to increase the specificity and reduce the toxicity of therapeutic agents by preferential uptake and accumulation in cancer cells but not in normal cells.
The cyanine dyes are water soluble, so they have rapid clearance and are unlikely to be trapped in the reticular endothelium of the liver, lung or spleen. They were found to be superior for cancer detection to other cyanine dyes such as indocyanine green and non-cyanine dyes such as rhodamine 123 (data not shown). Imaging with NIR dyes can yield much higher signal/noise ratios with minimal interfering background fluorescence. The fluorescence efficiency of cyanine dyes can increase by ~1,000-fold upon binding to proteins and nucleic acids (36
). The stable binding together with the shift toward increased fluorescence could be highly beneficial, accounting for the “trapping” of the NIR signals in cancer cells for prolonged periods (>5 days) and allowing tumor detection in live animals with high signal/noise ratios. The stability of these cyanine dyes after formalin fixation raises the possibility of developing new and sensitive means of detecting cancer cells in whole blood and in harvested surgical specimens by injecting the cyanine dyes prior to sampling at the time of surgery. In practice, these could help physicians and pathologists follow up patients with possible circulating cancer cells in blood and assess surgical margins at the time of surgery. Our study suggests the differential dye uptake and retention by cancer and normal cells and tissues can be demonstrated robustly by the use of a variety of detecting devices including Zeiss LSM 510 META, Kodak 4000MM, and Olympus OV100 systems. We adopted these different detecting methodologies based on their sensitivity and capability of allowing merging of images obtained via different detection modalities (e.g. X-ray and NIR imagings). The wide range of detecting devises used in our study supports the conclusion that IR-783 is preferentially taken up and retained by cancer but not normal cells.
The mechanisms by which these cyanine dyes cross the cytoplasmic membranes of cancer cells but not normal cells were investigated. We concluded that the uptake was mediated by proteins of the OATP family, because the active uptake could be effectively blocked by BSP. OATPs are well-recognized as channels for the transport of a diverse group of substrates including bile acids, hormones, xenobiotics and their metabolites (40
). Results from this study are consistent with published reports which indicate differences in the type and levels of OATPs between cancer and normal cells(43
). Moreover certain members of OATPs have recently been shown to be overexpressed in various human cancer tissues as well as in cancer cell lines (47
) and the confirmation of OATPs as the key mediator of heptamethine cyanine dye uptake and retention in tumor cells warrants further investigation.
The ability of mouse tumors to accumulate these cyanine dyes is of great significance. This will facilitate the use of these dyes in immune-intact syngenic and transgenic mouse models to study the fundamentals of cancer biology, metastasis and therapy. Since these dyes can be further explored as generalized ligands for all malignant cells, the synthesis of dye-antineoplastic drug conjugates, dye-radiolabeled drug conjugates and dye-toxin conjugates could immensely facilitate the development of new therapeutics to treat cancer and pre-cancerous conditions.
In summary, two heptamethine cyanine dyes were demonstrated to selectively target cancer but not normal cells, irrespective of their species and organ of origin. This class of dual imaging and targeting cyanine dyes holds great promise for novel therapeutics for future cancer therapy and imaging. Future application of NIR fluorescent dyes in the clinic could lead to important progress in the management of cancer patients on an individual basis.
Cancer mortality can be reduced by the development of non-invasive and effective imaging technologies that can detect tumors at metastatic sites and cancer cells in biologic fluids. We report our discovery of two heptamethine cyanine dyes with near-infrared fluorescence emission profiles that can detect the presence of human tumors grown in mice, spontaneous prostate and intestinal tumors in transgenic animals, and tumor cells in human blood, without the necessity of chemical conjugation. These unique heptamethine cyanine dyes can be further exploited for the detection of tumor cells in histopathologic specimens, circulating tumor cells in blood, and differentiating surgical margins in clinical specimens for improved diagnosis, prognosis and treatment of cancer patients.