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Int. J. Mol. Sci. 2018, 19, 1107 4 of 17 In the present study, we examined the photodynamic effect on these two HNSCC cell lines using methylene blue as photosensitizer and a diode laser at 660 nm at a fixed area dose of 95 J/cm2. We aimed to identify MB concentrations, MB exposure times and laser exposure times in order to achieve maximum tumor toxicity due to phototoxic effect at minimum tumor toxicity due to MB alone. 2. Results 2.1. Stem Cell Characteristics in the SCC-25 and Detroit 562 Cell Lines P75 neurotrophin receptor (p75NTR) and Nanog are described cancer stem cells markers in HNSCC. In epithelial cells, p75NTR characterized an immature keratinocyte subset, which was slow-cycling in vivo and presented a strong regenerative potential in vitro, demonstrating the main requirements for stem cells, and introducing the importance of this receptor for the maintenance of a stem cell pool through the induction of a quiescent state. In human adult oral mucosa lamina propria, p75NTR-positive stem cells are self-renewing cells that co-express Oct4 and partially express Sox2 and Nanog transcription factors [42]. In this regard, we have done immunohistochemical stainings in agarose and paraffin embedded SCC-25 and Detroit 562 cells using antibodies against CD-44, Nanog, and p75NTR. Using a combined immunohistochemical reaction for CD-44 (green) and Nanog (red), SCC-25 cells were frequently Nanog positive, but only scattered CD-44 positive (Figure 1A–C). Nearly all Detroit 562 cells were both Nanog and CD-44 positive (Figure 1E–G). Practically, all Detroit 562 cells were p75NTR positive (Figure 1H), while SCC-25 cells were only relatively scattered p75NTR positive (Figure 1D). Figure 1. Detection of cancer stem cell markers in SCC-25 and Detroit 562 cells. SCC-25 (A–D) and Detroit 562 (E–H) cells have been cultured to confluency, collected in pellets, embedded in agarose and in paraffin, sectioned and were subjected to combined indirect immunohistochemical reactions for Nanog (detected in red) and CD-44 (detected in green) (A–G) and to single immunohistochemical reaction for p75NTR (detected in green; D,H). Detroit 562 cells showed more frequent reactions for all cancer stem cell markers. Scale bars: 50 μm. 2.2. MB Effects on Cell Viability with and without Photoactivation A low level 600 nm diode laser with a constant area dose of 95 J/cm2 with up to 15 min exposure caused a nonsignificant loss of cells in HNSCC tumor cell culture, which is comparable with the viability of routine cell culture (Figure 2) [43].PDF Image | Photodynamic Low Level Laser Squamous Cell Carcinoma
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