Cryopreserved human HCC tissue samples
Tissue samples from the site of tumour and non-tumour liver parenchyma were harvested and cryopreserved from patients undergoing a curative liver resection at the University Hospital of Kyoto Prefectural University of Medicine (KPUM). A consort flow chart is provided in Figure 1a. The liver tumour data-base searched from January 2014 to December 2018 revealed 189 patients. Of these, 111 patients, from whom tissue samples could not be harvested, were excluded. Of the remaining 78 patients, those with metastatic liver cancer (n = 1), anisakiasis (n = 1), benign liver tumour (n = 3), cholangiocellular carcinoma (n = 4), and combined hepatocellular-cholangiocellular carcinoma (n = 1) were excluded. Finally, 68 consecutive patients were included in this study. The Institutional Review Board of KPUM examined and approved the study protocol (approval number: ERB-C-67) in accordance with the tenets of the Declaration of Helsinki. Written informed consent was acquired preoperatively from all patients. Among the 68 patients, alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKAII) could not be measured in four patients and two patients did not have ICG data.
Cell lines and culture
Four HCC cell lines and human umbilical vein endothelial cells (HUVECs) were used in this study: Hep-G2, HuH-7, and Li-7 cell lines (RIKEN Bioresource Center, Japan); PLC/PRF/5 cell line (JCRB Cell Bank, Japan); HUVECs (provided by T.K., Tokyo, Japan). HuH-7, Hep-G2, and PLC/PRF/5 cells were cultured in DMEM (Nacalai Tesque, Inc.,Japan). Li-7 cells were cultured in RPMI1640 (Nacalai Tesque) Both medium contained 10% heat-inactivated fetal bovine serum (Gibco, MA, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL) (Nacalai Tesque). HUVECs were cultured in Endothelial Cell Basal Medium-2 (Lonza, Switzerland) and Endothelial Cell Growth Medium-2 SingleQuots[TM] Supplements and Growth Factors (Lonza) in collagen I-coated dishes (AGC TECHNO GLASS, Japan). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
Preparation of cryopreserved HCC tissue lysates
Cryopreserved tissues were suspended in CelLytic M (Sigma-Aldrich, MO, USA) and finely chopped using scissors. Thereafter, the chopped tissues were homogenized using an ultrasonic homogenizer on ice. The lysed tissues were centrifuged (14,000 × g for 10 min at 4°C) to pellet cellular debris. The supernatant was then harvested and assessed for protein concentration using a BCA protein assay kit (Pierce, MA, USA), in accordance with the manufacturer’s instructions. Subsequently, the supernatant was diluted to 1 mg/mL with D-PBS (Nacalai Tesque).
Evaluation of β-Gal activity
β-Gal activity was evaluated in 96-well black plates (CORNING, MA, USA) using acetate buffer (pH 5.0) (Nacalai Tesque) or D-PBS (pH 7.4) and a FluoReporter lacZ/Galactosidase Quantitation Kit (Thermo Fisher Scientific, MA, USA), in accordance with the manufacturer’s instructions. First, tissue lysate samples (1 mg/mL protein concentration, 10 μL/well) were added to triplicate wells. Thereafter, 3-carboxyumbelliferyl β-D-galactopyranoside (1.1 mM) prepared in acetate buffer (pH 5.0; 100 μL/well) or D-PBS (pH 7.4) was added to the wells. Acetate buffer (10 μL/well) and 7-hydroxycoumarin-3-carboxylic acid (0.1 mM) diluted with acetate buffer (100 μL/well) or D-PBS (pH 7.4) (10 μL/well) and 7-hydroxycoumarin-3-carboxylic acid (0.1 mM) diluted with D-PBS (100 μL/well) were added to triplicate wells as a reference standard. For the β-Gal activity assay at pH 5.0, the plates were incubated for 30 min (5% CO2, 37°C). Then, 50 μL of Na2CO3 (0.2 M in H2O) was added to all wells to terminate the reaction and the fluorescence intensity (Ex/Em: 390/460 nm) was measured using a micro plate reader (SpectraMax M2, Molecular Devices, CA, USA). For the β-Gal activity assay at pH 7.4, the plate was incubated for 30 min (5% CO2, 37°C). Subsequently, the fluorescence intensity was measured. The intensity of each sample was normalized against that of the reference standard.
GGT activity assay
GGT activity was evaluated in 384-well black plates using fluorescence probes (gGlu-HMRG and HMRG). Tissue lysates (1 mg/mL protein concentration, 5 μL/well) and fluorescence probes (1.33 μM in D-PBS, final: 1 μM) were added to each well. HMRG was used as a reference standard, and gGlu-HMRG was used as a fluorescence probe. Fluorescence intensity was measured using an EnVision multilabel plate reader (Perkin Elmer, MA, USA) every minute for 120 min (FITC filter; Ex/Em: 485/535 nm). The results of the gGlu-HMRG assay were normalized to those of the HMRG assay, which was concurrently performed. GGT activity was determined using the following formula:
Activity = (fluorescence increase rate) / (fluorescence intensity of HMRG in lysate – fluorescence intensity of gGlu-HMRG just after lysate addition) / (protein concentration).
