Early detection and staging of chronic liver diseases with a protein MRI contrast agent

Abstract

Early diagnosis and noninvasive detection of liver fibrosis and its heterogeneity remain as major unmet medical needs for stopping further disease progression toward severe clinical consequences. Here we report a collagen type I targeting protein-based contrast agent (ProCA32.collagen1) with strong collagen I affinity. ProCA32.collagen1 possesses high relaxivities per particle (r₁ and r₂) at both 1.4 and 7.0 T, which enables the robust detection of early-stage (Ishak stage 3 of 6) liver fibrosis and nonalcoholic steatohepatitis (Ishak stage 1 of 6 or 1 A Mild) in animal models via dual contrast modes. ProCA32.collagen1 also demonstrates vasculature changes associated with intrahepatic angiogenesis and portal hypertension during late-stage fibrosis, and heterogeneity via serial molecular imaging. ProCA32.collagen1 mitigates metal toxicity due to lower dosage and strong resistance to transmetallation and unprecedented metal selectivity for Gd³⁺ over physiological metal ions with strong translational potential in facilitating effective treatment to halt further chronic liver disease progression.

Fig. 1

Development of ProCA32.collagen1 and its biophysical characteristics. a The model structure and development of ProCA32.collagen1 by engineering collagen type I targeting moiety (GGGKKWHCYTYFPHHYCVYG, red) at C-terminal of ProCA32 using a flexible hinge (green) and PEGylation. b Measurement of the dissociation constant of ProCA32.collagen1 to collagen type I using indirect ELISA assay. No collagen I binding was observed for ProCA32 (PEGylated, nontargeted agent). c The relaxation rates change of clinical contrast agents (black diamond Eovist; black circle Magnevist; white square MultiHance; white triangle Omniscan; blue square ProHance; yellow circle Dotarem) and red lozenge ProCA32.collagen1 in the presence of ZnCl₂ at different time points up to 4 days. d Pharmacokinetic of ProCA32.collagen1. Gd³⁺ concentration in serum collected after injection of ProCA32.collagen1 was determined by ICP-OES. Error bars indicate standard deviations of six separate measurements in n = 6, biologically independent animals

Fig. 2

Early stage detection of liver fibrosis in TAA/Alcohol model. a R1 maps and ΔR1 values of normal (Ishak stage 0 of 6), early-stage (Ishak stage 3 of 6), and late-stage (Ishak stage 5 of 6) liver fibrosis before and 24 h after injection of ProCA32.collagen1, and ProCA32. b ΔR2 values derived from T2 maps demonstrating the highest enhancement for late-stage liver fibrosis at 24 h post injection. c ProCA32.collagen1 can distinguish early-stage liver fibrosis from normal and late-stage fibrotic liver with ΔR1 derived from a R1 map at 24 h time point. d Correlation between Gd³⁺ concentration in liver with Ishak score at 24 h time point. e Percent injection dosage of ProCA32.collagen1 based on [Gd³⁺] in fibrotic and normal livers at 24 h time point. f Sirius red, H&E and αSMA staining of early- and late-stage fibrotic liver compared to normal liver confirms the stage of liver fibrosis. scale bar, 100 µm, *P < 0.05, student’s t test, all data are represented as mean ± SD, n = 4 biologically independent animals in each group

Fig. 3

Early stage detection of NASH in NASH diet model. a R1 maps of NASH liver (Ishak stage 1 of 6 or Mild-1A in NASH CRN system), late-stage liver fibrosis (Ishak stage 5 of 6 or 3 in NASH CRN system), and normal liver (Ishak stage 0 of 6) before and 24 h after injection of ProCA32.collagen1. b ΔR2 values derived from T2 map are consistent with ΔR1 values showing the highest enhancement for late-stage liver fibrosis. c ProCA32.collagen1 can distinguish early-stage NASH from normal and late-stage fibrotic liver with ΔR1 at 24 h time point. d Correlation between Gd³⁺ concentration in liver with Ishak scores at 24 h time point. e Percent injection dosage of ProCA32.collagen1 based on [Gd³⁺] in NASH, late-stage fibrotic and normal liver at 24 h time point. f αSMA staining of early-stage NASH and late-stage fibrosis demonstrates the degree of steatosis which is much higher in late-stage fibrosis (scale bar, 50 µm). g Sirius red, and H&E staining of early-stage NASH compared to normal liver demonstrate the presence of steatosis and collagen, as indications of the disease stage. scale bar, 100 µm; ^*P < 0.05, unpaired two-tailed student’s t test; all data are represented as mean ± SD, n = 4 biologically independent animals in each group

