StemCell Therapy

Cell culture and cell lines

The mouse cell lines MCA205 H-2b (MCA, methylcholanthrene derived sarcoma, provided by Dr. Guido Kromer, France) and B16F10 (B16, melanoma, kindly provided by Dr. Roger Chammas, ICESP) were maintained in a humidified incubator at 37C with 5% CO2 and cultivated in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Invitrogen) as well as 1X Anti-Anti (AntibioticAntimycotic -100X, Thermo Fisher Scientific). HEK293 cells were cultivated in Dulbeccos modified Eagle medium (both from Thermo Fisher Scientific), supplemented and maintained in the same conditions as above.

Here we use the MCA sarcoma cell line and employed an intratumoral (i.t) application model since it was demonstrated under these conditions the ability of Dox to unleash ICD and stimulate immune responses in vivo11. We also used the B16 cell line, as it was with this model that we revealed the cell death and immune stimulatory events of our p19Arf/IFN treatment. With regard to the treatment order, we based our approach on the work of Fridlender and collaborators (2010) that showed that association of an adenoviral vector encoding IFN with chemotherapy is more effective when gene transfer is applied first23.

The MCA-DEVD cell line was generated by transduction with a lentivirus reporter for caspase-3 activity and selection for puromycin resistance (0.5g/ml). This vector, previously described24, encodes a constitutively expressed luciferase-GFP protein separated from a polyubiquitin domain via a caspase-3 cleavage site and was generously provided by Dr. Chuan-Yuan Li (Department of Radiation Oncology, University of Colorado School of Medicine, Aurora, CO, USA).

Construction and production of AdRGD-PG adenoviral vectors (serotype 5) containing modification with the RGD motif in the fiber as well as the p53-responsive promoter (PGTx, PG) has been described previously14. Titration of adenoviral stocks was performed using the Adeno-X Rapid Titer Kit (Clontech, Mountain View, CA, USA) and titer yields were: AdRGD-CMV-LacZ (3.6109IU/mL, infectious units/milliliter), AdRGD-PG-LUC (11011IU/mL), AdRGD-PG-eGFP (51010IU/mL), AdRGD-PG-p19 (1.31010IU/mL) and Ad-RGD-IFN (51010IU/mL). This biological titer was used to calculate multipilicity of infection (MOI).

MCA or B16 cells (1105) were plated in 6 well plates containing 1mL of RPMI media and transduced with adenovirus at the desired MOI. After an overnight transduction period (1216h), 2mL of media was added and cells kept in culture until needed. When combining adenoviral transduction with chemotherapy, Dox (doxrubicin hydrochloride, Sigma, St. Louis, MO, USA) was added immediately after the overnight transduction using the concentration indicated for each experiment. Importantly, in the Dox single treatment condition, Dox was added at the same moment as in the association group, 12 to 16h after cell plating. After 12h treatment with Dox (1mg/mL) or Nutlin-3 (10M, Sigma), expression of eGFP from AdRGD-PG-eGFP was analyzed by flow cytometry (Attune, Life Technologies). Cell viability was assessed by MTT assay where, 8h after transduction in 6 well plates, 2104 cells/well were plated in 96 well plates, treated with Dox, and analyzed after 16h of incubation. Non-transduced cells were used as viable control and protocol was carried out as described previously25. Cell cycle analysis by propidium iodide (PI) staining was carried out 72h after p19Arf/IFN and Dox single treatment, as previously described16. Analysis of caspase 3 activity in vitro was performed 16h after combined treatment using the CellEvent Caspase-3/7 Green Reagent (Thermo Fisher Scientific) by flow cytometry, following manufacturers instructions. Last, analysis of ICD markers upon p19Arf/IFN+Dox was conducted as detailed previously14. Briefly, detection of calreticulin+ and PI- cells was made 14h after combined treatment, by staining with a CRT-specific antibody (1:100, Novus, Biologicals, CO, USA) and after cells were washed with PBS, they were incubated with Alexa488-conjugated anti-rabbit secondary antibody (1:500, Thermo Fisher Scientific) followed by PI staining to exclude dead cells, immediately before flow cytometry. Accumulation of ATP in the cell supernatant was detected using the ENLITEN ATP Assay (Promega, Madison, WI, USA), as per the manufacturer's instructions. Luminescence was observed using a GloMax Plate Reader (Promega). HMGB1 in cell supernatant was detected by Western blot after conditioned medium was supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). Then, 180l of the medium was concentrated (Concentrator PlusEppendorf, Hamburg, Germany) and subjected to western blotting. Unrelated, high molecular weight regions of the membrane were removed before detection was performed using anti-HMGB1 (Abcam ab79823, Cambridge, UK) and a secondary antibody conjugated with horseradish peroxidase before visualization using ECL (GE Healthcare, Chicago, IL, USA) and the ImageQuant LAS4000 imaging platform (GE Healthcare). See Supplementary Information Westerns S2 for original images from three independent assays. Additional Western blots were performed using cell lysates, high-molecular weight regions of the membranes were removed and then detection was performed using anti- PARP (Cell Signaling, Danvers, MA, USA, #9542), anti-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, #47778), anti-Caspase 3 (Cell Signaling, #9662), anti-Tubulin (Millipore, Burlington, MA, USA, #05-829) and the appropriate secondary antibodies conjugated with horseradish peroxidase (anti-mouseSigma #A9044 e anti-rabbitSigma #A0545). See Supplementary Information Westerns S2 for original images from two independent assays.

