# Raman Spectroscopy of Individual Cervical Exfoliated Cells in Premalignant and Malignant Lesions
## 4 Discussion
The results obtained in this study show that the increased signals in Raman spectroscopy bands of samples from patients with cervical lesions corresponded to proteins originating from the increased expression of genes that interfere with the repair and control processes of protein synthesis, as well as increased expression of genes encoding the viral structural proteins. The increase in viral protein expression plays an essential role in the activation of factors that stimulate the proliferation of epithelial cells of the basal stratum, allowing the dysregulated growth of the epithelium and thus, extending the lesion [37,38]. These metabolic alterations explain the Raman spectra obtained. In this study, bands corresponding to proteins had augmented intensity, which correlated to the degree of the cervical lesion. For example, the band intensity was higher in CIN2 and CIN3/CIS than in CIN1 because CIN2 and CIN3/CIS are higher degree lesions, where there is a higher expression of viral genes and therefore, the concentration of proteins, increasing the intensity of the corresponding bands in the Raman spectra. The intensity of protein bands increases in the nucleus since the virion assembly is carried out in this compartment. Therefore, the proteins are concentrated after their translation and before assembly [37,38]. The increment in the intensity of the bands corresponding to guanine and adenine, components of DNA and RNA, in the Raman spectra of samples from patients presenting both nuclear and cytoplasmic lesions, could be due to the synthesis of viral DNA (a circular, double-stranded form of DNA virus) [37,38]. In the nuclear compartment of the lower basal and suprabasal layers, this synthesis proceeds more slowly, with viral episomal replication occurring from 50 to 100 copies per cell. Still, in the upper layers of intermediate and superficial cells, the synthesis of the viral genome is increased (up to 1000 copies per cell) [37]. As the severity of the lesion progresses, as in CIN3/CIS (cancer in situ), the virus can insert part of its genome into that of the host cell (promoter P67, E6, and E7, which occurs in the nucleus), thus allowing higher viral expression and malignization [38]. Conversely, in the cytoplasm, the increased intensity of the nucleic acid bands in the Raman spectra could be explained because adenine and guanine are components of the viral DNA and RNA, which is expressed in the cells as mRNA. In the HPV-infected cells, in addition to the production of healthy cellular proteins, viral mRNA is also expressed and leads to new viral proteins [38]. The CIN group shows higher dispersion signals, indicating that both low and high-grade lesion patients were included in the same group. The HPV virus' effects on the epithelium cells also cause changes at the intracellular level. According to the discriminant analysis, the powers of classifying a sample as healthy or presenting a CIN using the measurements obtained from the cytoplasm or nucleus were 76.5% and 82.7%, respectively. It is essential to underline that in our study, the measurements and results obtained derived directly from individual cell compartments, that is, the cytoplasm and nucleus of single cells. Our findings are similar to previous studies, such as by [PERSON]. et al., where the measurements on cell pellets achieved an efficiency of classification of ~79% [18]. In our study, the classification efficiency in the nuclear measurements was near 80% (see Supplementary Material). In contrast to our study findings, [PERSON] et al. in 2015 and [PERSON] in 2017 [16; 39] reported a more substantial classification efficiency. However, while the authors used cell lines in these studies, our study was carried out on clinical samples. Furthermore, other studies, including that by [PERSON] et al. (2019), report excellent precision (~93.0%), despite the type of sample used for the spectroscopy measurements, tissue and not cells [40].