Canine adipose tissue-derived mesenchymal stem cells (MSC) were purchased from Cellider Biotech S.L. and cultured in MEM alpha (BioWest) supplemented with 20% FBS (Hyclone) . Commercial cell lines used for co-culture are A549 (human lung adenocarcinoma; ATCC CCL-185), Hs 888.T (human osteosarcoma; ATCC CRL-7622), RPMI 2650 (human nasal septum squamous cell carcinoma; ATCC CCL-30) and CLAC (canine lung adenocarcinoma; JCRB 1453). Except for RPMI 2650 that was maintained in EMEM (Lonza) supplemented with 10% FBS, all other commercial cell lines were cultured in DMEM (BioWest) supplemented with 10% FBS. Cells were maintained at 37 °C in humidified atmosphere and 5% CO2.
Passage 4 of MSCs were transfected with CD::UPRT::GFP plasmid as previously described . A total of 5.5 × 106 cells were seeded in a 500 cm2 dish (Corning 431,111) 24 h prior to transfection. Polyethylenimine MAX (PEI, Polyscience) was added to serum free MEM alpha at 4µL of PEI (1 mg/mL) to 1 µg of plasmid DNA. A total volume of 5 mL of the mixture was incubated at room temperature for 15 min. After incubation, the transfection mixture was supplemented with transfection Enhancer. The Enhancer consist of DOPE/CHEMS (9:2 molar ratio Fusogenic lipid, Polar Avanti Lipid) and 1 µM Bufexamac (Histone deacetylase inhibitor; HDACi, Sigma-Aldrich) . Cells were incubated for 24 h before analysis.
Cryopreservation of MSC overexpressing CD::UPRT::GFP
Cells were washed twice with Plasma-Lyte A (Baxter 2B2544X) and harvested using TrypLE Express. After centrifugation, the cell pellets were resuspended in cryopreservation media, CS10 (CryoStor10), at a concentration of 1 × 106 cells/mL, 2 × 106 cells/mL or 3 × 106 cells/mL in cryovials. The cryovials were transferred into a Mr. Frosty™ Freezing Container (Thermo Fisher Scientific) for overnight freezing in − 80 °C, before being stored in liquid nitrogen vapour for at least 5 months and up to 1 year. Before use, the cells were thawed using the ThawSTAR Automated Thawing System (Biolife Solutions), resuspended in 4 mL of Plasma-Lyte A, followed by centrifugation at 300 × g for 5 min. The pelleted cells were either resuspended in HypoThermosol Preservation Solution (Sigma-Aldrich) for cell viability assessment, administration for canine patients or suspended in culture medium for functionality analyses.
Cells were washed twice with Plasma-Lyte A (Baxter 2B2544X) and harvested using TrypLE Express. Single-cell suspension was obtained by passing through a 100 µm cell strainer (SPL). Percentage of fluorescent positive cells was quantified by CytoFLEX LX flow cytometer (Beckman Coulter) and the raw data was analyzed using non-modified MSCs as negative controls at < 0.9%, using Attune™ NxT Software (Version 3.1.2 Thermo Fisher Scientific). At least 10,000 cells were analyzed per sample.
Cell images were taken with EVOS FL Cell Imaging System (Thermo Fisher Scientific) equipped with fluorescent light cubes for viewing of DAPI (Ex357/Em447) and GFP (Ex470/Em510) fluorescence.
Cell viability assessment
To examine the stability of post-thaw modified cells, cryopreserved modified cells were thawed, centrifuged, and left in suspension at room temperature or 4 °C for up to 4 h. The cells were then stained with acridine orange (AO; 500 µM/mL; Sigma-Aldrich) and 4′,6 diamidino-2-phenylindole (DAPI; 250 µg/mL; Thermo Fisher Scientific) and quantified using the NucleoCounter® NC-3000 Automated Cell Counter. The viability was assessed using NucleoView (ChemoMetec).
To determine the effect of cryopreservation on MSC phenotype, unmodified, freshly modified, and cryopreserved modified cells were fixed in 4% formaldehyde (Sigma-Aldrich) and stained with antibodies. Suspension cells were centrifuged at 800 × g for 5 min; and resuspended in 1 mL of Blocking Buffer containing 15% FBS in 1XPBS (BioWest). Cells were kept on ice for 15 min; washed with Flow Wash Buffer consisting of 1% FBS in 1XPBS. The cell pellets were then resuspended at a concentration of 1 × 106 cells/mL in Flow Wash Buffer after removing the supernatant. Cells were either directly or indirectly stained with antibodies that recognize the specific cell surface markers. For direct staining, cells were stained with isotypic control (PE, Thermo Fisher Scientific), CD90 (PE, Thermo Fisher Scientific, clone YKIX337.217) and CD34 (PE, BD Biosciences, clone 1H6). For indirect staining, cells were stained with primary antibody of CD44 (Bio-Rad, clone YKIX337.8.7) and secondary antibody of PE (Thermo Fisher Scientific). All samples were washed, then resuspended in Plasma-Lyte A, and stored at 4 °C prior to analysis by flow cytometry.
