Cells lines and animals
B16-F10 (murine melanoma) cell line (ATCC, Manassas, VA, USA) used to obtain primary tumors and metastases of murine melanoma was maintained using RPMI 1640 medium (Gibco BRL, Paisley, UK) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, USA). GL261 (mouse glioma) cell line (PerkinElmer, Waltham, MA, USA) was maintained using DMEM (Gibco BRL, Paisley, UK) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, USA). Cells cultures were kept under standard conditions (37 °C, 5% CO2, 95% humidity).
Experiments on animals were carried out in accordance with standard procedures. The study was approved by the Local Ethics Commission at the Medical University of Silesia in Katowice (approval no. 54/2018). The study was carried out in compliance with the ARRIVE guidelines. Mice (6 to 8-weeks-old, C57Bl/6NCrl) were obtained from Charles River Breeding Laboratories. All mice were housed in the Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice Branch (Poland) in a HEPA-filtered Allentown’s IVC System (Allentown Caging Equipment Co, NJ, USA). The animals received a total pathogen-free standard diet (Altromin 1314, Altromin Spezialfutter GmbH & Co. KG, Germany) and water ad libitum throughout the whole study. The animals were monitored every day. This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments on animals were conducted in accordance with the 3R rule.
Mesenchymal stromal cells: isolation and phenotypic characterization of mesenchymal stromal cells
The mice (6–8-week-old) were sacrificed by cervical dislocation. Femoral bones were excised and soft tissues were removed.
The bones were cut, washed with saline and incubated with collagenase solution (3 h, 37 °C, 0.4 U/ml, Serva Electrophoresis, Heidelberg, Germany). The cells suspension was filtered through 70 μm strainer. The cells were maintained in IMDM (Sigma Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), antibiotics (penicillin and streptomycin, Sigma Aldrich, USA) and murine bFGF (1 ng/ml). After 72 h, the plates were rinsed with PBS and fresh culture medium was provided. The medium was changed every 2–3 days. Cell cultures were kept under standard conditions (37 °C, 5% CO2, 95% humidity). The cells were cryopreserved in FBS, DMSO and IMDM solution in − 80 °C until needed.
The phenotype of the cells adherent to plastic dishes was determined using a flow cytometer (FACS Canto BD and BD FACSAria III; BD, Franklin Lakes, NJ, USA). To obtain a single cell suspension the cells were treated with 0.25% trypsin (Gibco BRL, Paisley, UK). The cells were incubated with antibodies directed against the following murine antigens: CD44, CD105, Sca-1, CD29, CD90.1, CD45, CD11b, CD106 or isotype-matched control antibodies according to the manufacturer’s protocol (30 min., 4 °C, BioLegend, San Diego, CA, USA). 7-AAD (7-amino-actinomycin D) was used to stain nonviable cells (5 μl/106 cells; BioLegend, San Diego, CA, USA). The cells with phenotype 7-AAD–CD44+CD105+Sca-1+CD29+CD90.1+CD45–CD11b–CD106– were considered as MSC.
The ability of obtained cells to differentiate into adipocytes, chondrocytes and osteoblasts was assessed using Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D, Minneapolis, MN, USA). The procedure was performed in accordance with the manufacturer’s instructions. The differentiation of MSC into chondrocytes and osteocytes was visualized by histochemical staining using Safranin O and Alizarin Red (Sigma Aldrich, USA) and observed with Eclipse 80i microscope (Nikon Instruments Inc., Melville, NY, USA). The differentiation of MSC into adipocytes was assessed by immunofluorescence staining using an antibody directed against the FABP4 (Abcam; Ab205332, 1:100, Cambridge, UK). The sections were incubated with secondary antibodies conjugated with fluorochromes (FITC) (Vector Laboratories, FI-1200, 1:100; Burlingame, USA). Fluorescence imaging of the stained cells was performed using a LSM710 confocal microscope (Carl Zeiss Microscopy GmbH, Germany).
MSC tropism towards cancer cells, in vitro examination in Boyden Chambers
Tropism towards tumor cells was examined in vitro using Boyden chambers (Corning Life Sciences, NY, USA). MSC or MSC/IL-12 were placed on a cylindrical cell culture insert with porous bottom nested inside the well of a cell culture plate filled with tested medium. The tested media were collected from over the B16-F10 and GL261 cell cultures, as control media fresh IMDM media with and without FBS supplementation were used. The cells that migrated through the pores of the cellulose membrane were fixed in cold methanol and stained with Giemsa solution (Merck Milipore, Darmstadt, Germany). The cells were counted in 5 fields of view of Eclipse 80i microscope at × 10 magnification.
