Quantitative Image Analysis of Fibrillar Collagens Reveals Novel Diagnostic and Prognostic Biomarkers and Histotype-dependent Aberrant Mechanobiology in Lung Cancer.

Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). Yet, the potential of collagen fiber descriptors as a source of clinically-relevant biomarkers in NSCLC is mainly unknown. Likewise, our understanding of the aberrant collagen organization and associated tumor-promoting effects needs to be better defined. To address these limitations, we identified a digital pathology approach that can be easily implemented in pathology units based on the Curvelet Transform filtering and single Fiber Reconstruction (CT-FIRE) software analysis of picrosirius (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE settings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas (ADC), 89 squamous cell carcinomas (SCC)). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve AUC = 0.92) and fiber density as the single descriptor consistently associated with poor prognosis in both ADC and SCC independently of the gold standard based on tumor size, lymph node involvement and metastasis (TNM) staging (Hazard ratio HR = 2.69 (1.55-4.66), p < 0.001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumors compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (alpha-smooth muscle actin (α-SMA)), and immune evasion (programmed death-ligand 1 (PD-L1)). Likewise, transcriptional correlation analysis supported the potential involvement of the major Yes-associated protein 1 (YAP)/TAZ mechanobiology pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of surgical NSCLC patients. Our findings also unveil an aberrant stiff microenvironment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities.