Generation of iPSDCs
Three patients with colorectal cancer were enrolled in this project, each providing signed informed consent. They all underwent surgery for colorectal cancer at Wakayama Medical University Hospital. This study protocol was approved by the Institutional Review Board of the Ethical Committee on Human Research at WMUH (approval no. 2657 UMIN000038017). All research has been performed in accordance with the Declaration of Helsinki. All three patients have given their consent for publication in an online open access publication. We collected 15 ml of blood and 1.2 × 10^7 peripheral blood mononuclear cells (PBMCs) from these three donors. iPSCs were derived from donor PBMCs using CytoTune-iPS 2.0 (ID Pharma) in accordance with the manufacturer’s instructions14,15. iPSCs colonies were formed in about 20 days. We confirmed the undifferentiated and pluripotent status of iPSCs by evaluating alkaline phosphatase staining and fluorescent staining with undifferentiated markers (R&D Systems) according to the manufacturer’s instructions. The differentiation protocol of the iPSDCs was modified from a previously established protocol7,9. In brief, in step 1, undifferentiated iPSCs were disseminated onto a 100-mm culture dish coated with growth factor-reduced Matrigel (CORNING) in mTeSR1-cGMP medium (STEMCELL Technologies) supplemented with 80 ng/mL rhBMP4 (R&D Systems). In step 2, on day 4, mTeSR1 medium was replaced with StemPro-34 serum-free medium (Gibco) containing 2 mM L-glutamine supplemented with 80 ng/mL rhVEGF (R&D Systems), 25 ng/mL basic FGF (FUJIFILM Wako), and 100 ng/mL rhSCF (PEPROTECH). In step 3, on day 6, the cytokines in StemPro-34 were changed to cytokines mixed with 50 ng/mL rhSCF, 50 ng/mL rhIL-3 (R&D Systems), 5 ng/mL rhTPO (PEPROTECH), 50 ng/mL rhM-CSF (PEPROTECH), and 50 ng/mL rhFlt-3 ligand (PEPROTECH). In step 4, on day 13, the cytokines in StemPro-34 were changed to cytokines mixed with 50 ng/mL rhM-CSF, 25 ng/mL rhGM-CSF (PEPROTECH), and 50 ng/mL rhFlt3 ligand. CD14 positive monocytic lineage cells were sorted using an autoMACS Pro Separator with CD14 MicroBeads, human (Miltenyi) on days 16 to 28. In step 5, 1.5 × 10^6 CD14 positive monocytic cells/well in six-well Costar Ultra-Low Attachment Surface plates (CORNING) were cultured in the StemPro-34 medium containing 25 ng/mL rhGM-CSF and 40 ng/mL rhIL-4 (R&D Systems) for 5 days for differentiation into immature iPSDCs. Immature iPSDCs were matured in the presence of 100 ng/mL rhIL-6 (R&D Systems), 10 ng/mL rhTNFα (R&D Systems), 10 ng/mL rhIL-1β (R&D Systems), and 1 μg/mL Prostaglandin E2 (Sigma Aldrich) to induce final maturation for 48 h.
Tumor cell line preparation
For the primary culture of the cancer cells from patients with colorectal cancer, we established CTOSs, as described previously26. In brief, surgical specimens were minced into 2 mm cubes using scalpel blades and washed several times with Hank’s balanced salt solution (HBSS). Tumor fragments were digested with 0.26 U/mL Liberase DH solution (Roche Diagnostics) at 37 °C for 1 h 45 min, followed by adding Recombinant DNase I (Takara Bio) for 15 min. The partially digested tissue was filtered through a 500-μm mesh filter and a 250-μm mesh filter. The filtrate was filtered through a 100-μm strainer and a 40-μm strainer. The tumor tissue retained in the strainer was collected, washed twice with HBSS, and transferred to CTOS medium [18 mL DMEM/F-12 with GlutaMAX medium (Gibco) supplemented with 400 μL StemPro hESC SFM Supplement (Gibco), 8 ng/mL bFGF, 20 ng/mL EGF (FUJIFILM Wako), 0.1 mM 2-mercaptoethanol, 100 units/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL Amphotericin B]. CTOSs were cultured in suspension in a stem cell medium. After suspension culture, 3D culture was performed. For 3D culture, the CTOSs were embedded in Cellmatrix type I-A (Nitta Gelatin) droplets on untreated tissue culture dishes and overlaid with stem cell medium. The medium was changed two or three times a week. For further analysis or passage, CTOSs were released from the Cellmatrix after 1–2 weeks of cultivation by incubation with 0.2 mg/mL Collagenase Type IV (STEMCELL Technologies). For expansion, CTOSs were cut into cell clusters using 23-gauge needles, and the cell clusters were transferred to the fresh stem cell medium.
