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Abstract

Early diagnosing of skin cancer and investigation of metastatic potential of cancer cells are very important to treat it appropriately. Infrared spectroscopy of biological tissues is an emerging technique which gives the spectral differences between healthy and diseased cells. In this work, we have demonstrated that attenuated total reflectance Fourier transform (ATR-FTIR) spectroscopy can be used in diagnostic of skin cancer and in differentiating metastatic potential of cancer cells. Using IR spectroscopy, we can identify various types of cancer such as basal cell carcinoma, malignant melanoma, nevus and metastatic potential by alternations in hydration level and molecular changes. We examined biopsy of different types of cancer cells to diagnose skin cancer at early stages by using FTIR spectroscopy. To differentiate metastases we examined two human melanoma cells of same patient but at different metastatic potential and two murine melanoma cells with common genetic background but different metastatic potential.

Our findings revealed that melanoma changes the permeability of cell membrane and higher metastatic potential is related to the hydration level of cell membrane. Thus, ATR-FTIR spectroscopy is a potential technique to help in early diagnosing of skin cancer and to differentiate different metastatic potentials.

Keywords

Metastatic ATR-FTIR melanoma spectroscopy

1. Introduction

Skin protects our body from unhealthy environmental effects. Ultraviolet radiations from sun and other ionizing radiations cause sun burns, melanoma, skin carcinoma and premature skin aging. Cases of cutaneous melanoma are increasing largely with every passing year and its worst condition metastatic cancer is a major cause of deaths. Cutaneous melanoma (MM), Basal cell carcinoma (BCC) and squamous cell skin (SCC) are the three major types of skin cancer [1, 2, 3, 4, 5, 6]. Cutaneous melanoma is directly related to the sun exposure and studies show that sun shield sites show aggressive behavior as compared to sun exposed areas. A low serological level of vitamin D is observed in sun shield sites as compared to non shield sites and this low level of vitamin D is related to the low percentage of BRAF mutation in melanoma patients [7]. BCC and MM skin cancers are mostly localized and probability of metastatic stage is rare. However, SCC cancer can spread all around developing a metastatic stage which becomes a cause of death. While treating a cancer patient, it is very important that metastatic potential of the tumor should be correctly identified. Usually morphological and lymph node states methods are used for the classification of tumor. But these methods are not reliable in all types of cancer such as breast and pancreatic tumors. Other methods for metastatic classifications are time taking and cost effective such as genetic test for tumor [8, 9, 10].

Hence, the objective of this research was to propose an easy and new diagnostic technique for cancer and different metastatic potentials by FTIR spectroscopy. This technique is also used to identify other kinds of melanoma and non melanoma skin cancer. Mid infra range spectroscopy is a very useful technique for biological molecules and their relation with the surroundings. This technique has potential to investigate differences in biological cells but water contents in biological samples limit the efficiency of mid-IR spectroscopy. This problem can be overcome by equipping FTIR spectrometer with attenuated Total Reflectance (ATR) element. Using this method, we can obtain spectra of live biological cells in solution [11, 12, 13, 14, 15]. The ATR infrared spectroscopy enables total internal reflection phenomenon and large number of internal reflections produce evanescent wave. If we place a sample in contact with ATR than that evanescent wave loses its energy at similar frequencies to that of sample’s absorbance. This technique enables the direct measurement of absorption spectra of biological samples placed in contact with ATR element [16]. Previous research showed that the higher movement of metastatic cells and the fluidity of the cell’s membrane; both can be linked with each other [17, 18, 19, 20, 21, 22].

2. Methodology

For the study of different types of skin cancer we took different samples from 6 patients with BCC on the head (6 specimens), 4 patients with MM on the back (8 specimens) and 2 patients with nevus (NEV) on neck (3 specimens). For different stages of cancer, B16-F1 and B16-F10 cells (grown in growth medium composed of DMEM (D5796, Sigma-Aldrich, St. Louis, MO, USA)) were produced from the murine melanoma B16 by injecting into C57 mice. B16-F1 cells produced as lung metastases of B16 cells. Another cell B16-F10 with different metastases was generated by re-injecting into mice for several rounds of selection. This selection method gave two cell lines with a genetic background in common but at different metastatic potential. B16-F10 cells are at higher metastatic potential as compared to B16-F1 cells. Similarly, two human melanoma cells are taken from primary tumor but different metastases of same patient. These cells were spherical in medium solution having medium ratio 0.99 $\pm$ 0.02 and 0.094 $\pm$ 0.02 for human and mice samples respectively. FTIR spectra were taken using Bruker Tensor 27 spectrometer equipped with Germanium ATR device. Refractive index of ATR is 4 and dimensions are 47 mm $\times$ 5 mm. Other specimen of BCC, MM and NEV were also same in size and fixed in 10% in formulation of neutral-buffered formalin. The emitted radiations from IR source were focused into the Ge ATR and output radiations were focused onto detector. Matlab 2016a software was used to perform all the calculations and data processing. Non-linear least square method was used for curve fitting of Gaussians-Sum Model.

