A; currently, trained dermatologists perform visual- and epiluminescence-based macroscopical inspection, and suspect melanoma is then confirmed by histological examination. ESR-based in situ analysis of pigmented skin lesions has been suggested as a possible noninvasive diagnostic tool, according to results obtained on melanoma cell lines and melanoma tissues [29,39?1] and in animal models of melanoma [39,42]. As possible melanoma markers, stable melanin granule-connected [29] or cellular membrane-associated [43] or UV-induced short living melaninand oxygen-derived 11967625 ESR signals have been proposed [44,45].Melanoma Diagnosis via Electron Spin ResonanceFigure 3. ESR signal of nevi and melanomas groups and subgroups. Bars report the ESR mean value of each subgroup with SEM. A) ESR value recorded in nevi (white bars) and melanomas (grey bars) in the “Measuring Set” and “Validation Set”. B) Comparison of nevi (white bars) vs melanoma (grey bars) in each subgroup of the “All Set”; * indicates p#0.05 for “Female” and “Head and Neck”, in all other cases p,0.01; ns stands for “not significant”. doi:10.1371/journal.pone.0048849.gHowever, a statistical analysis of endogenous ESR signal on human melanomas and nevi samples collection has never been performed. In the present study different melanoma cell lines are reported to exhibit endogenous ESR signal at g = 2.00560.001 in the spectrum obtained under specific physical settings while other melanoma cell lines do not present the ESR signal. Such difference may be related to heterogenic features (e.g. primary or metastatic) and GR79236 manufacturer growth related properties (e.g. doubling time) of cultured melanoma cell lines, reflecting the clinical variability observed in melanoma patients [46]. Noteworthy in our experimental in vitro conditions the signal recorded in SKMEL-28 at the 5th GSK0660 biological activity culture passage was lost at the 10th culture passage. We hypothesize that this signal loss may be related to the cellsenescence and modification of oxidative stress, processes intrinsically linked to melanogenesis [47]. The physical parameters of the measured ESR signal strongly suggested its connection with melanin [17,20,48]. Eu- and pheomelanogenesis occur under low- and high- GSH levels conditions, respectively [49,50], and changes of GSH levels and increase of oxidative cellular stress are typically observed in melanoma [51]. The presence/absence of ESR signal in different cellular systems may thus depend on factors including senescence level, oxidative stress level, melanogenesis and eu/pheomelanin proportion. In human primary melanocytes (1.26106 cells) showing a dark cellular pellet, no ESR signal was recorded (Fig. 1B); on the contrary, melanoma cells SKMEL-28 and SKMEL-110 (1.06106 cells) show an intense ESR signal (Fig. 1A).Melanoma Diagnosis via Electron Spin ResonanceFigure 4. ESR signal within melanoma subgroups. A) Each subgroup was classified according to tumour thickness (High or Low Breslow’s depth). Bars report the ESR mean value of each subgroup with SEM; * indicates p#0.05. B) ANOVA analysis with Bonferroni Multiple Comparison Test, within the group containing nevi, melanomas “Low Breslow’s depth” (,1 mm) and melanomas “High Breslow’s depth” ( 1 mm); * indicates p,0.01 of melanomas “High Breslow” vs nevi and vs melanomas “Low Breslow”). C) ANOVA analysis with Bonferroni Multiple Comparison Test of the eu/ pheomelanin ratio (a/b), of nevi, melanomas “Low Breslow’s depth” (,1 mm) and melanomas “.A; currently, trained dermatologists perform visual- and epiluminescence-based macroscopical inspection, and suspect melanoma is then confirmed by histological examination. ESR-based in situ analysis of pigmented skin lesions has been suggested as a possible noninvasive diagnostic tool, according to results obtained on melanoma cell lines and melanoma tissues [29,39?1] and in animal models of melanoma [39,42]. As possible melanoma markers, stable melanin granule-connected [29] or cellular membrane-associated [43] or UV-induced short living melaninand oxygen-derived 11967625 ESR signals have been proposed [44,45].Melanoma Diagnosis via Electron Spin ResonanceFigure 3. ESR signal of nevi and melanomas groups and subgroups. Bars report the ESR mean value of each subgroup with SEM. A) ESR value recorded in nevi (white bars) and melanomas (grey bars) in the “Measuring Set” and “Validation Set”. B) Comparison of nevi (white bars) vs melanoma (grey bars) in each subgroup of the “All Set”; * indicates p#0.05 for “Female” and “Head and Neck”, in all other cases p,0.01; ns stands for “not significant”. doi:10.1371/journal.pone.0048849.gHowever, a statistical analysis of endogenous ESR signal on human melanomas and nevi samples collection has never been performed. In the present study different melanoma cell lines are reported to exhibit endogenous ESR signal at g = 2.00560.001 in the spectrum obtained under specific physical settings while other melanoma cell lines do not present the ESR signal. Such difference may be related to heterogenic features (e.g. primary or metastatic) and growth related properties (e.g. doubling time) of cultured melanoma cell lines, reflecting the clinical variability observed in melanoma patients [46]. Noteworthy in our experimental in vitro conditions the signal recorded in SKMEL-28 at the 5th culture passage was lost at the 10th culture passage. We hypothesize that this signal loss may be related to the cellsenescence and modification of oxidative stress, processes intrinsically linked to melanogenesis [47]. The physical parameters of the measured ESR signal strongly suggested its connection with melanin [17,20,48]. Eu- and pheomelanogenesis occur under low- and high- GSH levels conditions, respectively [49,50], and changes of GSH levels and increase of oxidative cellular stress are typically observed in melanoma [51]. The presence/absence of ESR signal in different cellular systems may thus depend on factors including senescence level, oxidative stress level, melanogenesis and eu/pheomelanin proportion. In human primary melanocytes (1.26106 cells) showing a dark cellular pellet, no ESR signal was recorded (Fig. 1B); on the contrary, melanoma cells SKMEL-28 and SKMEL-110 (1.06106 cells) show an intense ESR signal (Fig. 1A).Melanoma Diagnosis via Electron Spin ResonanceFigure 4. ESR signal within melanoma subgroups. A) Each subgroup was classified according to tumour thickness (High or Low Breslow’s depth). Bars report the ESR mean value of each subgroup with SEM; * indicates p#0.05. B) ANOVA analysis with Bonferroni Multiple Comparison Test, within the group containing nevi, melanomas “Low Breslow’s depth” (,1 mm) and melanomas “High Breslow’s depth” ( 1 mm); * indicates p,0.01 of melanomas “High Breslow” vs nevi and vs melanomas “Low Breslow”). C) ANOVA analysis with Bonferroni Multiple Comparison Test of the eu/ pheomelanin ratio (a/b), of nevi, melanomas “Low Breslow’s depth” (,1 mm) and melanomas “.
