Benchmarking Spatial Clustering Methods for Mass Spectrometry-Based Spatial Metabolomics

<b>Background:</b> Mass spectrometry imaging (MSI) enables in situ mapping of metabolite distributions within tissues, and spatial clustering is a key step for delineating metabolically distinct regions. Nevertheless, spatial clustering methods have not been systematically benchmarked for spatial metabolomics data. <b>Methods</b>: Here, we evaluated the effects of ion filtering and clustering method selection on clustering performance and established a dual-metric framework that jointly assesses the spatial continuity of cluster labels and inter-cluster metabolic heterogeneity. We benchmarked 30 clustering algorithms across 12 heterogeneous MSI datasets spanning three major ion sources, four mass analyzers, and multiple spatial resolutions, covering approaches from non-spatial methods to advanced spatially aware models. <b>Results</b>: Noise filtering markedly improved the spatial continuity of results generated by non-spatial methods (mean improvement, approximately 28%) but provided limited benefit for spatially aware methods. Across the 12 datasets, a median of only 11 methods satisfied both evaluation criteria simultaneously, whereas SSC and DRSC met the dual-metric thresholds in at least nine datasets. In the mbrain2_pos50 dataset, the top-ranked method based on the composite dual-metric score achieved 22% higher concordance between cluster assignments and cell-type annotations than the lowest-ranked method. <b>Conclusions</b>: Together, the proposed evaluation framework and the online platform SMcluster provide a standardized resource for benchmarking and selecting MSI clustering methods. Our results highlight the critical roles of preprocessing and method selection in determining spatial clustering performance and offer practical guidance for spatial metabolomics studies.