Abstract
MOTIVATION: The advent of highly multiplexed in situ imaging cytometry assays has revolutionized the study of cellular systems, offering unparalleled detail in observing cellular activities and characteristics. These assays provide comprehensive insights by concurrently profiling the spatial distribution and molecular features of numerous cells. In navigating this complex data landscape, unsupervised machine learning techniques, particularly clustering algorithms, have become essential tools. They enable the identification and categorization of cell types and subsets based on their molecular characteristics. Despite their widespread adoption, most clustering algorithms in use were initially developed for cell suspension technologies, leading to a potential mismatch in application. There is a critical gap in the systematic evaluation of these methods, particularly in determining the properties that make them optimal for in situ imaging assays. Addressing this gap is vital for ensuring accurate, reliable analyses and fostering advancements in cellular biology research. RESULTS: In our extensive investigation, we evaluated a range of similarity metrics, which are crucial in determining the relationships between cells during the clustering process. Our findings reveal substantial variations in clustering performance, contingent on the similarity metric employed. These variations underscore the importance of selecting appropriate metrics to ensure accurate cell type and subset identification. In response to these challenges, we introduce FuseSOM, a novel ensemble clustering algorithm that integrates hierarchical multiview learning of similarity metrics with self-organizing maps. Through a rigorous stratified subsampling analysis framework, we demonstrate that FuseSOM outperforms existing best-practice clustering methods specifically tailored for in situ imaging cytometry data. Our work not only provides critical insights into the performance of clustering algorithms in this novel context but also offers a robust solution, paving the way for more accurate and reliable in situ imaging cytometry data analysis. AVAILABILITY AND IMPLEMENTATION: The FuseSOM R package is available on Bioconductor and is available under the GPL-3 license. All the codes for the analysis performed can be found at Github.