Semi-automated approaches for interrogating spatial heterogeneity of tissue samples.

Tissues are spatially orchestrated ecosystems composed of heterogeneous cell populations and non-cellular elements. Tissue components' interactions shape the biological processes that govern homeostasis and disease, thus comprehensive insights into tissues' composition are crucial for understanding their biology. Recently, advancements in the spatial biology field enabled the in-depth analyses of tissue architecture at single-cell resolution, while preserving the structural context. The increasing number of biomarkers analyzed, together with whole tissue imaging, generate datasets approaching several hundreds of gigabytes in size, which are rich sources of valuable knowledge but require investments in infrastructure and resources for extracting quantitative information. The analysis of multiplex whole-tissue images requires extensive training and experience in data analysis. Here, we showcase how a set of open-source tools can allow semi-automated image data extraction to study the spatial composition of tissues with a focus on tumor microenvironment (TME). With the use of Lunaphore COMET platform, we interrogated lung cancer specimens where we examined the expression of 20 biomarkers. Subsequently, the tissue composition was interrogated using an in-house optimized nuclei detection algorithm followed by a newly developed image artifact exclusion approach. Thereafter, the data was processed using several publicly available tools, highlighting the compatibility of COMET-derived data with currently available image analysis frameworks. In summary, we showcased an innovative semi-automated workflow that highlights the ease of adoption of multiplex imaging to explore TME composition at single-cell resolution using a simple slide in, data out approach. Our workflow is easily transferrable to various cohorts of specimens to provide a toolset for spatial cellular dissection of the tissue composition.