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

Abstract

Imaging Mass Cytometry (IMC) is a novel, and formidable high multiplexing imaging method emerging as a promising tool for in-depth studying of tissue architecture and intercellular communications. Several studies have reported various IMC antibody panels mainly focused on studying the immunological landscape of the tumor microenvironment (TME). With this paper, we wanted to address cancer associated fibroblasts (CAFs), a component of the TME very often underrepresented and not emphasized enough in present IMC studies. Therefore, we focused on the development of a comprehensive IMC panel that can be used for a thorough description of the CAF composition of breast cancer TME and for an in-depth study of different CAF niches in relation to both immune and breast cancer cell communication. We established and validated a 42 marker panel using a variety of control tissues and rigorous quantification methods. The final panel contained 6 CAF-associated markers (aSMA, FAP, PDGFRa, PDGFRb, YAP1, pSMAD2). Breast cancer tissues (4 cases of luminal, 5 cases of triple negative breast cancer) and a modified CELESTA pipeline were used to demonstrate the utility of our IMC panel for detailed profiling of different CAF, immune and cancer cell phenotypes.

Keywords: breast cancer; cancer associated fibroblasts; imaging mass cytometry; microniches; tumor microenvironment.

Figure 1

Comparative side by side representation of IMC and IHC staining patterns. Image pairs consist of IMC-acquired images presented to the left, and IHC micrographs presented to the right, both obtained from the same cores of the “test-TMA”. Positive antibody staining is visualized with red in the IMC images, and brown (DAB) in the IHC images. Blue color corresponds to nuclear staining. Cl Caspase 3, Cleaved Caspase 3; PanCK, Pan Cytokeratin. Each scale bar corresponds to 100 µm.

Figure 2

Quantitative analysis of IMC staining. (A) IHC/IMC side-by-side image representation. Each scale bar corresponds to 100 µm. (B) Scatterplots showing associations of mean signal intensity between IHC and IMC staining for given antibody. (C) Scatterplots showing associations of positive cell detection between IHC and IMC staining for given marker. (D) Bland-Altman plots showing IMC/IHC concordance in detection of cells positive for a given marker.

Figure 3

Representation of “test-TMA” IMC staining Images show staining patterns of the level 1 classification markers: Vimentin (red), PanKeratin (PanCK) (green), CD31/34 (blue), and CD45 (turquoise). Lum, Luminal; TN, Triple-negative and HER2, HER2-enriched. Each scale bar corresponds to 100 µm. LN, lymph node; LN met, metastatic lymph node.

Figure 4

Representation of “pilot-TMA” IMC staining. Images show staining patterns of the level 1 classification markers: Vimentin (red), PanKeratin (PanCK) (green), CD31/34 (blue), and CD45 (turquoise). Lum, Luminal; TN, Triple-negative and HER2, HER2-enriched. Each scale bar corresponds to 100 µm.

Figure 5

Pseudo images showing cell phenotypes identified using stepwise CELESTA approach. (A) Representation of the main cell classes identified in the first step. (B) Representation of different CAF phenotypes identified in the second step. Label “fibroblasts und” corresponds to CAFs with undefined phenotypes (C) Representation of different immune phenotypes identified in the second step. Label “immune cells und” corresponds to immune cells with undefined phenotypes (D) Representation of different cancer phenotypes identified in the second step. Label “tumor cells und” corresponds to tumor cells with undefined phenotypes. Lum, Luminal and TN, Triple-negative.

Figure 6

Relative abundance of identified cell phenotypes demonstrated per core, per case and per subtype. (A) Relative abundance of identified main cell classes. (B) Relative abundance of identified CAF phenotypes. Label “fibroblasts und” corresponds to CAFs with undefined phenotypes (C) Relative abundance of identified immune phenotypes. Label “immune cells und” corresponds to immune cells with undefined phenotypes (D) Representation of identified cancer phenotypes. Label “tumor cells und” corresponds to tumor cells with undefined phenotypes. Lum, Luminal and TN, Triple-negative.