Development of an antibody panel for imaging mass cytometry to investigate cancer-associated fibroblast heterogeneity and spatial distribution in archival tissues

Abstract

AbstractIntroductionCancer associated fibroblast (CAF) mediated crosstalk with other tumor compartments is shown to modulate cancer development and progression. Conventionally, CAFs are known to promote tumorigenesis either by secreting a wide range of growth factors, cytokines, and chemokines or by remodeling the underlying stroma. However, recent studies aimed at depleting CAFs have shown progression to more aggressive tumors leading to poorer outcomes. This may be attributed to the heterogeneity and plasticity of CAFs. Understanding CAF heterogeneity has been limited due to the lack of specific markers. Imaging mass cytometry (IMC) can analyze up to 40 parameters at subcellular resolution simultaneously and hence it can be an ideal tool to unravel the heterogeneity of CAFs and decipher the role of various CAF subsets in cancer development and progression. The aim of this study was to develop a panel of antibodies (ABs) for IMC to identify different CAF phenotypes and their spatial distribution in tissues and other tumor components in formalin-fixed paraffin-embedded (FFPE) archival tissues.MethodsA comprehensive panel of ABs were chosen based on known literature on CAFs and other tumor and microenvironment-related markers. ABs were conjugated to rare earth metals using the protocol provided by the supplier (Fluidigm, CA, USA). FFPE tissue samples from different organs/lesions known to express certain markers were used for making control tissue microarrays (cTMAs) for verification of conjugated ABs. Immunohistochemistry (IHC) with metal-conjugated ABs was used as the “gold standard” for optimization of IMC. Tissue images were segmented using ImcSegmentationPipeline (Ilastik and CellProfiler). Downstream analysis of single-cell data was performed using a newly developed bioinformatics pipeline as well as ImaCytE, an existing software solution.ResultsThe final panel of ABs was decided based on a comparable performance of conjugated ABs validated using IHC and IMC. Different cell types were identified including various subsets of CAFs and immune cells using cell segmentation and unsupervised clustering.ConclusionThis study establishes an extensive panel of IMC ABs on archival FFPE tissue material for unravelling CAF heterogeneity. In addition, the panel described here, the downstream analysis and the newly developed pipeline can be readily applied to different tissue types in context of cancer research or other fibrotic disease.