Using Raman microscopy, we investigated epithelial cervical cells collected from 96 women with squamous cell carcinoma (SCC) or belonging to groups I, IIa, IIID-1 and IIID-2 according to Munich III classification (IIID-1 and IIID-2 corresponding to Bethesda LSIL and HSIL groups, respectively). All women were tested for human papillomavirus (HPV) infection using PCR. Subcellular resolution of Raman microscopy enabled to understand phenotypic differences in a heterogeneous population of cervical cells in the following groups: I/HPV−, IIa/HPV−, IIa/HPV−, LSIL/HPV−, LSIL/HPV+, HSIL/HPV−, HSIL/HPV+ and cancer cells (SCC/HPV+). We showed for the first time that the glycogen content in the cytoplasm decreased with the nucleus size of cervical cells in all studied groups apart from the cancer group. For the subpopulation of large-nucleus cells HPV infection resulted in considerable glycogen depletion compared to HPV negative cells in IIa, LSIL (for both statistical significance, ca. 45%) and HSIL (trend, 37%) groups. We hypothesize that accelerated glycogenolysis in large-nucleus cells may be associated with the increased protein metabolism for HPV positive cells. Our work underlines unique capabilities of Raman microscopy in single cell studies and demonstrate potential of Raman-based methods in HPV diagnostics.

Cellular lipid metabolism is significantly transformed during oncogenesis. To assess how dysplasia development influences lipid cellular metabolisms and what is the molecular background behind it, cervical epithelial cells of 63 patients assigned to seven groups (based on the cytological examination and HPVhr test results) were studied using a multimethodological approach including Raman microscopy and molecular methods. The consistent picture obtained studying the lipid content, cell inflammation, SREBF1 gene methylation (hence SREBP1 inhibition) and level of mitochondrial DNA copies (indirectly the number of mitochondria) showed that changes in lipid metabolism were multidirectional. Cells from patients classified as mildly dysplastic (LSIL) exhibited a unique behavior (the highest level of inflammation and SREBF1 methylation, the lowest lipid content and mitochondrial DNA). On the contrary, cells from severe dysplastic (HSIL) and cancer (SCC) groups showed the opposite characteristics including the lowest SREBF1 gene methylation as well as the highest level of mitochondrial DNA and lipid cellular concentration (for HSIL/HPVhr+ and SCC groups). Following dysplastic progression, the lipid content decreases significantly (compared to the control) for mildly abnormal cells, but then increases for HSIL/HPVhr+ and SCC groups. This intriguing dual switch in lipid metabolism (reflected also in other studied parameters) on the way from normal to squamous carcinoma cells is of potential diagnostic interest.