Mesenchymal stromal cells as carriers of IL-12 reduce primary and metastatic tumors of murine melanoma

Due to immunosuppressive properties and confirmed tropism towards cancer cells mesenchymal stromal cells (MSC) have been used in many trials. In our study we used these cells as carriers of IL-12 in the treatment of mice with primary and metastatic B16-F10 melanomas. IL-12 has confirmed anti-cancer activity, induces a strong immune response against cancer cells and acts as an anti-angiogenic agent. A major limitation of the use of IL-12 in therapy is its systemic toxicity. The aim of the work was to develop a system in which cytokine may be administered intravenously without toxic side effects. In this study MSC were used as carriers of the IL-12. We confirmed antitumor effectiveness of the cells secreting IL-12 (MSC/IL-12) in primary and metastatic murine melanoma models. We observed inhibition of tumor growth and a significant reduction in the number of metastases in mice after MSC/IL-12 administration. MSC/IL-12 decreased vascular density and increased the number of anticancer M1 macrophages and CD8+ cytotoxic T lymphocytes in tumors of treated mice. To summarize, we showed that MSC are an effective, safe carrier of IL-12 cytokine. Administered systemically they exert therapeutic properties of IL-12 cytokine without toxicity. Therapeutic effect may be a result of pleiotropic (proinflammatory and anti-angiogenic) properties of IL-12 released by modified MSC.


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% CO 2 , 95% humidity). The cells were cryopreserved in FBS, DMSO and IMDM solution in − 80 °C until needed.
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.
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 cm 3 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 × 10 5 MSC suspended in IMDM culture medium with FBS was placed on a 6 cm plate and incubated under standard culture conditions (37 °C, 5% CO 2 ). 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% CO 2 ) 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.

Results
The cells. Mesenchymal stromal cells were isolated from murine bone marrow. The cells isolation protocol was optimized. The obtained cells met the MSC-specific criteria. The cells were grown on plastic dishes, they had a slender, fusiform shape and a fibroblast-like morphology (Fig. 1A). Using a flow cytometer, their phenotype was examined. Isolated cells had a characteristic MSC phenotype; 80% of the cells expressed Sca-1 and CD29 antigens, 70% CD90 antigen, 60% CD44 and CD105 antigens. Cells had less than 3% CD45 hematopoietic antigen and did not have endothelial CD31 antigen (less than 1%) (Fig. 1B). The isolated cells had the ability to differentiate into three cell lines: adipocytes, chondrocytes and osteoblasts. Cells differentiated to adipocytes were determined by expression of fatty acid binding protein 4 (FABP4) ( Fig. 2A). Alizarin red revealed the presence of Ca 2+ calcium ions in the osteoblast preparation obtained from differentiated MSC (Fig. 2B). MSC differentiated into chondrocytes were stained with Safranin O solution (Fig. 2C).
The migration ability of MSC towards cancer cells was confirmed. The migration capacity of MSC and MSC/ IL-12 to cancer cells and to media collected from cancer cells (chemotactic agents secreted by the cells) was examined (Figs. 3, 4). The tropism towards the media containing chemotactic factors secreted by cancer cells was examined using Boyden chambers (Fig. 3). MSC migration was confirmed to both media collected from melanoma and glioma cells culture (Fig. 3A). MSC/IL-12 migration to media collected from melanoma cells culture was confirmed (Fig. 3A). The cells migrated through the pores of the sieve towards the tested medium. The number of the cells migrating to the media was counted and compared (Fig. 3B). More than sixfold more MSC as well as MSC/IL-12 migrated to the B16-F10 cell conditioned medium than to the control medium without serum (and fourfold more than to the control medium supplemented with FBS (Fig. 3B). More than 4 times more MSC migrated to the medium from GL261 than to the control medium without serum and 3 times more than to medium with serum (Fig. 3B).
Co-cultures of MSC and GL261eGFP glioma cells were observed to examine the cell mobility (Fig. 4). MSC were stained with PKH26 and showed red fluorescence. The cells were incubated in Matrigel (as a simulation of tissue conditions) under standard culture conditions. The 24-h culture and observations were performed in the www.nature.com/scientificreports/ microscope chamber for in vivo cell examinations. Over time, there was a gradual accumulation of MSC around cancer cells (Fig. 4). After the first 2.5 h, aggregation of MSC near cancer cells was noticeable (Fig. 4). After 6 h, all MSC visible in the field of view remained clustered around the glioma cells (Fig. 4).

