Highly aligned stromal collagen is a negative prognostic factor following pancreatic ductal adenocarcinoma resection

Abstract

Risk factors for pancreatic ductal adenocarcinoma (PDAC) progression after surgery are unclear, and additional prognostic factors are needed to inform treatment regimens and therapeutic targets. PDAC is characterized by advanced sclerosis of the extracellular matrix, and interactions between cancer cells, fibrillar collagen, and other stromal components play an integral role in progression. Changes in stromal collagen alignment have been shown to modulate cancer cell behavior and have important clinical value in other cancer types, but little is known about its role in PDAC and prognostic value. We hypothesized that the alignment of collagen is associated with PDAC patient survival. To address this, pathology-confirmed tissues from 114 PDAC patients that underwent curative-intent surgery were retrospectively imaged with Second Harmonic Generation (SHG) microscopy, quantified with fiber segmentation algorithms, and correlated to patient survival. The same tissue regions were analyzed for epithelial-to-mesenchymal (EMT), α-SMA, and syndecan-1 using complimentary immunohistostaining and visualization techniques. Significant inter-tumoral variation in collagen alignment was found, and notably high collagen alignment was observed in 12% of the patient cohort. Stratification of patients according to collagen alignment revealed that high alignment is an independent negative factor following PDAC resection (p = 0.0153, multivariate). We also found that epithelial expression of EMT and the stromal expression of α-SMA and syndecan-1 were positively correlated with collagen alignment. In summary, stromal collagen alignment may provide additional, clinically-relevant information about PDAC tumors and underscores the importance of stroma-cancer interactions.

Keywords: collagen; microenvironment; pancreatic cancer; quantitative pathology; stroma.

Figure 1. Representative pancreatic tissues visualized with H&E staining and SHG microscopy

H&E stained tissues cores representing normal and distinct PDAC regions (low grade, high grade, tumor core, infilitrating edge) were visualized holistically with SHG stitching. For each core, 1-2 pathology-confirmed regions of interest (ROIs) containing malignant cells and associated stroma were marked to omit stroma associated with non-malignant tissue components. Yellow boxes indicate representative ROI regions that were extracted from whole TMA cores and subsequently quantified for collagen alignment.

Figure 2. Collagen alignment significantly varies between individual PDAC tumors

A. Different collagen alignment values for the entire 114 patient cohort. Data points highlighted in red represent highly aligned tumors. B. Kaplan-Meier analysis shows the post-surgical survival of patients with high collagen alignment is decreased. The median survival of each patient cohort was compared using the Log-rank test (p = 0.0062, univariate).

Figure 3. Co-localization of EMT-positive PDAC cells with stromal collagen alignment

A. Sections adjacent to the H&E tissues from which SHG was measured were immunofluorescently labeled for E-cadherin, vimentin, and DAPI. The same regions were scored for double positivity using inForm analysis software as an indication of EMT (yellow overlays). A greater proportion of cells express EMT in the context of highly aligned collagen. Scale bars = 100 μm B. Correlation of EMT-expressing PDAC cells with the degree of collagen alignment. EMT expression indicates the fraction of total PDAC cells double positive for E-cadherin and vimentin. A total of 315 confirmed PDAC tissues cores representative of 114 patients were analyzed. Spearman r = 0.116, *p = 0.039

Figure 4. Co-localization of CAF markers (α-SMA and syndecan-1) with stromal collagen alignment

A. Sections adjacent to the H&E tissues from which SHG was measured were immunohistochemically labeled for α-SMA and syndecan-1 and scored for stromal positivity. Both markers co-localize and orient along aligned collagen fibers. Scale bars = 100 μm B. Correlation of α-SMA expression with the degree of stromal collagen alignment in the periductal stroma of PDAC cells. Spearman r = 0.121, *p = 0.022. C. Correlation of syndecan-1 expression with the degree of stromal collagen alignment in the periductal stroma of PDAC cells. Spearman r = 0.115, *p = 0.029. A total of 356 confirmed PDAC tissues cores representative of 114 patients were analyzed.