MSC tropism towards glioma cells in Matrigel
Co-culture of MSC and GL261eGFP cells in Matrigel medium. MSC were stained with the PKH26 Red Fluorescent Cell Linker Kit (Sigma Aldrich, USA) according to the manufacturer’s protocol. Single cells suspensions of GL261eGFP and stained MSC were mixed in equal numbers, suspended in IMDM (3 × 103 cells/100 µl medium/well) and placed in Matrigel coated 96-well plate (according to manufacturer’s protocol). The plate was placed in the Zeiss Cell Observer SD chamber and incubated for 24 h under standard culture conditions (37 °C, 95% CO2). Observations were recorded using a microscope camera. In the first 5 h of the experiment, photos were taken every 30 min and then every 1 h.
Cloning of cDNA encoding IL-12 in adenoviral vectors
pBCMGSNeo plasmid obtained from Dr. H. Yamamoto (Osaka University, Osaka, Japan) contains cDNA encoding the two subunits of IL-12. The cDNA was isolated and amplified with PCR reaction. The starters were designed based on IL-12 cDNA template with 15 bp extensions homologous to the ends of linearized adenoviral vector pAdenoX-DsRed-Express (Clontech, CA, USA). PCR product was purified and introduced into the adenoviral vector with In-Fusion cloning system (Clontech, CA, USA). The procedure was performed in accordance with the manufacturer’s instructions. Following the reaction a portion of In-Fusion mixture was transformed into E. coli in SOC medium (Stellar Component Cells, Clontech, CA, USA). The transformation mix was spread onto LB agar plates with ampicillin (100 μg/ml). Well-separated colonies arisen on the plates were subjected to PCR Colony Screening using Terra PCR Kit (Clontech, CA, USA). Positive clones were amplified and the plasmid was purified. Cloning correctness was confirmed by sequencing of DNA fragment incorporated into the adenoviral vector. The sequencing procedure was performed using 3500 HITACHI Genetic Analizer (Applayd Biosystem, Foster City, CA, USA). Obtained vector was linearized using PacI restriction enzyme according to manufacturer’s instructions (NewEngland Biolabs, UK).
Preparation of the viruses
Transfection of packaging AdenoX 293 cells (Clontech, USA) with obtained vector was conducted using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) according to manufacturer’s instructions.
Cell lysis was performed by rapid freezing and thawing the suspension in an alcohol bath (dry ice with 80% alcohol) and in a water bath (37 °C). For the amplification of the adenoviruses, AdenoX 293 cells were transduced with obtained virus particles on 75 cm3 bottles according to manufacturer’s instructions.
The procedure was repeated. Viruses isolated from the 7th amplification cycle were used for the experiment. The AdenoX GoStix system (Clontech, USA) was used to confirm the presence and concentration of viral particles in the supernatant medium according to the manufacturer instructions. The aliquots obtained were stored at -20 °C until needed.
Preparation of modified MSC
7.5 × 105 MSC suspended in IMDM culture medium with FBS was placed on a 6 cm plate and incubated under standard culture conditions (37 °C, 5% CO2). When the cells reached 70% confluence, the medium was replaced with 1.5 ml fresh IMDM, 200 µl of the adenovirus suspension obtained in the previous steps of the procedure and polybrene (8 µg/ml). Cells were incubated (4 h, 37 °C, 5% CO2) then 2 ml fresh IMDM culture medium with FBS was added and incubated until intense fluorescence (24–48 h). Cells fluorescence was observed under a microscope Zeiss Cell Observer. An ELISA assay (Platinum ELISA, eBioscience, USA) was used to confirm if the modified cells produce IL-12 protein. The procedure was carried out according to the manufacturer’s protocol.
Phenotypic characterization of modified MSC
Sterile cover glasses were placed on a 6 cm plate and 7.5 × 105 modified MSC suspended in IMDM culture medium with FBS were incubated under standard culture conditions (37 °C, 5% CO2) for 24 h. Cover glasses were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained with antibodies conjugated with fluorochrome: CD29-APC, CD90-FITC, Sca-1-APC, CD45-APC (BioLegend, USA). Microscopic observations were performed using an LSM 710 Zeiss confocal microscope (Carl Zeiss Microscopy GmGB, Gottingen, Germany).
Treatment of mice bearing primary melanoma B16-F10 tumors
6–8 weeks mice were used for the experiment. The mice were divided into 3 experimental groups: (1) PBS, (2) MSC, (3) MSC/IL-12. Nine days after inoculating the mice (lower flank) with B16-F10 melanoma cells (2 × 105 cells/100 μl PBS/mouse) MSC, MSC/IL-12 cells and PBS were administered intratumorally (5 × 105 cells/100 μl PBS/mouse). Growing tumors were measured with calipers, and tumor volumes were determined using the formula: volume = width2 × length × 0.52.
