Raman microspectroscopy for cervical cancer diagnosis: an ex-vivo cryopreserved tissue analysis

Cervical cancer was the fourth most common cancer worldwide in 2022. There is an urgent clinical need for new objective and reproductible methods aiming an early diagnosis. The aim of the present study is to characterize fresh cryopreserved human cervical tissue through Raman microspectroscopy to identify spectral biomarkers that highlight the chemical differences between healthy and malignant tissues. Normal and tumoral human cervical tissues samples were collected prospectively from patients and from pathology specimens and cryopreserved at -80°C without any treatment or cryoprotectant. Two parallel 10 µm sections were cut from each tissue block for histopathology and spectroscopy analysis. Raman microspectroscopy using a 532 nm laser was performed in the first minutes after defrosting the sample. Concerning the high-wavenumber region (2800-3600 cm^-1) the signals from lipids and proteins are those that most contribute to the spectra of the tumoral samples. In the fingerprint region (600-1800 cm^-1) the spectra of the malignant samples show characteristic nucleic acids signals and absence of features from either collagen or glycogen, which are usually present in healthy tissues. Distinct vibrational spectroscopic differences were obtained, and these signals may be considered as spectral biomarkers of tumoral tissues. A successful classification model was constructed to categorize the samples into either healthy or tumoral, with an accuracy higher than 90%. This report evidences the feasibility of Raman microspectroscopy as a reliable and non-invasive diagnostic tool for differentiating healthy from tumoral tissues with high accuracy. The use of snap-frozen tissue samples allows the translation to real-time in vivo fresh samples. Coupled to existing histopathological methods, this technique can is therefore a useful tool in the clinical field for early cancer diagnosis.