The influence of two independent variables, namely, MOI of adenoviral vectors encoding p19Arf/IFN and the concentration of Dox, was investigated on MCA and B16 cells using factorial experiments in five levels (Table S1), with the percentage of hypodiploid cells as the variable response. The experiments were carried out employing central composite rotational design (CCRD) where, for each cell line, a set of twelve combinatory assays containing a central composite factorial matrix plus rotation points, central points and controls was performed (Table S2, where the assays and conditions are provided in detail). To better visualize the effects and interactions of MOI and Dox concentration on the percentage of hypodiploid cells, assessed by PI staining after 20h of treatment, the results were plotted in response surface graphs.

Importantly, the statistical significance of the independent variables and their interactions was determined by Fishers post-test for an analysis of variance (ANOVA) and Pareto chart analysis, both at a confidence level of 95% (p0.05). Moreover, five repetitions at the central point (CP) assays were used to minimize the error term of the ANOVA. Experimental designs, data regression and graphical analysis were performed using the Statistica software v.7.0 (Statsoft, Inc., Tulsa, OK, USA).

Both C57BL/6 and Nude mice were female, 7weeks old, obtained from the Centro de Bioterismo da FMUSP and kept in the animal facility in the Centro de Medicina Nuclear (CMN) in SPF conditions, with food and water ad libitum. The methods are reported in accordance with ARRIVE guidelines. The well-being of the mice was constantly monitored and all methods, including vaccination protocols, in vivo gene therapy, imaging, echocardiographic assessments, anesthesia and euthanasia were carried out in accordance with relevant guidelines and regulations of Brazil and our institution whose ethics committee (Committee for the Ethical Use of Animals, CEUA, University of So Paulo School of Medicine, FMUSP) approved this project (protocol n 165/14).

In the first step of the immunotherapy model, nave C57BL/6 mice were inoculated (s.c) in the right flank (tumor challenge site) with fresh untreated MCA (2105) or B16 (6104) cells and in the second step, vaccinated (s.c) on days+3,+9 and+15 with 3105 ex vivo treated cells applied in the left flank (vaccine site). Ex vivo treatment was carried out as follows: MCA or B16 cells were seeded in 10cm plates with 2mL of media and co-transduced with the AdRGD-PG-p19 and AdRGD-PG-IFN (MOI 500 for each) for 4h before the addition of 8mL of fresh media. Then, cells were kept in culture for 16h and in the p19Arf/IFN+Dox or Dox groups, Dox (14M) was added for 6h, until cells were harvested, washed twice with cold PBS, counted and resuspended in 100 L of cold PBS. For the DEAD cell+GFP control group, cells were transduced with the AdRGD-PG-eGFP vector (MOI 1000) and after 16h, harvested, washed twice with cold PBS, resuspended and lysed by three cycles of freezing and thawing.

