FT-IR Spectral Signature of Sensitive and Multidrug-Resistant Osteosarcoma Cell-Derived Extracellular Nanovesicles

Abstract

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, the outcome is unsatisfactory, and multidrug resistance (MDR) is a pivotal process in OS treatment failure. OS-derived extracellular vesicles (EVs) promote drug resistance to chemotherapy and target therapy through different mechanisms. The aim of this study was to identify subpopulations of osteosarcoma-EVs by Fourier transform infrared spectroscopy (FT-IR) to define a specific spectral signature for sensitive and multidrug-resistant OS-derived EVs. EVs were isolated from sensitive and MDR OS cells as well as from mesenchymal stem cells by differential centrifugation and ultracentrifugation. EVs size, morphology and protein expression were characterized. FT-IR/ATR of EVs spectra were acquired in the region of 400–4000 cm−1 (resolution 4 cm−1, 128 scans). The FT-IR spectra obtained were consistently different in the EVs compared to cells from which they originate. A specific spectral signature, characterized by a shift and a new band (1601cm−1), permitted to clearly distinguish EVs isolated by sensitive and multidrug-resistant OS cells. Our data suggest that FT-IR spectroscopy allows to characterize and define a specific spectral signature for sensitive and MDR OS-derived EVs.

Machine Learning-Assisted FTIR Analysis of Circulating Extracellular Vesicles for Cancer Liquid Biopsy

“…A more in-depth analysis of Figure 2 shows the presence of changes in the spectral shape and in the relative intensity of several absorption bands, namely the carbohydrates and nucleic acids band at 1000–1200 cm −1 [ 16 , 19 ], the protein Amide I and II bands [ 13 , 36 , 37 ], the lipid C-H stretching band at 2800–3000 cm −1 [ 23 , 38 ], and the C=O stretching at approximately 1735 cm −1 , which has been assigned to purine base and ester groups of lipids in EV samples [ 9 , 36 , 37 ]. These bands are highlighted in green and red color in controls and patients, respectively, and are considered a major source of spectral biomarkers in EV research [ 4 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 23 ]. To investigate more in-depth the difference between the two groups, an enlarged detail of the four mentioned IR bands for controls (green) and oncologic patients (red) is reported in Figure 3 .…”

“…According to several papers investigating the spectral characteristics of EVs using ATR-FTIR, sample solution droplets were deposited on the ATR crystal and a thin film was obtained by slow evaporation of the solvent under ambient conditions. Specifically for each measurement, 5 μL of the EV solution was air-dried for at least 10–15 min on a high-throughput ZnSe crystal [ 4 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. This step was repeated three times, resulting in a total volume of 15 μL of dried solution.…”

“…Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) of spectra were carried out after subtracting a linear baseline from the analyzed absorption band. This analysis protocol was chosen as PCA alone, as well as PCA-LDA in combination, were successfully exploited in similar papers for discriminating EVs of different origins according to their vibrational fingerprints [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. Additionally, PCA-LDA has been widely employed in other FTIR-based classification problems in different fields, including diagnostics and forensic sciences [ 17 , 18 , 19 , 20 , 21 ].…”

“…In this regard, Fourier-transform infrared spectroscopy (FTIR), especially in the Attenuated Total Reflection (ATR) mode, is emerging as a promising tool for label-free molecular profiling of EVs, with several papers published on the subject [ 4 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. Most of these works involve in vitro experiments aimed at assessing sample composition and purity [ 15 , 16 ], distinguishing different EV classes [ 18 ] or EVs derived from cells under different states/phenotypes/culture conditions [ 4 , 8 , 10 , 12 , 21 , 22 , 23 ]. So far, very few FTIR papers have focused on EVs purified from biofluids of subjects enrolled in clinical studies.…”

Spectroscopic insight into breast cancer: profiling small extracellular vesicles lipids via infrared spectroscopy for diagnostic precision

“…Fourier transform infrared spectroscopy (FTIR), particularly in attenuated total reflection (ATR) mode, is a viable method for the label-free chemical profiling of small EVs, as evidenced by the publication of numerous papers on the subject 8 – 10 . Most of this research employs in vitro procedures to determine sample composition and purity 11 – 13 , classify various EVs 14 , or identify EVs produced by cells at different developmental stages, phenotypes, or culture conditions 15 .…”