Developing non-invasive and visual method for monitoring tumor cells in vivo is important for studies on the genesis, early diagnosis and treatment of tumors. Compared to conventional organic fluorescent dyes or fluorescent proteins, QDs have the excellent luminescent features of stability and high yield of fluorescent quantum. QDs technology has displayed its promising prospects in long-term and non-invasive monitoring and tracking tumor cells in vivo [7]. Currently, the approaches that are used for QDs labeling of live cells include liposome-mediated transduction, non-specific internalization, micro-injection, electroporation and monoclonal or peptide-mediated transduction [14–20]. Previous studies have demonstrated that liposome-mediated QDs internalization displayed vesicle-like compartmental aggregation within the cells, which makes it difficult to control the concentration of QDs [14]. Non-specific internalization is only suitable for the labeling of strong phagocytic cells [2]. Micro-injection is also suitable for the labeling of small amount of cells [15,16]. Electroporation causes damages on the cells, leading to the death of some cells [14,17]. Recent studies demonstrate that cell-penetrating peptide conjugated QDs are suitable for rapid and efficient labeling of various types of cells. More importantly, these conjugated QDs do not have damaging effect on the cells [18–20]. Lin et al. utilized near-infrared fluorescent quantum dots (Qtracker^TM QD800) to label mouse embryonic cells and demonstrated that QD800 not only had excellent in vivo imaging ability, but also did not affect the viability and differentiation of embryonic stem cells. These results provided fundamental bases for further studies on the imaging and tracking of stem cells by quantum dot labeling. Although studies have been reported with regard to the QD800 imaging of tumor cells to investigate the invasion and metastasis in vivo [12,13], the effect of QD800 on the invasion and metastasis has not been reported. We showed here that co-culture of QD800 with Tca8113 cells resulted in the rapid entrance of QD800 into the cells and the labeling rate of Tca8113 cells was as high as 93.18% after 6 h of co-culture. As the co-culture time increased, the cells continued to divide and the labeling rate was gradually decreased. Entrance of peptide conjugated QDs did not affect the proliferation, invasion, adherence and chemotaxis of Tca8113 cells. These results provided basis for the application of QDs in the non-invasive, in situ and visual studies of genesis, development and metastasis of tumor cells.

The human tongue squamous cell carcinoma cell line Tca8113 was provided by West China College of Stomatology, Sichuan University (Sichuan, China). Qtracker^TM 800 Cell Labeling Kit (Invitrogen, Carlsbad, CA, USA) uses a custom peptide to deliver QDs into the cytoplasm of live cells without cell-type specificity. Laser scanning confocal microscope was purchased from Leica (Tcs-sp5, Germany). Flow cytometry (BD Biosciences, San Jose, CA, USA) was used for detection of cell labeling. Cold low-speed centrifuge (Z233MK-2) was purchased from HERMILE (Germany). Transwell chamber (Corning Incorporated, Corning, NY, USA) was used for the chemotaxis experiment.

Labeling Tca8113 cells with QD800 was performed according to the manufacturer’s instructions. Briefly, trypsin-digested Tca8113 cells were transferred into 5 EP tube with 1 × 10⁶ cells in each tube and then 0.2 mL of QD800 labeling solution (10 nM) was added. After mixing, the cells were co-cultured with QD800 for 1 h and centrifuged (1,000 x g) for 5 min at 4 °C. The supernatant medium was discarded and the cell pellets were washed with PBS for twice to remove the uninternalized QDs. The QD800-labeled Tca8113 cells (Tca8113/QD800) in the 5 EP tubes were individually seeded in a 6-well plate for continuous culture. At 6 h, 1 d, 3 d, 5 d and 7 d post-labeling, the labeled cells were dispersed into suspension and the labeling efficiency was detected at these five time points. Laser-scanning confocal microscope was used to observe the distribution of QD within the cells after Tca8113 cells were labeled for 1 d. All the experiments were repeated for 3 times and the average of the labeling rate was calculated accordingly.

Well-growing Tca8113 cells (2 × 10⁴ cells/mL) were inoculated into four 24-well plates with 0.18 mL in each well. After 24 h of culture, synchronous growth was achieved and these four plates were divided into four groups. To the control group was added with 0.02 mL of RPMI1640 medium containing 15% FBS. Experimental groups A, B and C were added with different amount of QD800 to achieve a final concentration of 5 nM, 10nM and 15 nM, respectively. Three wells of cells were randomly selected from each group every day and were completely digested with 0.1% of trypsin. Single cell suspension was obtained by pipetting and cell number was counted by using a hemocytometer for a consecutive eight days. All the experiments were repeated for three times and the average value was reported.

Cell suspension (1 × 10⁶ cells/mL, 0.2 mL) was co-cultured with QD800 (10 nM) at 37 °C with 5% of CO₂ for 1h, then centrifuged at 1,000 r/min for 5 min at 4° C to wash off the QDs that did not enter the cells for twice. The obtained Tca8113/QD800 cells were used for the following experiments immediately. Suspensions of Tca8113/QD800 or Tca8113 were seeded into two 60 mm Petri dishes with 2 × 10² cells in each dish. The cells were dispersed evenly by slightly shaking the Petri dishes and then were incubated at 37 °C with 5% of CO₂ for 21 d until the visible clones appeared. The medium was discarded and the cells were carefully washed with PBS for twice. After fixed with methanol for 15 min, the cells were stained with Giemsa's solution for 15 min before washing with tapping water and air-drying. The clonies with more than 50 cells were counted with an inverted microscope and the clone formation rate was calculated with the following formula: Plate clone formation efficiency = (number of clonies/number of cells inoculated) × 100%. All the experiments were repeated for 3 times and the average values were reported.

Cell suspension (5 × 10⁶ cells/mL) of Tca8113/QD800 were prepared according to the procedure described above. The obtained Tca8113/QD800 cells were used for the following experiments immediately. Cell suspension (5 ×10⁶ cells/mL) was prepared for both Tca8113/QD800 and Tca8113 and was seeded in 24-well plates with 0.2 mL in each well and 12 wells for each experimental group. After 30 min, 60 min, 90 min and 120 min of incubation, three wells of cells in each group were washed with PBS to remove non-adherent cells and the adherent cells were digested with 0.25% of trypsin for cell counting using a microscope. The cell adherent rate was calculated using the following formula: cell adherent rate = (the number of adherent cells/total number of cells) × 100%. All the experiments were repeated for three times and the average values were reported.

Tca8113/QD800 cells that were prepared according to the procedure described above were used for the following experiments immediately. Cell invasion and reconstruction of basement membrane experiment was performed in Transwell that was separated into upper and lower chambers by a poly carbonate acid filter membrane containing multiple 8 μm-diameter pores. Fifteen micrograms of matrigel were poured in the upper and lower sides of the filter membrane for 1 h of reconstruction at 37 °C before uses. Tca8113/QD800 or Tca8113 cells (2 × 10⁵) were added into the upper chamber and incubated at 37 °C for 12 h. The cells that invaded into the lower side of the membrane were stained with HE solution and five fields of views (400 × magnitude) were randomly selected and the cell number was counted. All the experiments were repeated for three times and the average number was reported to indicate the invasive ability of Tca8113 or Tca8113/QD800 cells.

In cell chemotaxis experiment, the matrigel was not applied on the surface of the filter membrane. All the other procedures were identical to the cell invasion assay. All the experiments were repeated for 3 times and the average number of cells in the lower side of the filter membrane was reported to indicate the chemotactic ability of Tca8113 or Tca8113/QD800 cells.