Figure 1.

FT-IR spectra of skin tissues: normal healthy tissue (N), basal cell carcinoma (BCC), melanoma (MM) and nevus (NEV), in the region 4000–400 cm ${}^{-1}$ (a) FTIR spectra in range (900–800) cm ${}^{-1}$ for all types of tissues such as normal (N) subtracted from the spectra of melanoma (MM) tissue (MM-N) (c) FT-IR spectra of basal cell carcinoma (BCC) subtracted from the spectra of melanoma (MM) tissue (MM-BCC).

3. Results and discussion

3.1 Early diagnosing of cancer

FTIR spectra of normal (N), NEV, BCC and MM biopsies are given in Fig. 1a. This spectrum gives intensities measurements and shifts in frequencies of normal and cancerous tissues in spectral range 4000–400 cm ${}^{-1}$ . In region 4000–3000 cm ${}^{-1}$ , we can observe stretching vibrations of OH and NH groups of collagens and proteins of the skin. This region also gives the stretching vibration bands of water molecules [11, 23, 24, 25, 26, 27, 28]. In Fig. 1a, the broad band at 3484 cm ${}^{-1}$ due to OH group stretching is at higher level in spectra of N and NEV. These bands are decreased for BCC and MM which is because of the skin dehydration. The region 3062 cm ${}^{-1}$ contains most of the protein (amide B). In this region (3062 cm ${}^{-1}$ ), $\beta$ -sheets of protein structure is dominant as compared to amide A region [27, 29, 30]. These changes in spectra are very important to characterize a disease and its development. We can analyze spectral region 1800–1700 cm ${}^{-1}$ for the secondary structure of protein in Fig. 1a. The band at 1744 cm ${}^{-1}$ for N and BCC samples is at lower level but in the case of MM and NEV, this band is relatively at higher intensity. This shift in intensities might be due to lipid per oxidation in MM and NEV samples [28, 30]. In amide I region, high intensity band due to C $=$ O vibration is at 1650 cm ${}^{-1}$ but for MM and NEV this band splits up into two bands at 1650 cm ${}^{-1}$ and 1633 cm ${}^{-1}$ . This separation of peaks may be due to $\alpha$ -helix and conformations of random coil protein in MM and NEV samples. In amide II region, peak for normal cell was at 1544 cm ${}^{-1}$ and it shifts towards 1536 cm ${}^{-1}$ for BCC, 1548 cm ${}^{-1}$ for NEV and 1540 cm ${}^{-1}$ for MM. This secondary structure of protein $\beta$ -stretch and is shifted towards lower frequencies for malignancies [30, 31, 32]. In region (1250–1000) cm ${}^{-1}$ spectral changes can be observed at the band (1150–1165) cm ${}^{-1}$ due to C-O-C and at 1056 cm ${}^{-1}$ (C-C and C-O-H). The bands have increased intensities for BCC, MM and NEV tissues. These increases in frequencies might be due to access of carbohydrates and reduction in hydrogen group which might be due to glycosylation in cancer cells. In previous studies, these kinds of bands were also occurred in the other cancer types such as colon, breast cancer and metastatic bone cancer [28, 29, 33]. To analyze sugar-phosphate vibration, we plotted region 900–800 cm ${}^{-1}$ separately (Fig. 1b). Bands at 841 cm ${}^{-1}$ and 815 cm ${}^{-1}$ show B-DNA and Z-DNA and for MM type of cancer Z-DNA conformation was analyzed. B-DNA AND Z-DNA conformations were observed for BCC and for normal tissues B-DNA were distinguished from other absorptions.

