The particularly high mortality of epithelial ovarian cancer (EOC) is in part linked to limited understanding of its molecular signatures. Although there are data available on in situ N-glycosylation in EOC tissue, previous studies focused primarily on neutral N-glycan species and, hence, still little is known regarding EOC tissue-specific sialylation. In this proof-of-concept study, we implemented MALDI mass spectrometry imaging (MALDI-MSI) in combination with sialic acid derivatization to simultaneously investigate neutral and sialylated N-glycans in formalin-fixed paraffin-embedded tissue microarray specimens of less common EOC histotypes and non-malignant borderline ovarian tumor (BOT). The applied protocol allowed detecting over 50 m/z species, many of which showed differential tissue distribution. Most importantly, it could be demonstrated that α2,6- and α2,3-sialylated N-glycans are enriched in tissue regions corresponding to tumor and adjacent tumor-stroma, respectively. Interestingly, analogous N-glycosylation patterns were observed in tissue cores of BOT, suggesting that regio-specific N-glycan distribution might occur already in non-malignant ovarian pathologies. All in all, our data provide proof that the combination of MALDI-MSI and sialic acid derivatization is suitable for delineating regio-specific N-glycan distribution in EOC and BOT tissues and might serve as a promising strategy for future glycosylation-based biomarker discovery studies.

Head and neck squamous cell carcinoma (HNSCC) is often diagnosed at advanced stages. Due to pronounced intratumoral heterogeneity (ITH), reliable risk stratification and prediction of treatment response remain challenging. This study aimed to identify peptide signatures in HNSCC tissue that are associated with treatment outcomes in HPV-negative, advanced-stage HNSCC patients undergoing 5-fluorouracil/platinum-based chemoradiotherapy (CDDP-CRT). We integrated matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of tryptic peptides with univariate statistics and machine learning approaches to uncover potential prognostic patterns. Formalin-fixed, paraffin-embedded whole tumor sections from 31 treatment-naive, HPV-negative HNSCC patients were digested in situ with trypsin, and the generated peptides were analyzed using MALDI-MSI. Clinical follow-up revealed recurrence or progression (RecPro) in 20 patients, while 11 patients showed no evidence of disease (NED). Classification models were developed based on the recorded peptide profiles using both unrestricted and feature-restricted approaches, employing either the full set of m/z features or a subset of the most discriminatory m/z features, respectively. The unrestricted model achieved a balanced accuracy of 71% at the patient level (75% sensitivity, 66% specificity), whereas the feature-restricted model reached a balanced accuracy of 72%, showing increased specificity (92%) but reduced sensitivity (52%) in the CDDP-CRT cohort. In order to assess treatment specificity, models trained on the CDDP-CRT cohort were tested on an independent patient cohort treated with mitomycin C-based CRT (MMC-CRT). Neither model demonstrated prognostic performance in the MMC-CRT patient cohort, suggesting specificity for platinum-based therapy. Presented findings highlight the potential of MALDI-MSI–based proteomic profiling to identify patients at elevated risk of recurrence following CDDP-CRT. This approach may support more personalized risk assessment and treatment planning, ultimately contributing to improved therapeutic outcomes in HPV-negative HNSCC.

Despite the growing burden of osteoarthritis (OA), effective disease‐modifying treatments remain limited. Hyaluronic acid, platelet‐rich plasma, and hydrogels are in clinical use, but their efficacy beyond placebo is debated. Cell‐based therapies are continuously investigated for their disease‐modifying potential. Stromal vascular fraction (SVF), a minimally processed and clinically accessible cell product, has shown therapeutic potential, but remains poorly characterized in respect to its pro‐regenerative cell types and molecular signaling. To determine whether CD31 ⁺ cell enrichment enhances the regenerative efficacy of SVF, a comparative efficacy study was conducted using the Dunkin Hartley guinea pig model of spontaneous OA. Spatial proteomics reveals that both treatments modulate cartilage extracellular matrix (ECM) composition, reducing fibronectin and COL1A1 levels, with CD31 ⁺ SVF showing more pronounced effects. Both therapies attenuate cartilage fibrosis, increase aggrecan, suppress MMP‐13, and reduce TNF‐α and MCP‐1 expression. Compared to SVF, CD31 ⁺ SVF shows greater and more consistent improvements in subchondral and trabecular bone integrity. These findings demonstrate that CD31 ⁺ enrichment enhances the therapeutic potential of SVF, likely through more consistent modulation of ECM remodeling and osteochondral integrity. Our results provide a preclinical rationale for the testing of CD31 ⁺ ‐enriched SVF as a biologically refined cell therapy approach for OA, with potential disease modifying properties.