Live cell imaging
Cells (1.0 × 104 cells/dish) were seeded in the center of 35-mm glass-bottom dishes (Matsunami glass, Japan) and incubated in an atmosphere containing 5% CO2 at 37°C for 1–2 d. Thereafter, the cells were washed twice with Hanks’ Balanced Salt solution (HBSS; Nacalai Tesque). Next, SPiDER-βGal (1 μM) was added to the dishes, and the cells were incubated in 5% CO2 at 37°C for 60 min. Fluorescent images were obtained using a Keyence BZ-X800 with the TRITC filter (Ex: 545/25 nm, Em: 605/70 nm, Exposure time: 2 s). Bright-field images were captured simultaneously. As a control, we added an identical volume of HBSS to the dishes of cultured cells instead of SPiDER-βGal. The fluorescence intensities of 10 randomly selected cells were analyzed using Image J version 1.52a (NIH).
Mouse model imaging
All animal experiments were performed in compliance with both the ARRIVE guidelines and the institutional guidelines of KPUM, and approved by the animal experimental committee of KPUM (approval number: M30-554). Five-week-old female BALB/c nu/nu mice (average weight, 17 g) were purchased from SHIMIZU Laboratory Supplies, Japan. The mice were housed in plastic cages with stainless-steel grid tops in an air-conditioned environment with a 12-h light-dark cycle and were fed regular food and water ad libitum. Individual suspensions of four types of HCC cells in D-PBS (2.0×107 cells/mL) were mixed with an equal amount of Matrigel (CORNING, MA, USA) on ice. Under general anesthesia, the mixed suspension (100 μL) was then subcutaneously injected into the flanks of each mouse. After ≥4 weeks, tumour-bearing mice were euthanized using Isoflurane (Wako, Japan). Subcutaneous tumors and livers were dissected and divided into two using scissors. A solution of SPiDER-βGal (50 μM) in HBSS was then sprayed onto the cut surface of each tumour or the surface of normal liver. Sequence of fluorescent images were captured every 2 min for 30 min using IVIS Lumina Series III (Ex/ Em: 520/570 nm). Regions of interest (ROIs) were drawn for both the tumour and normal liver tissues, and the average radiant efficiency as fluorescence intensity was determined using Living Image version 4.4.
Histopathological analysis
Resected subcutaneous tumors from tumour-bearing mice were fixed with 10% neutral buffered formalin then embedded in paraffin. The paraffin blocks were sliced to a thickness of 5 μm, after which the paraffin-embedded sections were deparaffinized and stained with Mayer’s hematoxylin solution (Wako) and eosin Y (Wako) for histopathological analysis.
Freshly resected human specimens
Freshly resected human specimens were obtained from patients preoperatively diagnosed with HCC—through radiological examination—who received curative liver resection at the University Hospital of KPUM. Written informed consent was preoperatively acquired from all patients. The Institutional Review Board of KPUM examined and approved the research procedures (approval number: ERB-C-1470) in accordance with the tenets of the Declaration of Helsinki. Cases of cholangiocellular carcinoma without HCC components, as determined pathologically, were excluded. In total, 27 freshly resected human HCC specimens were examined prospectively from May 2019 to March 2020. Patient clinicopathological characteristics were described based on the General Rules for the Clinical and Pathological Study of Primary Liver Cancer, Edition 6, Revised Version 32.
Human specimen imaging
Fluorescence imaging of human specimens was performed within 1 h after liver resection. A solution of SPiDER-βGal (50 μM) prepared in HBSS was then sprayed onto the resected surface of the liver tissue samples. Sequences of fluorescent images were captured every 2 min for 30 min using IVIS Lumina Series III (Ex/Em: 520/570 nm). ROIs were drawn for both the tumour and normal liver, and the average radiant efficiency as a fluorescence intensity was determined using Living Image version 4.4.
Statistical analysis
A two-tailed paired t-test was used to compare β-Gal activity in cryopreserved human HCC samples at pH 5.0 and pH 7.4, and GGT activity in cryopreserved human HCC samples. A two-tailed Mann-Whitney U-test was used to compare the fluorescence intensity of live cells, fluorescence intensity of images from tumour-bearing mouse models, and clinicopathological characteristics of human HCC tissue samples that were either cryopreserved or freshly resected. A two-tailed Wilcoxon t-test was used to compare the fluorescence intensity of freshly resected HCC specimens. ROC and Youden index curves were used to determine a cut-off value for the increase in fluorescence intensity. The sensitivity, specificity, and area under the curve (AUC) were determined through ROC analysis. Results with p-values < 0.05 were considered significant. Statistical analysis was performed using the yStat 2013 software and JMP13 (SAS Institute, NC, USA).
Data Availability
The datasets of the current study are available from the corresponding author on reasonable request.