Total RNA isolation and amplification
Total RNA was isolated from CTOSs using the RNeasy plus micro kit and QIAshredder (Qiagen). Isolated RNA was amplified and prepared for in vitro transcription, as described previously12,13. Briefly, CTOSs mRNA was reverse transcribed using SuperScript II reverse transcriptase (Thermo Fisher Scientific). The first-strand cDNA synthesis was performed with 10 pmol of a modified oligo-dT primer [5′-AAGCAGTGGTATCAACGCAGAGTACT(30)VN-3′], where V is G, A, or C and N is G, A, T, or C (Sigma Aldrich). To this, we added DTT, reaction buffer, dNTP mixture (Takara Bio), SUPERase In RNase Inhibitor (Thermo Fisher Scientific). The reaction was incubated at 42 °C for 30 min, and then at 10 pmol the T7 strand switch primer [5′-CTAATACGACTCACTATAGGGCGGG-3′] was added. The reaction was continued for 30 min and was stopped by placing it on ice. Second-strand cDNA synthesis was performed using an Advantage 2 PCR enzyme system (Takara Bio) with RNase H, from E. Coli (Thermo Fisher Scientific). The cDNA was amplified by placing 2 μl of the RT reaction into a 100 μl PCR reaction containing 20 pmol of the following primers: T7 PCR (5′- CCATCCTAATACGACTCACTATAGGGC-3′) and 3′ PCR (5′- AAGCAGTGGTATCAACGCAGAGT-3′). Cycling conditions were as follows: an initial 1 min denaturing step at 95 °C, followed by cycling at 95 °C for 30 s, 65 °C for 30 s, 68 °C for 6 min, and a final extension at 68 °C for 7 min; this was performed for 18 cycles. The amplified cDNA was purified with a MinElute PCR Purification Kit (Qiagen).
In vitro transcription of amplified cDNA
In vitro transcription was performed using the T7 mMESSAGE mMACHINE Kit (Thermo Fisher Scientific)12,13. The reaction was carried out at 37 °C for 4 h, followed by the addition of TURBO DNase (Thermo Fisher Scientific) and incubation for 15 min. For the polyadenylation of in vitro transcriptional RNA, we used a Poly(A) Tailing Kit (Thermo Fisher Scientific). Amplified mRNA was purified with a RNeasy MinElute Cleanup Kit (Qiagen). RNA quality was verified by agarose gel electrophoresis and a Nanodrop 2000 (Thermo Fisher Scientific), and 2100 Bioanalyzer (Agilent Technologies), and then stored at − 80 °C.
Preparation of CEA and GFP in vitro transcription RNA
For CEA ivtRNA, we purchased Human CEACAM5/CEA/CD66e Gene ORF cDNA clone expression plasmid (Sino Biological). Linearization with Xba I (NIPPON GENE), followed by in vitro transcription with the T7 mMESSAGE mMACHINE Kit, yielded a transcript that contains 2109 nucleotides corresponding to the coding region of CEA.
For green fluorescent protein (GFP) ivtRNA, we purchased GFP mRNA (OZ Biosciences). This product is polyadenylated RNA at the 3′ end. It was therefore possible to prepare ivtRNA by the amplification and in vitro transcription protocol.
