Patient samples
Tumor tissues and peripheral blood were obtained from patients with a confirmed diagnosis of colorectal cancer. The fresh surgically resected tumor samples were collected in sterile 15mL tubes containing Advanced DMEM/F12 (GIBCO) supplemented with pencillin/streptomycin (GIBCO), follow resected into several fragments for TILs culture and generation of patient derived organoids (PDO). This study was approved by the Medical Ethical Committee of the Peking University Shougang Hospital and written informed consent was obtained from all patients.
PDO and TILs establishment
The establishment of PDO was performed in accordance with the previously described method (12). Briefly, the fresh tumor tissue and normal colon tissue was washed with ice-cold phosphate-buffered saline (PBS) buffer and minced into small fragments. The fragments were enzymatically digested using 10 mg/mL hyaluronidase type IV (Sigma-Aldrich), 1.5 mg/mL collagenase II (Sigma-Aldrich), and 10 mM Y-27632 (Sigma-Aldrich) for approximately 45 minutes before embedding in Matrigel (Corning). Tumor organoids were cultured in Advanced DMEM/F12 (Gibco) supplemented with 10mM HEPES (Gibco), 1x B27 supplement without vitamin A (GIBCO), 1x N2 (Gibco), 2 mM Ultra-glutamine I (Lonza), 500 ng/ml R-Spondin-1(Peprotech), 100 ng/ml Noggin (Peprotech), 10 nM prostaglandin E2 (Cayman Chemicals), 3 mM SB202190 (Cayman Chemicals), 10 nM Gastrin I (Sigma-Aldrich), 50 ng/ml human recombinant EGF (Peprotech), 0.5 µM A83-01(Tocris), 10 mM Nicotinamide (Sigma-Aldrich), 1 mM N-acetylcysteine (Sigma-Aldrich), 1x Pencillin/Streptomycin (Gibco) and 1x Primocin (Invivogen), 10 mM Y-27632 supplemented with medium for the first 3 days. Normal colon organoid medium is identical to tumor organoid medium, except that it needs to be supplemented with 50% Wnt3a-conditioned medium. The Wnt3a-conditioned medium were obtained from L-Wnt3a cells. PDO was passaged approximately every 7–10 days using TrypLE Express (GIBCO) for about 10 minutes at 37 ℃ to dissociate the PDO into single cells. After dissociation, the cells were embedded in Matrigel (Corning, USA), and the medium was changed every 3–4 days.
TILs were generated as previous described (21). Briefly, the tumor samples were minced into approximately 1–2mm fragments after washed with ice-cold PBS buffer, and then put in a digestion medium consisting of 1 mg/ml collagenase (Sigma-Aldrich, USA) and 30U/ml DNase (Sigma, USA) at 37℃ for about 60 minutes. Subsequently, cells after digestion were placed into a 6-well plate comprised of T cell media with X-VIVO 15 (Lonza, USA), 2 mM Glutamax (Life Technologies, USA), 6000 U/mL IL-2 (Perprotech, USA), 10 ng/mL IL-15 (Perprotech, USA), 10 ng/mL IL-21 (Perprotech, USA), and 30 ng/mL anti-human CD3 antibody (OKT-3, Miltenyi, Germany). The T cells were cultured at 37°C with 5% CO2 and passaged to maintain a density of 1× 106 cells/mL until there were enough TILs used for screening tumor-specific T cells.
Peripheral blood lymphocytes
PBLs were obtained from freshly peripheral blood through Ficoll Paque plus (GE, DN25) density gradient centrifugation. Subsequently, the isolated PBLs were cryopreserved in liquid nitrogen for future utilization.
Histological analysis and immunohistochemistry staining of PDO
Organoids were collected (e.g., 6-well plate, collection of 1–2 well organoids, about 300–800 organoids) and subsequently fixed in 4% paraformaldehyde for 30 minutes at 4°C, followed by gradient dehydration with ethanol and embedding in paraffin wax. The paraffin blocks were cut at a thickness of 5µm using a paraffin microtome (Leica, RM2245) and subjected to routine hematoxylin-eosin (H&E) staining.
For immunohistochemistry staining, paraffin sections were routinely deparaffinized and incubated for 30 minutes in 3% H2O2 to inactivate endogenous peroxidase. Heat-induced antigen retrieval was performed using citric acid for 15 minutes at 95°C. The sections were incubated with primary antibodies overnight at 4°C. Then the HRP-conjugated second antibodies were stained for 1 hour at room temperature (supplementary Table S1). The bound antibody was visualized using the DAB detection kit (ZSGB-BIO, ZLI-9019) and counterstained with hematoxylin. The results were recorded using a light microscope (Olympus).
For immunofluorescent analysis, the organoids were fixed in 4% paraformaldehyde (DingGuo) for 15 minutes at 4°C. Subsequently, they were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). Then the organoids were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hour (supplementary Table S1). Nuclear staining was performed using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma). The resulting images were captured using a confocal microscope (Zeiss, LSM780).