From our results it is clear that diseases effects the structure of skin at molecular level. To understand the behavior and spectral difference in diseases we subtracted the spectra of normal tissue (N) from melanoma (MM) (Fig. 1c). In the subtracted the spectra we observed $\upsilon$ OH and $\upsilon$ NH bands to be negative which suggest the dehydration of MM. On the other hand the absorption bands correspond to C-O-C and C-OH groups showed an increase in intensity. These findings lead to the suggestion that melanoma progression may cause an increase in the glycosylation of the tissue. To discuss the behavior of BCC diseases we subtracted the spectra of BCC from MM spectrum (MM-BCC) (Fig. 1d) in the region of 4000–2820 cm ${}^{-1}$ . The NH ${}_{2}$ at 3293 cm ${}^{-1}$ became negative in this subtracted spectra, whereas high intensity band at 1744 cm ${}^{-1}$ appeared for MM which is not observed in BCC. This higher intensity band indicate the production of peroxidation of lipids. The bands of amide I and amide II regions after subtraction show negative absorption for MM, which might be due to the reason that more collagen is produced in BCC which is in agreement with the literature [34, 35]. The other major difference was the reduction in the bands at 1048 cm ${}^{-1}$ in MM while these bands were strong for BCC. This band might be due to C-O-C and assigned to C-O vibrations of hyaluronic acid (HA). The absorption band at 1048 cm ${}^{-1}$ may be used as a biomarker of MM and due to the presence of many HA-chain breaks it might differentiate melanoma from non melanoma cancer. While discussing the DNA region (Fig. 1b) Z-DNA form is observed in MM, while in BCC both B-DNA and Z-DNA are present in the spectra of the skin.

Figure 2.

The graph represents absorption versus wave number in the range of 1000–1700 cm ${}^{-1}$ in amide II region. Absorption intensity spectra of B16-F1 and F16-F10 is shown in (a) intensity spectra of PT and MT is shown in (b). In the 3100–3500 cm ${}^{-1}$ range graph represent the absorption spectra of B16-F1 and B16-F10 (c) and absorption intensities for PT and MT.

3.2 Metastatic potential of melanoma cells

To analyze the ability of ATR-FTIR between the different metastatic potential of similar tumor we used a pair of human and mouse melanoma cells. The B16-F1 and B16-F10 cells from the same parental murine but at low and high metastatic potential respectively. Similarly, we investigated two biopsies of same patient such as PT (Primary tumor) and MT (metastatic Tumor), whereas PT is primary tumor and MT are at lower and higher metastatic potential respectively. For FTIR spectra, we used live cell solution and Germanium ATR element. The solution was homogenous, as the cells were hung in the medium. For background measurements, a small drop of solution was placed on Germanium ATR and measurements were made. That homogenous stage spectrum was subtracted from each measured spectrum of different cells. Metastatic cells are more mobile in the tissue and fluidity level in plasma membrane is higher in comparison with cells at less metastatic potential [17, 18, 19, 20, 21, 36]. Increase in fluidity level in plasma membrane cause an increase in hydration level [37, 38, 39]. So, metastatic of cancerous cell might be due to hydration levels and can be observed by absorption intensities of protein in different regions such as amide A, amide B, amide I, II, III and amide IV [40]. It is clear from previous study that the intensity of absorption peak in protein increases with the increase in hydration level [14]. It was assumed that protein of membrane show similar effects by an increase in hydration. Normalization of intensities of different peaks dominant in various experiments is a good way to compare the protein related absorption intensities. We normalized amide II peaks intensity of 1540 cm ${}^{-1}$ with intensity of strong peak at 1035 cm ${}^{-1}$ . We measured the amide II normalized intensity for human melanoma cell 0.33 $\pm$ 0.01 for PT and 0.9 $\pm$ 0.03 for MT cells and a similar trend was observed for murine melanoma cells such as 0.23 $\pm$ 0.03 for B16-F1 and 0.40 $\pm$ 0.03 for B16-F10. Graphs (Fig. 2) shows the differences in amide II peaks intensities for both cells types. These results show that changes in absorption intensity of the cell membrane is due to hydration level of cell membrane. Intermolecular structure of water molecules is another marker for the hydration level of cell membrane. Low density water molecules (LDW) are a crystal-like structure which can be formed by connecting hydrogen bonds with other water molecules. These hydrogen bonds can also be connected to non-water molecules or high density water (HDW) [14, 41, 42]. Different types of water structure gives different absorption frequencies because hydrogen bonding with different water molecules have effects on vibration rate of O-H stretch modes. From previous literature, it has been observed that liquid water is thought to be formed by a mixture of structural species [43]. Peaks for each form are different of O-H stretch modes as LDW has peak at 3200 cm ${}^{-1}$ and HDW shows peak at 3400 cm ${}^{-1}$ . Figure 2a and b shows the differences in absorption spectra for both states of cancer for human melanoma and murine cell line. In range 3100–3500 cm ${}^{-1}$ , we can measure the differences in hydration levels by taking ratio of HDW to LDW (Fig. 2c and d). This is because of the reason that at higher hydration levels it is easy for water molecules to break hydrogen bonds that are in structural order [44]. For analysis, we used 2-components mixture in a 4-component mixture model. We used a method which consists of two Gaussians along with non-linear least square error fit. $a_{1}\cdot\exp(-(x-\mu_{1})^{2}/2\sigma_{1}^{2})+a_{2}\cdot\exp(-(x-\mu_{2})^{% 2}/2\sigma_{2}^{2})+C$ was used where $\{a_{1},\mu_{1},\alpha_{1},\mu_{2},\alpha_{2},c\}$ are parameters. The values of parameter $\mu_{1}$ and $\mu_{2}$ were set at 3200–3400 cm ${}^{-1}$ while actual spectral range were 3229–3238 cm ${}^{-1}$ and 3354–3400 cm ${}^{-1}$ . In next step, we took ratio of two Gaussian centered around 3200 cm ${}^{-1}$ and 3400 cm ${}^{-1}$ given as $A_{3400}/A_{3200}=a_{3400}\cdot\alpha_{3400}/a_{3200}\cdot\alpha_{3200}$ . As we set the back ground homogenous so due to the penetration of cell in evanescent field absorption of water becomes smaller and negative peaks of water absorbance are obtained. As the area of Gaussian is inverse relation with LDW or HDW molecules, so we can say that $A_{3400}/A_{3200}\propto(N_{\text{HDW}}/N_{\text{LDW}})^{-1}$ . The box plot in Fig. 3 gives ratio ( $A_{3400}/A_{3200}$ ) values taken during saturation stage. In this figure, difference between two types of samples such as more metastatic and less metastatic can be clearly seen. The real ratio of less metastatic state (PT) is 54 and high metastatic state (MT) is 21, similarly the average real ratio for B16-F1 cell is 35.2 and for B16-F10 it is 21.4. These values of ratio indicate that PT state has more LDW as compared with MT and similarly B16-F1 has LDW in more quantity as compared to B16-F10. These results confirm our assumption that hydration level is major difference between different metastatic states of cells and it can be used as a tool to find the metastatic potential of different cancer cells.

