Local, Quantitative Morphometry of Fibroproliferative Lung Injury using Laminin

Abstract

Investigations into the mechanisms of injury and repair in pulmonary fibrosis require consideration of the spatial heterogeneity inherent in the disease. Most scoring of fibrotic remodeling in preclinical animal models rely on the modified Ashcroft score, which is a semi-quantitative scoring rubric of macroscopic resolution. The obvious limitations inherent in manual pathohistological grading have generated an unmet need for unbiased, repeatable scoring of fibroproliferative burden in tissue. Using computer vision approaches on immunofluorescent imaging of the extracellular matrix (ECM) component laminin, we generate a robust and repeatable quantitative remodeling scorer (QRS). In the bleomycin lung injury model, QRS shows significant agreement with modified Ashcroft scoring with a significant Spearman coefficient r=0.768. This antibody-based approach is easily integrated into larger multiplex immunofluorescent experiments, which we demonstrate by testing the spatial apposition of tertiary lymphoid structures (TLS) to fibroproliferative tissue. The tool reported in this manuscript is available as a standalone application which is usable without programming knowledge.

Figure 1.. Basement Membrane Remodeling Phenotype in Bleomycin Lung Injury

(A) Representative micrographs of serial sagittal sections of a left mouse lung two weeks post bleomycin treatment. Histochemical staining for Masson’s trichrome (A, left) and immunofluorescence for collagen I (anti-Col1a1) alone (A,middle) and with laminin (anti-laminin) (A, right). (B) Expanded insets of micrographs, taken from healthy (B, top) and fibroproliferative (B, bottom) regions of the same section. Asterisk indicates the collagen-rich, laminin-negative cuff space surrounding large airways. (C) Higher resolution fluorescence micrographs of laminin from a serial section of the same lung. Field from healthy (C, top) and fibroproliferative (C, bottom) regions of lung parenchyma. Scale bars = 100 microns.

Figure 2. Validation of Quantitative Remodeling Scorer: Association of Computed Laminin Features to Histopathologic Ground Truth

(A) Diagrammatic representation of the lab workflow for sequential staining of single section for laminin and subsequently Masson’s trichrome. (B) Two scoring modalities performed on the immunofluorescence and brightfield acquisitions, respectively. Immunofluorescence scoring (B, Top) is performed on subsampled tiles of the larger image, and consists of multiple independent computational measures on per-pixel values and multi-pixel relationships within each tile. Tiles are color-coded yellow based on their score, with higher yellow values indicating higher score. Histopathologic scoring via Modified Ashcroft (B, Bottom) is performed on simulated 10x Fields of View (FOV). Each field is given a mean score from four independent, blinded scorers. (C, top) An overlay of the brightfield trichrome image with the computed laminin feature map. (C, bottom left) Volcano plot of the r and p values for the spearman correlation testing on the various computed laminin features against the mean Ashcroft score (C, bottom right) The simple linear regression of the highest scoring laminin feature chosen for the quantitative remodeling scorer, Mean Peak Width (MPW) is shown. n = 10 lungs, with 9 or 10 random fields selected per lung (98 fields total) for histopathologic scoring and subsequent comparison and regression analysis.

Figure 3.. Quantitative Remodeling Scorer (QRS) Enables Testing of Spatial Hypotheses: Fibroproliferative Tissue Associate with Tertiary Lymphoid Structures (TLS).

(A) Representative micrograph of multicolor immunofluorescence panel + QRS heatmap. Nuclei (NucGreen), B Cells (anti B220), laminin (anti-Laminin) and QRS heatmap (computationally derived from laminin) are visible as single channels (A, left), and composite overlay (A, right). White triangles indicate tertiary lymphoid structures. Scale bar = 100 microns. (B) Parametrized tiles visualized using standard cytometric software, with the field from (A) bounded in red. Heatmap of whole section QRS (B, top). Distance field indicating distance from any tile to the nearest TLS (B, bottom). (C) Plot of the mean QRS at a given distance to the nearest TLS (in 25 micron bins). Individual colored lines represent individual lungs, black shaded line is the overall mean + SEM. (D) Histogram of tissue QRS, with regions gated as ‘Healthy’ and ‘Fibrotic’ at a QRS of 42. (E) Plot of the fraction of all tissue scoring fibrotic (QRS > 42) at a given distance from the nearest TLS. Individual colored lines represent, black shaded line is the overall mean + SEM.) (F) Scored lung sections split into ‘Healthy’ (QRS < 42) or ‘Fibrotic’ (QRS > 42). Plotted median distance of ‘Healthy’ or ‘Fibrotic’ tissue from nearest TLS. Scored lung sections are paired. Bars indicate the mean within each group. (n = 9, Wilcoxon matched-pairs signed rank test. **p < 0.01)