TILs and PBLs phenotypic characterization analysis
For CD39+CD103+ positive T cell analysis, TILs and PBLs from CRC patients were stained with anti-human CD3-FITC, anti-human CD8-PE, anti-human CD39-PE-Cy7, anti-human CD103-APC antibodies cocktails and 7AAD (Invitrogen). For anti-human CD8+PD1+ positive T cell analysis, TILs and PBLs were stained with anti-human CD3-FITC, anti-human CD8-PerCP5.5, anti-human CXCL13-PE antibodies cocktails and 7AAD. For CD8+PD1+ positive T cell analysis, TILs and PBLs were stained with anti-human CD3-FITC, anti-human CD8-PerCP5.5, anti-human PD-1-PE antibody cocktails and 7AAD (supplementary Table S2). Then samples were analyzed by flow cytometry, the data were analyzed using FlowJo software.
Generation of tumor organoid-enriched T cells (oeT) by tumor organoid–autologous TILs co-culture
Co-culture of single cell-derived organoids and autologous TILs were performed to enrich tumor-specific T cells. Briefly, two days before co-culture, tumor organoids embedded in Matrigel were stimulated with decitabine (DAC) cocktails (10 µM DAC (Sigma Aldrich), 10 ng/mL TNF-α (ACRO), 100 U/mL IFNγ (ACRO)) for 48 hours. DAC cocktails restored expression of HLA by inhibiting DNA methylation or enhancing expression of HLA-related molecular TAP or LMP genes (22–24).
Next, the tumor organoids were dissociated into single cells using TrypLE Express and then resuspended in T cell medium. Prior to the experiment, a 96-well U-bottom plate was prepared by coating with 5 µg/mL anti-CD28 antibody in PBS (50 µL per well) and subsequently wrapping in parafilm overnight at 4°C. The anti-CD28-coated plates were then washed twice with PBS. TILs were seeded at a concentration of 105 cells per well and co-cultured with autologous single cell organoids at a concentration of 104 cells per well at a 10:1 effector: target ratio. Co-cultures were performed in T cell medium (X-VIVO15 medium supplemented with 10% human AB serum, 2 mM glutamine, 150 U/ml IL-2, 20 ng/mL IL-15, 10 ng/mL IL-21, 1% penicillin-streptomycin, 20 µg/mL anti-PD-1 blocking antibody (Biolegend)) and cultured at 37°C with 5% CO2. On the 7th day, co-cultures TILs were re-stimulated with fresh organoids at a ratio of 10:1 for an additional 7 days. The medium was half-changed every 1–2 days, and co-culture ended on day 14. TILs without tumor organoids stimulation were set up as a control.
Three-dimensional (3D) organoid killing assays
Organoids killing assay was performed in accordance to previously described with slight modification (25). In brief, tumor organoids were stimulated with DAC cocktails for 48 hours, and part of the organoids were dissociated into single cells and then counted to infer the number of tumor cells at a effector: target = 10:1 ratio. 1 mM of (Invitrogen) celltrace yellow cell proliferation kit (Invitrogen, cat. no. C34567) was used to stain organoids for 20min. 3D tumor organoids and normal organoids were resuspended in T cell medium, and seeded in flat-bottom 96-well plate with 1×105 autologous oeT cells obtained by 14 days of tumor organoid co-culture. Green-fluorescent caspase 3/7 probe (Invitrogen, cat. no.C10723) was added during co-culture at 1:2000 dilution to visualize tumor cells undergoing apoptosis. After 20 hours of coculture, the images of the killed organoids were observed and photographed using fluorescence microscope. For MHC Ⅰ/Ⅱ blocking assay, tumor organoids were pretreated with 50 µg/mL pan-MHC Ⅰ antibody (Biolegend, 311428) and pan-MHC Ⅱ antibody (BD, 555556) blocking for 40 min before co-culture. Part of organoids dissociated into single cells with TrypLE Express, and the cells stained with anti-human CD3-FITC (Biolegend), anti-human CD8-PE (Biolegend), anti-human CD107a-PE-Cy7 (Biolegend) and 7AAD (supplementary Table S2). Then the samples were analyzed by flow cytometry.
Isolation of tumor-specific T cells after repeated stimulation with autologous tumor cells
To evaluate tumor reactivity of oeT cells, oeT cells were collected after 14 days of incubation with tumor organoids and resuspended in T cell medium. The oeT cells were then restimulated with tumor organoids and normal organoids at a 1:1 effector: target ratio, respectively. Subsequently, seeded in anti-CD28-coated plates in the presence of 20 mg/mL anti-PD-1 and co-cultured for 24 hours. TILs without organoids stimulation and normal organoids stimulation were set up as controls. Cells were washed twice in PBS and stained with the following antibodies: anti-human CD3-FITC (Biolegend), anti-human CD8-PE (Biolegend), anti-human CD137-APC (Biolegend) and 7AAD for 30 minutes at 4 ℃ (supplementary Table S2). Then the cells were washed twice with PBS. The expression of surface markers were quantified, and the CD8+CD137+ positive T cells were sorted into 96-well PCR plates using the Symphony S6 (BD) and analyzed using FlowJo software. Subsequently, the 96-well PCR plates were immediately stored at -80°C.