Transfection of iPSDCs with ivtRNA
Mature iPSDCs were harvested and washed once with PBS (all at room temperature). The cells were resuspended in Opti-MEM without phenol red (Thermo Fisher Scientific) at a concentration of 5 × 10^6/ ml. RNA was transferred to a 4-mm cuvette (80 μg/ ml final concentration). A volume of 200 μl of cell suspension was added and incubated for 3 min before being pulsed in a Gene Pulser Xcell (Bio-Rad). Pulse conditions were square-wave pulse, 500 V, 0.5 ms17. Immediately after electroporation, the cells were transferred to Step 5 differentiation medium (the StemPro-34 medium containing 25 ng/ ml rhGM-CSF and 40 ng/ ml rhIL-4).
Flow cytometric analysis
For BMDCs and iPSDCs staining, the following monoclonal antibodies were used: PE-conjugated anti-human CD11c, FITC-conjugated anti-human CD80, FITC-conjugated anti-human CD83, PE-conjugated anti-human CD40, PE-conjugated anti-human HLA-ABC, PE-conjugated anti-human HLA-DR (all from BD Biosciences), primary antibodies against HLA-A were Anti-HLA A antibody, and secondary antibodies were Goat Anti-Rabbit IgG H&L (FITC) (all from Abcam). For CTOSs staining, the following monoclonal antibodies were used: FITC anti-human EpCAM (Miltenyi), PE anti-human CD45 (BD Biosciences). Primary antibodies against CEA were anti-human CEA monoclonal antibody clone COL-1 (Leica Biosystems), and secondary antibodies were FITC-conjugated anti-mouse IgG polyclonal antibody (Agilent Technologies).
Assays for cytokine secretion
The BMDCs and iPSDCs were adjusted to a concentration of 2.0 × 10^5 cells/well and cultured on a 48-well plate for 48 h in AIM-V Medium (Thermo Fisher Scientific) (1 ml/well). The supernatants were then harvested, and the human IFN-γ and human IL-12 (p70) and human TNF-α levels were measured using an INF-γ ELISA kit and an IL-12 (p70) ELISA kit and a human TNF-α kit, respectively (all from Thermo Fisher Scientific). In the TNF-α assay, 100 ng/ ml of LPS (Sigma Aldrich) was used to stimulate DCs.
Induction of CD8+ cytotoxic T cells
For iPSDCs-ivtRNA, CD8+ cytotoxic T cells were induced from autologous PBMCs. On day 0, a total of 4 × 10^6 PBMCs and 2 × 10^5 iPSDCs-ivtRNA were mixed in AIM V Medium containing 10 ng/ ml rhIL-7 (CellGenix) and cultured in a 24-well plate at a total volume of 1 ml/ well. On day 2, AIM V Medium containing 20 U/ ml of rhIL-2 (PEPROTECH) was added at a total volume of 2 ml/ well. On days 7 and 14, the cultures were re-stimulated with iPSDCs-ivtRNA at a ratio of 20:1. After three cycles of re-stimulation, CD8+ cytotoxic T cells were sorted from the stimulated PBMCs on day 21 using an autoMACS Pro Separator and then used for the subsequent experiments9.
For iPSDCs-neoantigen peptide, iPSDCs were pulsed with 20 μg/ ml of the respective synthesized more than 95% purity neoantigen peptides (Hokkaido System Science) for 16 h at 37 °C, and treated with 30 μg/ ml of mitomycin C (Sigma Aldrich) at 37 °C for 30 min. Following washing out the residual peptides and mitomycin C, iPSDCs were cultured with autologous CD8+ T cells in 0.5 ml of CellGenix GMP DC Medium (CellGenix) with 5% Human serum AB male (ABS) (Biowest) supplemented with 30 ng/ ml rhIL-21 (CellGenix) on 48-well plate (each well contained 1.0 × 10^5 peptide pulsed iPSDCs, 5 × 10^8 CD8+ T cells) on day 1. Three days later (day 4), 5 ng/ ml rhIL-7 and 5 ng/ ml rhIL-15 (PEPROTECH) were added in the culture media31,32,33. On day 6, the cultures were transferred to a 12-well plate with DC Medium/5% ABS containing 5 ng/ ml IL-7 and 5 ng/ ml rhIL-1534. On day 8, cultures were supplemented with DC Medium/5% ABS containing 10 ng/ ml rhIL-7 and 10 ng/ ml rhIL-15. On day 11, neoantigen-specific T cells were assessed using an ELISpot assay.
