Primary cells and cell lines
Frozen human peripheral blood mononuclear cells (PBMCs) from various de-identified donors (STEMCELL Technologies, Vancouver, BC, Canada) were thawed, washed, and suspended in the Roswell Park Memorial Institute (RPMI)-1640 (HyClone Laboratories, Logan, UT, USA) complete medium containing 10% (v/v) fetal bovine serum (FBS, HyClone), 100 units/mL of penicillin (Life Technologies, Carlsbad, CA, USA), and 100 mg/mL of streptomycin (Life Technologies) to prepare for subsequent cultures.
Phoenix-GP cells stably expressing MoMLV gag-pol (American Type Culture Collection (ATCC) #CRL-3215, Bethesda, MD, USA) and PG13 cells (ATCC #CRL-10686) were maintained in Dulbecco's modified Eagle's medium (DMEM) (#08458-45, Nacalai Tesque, Kyoto, Japan) supplemented with 10% (v/v) FBS.
Luciferase-expressing cell lines, BT474 (breast ductal carcinoma) (a kind gift from Dr. Jeong Eon Lee from Samsung Medical Center) and SKOV3 (ovarian adenocarcinoma, #1594), were provided by the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan) Cell Bank. U87 glioma cells purchased from ATCC were engineered to express luciferase by lentiviral transduction with pLenti CMV Puro LUC (Addgene #17477, Watertown, MA, USA) and treated with puromycin (InvivoGen, San Diego, CA, USA) for at least 1 week. Subsequently, a single cell clone of U87 with high luciferase expression was obtained by limiting dilution. Parental U87 cells were maintained in Minimum Essential Media (MEM, Life Technologies), while luciferase-expressing U87, BT474, and SKOV3 cells were maintained in RPMI-1640 supplemented as above. All the cells were cultured at 37 °C and 5% carbon dioxide (CO2).
HER2-CAR vector construction and generation of CAR T cells
The HER2-CAR vector consists of a HER2-binding single-chain variable fragment (scFv) moiety bearing a 4D5 sequence derived from the humanized monoclonal antibody trastuzumab (trastuzumab) [32], a CD8a hinge and transmembrane region, and intracellular domains of CD3z and CD28 co-stimulatory receptors. Sequences were synthesized (Bio Basic, Markham, Ontario, Canada) and sub-cloned into the MSGV Hu Acceptor retroviral vector (Addgene #64269) after removal of human T-cell receptor (TCR) genes.
Phoenix-GP cells were co-transfected with the HER2-CAR vector and pCMV-VSV-G (Addgene #8454) using FuGENE 6 (Promega, Madison, WI, USA). CAR retroviral supernatant was collected 24–48 hrs post-transfection, filtered with a 0.45 µm filter, and applied in the presence of 8 mg/mL polybrene to PG13 cells by spinoculation at 600 ×g for 2 hrs to generate cells producing GaLV-pseudotyped HER2-CAR retrovirus (HER2-CAR PG13 cells), which were expanded and cryopreserved. One week before T cell activation, HER2-CAR and parental PG13 cells were thawed and cultured to generate HER2-CAR and mock viral supernatants. Non-tissue culture-treated plates were pre-coated with RetroNectin (rFN-CH-296, #T100B, Takara Bio Inc., Otsu, Japan) at 5.26 µg/cm2 as per the manufacturer’s instructions. The viral supernatant was filtered and added to RetroNectin-coated wells, followed by centrifugation at 1500 × g for 2 hrs. The cells were activated with 50 ng/mL soluble anti-CD3 (OKT3, eBioScience, San Diego, CA, USA) and 100 ng/mL soluble anti-CD28 (CD28.2, eBioScience) monoclonal antibodies (mAbs) in the presence of 20 IU/mL recombinant human interleukin-2 (IL-2, Peprotech, East Windsor, NJ, USA) for 2 d and then transduced with HER2-CAR virus-bound or unbound RetroNectin at 600 ×g for 30 min to generate HER2-CAR and mock T cells, respectively. Transduced T cells were expanded in the presence of 100 IU/mL IL-2 for 5–7 d before cryopreservation. HER2-CAR and mock T cells were thawed and rested overnight in complete medium prior to co-culture with 2D tumor cell culture or spheroids.
Flow cytometry
Cells were first treated with Human TruStain FcX (Fc receptor blocking solution, #422302, BioLegend) and then incubated with recombinant biotinylated protein L (#RPL-P814R, ACROBiosystems, Beijing, China) followed by PE-conjugated streptavidin (#12-4317-87, eBioscience) to assess HER2-CAR expression. Then, 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, St. Louis, MO, USA) was used to exclude the dead cells. Samples were acquired on a MACSQuant X cytometer (Miltenyi Biotec, Auburn, CA, USA) and analyzed using the FlowJo software (TreeStar Inc., Ashland, OR, USA).
Cytotoxicity assay of the mock and HER2-CAR T cells in 2D culture
Mock or HER2-CAR T cells were co-cultured with 1 × 104 luciferase-expressing tumor cells at an effector (CAR T) to target (tumor) ratio of 4:1 in 96-well plates for 24 hrs in triplicate. Surviving tumor cells were assessed for luciferase activity using the Bright-Glo Luciferase Assay System (#E2620, Promega) according to the manufacturer’s protocol, with the luciferase reagent diluted with phosphate-buffered saline (PBS, pH 7.4) at a 1:1 ratio prior to use. Cell culture media were removed from the wells, and 100 µL of the diluted luciferase reagent was added to each well. The plates were shaken for 5 min to allow complete lysis of the cells before measurement. Luminescence of the lysed mixture was measured using the Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA).