Trilineage differentiation assay
Cryopreserved cells were thawed, washed with cell culture media, centrifuged, and plated in media at 37 °C in humidified atmosphere and 5% CO2.
Cells were plated at 1 × 105 cells/well in a 24-well plate in culture media for 24 h. Media was removed and replaced with culture media (control wells) or StemPro™ Adipogenesis Differentiation Kit (Thermo Fisher Scientific). Media for each sample was changed every 3 days and cells were maintained for 14 days. Oil droplet was stained with Oil Red O solution and assessed by microscopy.
Cells were plated at 1 × 105 cells/well in a 24-well plate in culture media and permitted to adhere overnight before differentiation induction. Media was removed and replaced with culture media (control wells) or StemPro™ Osteogenesis Differentiation Kit (Thermo Fisher Scientific). Media was changed every 3 days. Calcium deposits were stained by Alizarin red S solution at day 21 post induction.
A total of 1 × 105 cells/well was seeded in an ultra-low attachment round bottom 96-well plate in culture media 24 h prior to induction. Media was removed and replaced with culture media (control wells) or StemPro™ Chondrogenesis Differentiation Kit (Thermo Fisher Scientific). Media was changed every 3 days and cells were maintained for 21 days. The formation of cartilage, aggrecan, was stained using copper-containing Alcian Blue dye.
Cell migration assay
MSC overexpressing CD::UPRT::GFP tumour tropism was assessed using 8.0 µm Transwell (Corning). A total of 4 × 105 cells/well of cancer cells, A549 and RPMI 2650, were seeded in complete media in the lower chamber of the 24-well plate. After 24 h of incubation, the cultures were washed twice with 1XPBS and replaced with serum free DMEM. Unmodified, freshly modified and/or cryopreserved MSC overexpressing CD::UPRT::GFP, were seeded at 1.5 × 105 cells/well into the upper chambers in serum free media. Cells that migrated to the bottom of the upper chamber were fixed with 4% PFA and stained with 10 µg/mL Hoechst 33,342 (Thermo Fisher Scientific). Three images per well were taken with EVOS FL Cell Imaging System (ThermoFisher Scientific) at 10 × magnification and the migrated cells were counted using ImageJ.
In vitro anticancer efficacy of cryopreserved CD::UPRT::GFP producing MSCs
The anticancer efficacy of MSC overexpressing CD::UPRT::GFP in vitro was determined by direct co-culture with 4 cancer cell lines (A549, Hs 888.T, RPMI 2650 and CLAC). Here, 2 × 103 cells/well of A549, Hs 888.T and CLAC, and 4 × 103 cells/well in RPMI 2650 were seeded in 96-well plate 5 h prior to the seeding of fresh or cryopreserved MSC overexpressing CD::UPRT::GFP at the ratios of 1 MSC to 1, 5, 10, 50 and 100 cancer cells. After 24 h, the culture media was replaced with DMEM supplemented with 10% FBS, with or without 5-FC (150 µg/mL). The anticancer efficacy was measured by MTS assay after 6 days of co-culture.
Use of GDEPT in canine patients
All the pet owners signed an informed consent authorizing treatment and were informed of the possible risks and side effects, and potential complications of the procedure. The body weight and tumor size of the canine patient were measured before and after the treatment. The x-rays or other diagnostic images of the pet patients were collected before, during and after the course of the treatment. The pet patients were given 500 mg per day of non-toxic prodrug 5FC orally for four consecutive days. The treatment was completed in 3 cycles. The health status, including daily activity and appetite of the treated pets was recorded by the owners. Follow-up of the patients’ well-being for at least 1 year included owners’ subjective observations and diagnoses from the veterinarian.
For intratumoural injection, cryopreserved modified cells were thawed, centrifuged, and resuspended in 0.5 mL of HypoThermosol Preservation Solution in an insulin syringe. The cryopreserved MSC overexpressing CD::UPRT::GFP (1 × 107 cells) were injected intratumourally at several spots around tumour mass.
For intravenous injection, cryopreserved modified cells were thawed, centrifuged, and resuspended in 7.5 mL of HypoThermosol Preservation Solution. The cryopreserved MSC overexpressing CD::UPRT::GFP (1.5 × 107 cells) were injected intravenously through a cephalic vein. The cephalic vein was cleaned with an alcohol swab for sterilization and better visualization. A 27 gauge was inserted into the vein valve stylet and was secured by pasting micropore tape on the catheter. The other end of the extension tube was then attached to the catheter. The whole extension tube was inserted into a syringe pump system of which the flow rate can be controlled. The rate of infusion was set at 3 mL/h. A washing step of 2 mL saline was implemented after the syringe was empty to ensure the remaining volume of modified MSCs in the extension tube to be infused into the vein. The canine patient was monitored for 1 h for potential side effects.
An unpaired two-tailed Student’s t-test was used, with the assumption that changes in the readout are normally distributed. All the graphs, calculations, and statistical analyses were performed using GraphPad Prism software version 9.4.1 for Windows (GraphPad Software, San Diego, CA, USA). All experiments were repeated at least thrice to determine reproducibility and to rule out random error.