The integration of spatial omics technologies can provide important insights into the biology of tissues. Here we combined mass spectrometry imaging-based metabolomics and imaging mass cytometry-based immunophenotyping on a single tissue section to reveal metabolic heterogeneity at single-cell resolution within tissues and its association with specific cell populations such as cancer cells or immune cells. This approach has the potential to greatly increase our understanding of tissue-level interplay between metabolic processes and their cellular components.

Brain iron accumulation has been found to accelerate disease progression in amyloid-β(Aβ) positive Alzheimer patients, though the mechanism is still unknown. Microglia have been identified as key players in the disease pathogenesis, and are highly reactive cells responding to aberrations such as increased iron levels. Therefore, using histological methods, multispectral immunofluorescence and an automated in-house developed microglia segmentation and analysis pipeline, we studied the occurrence of iron-accumulating microglia and the effect on its activation state in human Alzheimer brains. We identified a subset of microglia with increased expression of the iron storage protein ferritin light chain (FTL), together with increased Iba1 expression, decreased TMEM119 and P2RY12 expression. This activated microglia subset represented iron-accumulating microglia and appeared morphologically dystrophic. Multispectral immunofluorescence allowed for spatial analysis of FTL⁺Iba1⁺-microglia, which were found to be the predominant Aβ-plaque infiltrating microglia. Finally, an increase of FTL⁺Iba1⁺-microglia was seen in patients with high Aβ load and Tau load. These findings suggest iron to be taken up by microglia and to influence the functional phenotype of these cells, especially in conjunction with Aβ.

Imaging mass cytometry (IMC) allows the detection of multiple antigens (approximately 40 markers) combined with spatial information, making it a unique tool for the evaluation of complex biological systems. Due to its widespread availability and retained tissue morphology, formalin-fixed, paraffin-embedded (FFPE) tissues are often a material of choice for IMC studies. However, antibody performance and signal to noise ratios can differ considerably between FFPE tissues as a consequence of variations in tissue processing, including fixation. In contrast to batch effects caused by differences in the immunodetection procedure, variations in tissue processing are difficult to control. We investigated the effect of immunodetection-related signal intensity fluctuations on IMC analysis and phenotype identification, in a cohort of 12 colorectal cancer tissues. Furthermore, we explored different normalization strategies and propose a workflow to normalize IMC data by semi-automated background removal, using publicly available tools. This workflow can be directly applied to previously acquired datasets and considerably improves the quality of IMC data, thereby supporting the analysis and comparison of multiple samples.

Cancers are characterized by extensive heterogeneity that occurs intratumorally, between lesions, and across patients. To study cancer as a complex biological system, multidimensional analyses of the tumor microenvironment are paramount. Single-cell technologies such as flow cytometry, mass cytometry, or single-cell RNA-sequencing have revolutionized our ability to characterize individual cells in great detail and, with that, shed light on the complexity of cancer microenvironments. However, a key limitation of these single-cell technologies is the lack of information on spatial context and multicellular interactions. Investigating spatial contexts of cells requires the incorporation of tissue-based techniques such as multiparameter immunofluorescence, imaging mass cytometry, or in situ detection of transcripts. In this Review, we describe the rise of multidimensional single-cell technologies and provide an overview of their strengths and weaknesses. In addition, we discuss the integration of transcriptomic, genomic, epigenomic, proteomic, and spatially-resolved data in the context of human cancers. Lastly, we will deliberate on how the integration of multi-omics data will help to shed light on the complex role of cell types present within the human tumor microenvironment, and how such system-wide approaches may pave the way toward more effective therapies for the treatment of cancer.

Objective: A comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC. Design: Thirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence. Results: We discovered that a previously unappreciated innate lymphocyte population (Lin^-CD7⁺CD127^-CD56⁺CD45RO⁺) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103⁺CD69⁺) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδT cells with highly similar activated (HLA-DR⁺CD38⁺PD-1⁺) phenotypes. Remarkably, activated γδT cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδT cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes. Conclusion: This work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.

Background: The clinical benefit of immunotherapeutic approaches against cancer has been well established although complete responses are only observed in a minority of patients. Combination immunotherapy offers an attractive avenue to develop more effective cancer therapies by improving the efficacy and duration of the tumor-specific T-cell response. Here, we aimed at deciphering the mechanisms governing the response to PD-1/PD-L1 checkpoint blockade to support the rational design of combination immunotherapy. Methods: Mice bearing subcutaneous MC-38 tumors were treated with blocking PD-L1 antibodies. To establish high-dimensional immune signatures of immunotherapy-specific responses, the tumor microenvironment was analyzed by CyTOF mass cytometry using 38 cellular markers. Findings were further examined and validated by flow cytometry and by functional in vivo experiments. Immune profiling was extended to the tumor microenvironment of colorectal cancer patients. Results: PD-L1 blockade induced selectively the expansion of tumor-infiltrating CD4⁺ and CD8⁺ T-cell subsets, co-expressing both activating (ICOS) and inhibitory (LAG-3, PD-1) molecules. By therapeutically co-targeting these molecules on the T_AI cell subsets in vivo by agonistic and antagonist antibodies, we were able to enhance PD-L1 blockade therapy as evidenced by an increased number of T_AI cells within the tumor micro-environment and improved tumor protection. Moreover, T_AI cells were also found in the tumor-microenvironment of colorectal cancer patients. Conclusions: This study shows the presence of T cell subsets in the tumor micro-environment expressing both activating and inhibitory receptors. These T_AI cells can be targeted by combined immunotherapy leading to improved survival.