The cells modification.
The procedure of introducing IL-12 DNA into MSC was successful. First, two subunits of IL-12 cDNA were introduced into pAdenoX-DsRed-Express adenoviral vector (Fig. 5) by cloning. Positive clones were chosen using Colony PCR reaction (Fig. 6) and purified. The sequencing confirmed the cloning efficiency.  www.nature.com/scientificreports/ A transfection of AdenoX 293 packaging cells using obtained plasmid DNA was conducted and virus isolation and amplification were performed. MSC were transduced with obtained viral particles and incubated until intense red fluorescence was observed (Fig. 7A). The ability of MSC/IL-12 cells to secrete IL-12 was confirmed by ELISA test. Modified cells produced IL-12 in opposite to unmodified cells that do not secrete IL-12 at all (Fig. 7B). The phenotypic characteristic of the transduced cells was determined. Typical markers of MSC: CD90, CD29 and Sca-1 and the absence of the CD45 marker were shown in immunofluorescent staining and microscopic analysis (Fig. 7C).

Experiments on animals. The modified cells (MSC/IL-12) were used in experiments on animals bearing
primary and metastatic B16-F10 melanoma.
The single-dose administration of MSC/IL-12 cells significantly reduced the volumes of primary melanoma tumors. IL-12 producing MSC were used in the therapy of mice bearing primary B16-F10 melanoma tumors. 9 days after inoculation of mice with B16-F10 cells MSC/IL-12 were administered intratumorally (Fig. 8). On the 20th day of the experiment, the tumor volumes in mice treated with modified MSC were eightfold lower than in control mice receiving PBS - (Fig. 9A,B). The animals were lively and showed any side effects.
A single administration of MSC/IL-12 to the tail vein of animals resulted in a significant reduction in melanoma lung metastases. MSC/IL-12 cells were used in the treatment of mice with B16-F10 melanoma lungs metastases. 5 days after B16-F10 cells iv administration MSC/IL-12 were given to the tail vein. In 21st day after cells administration the number of lung metastases in mice treated with modified MSC was approximately fourfold lower than in mice receiving PBS - (Fig. 10A). Collected lungs were weighed, and it was noted that the lungs of mice treated with MSC/IL-12 had significantly lower mass than lungs of mice receiving PBS - (Fig. 10A). Metastases in the lungs of treated mice were small, not assembled, did not cause tissue deformation (Fig. 10B). Lung metastases in control mice were large and occupied a significant proportion of lung tissue (Fig. 10B).

Post-therapeutic analyses.
After the experiments on animals, the tumors were excised, collected and the post-therapeutic analyses were performed. To assess the effect of modified MSC on the two most important fac-   www.nature.com/scientificreports/ received MSC/IL-12 was twice lower than in controls both on days three (Fig. 11A) and eight (Fig. 11B) after MSC/IL-12 administration. In tumors collected from treated mice, the vessels were rare and small (Fig. 11C). In control mice tumors, a dense network of mature vessels with significant lumen was observed (Fig. 11C).

Single, intratumoral administration of MSC/IL-12 cells resulted in a change in the composition of the macrophage population in tumors.
The number of M1 and M2 macrophages in tumors collected from mice treated with MSC/IL-12 compared to controls by flow cytometry. On day 8 after cells administration, mice were sacrificed and tumors were excised for analysis. Tumor cell suspension was stained with antibodies directed against the CD45, F4/80, CD86, CD206, antigens. In tumors collected from mice treated with modified cells, the M1/M2 ratio was 14 times higher than in tumors of control mice (Fig. 12A).
The above observations were confirmed by immunohistochemical analyzes of tumor sections. MSC/IL-12 cells were administered intratumorally 8 days after the inoculation with B16-F10 cells. The tumors were collected 8 days after the cells administration; tissue sections were prepared and incubated with antibodies directed against the antigens M1 (F4/80 + /CD206 -, AlexaFluor594-red), M2 (F4/80 + /CD206 + , AlexaFluor594-red, FITC-green). Macrophages isolated from B16-F10 tumors were collected 8 days after the cells administrations and analysed by flow cytometry (Fig. 12A). The same analysis was performed in the frozen sections (Fig. 7B). The ratio of M1/M2 macrophages in tumors collected from mice treated with MSC/IL-12 was over fourfold higher comparing to controls (Fig. 12B,C). Selected photos of the preparations show the prevalence of M1 macrophages in tumor sections from treated mice. (Fig. 12D).

Single, intratumoral administration of MSC/IL-12 cells resulted in an increase of CD8 + T lymphocytes.
MSC/ IL-12 cells were administered intratumorally 8 days after the inoculation with B16-F10. Three days after the administration the tumors were collected, fixed and the number of CD8 + T cell was determined by immunohistochemistry (Fig. 13B). The number of CD8 + T cells (per 1 mm 2 of the preparation) in group that received MSC/IL-12 was over eightfold higher comparing to control and over 1.5-fold higher compared to MSC group (Fig. 13A).

