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Noninvasive imaging of cancer metastasis through the efficient cell labeling constitutes a major technological breakthrough for cancer research and patient monitoring post-surgery. In the current work, we expanded our cell surface labeling technique on the whole-body fluorescence imaging of tumor metastasis in BALB/c nude mice. Four kinds of human cancer cells (two cancer cell lines, MKN45 and HCT116, and their transfected versions expressing surface glycan-related genes, MKN45-GnT-V and HCT116-GMDS) were labeled by azaelectrocyclization with Hilyte Fluor 750 for 10 min and without affecting cell viability. Fluorescence-labeled cancer cells were injected into the abdominal cavities of BALB/c mice and whole-body scans were performed with an eXplore Optix device. In accordance with previous findings, the fluorescence imaging clearly showed that tumor metastasis was dependent upon the cell surface glycans: A larger polylactosamine structure or the loss of fucosylation on the cancer cell surfaces, respectively, enhanced the metastatic potential of the tumor cells. Our noninvasive technique provides the landmark opportunity for sensitively monitoring the dynamics of the cancer cells depending on their surface structures and/or the host environments, thus impacts on the cancer prognosis and the therapeutic applications.

We determined photodynamic therapy (PDT) efficacy using a flash wave (FW) and a continuous wave (CW) light, of which the fluence rate was 70 μW/cm2, for murine thymic lymphoma cells (EL-4) cultivated in vitro. The irradiation frequency and the pulse width of the FW light were in the range of 1–32 Hz and less than one millisecond, respectively. 5-Aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) was used as a photosensitizer. When EL-4 with ALA administration was irradiated by the light for 4 h (irradiation fluence: 1.0J/cm2), the survival rate of EL-4 by the FW light was lower than that by the CW light, except for the FW light with irradiation frequency of 32 Hz, and decreased gradually with decreasing irradiation frequency. Moreover, the FW light, especially at lower irradiation frequency, was superior to the CW light for the generation of singlet oxygen in an aqueous PpIX solution. Therefore, thehigher PDT efficacy for EL-4 of the FW light would be caused by the greater generation of singlet oxygen in the cells.

Versatile method for living cell labeling has been established. Cell surfaces are initially biotinylated by azaelectrocyclization, and then treated with the fluorescence-labeled avidin or the anti-biotin antibody.

To determine free iron in human serum, we first determined total concentrations of iron (It μmol/liter) and protein (Pt μg/ml) in the serum precisely with bathophenanthroline and Coomassie Brilliant Blue G-250 (Bio-Rad Protein Assay) by colorimetry. Then, 1.0ml of the serum was centrifuged on a membrane cone filter (Centriflo CF-25) at 1, 050×g for 60min at 4°C, and the serum protein fraction on the membrane cone filter was dissolved in saline and centrifuged again in the same way. The washed serum protein fraction was dissolved in saline, and an aliquot of the solution was subjected to analyses of iron (Iw μmol/liter) and protein (Pw μg/ml). To correct for the free iron adsorbed on the membrane cone filter during the centrifugation and to obtain the free iron concentration (If μmol/liter) in the serum, we used the following equation:If(μmol/liter)=It-(Iw×Pt/Pw).The average values of total and free iron in 16 different samples of human fresh sera were determined to be 21.5±6.1μmol/liter and 1.24±0.89 μmol/liter, respectively, by the present standard method; and the values were compared with those of the same sera frozen for 1 week. Free iron in human serum was found to exist as Fe2+.