Multidimensional spatial profiling of immune landscapes in colorectal cancer across Consensus Molecular Subtypes

Marieke Ijsselsteijn

Department of pathology, Leiden University Medical Centre, Leiden, The Netherlands

Colorectal cancer (CRC) is a heterogeneous disease entity where groups with clinical relevance can be formed based on genetics, transcriptomic profiles and/or immune contextures. Most large studies have relied on single cell or bulk RNA sequencing to provide a comprehensive overview of the biological complexity of the CRC microenvironments and, specifically, to study immunity. However, our understanding of anti-tumour immune responses in CRC would be greatly enhanced when retaining information on (immune)_cell localization and cellular neighbourhoods. We applied a 40-marker imaging mass cytometry panel to characterise the microenvironment of colorectal cancers in a cohort stratified by consensus molecular subtypes (CMS). We observed large heterogeneity in immune and stromal cell content between the CMS as well as differences in cancer cell protein expression. CMS1 was characterized by prevalent immune infiltration and IDO1and HLA-DR expression on cancer cells, while CMS2 cancers were characterized by strong expression of B-catenin in cancer cells and relative low levels of immune infiltration. Interestingly, CMS4 exhibited relatively high amounts of CD4+ T-cells, M1-macrophages and dendritic cells, suggesting ongoing anti-tumour immune responses in these tumours. In contrast, these cancers also contained high numbers of PDPN+ fibroblasts, which have been associated with poor prognosis. The use of IMC allowed the investigation of the spatial relationship between cell types within the cancers and distinct neighbourhood relationships were observed across CMS, with the majority of unique relationships occurring in CMS1 and CMS2 subtypes. Finally, combining the previous and a TLS focused IMC panel, we identified that CD31+CD38+ plasmablasts occur in high frequencies in about half of the cancers, regardless of CMS status. Furthermore, the plasmablasts form neighbourhood relationships with other plasmablasts, B cells, CD4+ T cells, monocytes and M2-macrophages. Lastly, cancers with high numbers of plasmablasts exhibited increased transcription of immunoglobulins and IL12A compared to plasmablasts-low cancers, suggesting a pro-inflammatory environment which should be further investigated for future therapeutic interventions. Overall, the characterisation of the spatial tumour immune microenvironment  demonstrates the complexity and heterogeneity of immune responses in CRC and the potential to identify new aspects of CRC immunity.