Fig. 4

Detection of portal hypertension during liver cirrhosis in TAA/alcohol- and NASH diet-induced cirrhosis. a R1 map and ΔR1 values of early-stage (Ishak stage 3 of 6), late-stage (Ishak stage 5 of 6), and normal liver (Ishak stage 0 of 6) before and 3 h after injection of ProCA32.collagen1 and Eovist (30 min) in TAA/alcohol model. b R1 changes of liver over different MRI time points after injection of ProCA32.collagen1, ProCA32, and Eovist in TAA/Alcohol model (P < 0.05, paired student’s t test). c R1 map and ΔR1 values of NASH (Ishak 1 of 6), late-stage (Ishak stage 5 of 6), and normal liver (Ishak stage 0 of 6) before and 3 h after injection of ProCA32.collagen1 and Eovist (30 min) in NASH diet model. d R1 changes of liver over different time points after injection of ProCA32.collagen1 and Eovist in NASH diet model (P < 0.05, paired student’s t test). e Representative SEM images of sections from mice with late-stage liver fibrosis in TAA/alcohol model. Quantitation of number and size of fenestrations of liver sinusoids in mice with late-stage liver fibrosis measured by manually counting/measuring number and the diameters of fenestration in the SEM images (scale bar, 500 nm). f Velocity of portal vein blood flow as measured by Doppler ultrasound imaging shows high-portal hypertension detected at 3 h after injection of ProCA32.collagen1 in late-stage liver fibrosis in TAA/alcohol model. g Representative images of IHC stains of CD31 and quantitation of CD31 IHC stains of liver tissue in mice with late-stage liver fibrosis in TAA/alcohol model confirming intrahepatic angiogenesis. scale bar, 100 µm; *P < 0.05, ***P < 0.001, unpaired two-tailed student’s t test; all data are represented as mean ± SD, n = 4 biologically independent animals in each group

Fig. 5

Demonstration of time-dependent mapping of collagen heterogeneity in DEN model. a T1-, T2-weighted, and T1-inversion recovery images of DEN-induced diseased liver before and 3 and 24 h post injection of ProCA32.collagen1 demonstrating collagen heterogeneity of liver in Area 2 (A2). b Sirius red staining and CPA analysis confirmed that A2 has more collagen than A1 which correlates with MRI (scale bar, 100 µm). c T1-, T2-weighted, and T1-inversion recovery images all demonstrated a higher contrast to noise ratio (CNR) in A2 of liver 3 h after ProCA32.collagen1 injection compared to A1. Data are represented as mean ± SD, n = 4 independent images. d Percentage of relative enhancement and quantitative voxel analysis of T1-inversion recovery images demonstrating liver regions enhanced at 3 and 24 h after injection of ProCA32.collagen1 compared to pre-injection (color scale represents number of voxels). e Percentage of relative enhancement and quantitative voxel analysis of T2-weighted images of liver demonstrating regions enhanced at 3 and 24 h after injection of ProCA32.collagen1 compared to pre-injection (color scale represents number of voxels)

Fig. 6

Assessment of contrast agent uptake and staging of NASH and liver cirrhosis in NASH diet model. a, b Area under the curve (AUC_0–48 R1 and R2) analysis with ProCA32.collagen1 enhanced MR in NASH diet model. c, d Area under the curve (AUC_3–48 R1 and R2) analysis demonstrates the washout rate of ProCA32.collagen1 3 h post injection in late-stage fibrosis based on decreasing R1 and R2 values and negative value of AUC compared to normal liver and early stage fibrotic liver. e AUC_3–48 h boxplot analysis demonstrating the ability of ProCA32.collagen1 to distinguish normal vs. early stage NASH and early stage NASH vs. late-stage fibrosis in NASH diet model (P < 0.01, unpaired two-tailed student’s t test; the midline is the median of the data, with the upper and lower limits of the box being the third and first quartile, 75th and 25th percentile, respectively. The whiskers extends up to 1.5 times the interquartile range and show the minimum and maximum as they are all within that distance). f Scatter-plots of Logit-transformed CPA against AUC_0–48 showed that AUC_0–48 and CPA levels are well correlated in NASH diet model. P < 0.001, unpaired two-tailed student’s t test; all data are represented as mean ± SD, n = 4 biologically independent animals in each group

Fig. 7

Human translational potential of ProCA32.collagen1. a ProCA32.collagen1 enhanced MRI can demonstrate different collagen distribution in DEN-, NASH diet-, and TAA/alcohol-induced models. b Schematic illustration of ProCA32.collagen1 distribution and suggested mechanism in different stages of fibrosis. c Demonstration of three hepatocellular carcinoma (HCC) and normal human liver tissue microarray stained with Sirius red (red) and ProCA32.collagen1 (brown) exhibiting the strong binding of the contrast agent to collagen human patient tissue (all scale bars, 100 µm)