.png)
Oral Contributed Presentation
RAM
Victor Alcolea-Rodriguez, PhD (he/him/his)
Marie Skłodowska-Curie Postdoctoral Fellow
POLITECNICO DI MILANO // CNR-IFN
Milan, Lombardia, Italy
Anna Mühlig
3Department of Otorhinolaryngology, Jena University Hospital
Jena, Thuringen, Germany
Chiara Agrimi
Politecnico di Milano, Department of Physics
Milan, Lombardia, Italy
Nadja Ziller
3Department of Otorhinolaryngology, Jena University Hospital
Jena, Thuringen, Germany
Nicolas Coca-Lopez
CSIC-ICP, Instituto de Catálisis y Petroleoquímica (CSIC)
Madrid, Madrid, Spain
Ayman Bali
3Department of Otorhinolaryngology, Jena University Hospital
Jena, Thuringen, Germany
Morteza Behrouzitabar
Università degli Studi di Milano-Bicocca
Milan, Lombardia, Italy
Giuseppe Antonacci, PhD
CEO
Specto Photonics
Milan, Lombardia, Italy
Giulio Cerullo
Politecnico di Milano, Department of Physics//CNR-Institute for Photonics and Nanotechnologies
Milan, Lombardia, Italy
Dario Polli, PhD
Professor of Physics
Politecnico di Milano, Department of Physics//CNR-Institute for Photonics and Nanotechnologies
Milan, Lombardia, Italy
Orlando Guntinas-Lichius
Department of Otorhinolaryngology, Jena University Hospital
Jena, Thuringen, Germany
Renzo Vanna
CNR-Institute for Photonics and Nanotechnologies //Politecnico di Milano, Department of Physics
Milan, Lombardia, Italy
Immune checkpoint inhibitors (ICIs) have emerged as a promising immunotherapy strategy for Head and Neck cancer, enhancing the immune system’s ability to recognize and eliminate tumor cells by blocking inhibitory programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) interactions. However, only 20–40% of patients respond to therapy, while tumor resistance remains poorly understood, underscoring the need for predictive biomarkers. Recent evidence implicates lipid metabolism and tumor microenvironment (TME) heterogeneity.
Raman microscopy, a label-free, non-destructive technique, enables high-resolution imaging of biochemical features within tissues, including lipid distribution and TME biochemical landscape. In this work, we present for the first time a methodology for chemical imaging of cultured patient-derived tumors after in vitro immunotherapy treatment, using spontaneous Raman microscopy.
Tumors were obtained from patients diagnosed with head and neck squamous cell carcinoma (HNSCC), sliced into 100 μm sections, and cultured in vitro. Slices were treated with Durvalumab (anti–PD-L1) for five days. Samples were collected at three timepoints (control, days 2–3, and 4–5) and cryosectioned into 10 μm slices on quartz slides. Adjacent sections were stained with H&E to identify tumor and healthy regions. Raman chemical images were acquired using a custom-built spontaneous Raman system (660 nm excitation, 50× objective, 35 mW, 0.5 s exposure), covering 120×120 pixels over a 0.6×0.6 mm area. Both fingerprint and CH regions (600-3200 cm-1) were analyzed.
Preliminary principal component analysis (PCA) of data from the first four patients (~120 images, ~1.5 million spectra) after preprocessing (background removal, baseline correction, normalization) revealed components corresponding to proteins (2930, 1661, 1004 cm⁻¹), lipids (2844, 1653, 1440 cm⁻¹), and nucleic acids (808, 2985 cm⁻¹). Lipid-related PCs distinguished tumor from healthy areas, and spectral subtraction indicated increased lipid content (2844 cm⁻¹) in tumor tissue following Durvalumab exposure. In contrast, healthy tissue exhibited high biochemical heterogeneity depending on sample location. Overall, this approach provides a powerful tool to visualize treatment-induced biochemical changes in patient-derived tumors, offering new insights into resistance mechanisms and supporting the development of predictive biomarkers for immunotherapy response.