Spheroid formation in agarose-coated wells
Agarose-coated wells were prepared by adding 50 µL of 1.5% (w/v) agarose (Sigma-Aldrich) at 70 °C in distilled water into 96-well plates (Corning Inc., Corning, NY, USA) and solidifying the agarose at 25 °C room temperature in a biosafety cabinet according to a previously described method [9,10]. A tumor cell suspension was prepared in RPMI-1640 at a density of 20,000 cells/mL, and 150 µL of the suspension was poured into each well. Then, the cells in the well were incubated at 37 °C with 5% CO2 for 48 hrs until they formed a spheroid with a diameter of approximately 300 µm.
When approximately 3,000 cells (BT474) were incubated in the agarose-coated wells, they started to form a single spheroid at 24 hrs and became larger up to approximately 300 mm in diameter after 48 hrs.
Cytotoxicity assay of the mock and HER2-CAR T cells in agarose-coated wells
A spheroid in each well was mixed with 10 μL of RPMI-1640 containing either 12,000 mock or HER2-CAR T cells. A single spheroid in each well was then mixed with either mock T or HER2-CAR T cells at an effector to target ratio of 4:1 for 24 hrs. The wells were incubated at 37 °C in 5% CO2 for 24 hrs. Then, 10 μL of LIVE/DEAD® Viability/Cytotoxicity Kit reagent (Molecular Probes, Eugene, OR, USA) was added to the wells and incubated at 37 °C with 5% CO2 for 30 min. Optical and fluorescent images were captured using a fluorescent microscope (DeltaVision Elite, GE Healthcare, Chicago, IL, USA). Images were processed and analyzed using the ImageJ software (NIH, Bethesda, MD, USA).
Fabrication of 3DHSP
The hanging dripper was designed using Inventor 2020 (Autodesk, San Rafael, CA, USA) and printed using a 3D printer (Cubicon, Seongnam, Korea) with acrylonitrile butadiene styrene (ABS) filaments. The plate consisted of two layers, of which the upper layer was made of poly (methyl methacrylate) (PMMA) (ENGP, Incheon, Korea), and the lower layer was made of polydimethylsiloxane (PDMS) (Dow Corning Co., Midland, MI, USA) [33]. A PMMA sheet with a thickness of 8 mm was ablated into a layer of 75 mm (L) ´ 58 mm (W) and holes with a diameter of 6 mm using a laser cutter (Zing™ 24 Laser, Epilog Laser, Golden, CO, USA). A PDMS layer of 2 mm thickness was fabricated by soft lithography using a silicon mold [33], and holes were punctured into the layer using a 6 mm biopsy punch (Kai Industries Co., Gifu, Japan). Both the PDMS layer and glass slide were treated with oxygen plasma for 30 s and then bound to each other. Subsequently, the PMMA layer was pasted with a mixture of PDMS and the curing agent (10:1 ratio) (w/w) on the bottom side and then placed onto the PDMS layer at 80 °C for 2 hrs until both layers were bounded.
Spheroid formation in 3DHSP
Before seeding tumor cells, the 3DHSP cells were thoroughly washed with 70% ethanol, rinsed twice with PBS, and irradiated with ultraviolet light for 30 min, detached from a Petri dish using trypsin/ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich), and then neutralized with RPMI-1640.
A cell suspension was prepared in RPMI-1640 at a density of 120,000 cells/mL. Approximately, 3,000 cells in 25 mL of RPMI-1640 were seeded into the 3DHSP through a hanging dripper using a pipette. Parafilm (Heathrow Scientific, Vernon Hills, IL, USA) was used to wrap the 3DHSP to prevent evaporation of the medium. The 3DHSP were incubated at 37 °C and 5% CO2 for 48 hrs until the cells formed a spheroid with a diameter of 300 mm in each hanging drop, while fresh RPMI-1640 was added to it by removing 7 µL of media from the 3DHSP through the dripper and adding 10 µL of fresh RPMI-1640 into the dripper [23].
Cytotoxicity assay of the mock and HER2-CAR T cells in 3DHSP
The 3DHSP assay was similarly performed as in agarose-coated cells (Fig. 4E). Each spheroid with a diameter of approximately 300 mm in the well was mixed with 10 μL of RPMI-1640 containing 12,000 either mock or HER2-CAR T cells. The 3DHSP was incubated at 37 °C in 5% CO2 for 24 hrs and then fixed on a tilting mechanical stage (Thorlabs, Newton, NJ, USA) with a custom-built computer program Kinesis® (Thorlabs) (Movie S3). A spheroid in the hanging dripper was deposited into the spheroid separation plate by injecting 100 μL of RPMI-1640 into the hanging dripper using a pipette, while dead and detached cells were separated from the spheroid by tilting the 3DHSP by 20°–40°. The remaining spheroid in the well was stained with 10 μL of LIVE/DEAD® Viability/Cytotoxicity Kit reagent as described above. Imaging of spheroids and analysis were performed as described above.
Rate of loss of spheroids after washing in agarose-coated wells and 3DHSP
To compare the rate of loss of spheroids after washing in agarose-coated wells and the 3DHSP, 3,000 BT474 cells were cultured in agarose-coated wells and the 3DHSP for 48 hrs to form spheroids, which were treated with 12,000 HER2-CAR T cells for 24 hrs. For the agarose-coated wells, 100 µL of PBS was added to the wells and then gently pipetted twice. After waiting for 30 s to allow the spheroid to settle down, the upper cell suspension was carefully removed. In the 3DHSP, 100 µL of PBS was added to the hanging drop, the 3DHSP was then tilted 30°, and the cell suspension was removed from the waste well.
Statistical analysis
All data are expressed as the mean ± standard deviation (SD) from three or more independent experiments. Statistical significance was determined using Student’s t-test.