Background
Mass spectrometry imaging (MSI) derives spatial molecular distribution maps directly from clinical tissue specimens and thus bears great potential for assisting pathologists with diagnostic decisions or personalized treatments. Unfortunately, progress in translational MSI is often hindered by insufficient quality control and lack of reproducible data analysis. Raw data and analysis scripts are rarely publicly shared. Here, we demonstrate the application of the Galaxy MSI tool set for the reproducible analysis of a urothelial carcinoma dataset.
Conclusion
Here, we show that translational MSI data analysis in a fully transparent and reproducible manner is possible and we would like to encourage the community to join our efforts.
Methods
Tryptic peptides were imaged in a cohort of 39 formalin-fixed, paraffin-embedded human urothelial cancer tissue cores with a MALDI-TOF/TOF device. The complete data analysis was performed in a fully transparent and reproducible manner on the European Galaxy Server. Annotations of tumor and stroma were performed by a pathologist and transferred to the MSI data to allow for supervised classifications of tumor vs. stroma tissue areas as well as for muscle-infiltrating and non-muscle infiltrating urothelial carcinomas. For putative peptide identifications, m/z features were matched to the MSiMass list.
Results
Rigorous quality control in combination with careful pre-processing enabled reduction of m/z shifts and intensity batch effects. High classification accuracy was found for both, tumor vs. stroma and muscle-infiltrating vs. non-muscle infiltrating urothelial tumors. Some of the most discriminative m/z features for each condition could be assigned a putative identity: stromal tissue was characterized by collagen peptides and tumor tissue by histone peptides. Immunohistochemistry confirmed an increased histone H2A abundance in the tumor compared to the stroma tissues. The muscle-infiltration status was distinguished via MSI by peptides from intermediate filaments such as cytokeratin 7 in non-muscle infiltrating carcinomas and vimentin in muscle-infiltrating urothelial carcinomas, which was confirmed by immunohistochemistry. To make the study fully reproducible and to advocate the criteria of FAIR (findability, accessibility, interoperability, and reusability) research data, we share the raw data, spectra annotations as well as all Galaxy histories and workflows. Data are available via ProteomeXchange with identifier PXD026459 and Galaxy results via https://github.com/foellmelanie/Bladder_MSI_Manuscript_Galaxy_links .