A total of 44 participants were considered for inclusion in this study from May 2021 to September 2022, including 24 healthy volunteers without any hepatitis (blank control) and 20 advanced HCC patients evaluated by clinical history, AFP and imaging findings. The inclusion criteria for HCC patients in this study were (a) availability of CTCs, frozen biopsy and/or resected primary and metastatic HCC tissues; (b) pathologically proven HCC based on WHO criteria; (c) gene mutation detection for CTCs and HCC tissues; and (d) availability of follow-up data. Advanced HCC patients received conversion therapies, including immunotherapy, targeted therapy (sorafenib/lenvatinib), transcatheter arterial chemoembolization (TACE), traditional Chinese medicine (Huaier granule) , and liver protection therapy. The therapeutic schedule was permitted to be adjusted according to the actual condition of individuals. Tumor samples were collected by resection or biopsy, and 10 mL blood samples were obtained each time for CTC counts at different time points: 1 day before surgery and 2 h, 1 d, 3 d, 1 w, 2 w, 4 w, 8 w, and 16 w after surgery. The matched specimens (n = 39), including primary lesions of HCC (n = 17), preoperative CTC samples (n = 17) and metastatic tissues (n = 5), were used for 610 gene mutation detection by NGS. HCC patients were followed up every month until September 30, 2022, by monitoring serum AFP levels, abdominal ultrasonography, chest X-ray or computed tomography depending on the patient’s condition. General data, metastatic characteristics, pathologic characteristics and survival were compared among the groups. This study was approved by the ethics committee of our hospital (No. 2022-020; China Clinical Trial Registration Center-Registration No. ChiCTR2200055847), and informed consent was obtained from participants in accordance with respective committee regulations.
Consumables and instruments
Fe3O4 solution, carboxymethyl chitosan hexadecyl quaternary ammonium salt (HQCMC), CK8/18/19 (CK)-FITC (Fluorescein Isothiocyanate), CD45-PE, DAPI, Ep-LMS, and Vi-LMS were purchased from Huzhou Lieyuan Medical Laboratory Co., Ltd. A Prussian blue staining kit was purchased from Solarbio. DMEM, fetal bovine serum and trypsin were purchased from Gibco, and GPC3 antibody was purchased from Abcam. 1,2-Dioleoyl phosphatidylcholine (DOPC), dimethyl octadecyl epoxy propyl ammonium chloride (GHDC), cholesterol, dichloromethane, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and other commonly used reagents were all purchased from Sinopharm Group. The TIANamp Genomic DNA Kit was purchased from Tiangen Biotech (Beijing).
Cell lines and culture
At the authors’ institution, a stepwise metastatic human HCC model system was established, which included a metastatic HCC model in nude mice (LCI-D20) , an HCC cell line with high metastatic potential that originated from LCI-D20 tumor (MHCC97) , a highly metastatic subclone (MHCC97H with a lung metastasis rate up to 100% using orthotopic inoculation) and a subclone with lower metastatic potential (MHCC97L with a pulmonary metastasis rate up to 40% using orthotopic inoculation) established through in vivo selection of MHCC97 cells . The HCC cell lines Hep3B and Huh7 with low metastatic potential and human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cell lines were cultured following procedures stated in our previous reports . HUVECs were grown in RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). The remaining cell lines were maintained in antibiotic-free Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL, Gaithersburg, MD). All cell lines were cultured without antibiotics in a humidified incubator containing 5% CO2/95% air at 37 °C.
Preparation of GPC3 magnetic spheres
Cholesterol (6 mg), DOPC (500 µL, 10 mg/mL) and emulsifier HQCMC (500 µL, 10 mg/mL) were codissolved in dichloromethane as the solvent. Then, 1 mL 20 mg/mL Fe3O4 solution and 20 mL 0.1 mol/L phosphate buffer saline (PBS) (pH 7.4) were added, followed by ultrasonic oscillation under a power of 100 W at 25 °C for 6 min and rotary evaporation for 30 min to remove dichloromethane and obtain LMS solution (1.8 mg/mL). Subsequently, 0.1 mg surfactant GHDC was dissolved in 1 mL isopropyl alcohol, and 60 µg GPC3 was dissolved in 1 mL GHDC solution (GPC3-GHDC), which was added to the coupling agents NHS (0.1 mg) and EDC (0.1 mg). After incubation overnight at 4 °C, 1 mL GPC3-GHDC was added into 1 mL LMS solution, which was subjected to vortex oscillation for 5 min and stored at 4 °C. Finally, the sample was taken out of the vortex every 1 h for 5 min for 24 h to obtain GPC3-LMS (2.16 mg/mL).
The sample (10 μL) was diluted in 1 mL of distilled water, and the particle size and potential of the magnetic spheres were measured by using a BI-90Plus laser particle sizer/Zeta potentiometer (Brookhaven), 50 μL of which was coated on mica plates. The morphology of LMS was observed under an AFM. Then, 50 μL of diluent was dropped on a copper mesh, and the morphology of LMS was observed under TEM after drying. After freeze-drying of a 1 mL sample, the infrared spectrum obtained by the KBr tablet method was measured on a Bio-Rad FTS 6000 spectrometer using FT-IR spectroscopy. Afterward, 10 μL samples were taken for magnetic separation and diluted in 1 mL distilled water, and the LMSwere scanned by ultraviolet absorption spectroscopy with an ultraviolet spectrophotometer. After a 1 mL sample was freeze-dried into powder, the magnetization curve was determined using a vibrating sample magnetometer (VSM) at room temperature.
