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Page 8 of 14 Liu et al. J Cancer Metastasis Treat 2019;5:4 I http://dx.doi.org/10.20517/2394-4722.2018.55

Figure 4. The relative concentration changes in biomarkers of tryptophan (left), carotenoids (middle) and lipids/lipoproteins (right) in the
normal skin tissue and BCC skin tissues with depth-dependence. Dramatic changes on relative concentrations are shown for the lesion
slices at the depth of 100 µm compared with other depths of 200 µm, 1,100 µm and 1,600 µm. Based on student’s t-test, the difference
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between normal and BCC samples is statistically significant (P < 0.05) for biomarkers: 752, 1,161 and 1,521 cm . The units of the numbers
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in the figure legends are cm . BCC: basal cell carcinoma

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Figure 5. The changes in the resonance Raman fingerprints represented by the ratios of peak intensities of biomarkers. The 1,589 cm
mode is supposed to be arising from tryptophan; 1,452 cm mode is assigned to fatty acids-lipid protein molecules; 1,662 cm mode is
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amide I combined with type I collagen; the largest ratio (I1589 to I1452) changes occurred at a depth of 100 µm in the BCC tissue (left).
Based on student’s t-test, the difference between normal and BCC samples is shown to be statistically significant (P < 0.05) for all
biomarkers. BCC: basal cell carcinoma
for about 60%-70% of the total of the five most concentrated carotenoids content in human organisms),
at 1,161 cm and 1,521 cm are thought to play a significant role in the normal dermis skin anti-oxidant
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defense system, as shown in Figure 2 (top) and Figure 3 (left). These two resonance bands are active because
carotenoids have a pre-resonance absorption band which falls in the pre-resonance range of the excitation
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wavelength of 532 nm [37,63-67] . The RR peaks of 1,161 cm and 1,521cm disappeared in the BCC sliced sample
at a depth of 100 µm as shown in Figure 2 (middle) and Figure 3 (left). This shows a clear progression of
the BCC tissue mutation process in comparison with normal skin tissue, which suggests it may be a lesion
with very serious status. The spectrum shown in Figure 2 (bottom) is from vertically sliced BCC sample at
a depth of 1,100 µm, substantially similar to the BCC sliced at a depth of 100 µm, with RR peaks present at
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1,012 cm , 1,452 cm , 1,662 cm , 2,861 cm , 2,898cm and 2,932 cm . But carotenoids peaks occurring at
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1,161 cm and 1,521 cm are obviously weaker than normal tissue sample [Figure 2 (top)], which suggest
this status may be a mildly lesion. Thus, we propose that the evolution of the RR spectra of biomarker
carotenoids with the depth dependence revealed the correlation between depth and the process of status
change. Carotenoids are the organic and natural fat-soluble pigments and exist in plants. Human beings
can obtain carotenoids from diet, such as fruits and vegetables, and its concentration depends on their daily
diet and stress factor [67,68] . Carotenoids accumulate in the epidermis through (1) diffusion from the fat tissue,
blood and lymph flows, or (2) secretion via sweat glands, and sebaceous glands onto the surface of the skin
and subsequent penetration. The specific importance of carotenoids is to serve as a marker substance for the
entire anti-oxidative network of human skin [69,70] . Because antioxidants form protective chains in skin tissue,
they act synergistically to protect each other against the destructive action of the free radicals, and mainly
reactive oxygen species [69-73] .

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RR spectral fingerprints of tryptophan: in contrast, the RR peaks of 1,161 cm and 1,521 cm diminished
sharply for BCC tissues at a depth of 100 µm while the fingerprints of tryptophan peak at 753 cm and 1,589 cm
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