Utilisation of spatial methodologies for the investigation of the tumour microenvironment

The tumour microenvironment (TME) is a complex and dynamic component of cancer progression, significantly influencing tumour development and response to therapy. Recent advancements in spatial-omics, such as Spatial Transcriptomics and Multiplex Imaging, have provided critical insights into TME organisation. For instance, the spatial distribution, abundance, and proximity of macrophages and cytotoxic T lymphocytes (CTLs) have been identified as key biomarkers of immune function, prognosis, and treatment response. Additionally, spatial studies have shown that phenotypic changes in tumour cells often correlate with shifts in immune cell populations, promoting tumour progression and immune evasion. This chapter serves as a practical guide for researchers working with spatial transcriptomic technologies. We focus on three leading platforms: Merscope, CosMx, and Xenium, explaining their principles and highlighting their key differences. An overview of each technology is provided to help researchers understand their applications and potential advantages. Beyond theoretical insights, we detail step-by-step experimental protocols, covering all aspects from sample preparation to data analysis. To support reproducibility and troubleshooting, we also include practical tips and lessons learned from our own experience, helping researchers optimise workflows and enhance data quality. By offering clear guidance and detailed methodologies, this chapter aims to assist researchers in successfully implementing spatial transcriptomics in their studies. Whether you are new to these technologies or seeking to refine your techniques, this resource provides essential information to advance your research in TME characterisation and precision oncology.