Abstract 1899: Non-invasive quantification of cross-linked type III collagen products - A measure of extracellular matrix stiffness related to TGF-β signaling in colorectal cancer
Authors: Jensen Christina , Rønnow Sarah , Sand Jannie M. , Jørgensen Lars N. , Karsdal Morten A. , Willumsen Nicholas 
Published: August 2019

Abstract
Background: Extracellular matrix (ECM) deposition and stiffening have emerged as an important player in cancer progression. ECM deposition is stimulated by transforming growth factor-beta (TGF-β) that is being widely evaluated as a new therapeutic target in cancer. ECM stiffness is characterized by type III collagen deposition and cross-linking in the tumor microenvironment. Cross-linked type III collagen products reflecting ECM stiffness and TGF-β signaling may therefore have biomarker potential in cancer. By developing an assay to quantify cross-linked type III collagen pro-peptides and applying the “Scar-in-a-jar” (SiaJ) model, we evaluated the impact of TGF-β on this novel biomarker and assessed the biomarker potential in colorectal cancer (CRC).
Methods: A monoclonal antibody was raised against the N-terminal pro-peptide of type III collagen and a sandwich ELISA was developed and technically validated. The cross-linked type III collagen product levels were assessed in supernatants from the SiaJ culture model in which primary human healthy lung fibroblasts were cultured up to 12 days in the presence of ficoll and 1ng/ml TGF-β, with or without addition of 1µM ALK-5/type I TGF-β receptor kinase inhibitor (iTGF-β). The pathological relevance of cross-linked type III collagen products was assessed in serum from patients with CRC (n=34) and healthy controls (n=39). Levels were compared using unpaired, two-tailed Mann-Whitney test.
Results: The newly developed sandwich ELISA was shown to be technically robust and specific towards cross-linked type III collagen pro-peptides. Stimulating lung fibroblasts with TGF-β induced the production of cross-linked type III collagen with levels increasing up to 22-fold compared to without TGF-β (p=0.029), and iTGF-β reduced this induction. Levels of cross-linked type III collagen were significantly elevated in CRC patients compared to healthy controls (7.0 ng/ml vs. 5.6 ng/ml, p=0.0009). Furthermore, the diagnostic accuracy to separate CRC patients from healthy controls was 72% (p=0.0003, AUC: 0.72) with an odds ratio of 8 (95%Cl=2.4-23.2, p=0.0006) for having CRC.
Conclusions: Cross-linked pro-peptides of type III collagen can be quantified in serum from CRC patients as a potential surrogate marker of tissue stiffness and stromal reactivity driving tumor progression. In addition, TGF-β induces increased production of cross-linked type III collagen suggesting that this novel biomarker could potentially interrogate the effect of anti-TGF-β therapy. Future studies are needed to investigate the prognostic and predictive potential.
Citation Format: Christina Jensen, Sarah Rønnow, Jannie M. Sand, Lars N. Jørgensen, Morten A. Karsdal, Nicholas Willumsen. Non-invasive quantification of cross-linked type III collagen products - A measure of extracellular matrix stiffness related to TGF-β signaling in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1899.