Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues

Abstract

IntroductionCancer 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.

Development of 42 marker panel for in-depth study of cancer associated fibroblast niches in breast cancer using imaging mass cytometry

“…While recently published CAF-focused studies performed using IMC platform report detection of various CAF populations ( 30 , 50 , 51 ), in our work we were able to identify a broader spectrum of different CAF subsets. Differently to the panel reported by Tornaas et al.…”

“…Activation of TGF-b pathway in tumor stroma is known to increase production of collagen rich extracellular matrix, to induce expression of FAP and aSMA, to mediate crosstalk between CAFs and cancer cells and therefore support CAF governing of stemness, EMT, proliferation, metastasis and chemoresistance, and is generally indicative of bad prognosis ( 65 – 70 ). Although our panel had a smaller number of CAF associated markers in comparison with the panel reported by Tornaas et al., ( 30 ) focusing on 6 CAF markers allowed implementation of more extensive set of immune and cancer cell markers, enabling more thorough CAF niche profiling.…”

“…Differently to the panel reported by Tornaas et al. ( 30 ), where the authors used higher number of CAF associated markers, we focused our panel on aSMA, FAP, PDGFRa and PDGFRb, which have been proven useful in the previous studies for classification of different CAF populations with demonstrated prognostic values ( 11 , 52 ). Driven by our previous study ( 52 ), we have reasons to believe that subclassifying CAFs by their expression profiles of these four markers holds significant prognostic value and that this classification approach could be complemental to functional CAF classification described in literature ( 53 , 54 ).…”

“…Although there are several methodological studies published recently reporting the various antibody panels developed for IMC use ( 3 – 5 , 29 , 30 ), almost all of these studies (aside from study by 30 ) are focused primarily on the immune contexture of the TME and largely neglect the mesenchymal component of the TME. In this paper, we are reporting a panel that can be used for a thorough CAF niche-oriented study of breast cancer TME in relation to both immune and breast cancer cell communications.…”