Figure 3.

A boxplot description of the distribution of values of ratio between the Gaussian centered around 3400 cm ${}^{-1}$ and 320 cm ${}^{-1}$ . A boxplot of cells B16-F1 and F16-F10 are shown in figure (a) and results for PT and MT are shown in figure (b).

In this study, we describe a promising ATR-FTIR method to measure the spectra of biological cells. We have analyzed the hydration level of the plasma membrane by using two human melanoma cells and two variant of murine B16 to investigate their metastatic potential. We have analyzed two main evidences that show relation of metastatic potential with level of hydration of plasma membrane. In first observation, the higher level of absorption intensity in amide II region for higher metastatic potential show the higher hydration level of plasma membrane. Second evidence is that higher metastatic state had more HDW molecules in cell’s membrane. Both of our tests gave regular results that may allow us to identify metastatic potential of various cells by examining spectral differences and relate to previous literature. Ghimire et al. [45] differentiated metastases of prostate cancer in lymphoma spectra and melanoma serum of mouse model by using FTIR spectroscopy. The major differences found between the two serum samples were absorption values of protein, carbohydrates and nucleic acid. Wu et al. [46] used FTIR spectroscopy to classify the metastatic and non metastatic lymph nodes. Using FTIR spectroscopy along with Support vector machine they found accuracy, sensitivity and specificity 88.9%, 75.0% and 95.3% respectively. Hands et al. [47] used 1 $\mu$ l of human blood serum and provided improved spectral signature to differentiate cancer versus non-cancer, metastatic cancer versus organ confined, brain cancer severity and other metastatic diseases. They found sensitivity and specificity 91.5% and 83% respectively. They observed the statistic of lipids and nucleic acid region for metastatic and non metastatic lymph nodes. Chrabaszcz et al. [48] used FT-IR and Raman spectroscopy for biochemical profiling of the early stages of the pulmonary metastases in the breast cancer of mice. They observed retinoids, lipids and carbohydrates constitute to a potential set of biomarker for early lung metastasis. In our work, we took cells from tissue and suspended in solution. These results can also be obtained by analyzing cancerous tissue directly with an ATR-FTIR microscope.

In this research we found remarkable differences between ATR-FTIR spectra of cells at different metastatic potential which may lead to develop a clinical diagnostic tool by analyzing the metastatic biomarker such as hydration level and high density water in cell membrane. Clinical FTIR spectroscopy may provide an accurate and improved diagnostic method for cancer by replacing the difficult and expensive existing techniques.

4. Conclusion

The ATR-FTIR method is a potential tool to diagnose various types of cutaneous melanoma by spectral differences. This method can also be used to identify metastatic potential of cancer cells. The comparison of less metastatic and more metastatic cells showed that hydration level of plasma membrane is a major difference between both states of cancer. ATR-FTIR method is a useful technique to identify different type of cancer cells and their metastatic potentials.

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Attenuated total reflectance spectroscopy to diagnose skin cancer and to distinguish different metastatic potential of melanoma cell

Abstract

Keywords

1. Introduction

2. Methodology

3.1 Early diagnosing of cancer

References