Discussion
In the last over a dozen years IL-12 has become the subject of interest of researchers as one of the most effective antitumor cytokine. IL-12 as a mediator of inflammation interacts with a number of cells of the immune system, acting as a bond between the adaptive and innate immune responses. IL-12 as a potent immunostimulant activates T lymphocytes and NK cells as well as triggers release of IFN-γ, all of which induce a strong immune response directed against cancer cells 22,23 . This effect is considered responsible for stimulating IL-12 related production of IFN-γ. IFN-γ also reduces the ability of tumor cells to produce VEGF 24 .
Our group is exploring the IL-12 based therapeutical approach for almost 20 years. Anti-tumor effects of IL-12 have been proved in models of renal cancer 25 and in anti-melanoma combination therapies with cyclophosphamide 26 , endoglin-based DNA vaccine 27 , CAMEL peptide 28 , D -K 6 L 9 peptide 29 , anti-vascular ABRaA-VEGF121 chimeric protein 30 and tumor cell lysate 31 . Similar studies have been carried out in many other laboratories, where the efficacy of therapy using adenoviral plasmid with IL-12 in liver, colon and pancreatic cancer models was tested 32 .
All the above therapeutic solutions have proved effective in inhibiting the growth of tumors and showed satisfactory effects only after administration of IL-12 directly in the vicinity of the tumor. However, there are www.nature.com/scientificreports/ serious limitations to use IL-12 in therapy. Long-term systemic administration of high doses of IL-12 caused side effects like fever, fatigue, hematological disorders, hyperglycemia, liver damage, and acute colitis. There have been deaths among patients undergoing clinical trials with its use [33][34][35] . There is still a need to find a way to administer the cytokine intravenously without overall toxicity. It would be necessary to treat the patients bearing with hard-to-reach tumors. The aim of the work was to develop a system in which cytokine may be administered intravenously without toxic side effects. In this study, mesenchymal stromal cells were used as carriers of IL-12 cytokine.
MSC are immunologically privileged, do not induce an immune response in the recipient after transplantation 9,36-39 and show strong tropism to tumors sites [40][41][42][43] . Inflammation and hypoxia are attractors to MSC, causing their migration into the damage zone, in which a number of chemokines, adhesion molecules and  48 . Similar results were presented by Liu et al. 49 .
In our study, we confirmed the ability of MSC to migrate towards the media collected from cancer cells (Fig. 3) More than sixfold more MSC migrated to the B16-F10 cell conditioned medium than to the control medium. More than 4 times more MSC migrated to the medium from GL261 than to the control medium. MSC migration was also observed towards cancer cells themselves (Fig. 4). The tropism was examined on Matrigel in co-culture of MSC with GL261 glioma cells. After the first 2 h of incubation, aggregation of MSC near cancer cells was noticeable. After 6 h, all MSC visible in the field of view remained clustered around the glioma cells. Owing to these specific migratory abilities MSC are an excellent vehicle for transferring therapeutic factors.
The employment of mesenchymal stromal cells for the transfer of anti-cancer agent as IL-12 appears to be a promising therapeutic strategy against melanoma. The use of genetically modified IL-12 producing cells has many benefits. Transfection or transduction may be optimized ex vivo without exposing the recipient organism to high, toxic doses of the cytokine. It is possible to use as gene carriers the cells showing natural tropism to tumors 50,51 . In our opinion, the most important is that IL-12-modified MSC trigger the cascade reaction activated by IL-12 protein. Previously, using the adenovirus vectors, the IL-12 gene was introduced into fibroblasts 52 , dendritic cells in therapies against leukemia 53 and melanoma 54 and T cells in therapy against the thymoma 55 . Mesenchymal stromal cells seem to be an effective carrier of the IL-12 gene. MSC secreting IL-12 were used in the therapy of mice bearing glioblastoma. Intracranial administration inhibited the growth of tumors and increased the survival of animals 51 . IL-12 secreting MSC were used in the treatment of mice with Ewing sarcoma, where tumor growth inhibition was also observed 56 .
In our study MSC secreting IL-12 (MSC/IL-12) administered directly to the B16-F10 melanoma tumors significantly inhibited their growth. Ten days after the administration of the cells, the volumes of tumors in mice that received MSC/IL-12 cells were eightfold lower than in mice treated with PBS - (Fig. 9).