MCA (2105) or B16 (5105) cells were harvested, washed twice with cold PBS, resuspended in 100 L of PBS per mouse and then inoculated subcutaneously (s.c) in the left flank of immune competent C57BL/6 or immune deficient Balb/c Nude (Foxn1n) mice. While mice were not randomized after injection of cells, but there was no specific selection of animals for each treatment group. No blinding of group allocation was performed at any phase of experimentation. No animals were excluded from the data. Approximately 8days later, palpable (60 mm3) tumors were treated three times, once every 2days, with intratumoral (i.t) injections (administered with precision Hamilton glass syringes (volume 100L) and 26G needles) of the following adenoviral vectors, AdRGD-CMV-LacZ or AdRGD-PG-LUC (4108IU, resuspended in 25 L final volume of PBS/mouse) or treated with the combination of AdRGD-PG-p19 and AdRGD-PG-IFN (2108IU, for each vector and maintaining the 25 L final volume per mouse). For the Dox single treatment model, chemotherapy was applied (i.t) once on day 12 with the following doses: 60, 20, 10 or 5mg/kg (in the final volume of 30 L of PBS/mouse). Whereas in the association model, adenoviral vectors were injected as explained above and Dox given 2days after the last viral injection (day 14), following the injection method as the Dox single treatment group. Tumor progression was measured by calipers every two days and volume calculated as described17. For the survival analysis comparing C57BL/6 and Nude mice, treated mice were euthanized by anesthesia with ketamine/xylazine followed by CO2 inhalation when tumor volume reached 1000 mm3 unless otherwise noted. See figure legends for the number of animals in each experimental group.

For the analysis of caspase 3 in vivo, MCA-DEVD tumors were treated in situ as described above and 24 and 48h after the last treatment injection, mice were submitted to bioluminescence imagining (IVIS Spectrum, Caliper Life Science) to detect the luciferase activity from the DEVD reporter. To this end, 10mg/kg luciferin (Promega) was administered by intraperitoneal (i.p) injection of each mouse and these were anesthetized with isoflurane (Cristalia, So Paulo, Brazil) using the Xenogen anesthesia system before imaging. Images were captured and only the strongest signal from each tumor was included in the analysis with Living Imaging 4.3 software (PerkinElmer, Waltham, MA, USA). Luciferase activity was obtained from the average radiance value [p/s/cm2/sr]. To calculate the fold activity overtime, average radiance values obtained for each mouse 48h post-treatment were divided by its respective value at 24h. Parental MCA tumors were used as negative control and no emission was detected (data not shown).

The systolic cardiac function was assessed by echocardiography. Exams were performed 10days after treatments with AdRGD-PG-eGFP (adenovirus control), Dox 10mg/kg, Dox 20mg/kg and p19Arf/IFN+Dox 10mg/kg. Mice were anesthetized with 1.5 to 2.5% isoflurane (in 100% oxygen ventilation). They were trichotomized and placed in supine decubitus to obtain cardiac images. Parasternal-long and short axis images were captured using VEVO 2100 ultrasound equipment (Vevo 2100 Imaging System, VisualSonics, Toronto, Canada) with a 40MHz linear-transducer. Analyses were performed off-line using VevoCQ LV Analysis software (VisualSonics). Parameters such as systolic and diastolic volumes were calculated using Simpsons modified algorithms present in the analysis software (parasternal-long axis images). Based on these volumes, stroke volume (L) and left ventricle ejection fraction (LVEF, %) were calculated. Also, linear measurements were obtained from parasternal short axis images. Left ventricle shortening fraction (LVSF, %) was calculated, using systolic and diastolic diameters. Left ventricle mass (LV mass, mg) was estimated by linear measurements. Beating rate (beats per minute, BPM) was recorded directly by an animal table-ECG system connected to the VEVO 2100 system. Echocardiographic results were interpreted considering the American Society of Echocardiography recommendations concerning the mouse model26. All parameters were shown as the mean values of three consecutive cardiac cycles. Transthoracic echocardiography image acquisition and analysis was performed by an expert investigator who was blind to the experimental groups.

Data are presented as meanSEM. Statistical differences between groups are indicated with p values, being *p<0.05, **p<0.01 and ***p<0.001. Statistical tests are indicated in each figure legend along with the number of independent experiments performed or number (n) of mice in each group. These analyses were made using the GraphPad Prism 5 (La Jolla, CA, USA) software, with the exception of the CCRD analysis (explained above).

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Potentiation of combined p19Arf and interferon-beta cancer gene therapy through its association with doxorubicin chemotherapy | Scientific Reports -...

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