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Review
. 2021 Jun;18(6):423-436.
doi: 10.1080/14789450.2021.1941893. Epub 2021 Jul 24.

Mass Spectrometry Imaging of Fibroblasts: Promise and Challenge

Peggi M Angel  1 , Denys Rujchanarong  1 , Sarah Pippin  1 , Laura Spruill  2 , Richard Drake  1
Affiliations
Review

Mass Spectrometry Imaging of Fibroblasts: Promise and Challenge

Peggi M Angel et al. Expert Rev Proteomics. 2021 Jun.

Abstract

Introduction: Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast signaling contributes to many disease states and extracellular matrix secreted by fibroblasts has been used to stratify patient by outcome, recurrence, and therapeutic resistance. Recent advances in imaging mass spectrometry allow access to single cell fibroblasts and their ECM niche within clinically relevant tissue samples.

Areas covered: We review biological and technical challenges as well as new solutions to proteomic access of fibroblast expression within the complex tissue microenvironment. Review topics cover conventional proteomic methods for single fibroblast analysis and current approaches to accessing single fibroblast proteomes by imaging mass spectrometry approaches. Strategies to target and evaluate the single cell stroma proteome on the basis of cell signaling are presented.

Expert opinion: The promise of defining proteomic signatures from fibroblasts and their extracellular matrix niches is the discovery of new disease markers and the ability to refine therapeutic treatments. Several imaging mass spectrometry approaches exist to define the fibroblast in the setting of pathological changes from clinically acquired samples. Continued technology advances are needed to access and understand the stromal proteome and apply testing to the clinic.

Keywords: Fibroblast; extracellular matrix; imaging mass spectrometry; imaging proteomics; mass spectrometry; proteomics; stroma; tissue.

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Conflict of interest statement

COI: The authors declare no potential conflicts of interest.

Figures

Figure 1.
Figure 1.
Heterogeneity in stromal patterning linked to fibroblast cells within a single tissue section. Tissue is a section of triple negative breast cancer stained by hematoxylin and eosin (H&E). The pink eosin stain highlights collagen stroma. Purple stain is for cell nuclei. A-D highlight specific areas where stromal organization changes by directionality, packing density (high density, darker pink; low density light pink, demonstrated in (A). Comparison of A and D demonstrates changes from “wavy” collagen stroma (A) to linearly packed collagen stroma (D).
Figure 2.
Figure 2.
The matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry workflow. A) Stained tissue sections (hematoxylin and eosin shown) are prepared by spraying enzymes and matrix (B) onto tissue sections. C) Imaging scanning uses a laser to collect data points at discrete x and y coordinates. D) Each data point is linked to a single pixel. E) A total average spectrum contains all signal across the tissue. F) Specific peaks can be explored to see changes in peak intensity across the tissue by image patterns.
Figure 3.
Figure 3.
A schematic of approaches that can be used towards imaging or profiling the fibroblast proteomic niche from global tissue patterns to single cell profiles. The fibroblast proteome is accessed for imaging analysis using enzyme specificity, examples include trypsin (cellular content), collagenase (extracellular matrix proteins) and PNGase F (N-linked glycans).
Figure 4.
Figure 4.. Integrated immunohistochemistry with MALDI IMS with staining and imaging done on the same tissue section.
A) staining for prolyl-4-hydroxylase 3 (P4HA3) that catalyzes collagen hydroxylated proline (HYP). Collagen HYP ultimately controls how collagen organizes within the tissue microenvironment and presents cell-recognition sites. B) Collagen targeted imaging done on the same section after P4HA3 staining. The peptide mapped belongs to COL1A1, amino acids 405–420. Site specific probabilities of HYP are given in parentheses. Notably, proline 418 is not hydroxylated, whereas proline 410 and 413 are hydroxylated. The combined staining and imaging experiments represent the potential for disease and cell specific understanding of collagen regulation. C) Location of the peptide (amino acids 405–420) maps to COL1A1 triple helical structure.
Figure 5.
Figure 5.. Histology directed workflow on immunostained tissue to understand the single cell fibroblast niche.
A) The tissue was stained for phosphatase and tensin homolog (PTEN) an oncogenic stroma regulator. The tumor displays heterogenous PTEN expression. B) Fibroblasts with low PTEN (high oncogenic signaling, n=50) and high PTEN (attenuated oncogenic signaling, n=50) were marked on the tissue. After preparing the same tissue prepared for ECM targeted proteomics, the MALDI laser is directed to specific fibroblast x,y coordinates for profiling, marked here by black or red circles. C) Example spectrum from targeting a single fibroblast with positive PTEN stain. D) Example spectrum from targeting a single fibroblast with no detectable PTEN stain. E) Example hydroxylated proline (HYP) modified COL1A2 peptide found with significantly higher expression in the PTEN positive fibroblasts compared to PTEN negative fibroblasts.
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