In the study MSC/IL-12 cells were administered to the tail vein of mice 4-5 days after experimental B16-F10 metastases initiation. The lungs were collected and analyzed after 21 days from administration of the modified MSC. In the lungs of mice treated with MSC/IL-12, fourfold less metastases were formed than in the lungs of control mice (Fig. 10). MSC are larger than hematopoietic cells, so as many as 80% of them are retained in the pulmonary capillaries a few minutes after iv administration 57 . At the basis of communication between MSC and endothelial cells are adhesive interactions between molecules on the surface of MSC and endothelial cell receptors. The adhesion of MSC to the pulmonary vascular walls is mainly due to the VCAM-1 adhesion-protein ligand 58 . After administration of the MSC to the bloodstream of the animal, the cells are located in the pulmonary alveoli. Due to the presence of adhesins and integrins (CD29, CD44) on the surface of the MSC membrane ( Fig. 1) they adhere to the walls of the blood vessels. MSC are located in the vicinity of macrophages residing in the lungs and in the vicinity of tumor cells 58 . Studies indicate that MSC remain in the lungs for up to 4 days after administration 57  IL-12 works comprehensively, modifies the microenvironment of tumor. Single, intratumoral administration of MSC/IL-12 cells, 8 days after the inoculation with B16-F10 resulted in a significant reduction in vascular density in melanoma tumors. Three and eight days after the cells administration the vascular density in group that received MSC/IL-12 was over twice lower than in controls (Fig. 11).
Tumor blood vessels are abnormally tortuous-their chaotic architecture greatly affects fluctuating slow blood flow and exacerbate metabolic mismatch between supply and demand what leads to progressive hypoxia. IL-12 modifies the tumor microenvironment: reduces the number of blood vessels. The anti-angiogenic efficacy of IL-12 is probably due to the stimulation of IFN-γ production 24,63 . IFN-γ induced by IL-12 reduces the secretion of VEGF by tumor cells. Therapy with the use of IL-12 lowers the production of metalloproteinases, important www.nature.com/scientificreports/ in tissue remodeling during neoangiogenesis. IL-12-stimulated production of IFN-γ also contributes to inhibiting the activation of α V β 3 integrins and downregulating the expression of ICAM-1 and VCAM-1 adhesion proteins on the surface of endothelial cells. IL-12 stimulates the production of cytokines and anti-angiogenic chemokines [64][65][66] . NK cells that accumulate around the blood vessels in the presence of IL-12 have cytolytic properties toward endothelial cells 67 . IL-12 modifies the tumor microenvironment-changes the proportion of the macrophage populations. Macrophages are the most plastic population of cells in the immune system. They undergo specific activation and occur in two functionally different phenotypes presented in response to environmental conditions 68 . These are the classically activated macrophages M1 (proinflammatory) and alternatively activated M2 macrophages (immunosuppressive). The specific plasticity allows them to functional reprogramming depending on the stimulus available 69 . M1 macrophages are associated with the initiation and maintenance of inflammatory response, M2 with inflammation quenching and tissue regeneration 70,71 . Tumor-Associated Macrophages (TAM) phenotype resembles the M2 macrophage phenotype. TAM (M2) produce tumor growth and invasive factors such as growth factors (EGF and VEGF), cytokines, enzymes supporting the process of angiogenesis (MMP9), inhibit the expression of anti-cancer factors such as IL-12 and accumulate at hypoxic sites 72,73 . TAMs show strong immunosuppressive properties through the production of anti-inflammatory cytokines and chemokines 73,74 .
In our study MSC/IL-12 cells elicit changes in the proportion of macrophage populations in tumors. A 14-fold increase in the ratio of M1 to M2 macrophages was observed in tumors after MSC/IL-12 treatment comparing to control (Fig. 12). These cells are probably co-responsible for therapeutic effect stimulated after MSC/IL-12 cells injection.

Summary
The effectiveness of the proposed system, i.e. the inhibition of melanoma progression, results from the use of both: the characteristics of the carrier and the drug transported. MSC show tropism to cancer cells and release the therapeutic protein in their vicinity. IL-12 has anti-angiogenic and immunostimulatory activity and leads to inhibition of tumor progression. Modified mesenchymal stromal cells secrete IL-12 that inhibit the progression of murine B16-F10 melanoma. The proposed therapeutic system inhibits the growth of primary tumors and metastases in the lungs. The probable mechanism responsible for the effectiveness of the therapy is the inhibition of angiogenesis evoked by MSC/ IL-12 cells and the increase in the percentage of M1 macrophages and CD8 T lymphocytes in the tumor tissue. The number of CD8 + surface area T cells (per mm 2 of the preparation) in group that received MSC/IL-12 was over eightfold higher comparing to control and over fivefold higher compared to MSC group (A). Representative images of tumor sections from mice treated with PBS, unmodified MSC or MSC/IL-12 (B). CD8 + T lymphocytes (AlexaFluor594-red), cell nuclei (DAPI-blue), lens magn. 20x, *p < 0.05, **p < 0.005.