The study was carried out in compliance with the ARRIVE guidelines. All methods were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by Institutional Review Board (G2018-132C5, Medical Research Ethics Committee for Genetic Research of Tokyo Medical and Dental University; A2019-263C2, Institutional Animal Care and Use Committee of Tokyo Medical and Dental University).
Human HCC cell line HuH7 was purchased from the American Type Culture Collection (Manassas, VA). Mouse cell line 3H3 was derived from HCC tumor grown in a C57BL/6J MC4R-KO mouse fed with high fat diet19. They were cultured in RPMI-1640 and DMEM (Wako, Osaka, Japan) medium containing 10% fetal bovine serum (FBS), and 1% penicillin, streptomycin and amphotericin B (Wako), maintained in a humidified incubator at 37 ℃ in 5% CO2, and harvested with 0.05% trypsin-0.03% EDTA (Wako).
Exon 3 skipping of β-catenin by multiplex CRISPR/Cas9-based genome engineering system
To generate the backbone plasmid for the CRISPR/Cas9 system, the lentiGuide-Puro (Addgene #52963) was modified by inserting a KpnI site in front of the U6 promoter and replacing the HindIII site behind the sgRNA scaffold with an EcoRI site, named as LG-U6. The LG-H1 plasmid was also produced by replacing the U6 promoter with the H1 promoter in the LG-U6 plasmid. The LG-U6 and LG-H1 plasmid for expressing sgRNAs targeting intron 2 and intron 3 of β-catenin (sgRNA-in2 and sgRNA-in3) were constructed following the manufacture’s manual (Supplementary Table 2). The H1-sgRNA-in3 sequence was tandemly cloned into the EcoRI site of the LG-U6-sgRNA-in2 plasmid (Figure 1A). The HuH7 and 3H3 cells were sequentially infected with the lentiviral vectors for constitutively expressing SpCas9 (lentiCas9-Blast; Addgene #52962) and simultaneously expressing sgRNA-in2 and sgRNA-in3, and then treated with 10 μg/mL blasticidin and 10 μg/mL puromycin, respectively. The subclones with β-catenin alleles lacking exon 3 were isolated by limiting dilution.
DNA extraction and PCR analysis
Cell pellets were suspended in TNE Buffer (10 mM Tris–HCl, pH 8.0; 150 mM NaCl; 2 mM EDTA; 0.5% SDS) with 1% proteinase K (TaKaRa Bio, Shiga, Japan) at 55 ℃ overnight. Genomic DNA was obtained from cells by phenol-chloroform extraction. The primer sets and amplification conditions for PCR are listed in Supplementary Table 3.
Total RNA was extracted from cells by using RNeasy Plus Mini Kit (QIAGEN, Germantown, MD). Contaminating DNA was removed by digestion with RNase-Free DNase Set (QIAGEN).
Quantitative RT-PCR analysis
For single-stranded complementary DNA synthesis, 1 μg of total RNA was reverse-transcribed by SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA). Quantitative RT-PCR analysis was performed by using TB Green Premix Ex Taq II (TaKaRa Bio) with StepOne real-time PCR system (Thermo Fisher Scientific) according to the manufacturer’s instructions, and the ΔΔCt method was used for quantification. GAPDH was used as an internal control. The primer sets and amplification conditions for PCR are listed in Supplementary Table 4.
RNA sequencing analysis
Sequencing libraries were prepared from total RNA with the TruSeq Standard mRNA Library Kit (Illumina, San Diego, CA), and RNA sequencing was run on an Illumina NovaSeq 6000. Sequence reads were aligned to the human and mouse reference genome (GRCh38 and GRCm38) by STAR (2.7.0d), and transcript quantification was performed by RSEM (1.3.1). Differentially expressed genes were determined by using DESeq2 (1.14.1).
After whole cell lysates were collected by using ice-cold RIPA buffer (Thermo Fisher Scientific), 30 μg of protein from each sample was subjected to electrophoresis through 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto Immobilon polyvinyldifluoride membranes (Millipore, Bedford, MA). The membrane was blocked with 5% skimmed milk or bovine serum albumin for an hour at room temperature, and then incubated overnight at 4 °C with primary antibodies as follows; β-catenin (D10A8, 1:1000), GAPDH (14C10, 1:1000) and lamin B1 (D6V6H, 1:1000), all of which were purchased from Cell Signaling Technology (Danvers, MA). Secondary antibodies were added, and signals were detected by using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA) with LAS-3000 (Fujifilm, Tokyo, Japan).
Subcellular fractionation analysis
Cytoplasmic and nuclear proteins were separately extracted by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions, and then Western blotting analysis was performed. GAPDH and lamin B1 were used for detecting cytoplasmic and nuclear fractionated protein, respectively.
Isolation of T cells
Eight-week-old male C57BL/6J mice were euthanized, and spleens were resected and disrupted with a flat plunger tip of a 5 mL syringe. After hemolysis, whole splenocytes were incubated in a nylon wool fiber column to remove B lymphocytes for an hour at 37℃. T lymphocytes were collected and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% ITS supplement (Thermo Fisher Scientific), 100 U/ml murine IL-2 (Peprotech, Cranbury, NJ) and 10 ng/ml murine IL-7 (Peprotech).
Isolation of mouse bone marrow and differentiation of DCs was performed as previously described25. Briefly, eight-week-old male C57BL/6J mice were euthanized, and bone marrow was flushed out from femur and tibia by using a 1 ml syringe and a 27G needle. Bone marrow-derived monocytes (BMDMs) were washed, and then cultured in DC differentiation medium as follows; RPMI-1640, 10% FBS, 1% penicillin-streptomycin-amphotericin B, 20 ng/ml murine GM-CSF (Peprotech) and 5 ng/ml murine IL-4 (Peprotech). Six days after preculture, differentiated bone marrow-derived dendritic cells (BMDCs) were further cultured in conditioned medium collected from the 3H3-Ctrl cells or 3H3-CTNNB1Δex3 cells for 24 hours to stimulate with cancer antigens.
T cell killing assay
A day after T lymphocytes were co-cultured with BMDCs for priming, the 3H3 cells or the 3H3-Ctnnb1Δex3 cells were plated at 5×103 cells per well in a 24-well tissue culture plate (for two-dimensional culture) or ultra-low attachment plate (for sphere culture) with primed immune cells for 48 hours. Advanced DMEM/F12 (Thermo Fisher Scientific) with 0.5% B-27 Supplement (Thermo Fisher Scientific), 20 ng/ml human EGF (Peprotech) and 1 μg/ml human FGF-basic (Peprotech) was used for sphere formation. To evaluate cytotoxic activity of immune cells in two-dimensional culture, cell viability was estimated by using CellTiter-Glo 2.0 reagent (Promega, Madison, WI) with FLUOstar OPTIMA-6 microplate reader (BMG Labtech, Durham, NC) according to the manufacturer’s instructions. For sphere culture, cancer cell area was measured by using ImageJ software.
Gene set enrichment analysis was performed with the MSigDB gene sets. Public genome and transcriptome data of 373 HCC samples were provided from the Cancer Genome Atlas Research Network, and downloaded from the cBioPortal site. Genome data divided them into 77 and 296 tumors with and without CTNNB1 hotspot mutations.