Multiparametric analysis of colorectal cancer immune responses

Abstract

Colorectal cancer (CRC) is a heterogeneous disease, with a diverse and plastic immune cell infiltrate. These immune cells play an important role in regulating tumour growth - progression or elimination. Some populations of cells have a strong correlation with disease-free survival, making them useful prognostic markers. In particular, the infiltrate of CD3⁺ and CD8⁺ T cells into CRC tumours has been validated worldwide as a valuable indicator of patient prognosis. However, the heterogeneity of the immune response, both between patients with tumours of different molecular subtypes, and within the tumour itself, necessitates the use of multiparametric analysis in the investigation of tumour-specific immune responses. This review will outline the multiparametric analysis techniques that have been developed and applied to studying the role of immune cells in the tumour, with a focus on colorectal cancer. Because much of the data in this disease relates to T cell subsets and heterogeneity, we have used T cell populations as examples throughout. Flow and mass cytometry give a detailed representation of the cells within the tumour in a single-cell suspension on a per-cell basis. Imaging technologies, such as imaging mass cytometry, are used to investigate increasing numbers of markers whilst retaining the spatial and structural information of the tumour section and the infiltrating immune cells. Together, the analyses of multiple immune parameters can provide valuable information to guide clinical decision-making in CRC.

Keywords: Colorectal cancer; Flow cytometry; Immune cells; Immunohistochemistry; Mass cytometry; Microscopy; Multiparametric analysis.

Figure 1

Diagram summarising the methodology and analysis techniques involved in multiparametric analysis of immune cells. Briefly, tissues of interest are taken and either dissociated into a single cell suspension or mounted onto slides. Cells in suspension are stained with fluorescently labelled antibodies for flow cytometry, and then acquired using a flow cytometer. This data is often presented as populations positive or negative for 2 markers. For mass cytometry, cells are stained with antibodies labelled with heavy metal isotopes, and then acquired using a mass cytometer. The high dimensionality of this data means that clustering analyses are preferred to analyse this data. For imaging techniques, the tissue slides are also labelled with antibodies, and can be imaged using IHC, IFM, IMC or MIBI. Imaging techniques enable location of immune cells in situ, as well as enumeration of cell types. IMC and MIBI use more parameters, meaning more cell types can be distinguished, and clustering algorithms can be used on this data too, and related back to the geographical location of each cell. IHC: Immunohistochemistry; IFM: Immunofluorescent microscopy; IMC: Imaging mass cytometry; MIBI: Multiplex ion beam imaging.