Cell lines, media and reagents
Colorectal cancer cell line Caco-2 was obtained from ATCC (catalogue number: HTB-37, USA). Cell line was tested for mycoplasma contamination using a PCR based detection kit (Venor®GeM, Minerva Biolabs). Caco-2 cells were cultured in DMEM (Gibco, Thermo Fisher Scientific, USA) supplemented with 15% Fetal calf serum (FCS) (Gibco, Thermo Fisher Scientific, USA).
The study was approved by the Regional Committee for Medical Research Ethics (Oslo, Norway) (REC approval no: 2013/624, 2016/2247). T cells from healthy donors were expanded using a protocol adapted for pre-clinical production of T cells employing CD3/CD28 Dynabeads essentially as previously described . In brief, PBMCs were isolated from Buffy coats by density gradient centrifugation and cultured with Dynabeads (CTS™ Dynabeads™ CD3/CD28, provided by Gibco, Life Technologies AS, Norway) at a 3:1 ratio in complete X-Vivo 15 medium with 100 U/mL IL-2 (Proleukin, Novartis Healthcare, USA) for 10 days. After 10 days’ expansion, CD3/CD28 Dynabeads were removed and T cells were frozen or used directly.
Retroviral particle production
Viral particles were produced as described in  and used to transduce T cells and Caco-2 cells. In brief, 1.2.106 Phoenix-AMPHO (ATCC, Catalogue number: CRL-3213) cells were plated. Transfection was performed using Extreme-gene 9 (Roche) with a mix of DNA including the retroviral packaging vectors and the expression vector to an equimolar ratio. After 24 h, the medium was replaced with 1% HyClone FCS-containing DMEM and the cells were transferred to a 32 °C incubator. Supernatants were harvested after 24 h and 48 h of incubation.
Transduction of cells
PBMCs were incubated for 2 days in a 24 wells-plate coated with CD3 and CD28 at 1.106 cells/mL. A 24-well plate was coated with 50 μg/mL of retronectin (Takara, Shiga, Japan) during 3 h at room temperature before being washed with PBS and blocked with a solution of 1 mg/mL of FBS during 30 min. One milliliter of virus solution was deposited in each well and topped with 500 μL of activated T cells or Caco-2 cells at a concentration of 0.3.105 cells/mL. The plate was then incubated for 30 min at 37 °C, 5% CO2 for 30 min, sealed and then spun down at 750 g, 32 °C during 60 min before being placed back in the incubator. The same spinoculation step was repeated the following day before the cells being collected, spun down, washed and resuspended in complete X-Vivo 15 medium (for CD19CAR cells) or complete DMEM medium (for Caco-2 cells) for 2 days before the expression of the construct was checked and the cells expanded using the procedure described above.
Antibodies and flow cytometry
CAR expression was detected by anti-mouse Fab antibody (Jackson ImmunoResearch, West Grove, PA, USA). CD19 expression was detected by anti-human CD19 APC (Invitrogen, Thermo Fisher Scientific, Germany). Cells were acquired on a BD FACSCanto flow cytometer and the data analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA).
Bioluminescence-based cytotoxicity assay
Luciferase-expressing Caco-2 cells were counted and resuspended at a concentration of 3.105 cells/mL. Cells were given D-Luciferin (75 μg/ml; Perkin Elmer) and were placed in 96-well white round-bottomed plates as 100 μl cells/well. CAR T cells were added at a 10:1 E:T ratio. In order to determine spontaneous and maximal killing, wells with target cells only or with target cells in 1% Triton™ X-100 (Sigma-Aldrich) were seeded. Cells were left at 37 °C and the bioluminescence was measured with a luminometer (VICTOR Multilabel Plate Reader) as relative light units (RLU) at indicated time points. Target cells incubated without any effector cells were used to determine baseline spontaneous death RLU at each time point. Triplicate wells were averaged and lysis percentage was calculated using the following equation: % specific lysis = 100x (spontaneous cell death RLU- sample RLU)/(spontaneous death RLU – maximal killing RLU). Sigmoid curves (no Hill equation) were fitted for every set of points (using Igor Pro 8.1, Wavemetrics, USA) as guide for the eye with standard deviation as the weighting factor.
3D structure formation
Caco-2 cells (GFP+ with or without CD19) were trypsinized and resuspended as a single cell suspension at a concentration of 1.106 cells/mL. For 1 mL of cysts mixture, 400 μL of Caco-2 cells were mixed with 400 μL of matrigel (BD Bioscience) and 200 μL of Rat collagen I (final concentration of 1 mg/mL, Cultrex). One hundred microliters of the mixture was dropped per well either into 96 wells plate for BLI assays and high throughput imaging or in Ibidi u-Slide 8 well treated with ibiTreat #1.5 polymer (Ibidi) before being covered with 100 μL of complete DMEM. Plates for live imaging were incubated into an Incucyte S3 (Essen Biosciences, UK) and imaged every 4 h. In all case, cysts were let to grow for 6 days prior to use.
For BLI assay, cysts were let to grow in 96-well white round-bottomed plates. At day 6, cysts were incubated with 100 μg/ml of D-Luciferin for 16 h. T cells were then added on top of the collagen-matrigel mixture by adding 5.105 cells/well (roughly at a 1:10 E:T ratio) in 50 μL of complete DMEM medium. Killing was assessed in a similar way as described above.
For high throughput imaging assay, at day 6, 50 μL of a 1:200 solution of Annexin V red (Essen Biosciences, UK) diluted in complete RPMI 1640 was added per well and the plate was subsequently incubated at 37 °C, 5%CO2 for 15 min. Mock and CD19CAR T cells previously washed and resuspended in complete DMEM medium were introduced in each well at a final concentration of 5.105 cells/well. The plate was then put into an Incucyte S3 with the same settings as described above. Analysis of cytotoxicity was performed using Incucyte software. Metrics were then extracted and corrected using Igor Pro 8.1 (Wavemetrics, USA).
For confocal imaging, at day 4, 2.104 Mock or CD19CAR T cells were placed on top of each well. Then at day 6, cysts were fixed in 4% formaldehyde (Sigma-Aldrich) for 30 min. After PBS rinsing, unreacted aldehyde groups were quenched with 50 mM of NH4Cl (Sigma-Aldrich) for 20 min. Cysts were permeabilized with 0.5% Triton X-100 for 15 min before being incubated with aPKC zeta (C-20) (Santa Cruz) and E-cadherin (32A8) (Cell Signaling) overnight at 4 °C in 0.05% of saponin in PBS. Cysts were then washed three times with 0.05% of saponin in PBS before being marked with secondary antibodies Alexa488 donkey-anti-mouse (Jackson ImmunoResearch) and Alexa568-donkey-anti-rabbit (Molecular Probes). Slides were then washed extensively with PBS before nuclei being marked with 20 μM of Hoechst 33352 for 5 min. After a final PBS wash, cysts were imaged with a Zeiss LSM 880 Airyscan with a 1.4 NA water immersion 40x objective.
Statistics were made with 1-way ANOVA with Brown-Forsythe and Welch and Dunett T3 corrections (single timepoint comparison) or 2-way ANOVA with Greenhouse-Geisser and Tukey corrections (multiple timepoints comparison). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All statistical analyses were performed using R.