T cell receptor sequencing and analysis
Single-cell RT-PCR is based on a previously described method (26). The PCR primer sequences are listed in supplementary Table S3. Briefly, the first round of RT-PCR produces single-cell cDNA. The reaction conditions for the first-round RT-PCR were as follows: the reverse transcription reaction for 30 minutes at 50℃; then 95℃ for 15 minutes and 30 cycles of 94℃ for 30 seconds, 52℃ for 30 seconds, 72℃ for 1 minute; 72℃ for 10 minutes. Subsequently, 2 µl of the first-round RT-PCR cDNA product was used as a template for the second round of TCRα/β PCR. The reaction conditions for the second round PCR are as follows: 98°C for 1 min and 43 cycles of 98°C for 10s, 52°C for 10s, 72°C for 45s; 72°C for 10 min. The PCR products were sequenced after gel cutting and purification. The TCR sequences were analyzed using the IMGT/V-Quest tool (http://www.imgt.org/).
TCR construction and transduction of PBLs
Construction of retroviral vectors encoding TCRs was used the methods as described previously (5, 27). Briefly, the complete sequences encoding the TCR were constructed in a β chain-α chain order, the TCRs was done by fusing the TCRβ V-D-J of the Vβ regions to the mouse TCRβ constant chain and the TCRα V-J regions to the mouse TCRα-constant chain. The mouse constant region that had been modified with interchain disulfide bond and hydrophobic substitution, as previously described (28–30). Additionally, furin-SGSG-P2A sequence was introduced between the TCRβ chain and TCRα chain. The TCR sequences were synthesized after codon optimization (GenScript) and subsequently inserted into the lentivirus vector.
TCR transduction was performed in accordance to previously described with slight modification (22, 31). In brief, human PBLs from autologous or healthy donors were stimulated in T cell media supplemented with 50 ng/ml OKT3 for 2 days before transduction. To generate transient retroviral supernatants, HEK293T cells were transfected with the lentivirus plasmid encoding TCRs and the envelope-encoding plasmid using CaCl2. After 48 hours, the viral supernatants were collected and used to infect PBLs in the presence of polybrene (8 µg/mL). 3 days after infection, TCR-positive T cells were isolated through flow cytometry using a mouse TCR beta monoclonal antibody (Invitrogen, 17-5961-82). The sorted cells were subsequently confirmed using anti-human CD3 FITC (Biolegend, USA), anti-human CD8 PE (Biolegend, USA) and mouse TCR beta monoclonal antibody (eBioscience, USA).
Enzyme-linked immunospot assay (ELISPOT)
The human IFN-γ ELISPOT assay was performed according to the instructions provided by the manufacturer of the commercially available human IFN-γ ELISPOT kit (Dakewe, cat. no. 2110005, China). Briefly, T cells were rested in cytokine-free medium for 24 hours, and tumor organoids were treated with DAC cocktails for 48 hours before co-culture. A total of 1 × 104 T cells and 1 × 104 autologous organoids were incubated together for 20 hours in cytokine-free medium at 37°C with 5% CO2. Phytohemaggluinin (PHA) was added as a positive control, and medium was added as a negative control. The number of IFN-γ positive spots was analyzed using an Immunospot plate reader (Cellular Technologies, USA).
Enzyme-linked immunosorbent (ELISA) assay
Human IFN-γ cytokine release was quantified using the human ELISA kit (Dakewe Biotech cat. no. 1110002, China). Briefly, T cells were rested in cytokine-free medium for 24 hours, and organoids were stimulated with DAC cocktails for 48 hours prior to co-culture. A total of 5 × 104 T cells and 5 × 104 autologous organoids were incubated together for 24 hours in cytokine-free medium, and then the co-cultured supernatant was collected. IFN-γ cytokine secretion was measured using a commercially available human IFN-γ ELISA kit, following the manufacturer's protocols. In the MHC I blocking experiment, tumor organoids were pretreated with 50 µg/mL of pan-MHC I antibody (Biolegend, 311428) for 40 minutes before co-culture. The tumor recognition was tested in technical replicate experiments.
Statistics analysis
Data statistical analysis was performed using GraphPad Prism 7.0. The statistical analysis was conducted with a student's t-test. p-values < 0.05 were considered statistically significant, denoted by * (p < 0.05), ** (p < 0.01), and *** (p < 0.001). All experiments were conducted more than three times independently.