Plasma Kallikrein-Activated TGF-β Is Prognostic for Poor Overall Survival in Patients with Pancreatic Ductal Adenocarcinoma and Associates with Increased Fibrogenesis

Rasmus S Pedersen, Neel I Nissen, Christina Jensen, Jeppe Thorlacius-Ussing, Tina Manon-Jensen, Majken L Olesen, Lasse L Langholm, Hadi M H Diab, Lars N Jorgensen, Carsten P Hansen, Inna M Chen, Julia S Johansen, Morten A Karsdal, Nicholas Willumsen, Rasmus S Pedersen, Neel I Nissen, Christina Jensen, Jeppe Thorlacius-Ussing, Tina Manon-Jensen, Majken L Olesen, Lasse L Langholm, Hadi M H Diab, Lars N Jorgensen, Carsten P Hansen, Inna M Chen, Julia S Johansen, Morten A Karsdal, Nicholas Willumsen

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a hard-to-treat cancer due to the collagen-rich (fibrotic) and immune-suppressed microenvironment. A major driver of this phenomenon is transforming growth factor beta (TGF-β). TGF-β is produced in an inactive complex with a latency-associated protein (LAP) that can be cleaved by plasma kallikrein (PLK), hereby releasing active TGF-β. The aim of this study was to evaluate LAP cleaved by PLK as a non-invasive biomarker for PDAC and tumor fibrosis. An ELISA was developed for the quantification of PLK-cleaved LAP-TGF-β in the serum of 34 patients with PDAC (stage 1−4) and 20 healthy individuals. Biomarker levels were correlated with overall survival (OS) and compared to serum type III collagen (PRO-C3) and type VI collagen (PRO-C6) pro-peptides. PLK-cleaved LAP-TGF-β was higher in patients with PDAC compared to healthy individuals (p < 0.0001). High levels (>median) of PLK-cleaved LAP-TGF-β were associated with poor OS in patients with PDAC independent of age and stage (HR 2.57, 95% CI: 1.22−5.44, p = 0.0135). High levels of PLK-cleaved LAP-TGF-β were associated with high PRO-C3 and PRO-C6, indicating a relationship between the PLK-cleaved LAP-TGF-β fragment, TGF-β activity, and tumor fibrosis. If these preliminary results are validated, circulating PLK-cleaved LAP-TGF-β may be a biomarker for future clinical trials.

Trial registration: ClinicalTrials.gov NCT03311776.

Keywords: ECM remodeling; LAP-TGF-β; PDAC; TGF-β; TGF-β activation; plasma kallikrein; serum biomarker; tumor fibrosis; tumor microenvironment.

Conflict of interest statement

R.S.P., H.M.H.D., L.N.J., C.P.H., I.M.C. and J.S.J. have no conflict of interest to declare. N.I.N., C.J., J.T.-U., T.M.-J., M.L.O., L.L.L., M.A.K. and N.W. are employed at Nordic Bioscience, and are involved in the discovery and development of serological biomarkers. T.M.-J., M.A.K., L.L.L. and N.W. own stocks in Nordic Bioscience.

Figures

Figure 1
Figure 1
Illustration of plasma kallikrein (PLK)-mediated TGF-β activation: TGF-β in its latent state in complex with latency-associated peptide (LAP) (LAP-TGF-β) is bound to the extracellular matrix (ECM) via latent TGF-β binding protein (LTBP). PLK cleaves LAP between R58 and L59, resulting in the release of active TGF-β and the PLK-cleaved LAP-TGF-β fragment. The active TGF-β will induce multiple biological functions, including tumor progression, metastasis, and collagen formation, leading to tumor fibrosis. The PLK-cleaved LAP-TGF-β fragment is released into circulation, and as a result, becomes detectable in serum and plasma samples through antibody binding.
Figure 2
Figure 2
Assay specificity towards PLK-cleaved LAP-TGF-β: (a) Inhibition curve for the selection peptide (LASPPSQGEV), elongated peptide (RLASPPSQGEV), truncated peptide (ASPPSQGEV), non-sense selection peptide (PNASPLLGS), and a non-sense coater peptide (YPNASPLLGS-K-(Biotin)). The peptides were diluted in 2-fold dilution series starting from 25 ng/mL. (b) PLK-cleaved LAP-TGF-β measured after one hour incubation of LAP-TGF-β with or without kallikrein.
Figure 3
Figure 3
Biomarker levels of PLK-cleaved LAP-TGF-β in matched serum and citrate plasma samples: (a) Matched samples are connected by lines. (b) Correlation between the serum and plasma PLK-cleaved LAP-TGF-β levels shown in (a) was tested with Pearson test; Pearson’s r (r) and p-values are shown. The line represents simple linear regression.
Figure 4
Figure 4
Diagnostic potential of PLK-cleaved LAP-TGF-β: (a) Serum levels of PLK-cleaved LAP-TGF-β in healthy individuals (n = 20) and patients with PDAC (n = 34). Black lines indicate median values. Patients in stages 1, 2, 3, and 4 have shape and color codes being green square, purple diamonds, light blue triangles, and orange circles, respectively. (b) Receiver operating characteristic curve for the patients and healthy individual mentioned in (a), area under curve (AUC).
Figure 5
Figure 5
Prognostic properties of PLK-cleaved LAP-TGF-β: Kaplan-Meier survival curves showing the association between serum PLK-cleaved LAP-TGF-β levels (dichotomized into low (median (orange curve)), and overall survival for patients with PDAC. Median survival and log-rank test are shown.
Figure 6
Figure 6
PLK-cleaved LAP-TGF-β levels associate with collagen formation: Biomarker levels of PRO-C3 (a) and PRO-C6 (b) from patients with PDAC dichotomized into low PLK-cleaved LAP-TGF-β levels (<median, n = 17) and high PLK-cleaved LAP-TGF-β levels (>median, n = 17). The groups were compared using Mann-Whitney test; p-value is shown.

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