The cells were prepared into a single-cell suspension with trypsin and cultured in 96-well plates at 8000 cells per well. When the cells grew to 80% confluence, the medium was replaced with 100 μL of complete DMEM containing different amounts of Ep-LMS, Vi-LMS and GPC3-LMS. In the blank control group, 100 μL of complete DMEM was added for further culture. After adding 20 μL MTT reagent (5 mg/mL), incubation was performed in a CO2 incubator for 3 h. Finally, 150 μL dimethyl sulfoxide (DMSO) was added to dissolve the prepared blue and purple crystalline formazans. The experimental results were read in a microplate reader and counted (wavelength, 490 nm), and 3 parallel tests were carried out in each group.
Distribution of magnetic spheres
Single-cell suspensions of Hep3B, Huh7, MHCC97L and MHCC97H cells were prepared. After counting, 100 cells were added to 7.5 mL PBS solution to simulate CTC suspension by separation with LMS, Ep-LMS, Vi-LMS and GPC3-LMS. The isolated cells were then stained with Prussian blue dye and observed under a fluorescence microscope after fixation on porous slides. Additionally, the captured cells were smeared on the sample mirror, sprayed with gold after drying, and observed under a SEM.
Cell capture efficiency
Using the Ep-LMS/Vi-LMS/GPC3-LMS capture scheme, HUVECs, Hep3B, Huh7, MHCC97L and MHCC97H cells were adjusted to different cell gradients of 10, 50, 100, 500, 1000, 5000 and 10,000 cells, respectively. The sensitivity and specificity of this scheme were determined in the PBS system and the simulated blood system, respectively. Finally, magnetic spheres with antibodies at 0, 20, 40, 60, 80 and 100 μg/mL were used to capture Huh7 cells. The cell capture efficiency of magnetic spheres was investigated in the above systems.
Cell capture time
Huh7 cells (1 × 104) were incubated in a culture dish, which was supplemented with 1 mL cell culture medium and then cultured in a constant-temperature 5% CO2 incubator at 37 °C for 24 h. After replacing the culture medium, 20 μL Ep-LMS-FITC or Vi-LMS-FITC, GPC3-LMS-FITC, 100 μL DAPI and 100 μL Dil were added. Finally, the culture dish was fixed on a fluorescence microscope and photographed at 0, 5, 10, 15 and 20 min.
Mouse grouping and treatments
Male athymic BALB/c nu/nu mice of 18–20 g at 5 weeks of age were obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Science. All mice were handled according to the recommendations of the National Institutes of Health Guidelines for Care and Use of Laboratory Animals. Human HCC tumor models produced by MHCC97H were established in nude mice by orthotopic inoculation, as described in our previous publications [42,43,44,45]. Blood samples were obtained for further examination, and lungs and other organs suspected of tumor involvement were sampled for HE staining. Paraffin blocks of 10% buffered formalin-fixed samples of tumor and various organ tissues were prepared, serial sections were cut at 5 μm, and lung metastatic nodules were verified with HE staining.
In the subcutaneous implantation tumor model group (n = 15), 3 nude mice were randomly selected at the end of weeks 0, 2, 3, 4 and 5 for weighing and then sacrificed for sample collection and CTC capture. Tumor volume was calculated by the formula V = a × b2/2, where a is the long tumor axis and b is the short tumor axis. Then, 72 mice were randomized into 4 groups (54 mice in the resection groups received orthotopic tumor transplantation, and 18 mice in the black control group only received liver exposure without tumor transplantation). Three nude mice in each group were randomly sacrificed for sample collection and CTC isolation at the end of the 0, 1st, 2nd, 3rd, 4th and 5th weeks. Resection started on Day 14 after HCC tissue implantation. In the blank control group (BC group, n = 18), the mice underwent liver exposure without resection; in the RR group (n = 18), the mice underwent radical HCC resection with a negative surgical margin; in the PR group (n = 18), mice underwent partial HCC resection with preservation of 2 mm of tumor pedicles [42, 44,45,46]; in the SO group (n = 18), mice underwent exposure of the liver but no resection.
CTC isolation and identification in HCC patients
Clinical samples were collected from patients with HCC. Blood samples (10 mL) were taken at different time points, and each sample was divided into five 2 mL aliquots. Then, 15 μL Ep-LMS, Vi-LMS, and GPC3-LMS was added to the Ep-LMS group, Vi-LMS group, and GPC3-LMS group, respectively, and incubated for 15 min. The Ep-LMS/Vi-LMS group was treated with 7.5 μL of Ep-LMS and Vi-LMS successively and incubated for 15 min. The Ep-LMS/Vi-LMS/GPC3-LMS group was treated with 5 μL of Ep-LMS, Vi-LMS and GPC3-LMS successively (the combination scheme). After incubation, the centrifuge tube was inserted into the magnetic separation rack for adsorption for 10 min. Subsequently, blood was removed, and 20 μL of CK-FITC, DAPI staining solution and CD45-PE were added for staining in the dark for 15 min. After staining, washing was performed twice using 1 mL of distilled H2O. Finally, 20 μL of distilled H2O was added into the centrifuge tube and mixed well. The mixed solution was evenly coated on the anti-off slide treated with polylysine. After the droplets were naturally dried, observation and counting were carried out under a fluorescence microscope.
Gene detection and analysis
Total DNA from tumor tissues and CTCs was extracted by the TIANamp Genomic DNA Kit (total tissue DNA > 200 ng, total CTC-DNA > 20 ng). The samples (n = 610) were detected via target region capture and NGS technology . Furthermore, TMB was calculated and compared among groups. The reference TMB value was 3.6 Muts/Mb, and if the detection value was greater than the reference value, immunotherapy was recommended.
Statistical analyses in this study were performed by SPSS 21.0. The data are expressed as the mean value (mean) ± standard deviation (SD). One-way ANOVA was used for comparisons between different time points, and the SNK test was used for pairwise comparisons. The results were considered statistically significant at P < 0.05.