Glypican-3-targeted precision diagnosis of hepatocellular carcinoma on clinical sections with a supramolecular 2D imaging probe

Abstract

The ability of chemical tools to effectively detect malignancy in frozen sections removed from patients during surgery is important for the timely determination of the subsequent surgical program. However, current clinical methods for tissue imaging rely on dye-based staining or antibody-based techniques, which are sluggish and complicated. Methods: Here, we have developed a 2D material-based supramolecular imaging probe for the simple, rapid yet precise diagnosis of hepatocellular carcinoma (HCC). The 2D probe is constructed through supramolecular self-assembly between a water soluble, fluorescent peptide ligand that selectively targets glypican-3 (GPC-3, a specific cell-surface biomarker for HCC) and 2D molybdenum disulfide that acts as a fluorescence quencher as well as imaging enhancer. Results: We show that the 2D imaging probe developed with minimal background fluorescence can sensitively and selectively image cells overexpressing GPC-3 over a range of control cells expressing other membrane proteins. Importantly, we demonstrate that the 2D probe is capable of rapidly (signal became readable within 1 min) imaging HCC tissues over para-carcinoma regions in frozen sections derived from HCC patients; the results are in accordance with those obtained using traditional clinical staining methods. Conclusion: Compared to conventional staining methods, which are laborious (e.g., over 30 min is needed for antibody-based immunosorbent assays) and complex (e.g., diagnosis is based on discrimination of the nucleus morphology of cancer cells from that of normal cells), our probe, with its simplicity and quickness, might become a promising candidate for tumor-section staining as well as fluorescence imaging-guided surgery.

Keywords: 2D material; diagnostics; frozen section; glypican-3; hepatocellular carcinoma.

Figure 1

(A) Structure of the fluorophore-tagged peptide probe (P-probe) for glypican-3 (GPC-3). (B) High-resolution transmission electron microscopy images of 2D MoS₂, P-probe (inset) and 2D probe (P-probe/2D MoS₂ = 2 μM/40 μg mL^-1). The enlarged images show the surface structure of the materials in the blue frames of the images above. (C) Energy dispersive X-ray spectrometry mapping analysis of 2D MoS₂ and 2D probe.

Figure 2

(A) Raman spectra of 2D MoS₂ (40 μg mL^-1) and 2D probe (P-probe/2D MoS₂ = 2 μM/40 μg mL^-1). (B) Zeta potential of P-probe (2 μM), 2D MoS₂ (40 μg mL^-1) and 2D probe (P-probe/2D MoS₂ = 2 μM/40 μg mL^-1). (C) Dynamic light scattering of P-probe (2 μM), 2D MoS₂ (40 μg mL^-1) and 2D probe (P-probe/2D MoS₂ = 2 μM/40 μg mL^-1). (D) Fluorescence spectra of P-probe (1 μM) with increasing 2D MoS₂ (0-12.3 μg mL^-1; interval: 1.2 μg mL^-1). (E) Fluorescence spectra of 2D probe (P-probe/2D MoS₂ = 1 μM/5 μg mL^-1) with increasing GPC-3 (0-0.3 μM; interval: 0.03 μM). (F) Fluorescence change (where I₀ and I are the fluorescence intensity of 2D probe in the absence and presence of an analyte, respectively) of 2D probe (P-probe/2D MoS₂ = 1 μM/5 μg mL^-1) with GPC-3 (0.3 μM) or control analytes (from left to right: 1 μM of proteins including human serum albumin, bovine serum albumin, pepsin, immunoglobulin G, lysozyme, and 7.5 μM of ions including Na⁺, K⁺, Ca²⁺, Fe³⁺, Mg²⁺, I^-, Br^-, CO₃^2-, HCO₃^-, HSO₄^-, H₂PO₄^-). All fluorescence spectra were measured in phosphate buffered saline (0.01 M, pH 7.4) with excitation at 510 nm.

Figure 3

Fluorescence imaging (A) and quantification (C) of Hep-G2 (human hepatoma) cells in the presence of P-probe (20 μM) with increasing 2D MoS₂ (***P< 0.001 with respect to P-probe alone). Fluorescence imaging (B) and quantification (D) of HEK293T (human embryonic kidney) cells overexpressing different membrane proteins including glypican-3 (GPC-3), glypican-1 (GPC-1), glypican-5 (GPC-5), golgi membrane protein-1 (GOLM-1) and dickkopf-like-1 protein (DKK-1) in the presence of 2D probe (P-probe/2D MoS₂ = 20 μM/10 μg mL^-1) (p-Enter is the empty plasmid). (E) Western blot analysis of the expression level of different membrane proteins including GPC-3, GPC-1, GPC-5, GOLM-1 and DKK-1 in HEK293T cells after transfection. Viability of (F) Hep-G2 and (G) L02 (human liver) cells in the presence of increasing concentrations of P-probe and 2D MoS₂. All fluorescence images were recorded using an Operetta high-content imaging system (Perkinelmer, US) (excitation and emission channels used were 520-550 and 580-650 nm, respectively; cell nuclei were stained by Hoechst 33342), and quantified and plotted by Columbus analysis system (Perkinelmer, US).

Figure 4

(A) Fluorescence imaging of frozen tissue sections removed from HCC patients in the presence of 2D probe (P-probe/2D MoS₂ = 40 μM/20 μg mL^-1) with time. (B) Fluorescence imaging of four independent frozen tissue slides of HCC after incubation with 2D probe for 5 min. (C) Fluorescence imaging of four independent frozen tissue sections of para-carcinoma after incubation with 2D probe for 5 min. (D) Double-blind fluorescence imaging of different kinds of frozen tissue sections with 2D probe for 5 min (the dashed curves divide HCC-positive from para-carcinoma regions). Hoechst 33342 and H&E (hematoxylin-eosin) staining were used as reference methods. The fluorescence images in (A-C) were obtained on a fluorescence microscope (Olympus, Japan; excitation and emission channels used were 535-555 and 570-625 nm, respectively) and those in (D) on a confocal laser scanning microscope (Olympus, Japan; excitation and emission channels used were 512 and 570-590 nm, respectively).