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Abstract
Background Early-stage pT1 colorectal cancer (CRC) presents with regions of various degrees of malignant transformation (figure 1A) – ranging from normal colon tissue, transition areas, different degrees of dysplasia, to carcinoma – and can thus provide valuable new insights into the mechanisms of CRC initiation and progression. Indeed, recent evidence has highlighted transcriptional differences supporting an early onset of inflammatory responses during malignant transformation and marked alterations in immune cell composition between histologies.1 However, the exact multicellular interactions that underly these differences have yet to be elucidated. The central role of such spatial interactions in cancer is becoming increasingly evident, with several multicellular collectives now linked to immunotherapy outcomes.2 3 Characterizing the dynamics of these multicellular interaction networks during the early stages of CRC development will be crucial for improving our biological understanding of CRC, as well as identifying new (multi)cellular and molecular targets for immunotherapy and biomarker discovery.
Methods Here, we performed spatial transcriptomics on six human early-stage pT1 tumors that were resected by en bloc endoscopic submucosal dissection at Leiden University Medical Center. Spatial transcriptomics was performed on the Xenium (10x Genomics) platform using a probe set that we specifically designed to allow for the discrimination of distinct immune cell populations and their functional states in CRC. We validated our approach using CRC samples for which we ran Xenium and Imaging Mass Cytometry on the same slide (figure 1B). Cell segmentation, cell type annotation, and neighborhood analysis were performed using an in-house custom analysis pipeline.
Results Spatial profiling of pT1 tumors enabled us to investigate the variations in cellular behavior and spatial organization within multicellular communities across different stages of malignant transformation (figure 1C). Our analyses revealed marked differences between these histologies, identifying several types of multicellular communities, each characterized by unique cell types and transcriptional programs. Notably, several of these were found to originate in transition regions and to evolve during malignant transformation. This process was characterized by extensive remodeling of immune cell functionality and their spatial behavior within these multicellular communities, implying a role for these communities in CRC progression.
Conclusions Altogether, we demonstrate the power of spatial transcriptomics to dissect multicellular communities in cancer. We identified cellular neighborhoods that were altered during CRC tumorigenesis, implying their critical role in CRC initiation and progression. These multicellular interaction networks and their specific dynamics may offer new targets and biomarkers for CRC prevention.
References
J Roelands, et al. ‘Transcriptomic and immunophenotypic profiling reveals molecular and immunological hallmarks of colorectal cancer tumourigenesis,’ Gut, 2023-07-01;72(7). doi: 10.1136/gutjnl-2022-327608.
JH Chen, et al. ‘Human lung cancer harbors spatially organized stem-immunity hubs associated with response to immunotherapy,’ Nature Immunology, 2024-03-19; 25:4. doi: 10.1038/s41590-024-01792-2.
A Magen, et al. ‘Intratumoral dendritic cell–CD4+ T helper cell niches enable CD8+ T cell differentiation following PD-1 blockade in hepatocellular carcinoma.’ Nature Medicine 2023-06-15;29:6. doi: 10.1038/s41591-023-02345-0.
Ethics Approval This study was approved by the medical ethical committee METC Leiden-Den Haag-Delft (protocol B20.039 and B22.036).
Identifying and characterizing multicellular interaction networks in early-stage pT1 human colorectal cancer. (A). Schematic representation of the different histologies of pT1 CRC tumors. (B). Co-registration of IMC and Xenium data. (C). Spatial projection of cells, ranging from normal epithelium (top) to carcinoma (bottom)
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