4-h 51Cr release assay
The cytotoxic activity of CD8+ cytotoxic T cells was tested using a 4-h 51Cr release assay, as described previously35,36. CTOSs were used as target cells. Spontaneous release was determined by incubating target cells alone in CTOS medium. The total release was determined by incubating target cells with 150 μl of 1 M HCl (Sigma Aldrich). CD8+ cytotoxic T cells were incubated at different ratios with the 51Cr-labeled CTOSs (E:T ratios of 50:1, 25:1, 12.5:1). Percentage cytotoxicity was calculated according to the formula: % Lysis = (experimental cpm – spontaneous cpm/total cpm – spontaneous cpm) × 100. Duplicate measurements of three-step titrations of effector cells were used for all experiments.
Whole-exome and transcriptome analysis
Case 3 underwent neoantigen analysis. Whole-exome and transcriptome were outsourced to Cancer Precision Medicine (CPM, https://www.cancerprecision.co.jp). Genomic DNAs and total RNAs were extracted from frozen tumors using the AllPrep DNA/RNA mini kit (Qiagen) according to the manufacturer’s instructions. Control genomic DNAs were extracted from peripheral blood samples using QIAamp DNA Blood Midi Kit (Qiagen). Whole-exome libraries were built up, as previously described37 and sequenced by 100-bp paired-end reads on HiSeq2500 Sequencer (Illumina). The obtained sequence data were analysed by CPM pipeline, as previously described24. Somatic variants (single nucleotide variations (SNVs) and indels) were called using the following parameters, (i) base quality of ≥ 15, (ii) sequence depth of ≥ 10%, (iii) variant depth of ≥ 2, (iv) variant frequency in tumor of ≥ 10%, (v) variant frequency in normal of < 2%, and (vi) Fisher P value of < 0.0538. SNVs and indels were annotated based on RefGene using ANNOVAR.
Identification of potential neoantigens
Human Leukocyte Antigen (HLA) class I genotypes were determined by OptiType algorithm39 using whole-exome data of possible 8- to 11-mer peptides harboring each amino acid substitution to HLA class I molecules. They were then filtered out with the predicted binding affinity to HLA molecules lower than 500 nM, using NetMHCv3.4 software. In addition, we used the transcriptome data to further select non-synonymously mutant peptides with a defined level (at least 10 reads among ~ 20,000,000 sequence reads) of gene expressions in tumor cells. For experimental validation, candidates were further prioritized based on the following criteria: (1) high predicted affinity (< 10 nM) somatic single nucleotide variations, (2) affinity of mutant type peptides ≥ wild type peptides, and (3) high mutant RNA expression (≥ 100)20,21,22,23,24.
IFN-γ ELISpot assay
ELISpot assay was performed using Human IFN-γ ELISpotPRO kit (Mabtech) according to the manufacturer’s instructions. Briefly, plates were washed four times with PBS and blocked with CellGenix GMP DC Medium containing 10% of ABS at least 30 min before use. For co-culture, 1 × 10^4 responder CD8+ T cells for detection of CD8+ T-cell responses were co-cultured with 5 × 10^3 stimulator autologous BMDCs. Responder CD8+ T cells were educated by iPSDCs-neoantigen peptide or iPSDCs-CTOS ivtRNA. Autologous PBMCs were generated using the plastic adherence method, as described previously34 and then pulsed with the respective neoantigen peptides (20 μg/ ml). After that, they were directly added to the ELISpot wells and incubated with responder CD8+ T cells overnight in DC Medium containing 10% of ABS at 37 °C. The mAb CD3-2 was used as a positive control. After 24 h of co-culture, cells were removed from the plate, washed five times with PBS, and then diluted with anti-human IFN-γ mAb (7-B6-1-Biotin, Mabtech) 1:200 in filtered PBS containing 0.5% ABS. After rinsing, TMB substrate solution was used to develop the immunospots. Spots were captured and analyzed by an automated ELISpot reader, ImmunoSpot S4 Software package, Version 6.0.0.2 (Cellular Technology Limited).