Treatment of mice with experimental lung metastasis of B16-F10 melanoma
6–8 weeks mice were used for the experiment. To obtain metastases in mice B16-F10 cells (2 × 105 cells/100 μl PBS/mouse) were administered to the tail vein. 3 experimental groups were formed: (1) PBS, (2) MSC, (3) MSC/IL-12. On day 5th after administration of the cancer cells the mice were given MSC, MSC/IL-12 cells (5 × 105 cells /100 μl PBS/mouse) and PBS to the tail vein. On day 21st the mice were sacrificed, the lungs collected and fixed in Bouin’s solution. After 24 h the lungs were weighed and metastases were counted.
Post-therapeutic analyses
Tumor vascularization analysis
On days 3 and 8 after MSC/IL-12 cells administrations tumors were excised, embedded in liquid nitrogen and sectioned into 5 µm slices. To determine the presence of the blood vessels in collected tumors, frozen sections were stained using antibody directed against CD31 antigen (Abcam; Ab7388, 1:50, Cambridge, UK). The sections were incubated with secondary antibodies conjugated with fluorochromes (AlexaFluor 594) (Abcam; ab150168, 1:100, Cambridge, UK) and cover slipped with Vectashield mounting medium containing DAPI (Vector Laboratories, USA). Area occupied by blood vessels was counted with ImageJ software (NIH). Stained blood vessels were counted in 5 randomly chosen fields (magn. ×20) per section in 5 tumors of each group. Microscopic observations were performed using an LSM 710 Zeiss confocal microscope (Carl Zeiss Microscopy GmGB, Gottingen, Germany).
Determination of the M1 and M2 macrophages presence in B16-F10 melanoma tumors after the therapy
Flow cytometry
On day 8 after MSC/IL-12 cells administration tumors were excised, single-cell suspension was obtained using collagenase II solution (500 U/ml; Gibco BRL, Paisley, UK). Red blood cells were lysed using 0.15 M ammonium chloride (Sigma Aldrich, St Louis, MO, USA). Dead cells were removed by centrifugation using Histopaque-1077 gradient (Sigma Aldrich, USA). To identify the subpopulations of macrophages, antibodies against the following antigens were used: CD45, F4/80, CD206 and CD86 (eBioscience, USA) or isotype-matched control antibodies. All FACS-analyzed (BD FACSCanto, BD, USA) populations were gated in a 7-AAD window to enrich for viable cells. 7-AAD–CD45+ F4/80+ CD86+ cells were considered as M1 macrophages, 7-AAD–CD45+ F4/80+ CD206+ cells were considered as M2 macrophages.
Immunohistochemistry
On day 8 after intratumoral MSC/IL-12 cells administration the tumors were collected, embedded in liquid nitrogen and sectioned. Frozen sections were stained using antibodies directed against CD206 antigen (1:100, Abcam, Cambridge, UK) and F4/80 antigen (1:100, Abcam; Cambridge, UK). The sections were incubated with the secondary antibodies conjugated with fluorochromes (FITC, AlexaFluor 594) (Abcam, UK; ab150168, 1:100, Vector Laboratories, Burlingame, USA UK UK). Sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, USA). The fluorescence intensity was measured with ImageJ software (NIH). Microscopic observations were performed using an LSM 710 Zeiss confocal microscope (Carl Zeiss Microscopy GmGB, Gottingen, Germany).
Determination of CD8+ T lymphocytes in B16-F10 melanoma tumors
On day 3 after intratumoral MSC/IL-12 cells administration the tumors were collected, embedded in liquid nitrogen and sectioned. Frozen sections were stained using antibody directed against CD8+ antigen (1:50, Abcam, Cambridge, UK). The sections were incubated with the secondary antibody conjugated with fluorochromes (Alexa Fluor 594) (1:100, Vector Laboratories, Burlingame, USA). Sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, USA). The number of the of CD8+ T lymphocytes in each group was determined in 5 randomly chosen fields (magn. 20 ×) per section in 5 tumors of each group and calculated per 1 mm2 of the tumor. Microscopic observations were performed using an LSM 710 Zeiss confocal microscope (Carl Zeiss Microscopy GmGB, Gottingen, Germany).
Statistics
Statistical analyses were performed using Statistica software (Dell Inc. (2016), version 13. software.dell.com.). The Shapiro–Wilk test was used to verify the normality of the distribution. The statistical significance of differences between the experimental and control groups were evaluated with Kruskal–Wallis and multiple comparisons of mean ranks for all groups tests using Statistica software. Differences in p values of 0.05 or less were considered significant.
