The Inhibitory Effects Of Interferon-? Gene-modified Human Gingiva-derived Mesenchymal Stem Cells On Tongue Squamous Cell Carcinoma

Objective:Tongue squamous cell carcinoma(TSCC)is a common cancer type in oral cancer.Despite the improvement made in the treatment strategies for TSCC during the past decades,currently the disease still produces high morbidity and mortality,and patients are deprived of effective therapies for it.Therefore,it is clear that novel treatment strategies for TSCC are urgently needed.One possible therapeutic molecule for TSCC is interferon-?(IFN-?),which has been proven to significantly inhibit tumor cells growth in vitro.Howe-ver,the short half-life and higher effective dose with multiple side effects of IFN-? hinder its in vivo studies.Previous studies showed that IFN-? gene therapy using adenoviral vectors is effective in such cancers as bladder cancer and lung cancer.However,viral vector-based gene delivery administration has been limited by vector safety,organ toxicity,and difficulty in selective delivery of the therapeutic gene to metastatic sites.To overcome this problem,human bone marrow mesenchymal stem cells(BMSCs)have been utilized as biological vehicles for gene delivery.BMSCs-based IFN-(3 therapy via systemic administration successfully migrated to tumor sites and attenuated growth of tumor.However,reports demonstrated that human BMSCs can promote the growth of the TSCC,as well as the proliferation and invasion of TSCC cells.One recent report suggested that human gingiva-derived mesenchymal stem cells(GMSCs)can suppress oral cancer cell growth in vitro and in vivo.Human GMSCs have nontumourigenicity and can be obtained from gingival tissues that were easily accessible from the oral cavity with minimal discomfort.These observations suggest that human GMSCs have high potential as biological vehicles for tumor tissue-targeted delivery of IFN-P therapy.In this study,we isolated human GMSCs by limiting dilution method.We constructed a lentivial vector encoding IFN-? and transfected it into human GMSCs to obtain GMSCs/IFN-? in order to investigate the biological effects of GMSCs and GMSCs/IFN-P on TSCC cells in vitro,and explore the role of GMSCs and GMSCs/IFN-? in engrafting selectively in TSCC and controlling tumor progression in xenografted nude mouse tumor model in vivo.Materials and methods:1.Isolation and characterization of human GMSCs by limiting dilution methodHuman gingival tissues were obtained from patients undergoing crown lengthening surgery with no history of periodontal disease and the tissues were used to isolate GMSCs by limiting dilution method.Human GMSCs were evaluated in terms of their colony-forming abilities and population doubling capacities.Surface markers for human GMSCs were quantified by flow cytometry.For osteogenic differentiation,human GMSCs were cultured on 6 and 24 well plates in osteogenic inductive medium[DMEM(Dulbecco's Modified Eagle's Medium)supplemented with 10%FBS(Fetal bovine serum),0.1?M dexamethazone,10mmol/L ?-sodium glycerophosphate and 50mg/L vitamin C].After 28 days incubation,mineral deposition was detected in 24 well plates by Alizarin Red S staining.For adipogenic differentiation,human GMSCs were cultured on 6 and 24 well plates in adipogenic inductive medium(DMEM supplemented with 10%FBS,2mmol/L insulin 0.5m mol/L methylisobutylmethylxanthie,0.5?mol/L hydrocortisone and 60?mol/L indomethacin).And after 28 days incubation,the presence of lipid drops was detected in 24 well plates by staining the cells with Oil Red O.For chondrogenic differentiation,human GMSCs were transferred into centrifuge tube and centrifuged at 4? to form cell pellets at the bottom of the tube.Cell pellets were cultured in chondrogenic induction medium(in serum free DMEM containing 0.1W?mol/L dexamethazone,50p.g/ml ascorbic acid,1mmol/L sodium pyruvate,40p.g/ml L-proline,l0ng/ml Transforming growth factor-?3 and 6.25p.g/ml ITS)for 28 days.After 28 days incubation,the cell pellets designated for histological assessment were fixed in 4%paraformaldehyde at 4? overnight,then embedded in paraffin and sectioned for immunohistochemical staining with mouse anti-human collagen type II antibody.As controls,GMSCs were cultured in basic medium.6 well plates designated for quantitative real time polymerase chain reaction(qRT-PCR)were rinsed three times with phosphate buffered saline(PBS)and RNA extracted using TRIzol according to manufacturer's instructions.The expression of osteogenic,adipogenic and chondrogenic differentiation related genes of GMSCs were analyzed by qRT-PCR.2.Construction of IFN-? gene-modified human GMSCsA lentivial vector encoding IFN-?(Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin-IFN-?)was constructed and transfected into human GMSCs to obtain GMSCs/IFN-?.Parallel control cultures of GMSCs were transfected with blank vector(Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin).To determine the transduction efficiency of IFN-? in GMSCs,cells and condition medium(CM)were collected and detected by qRT-PCR and Enzyme-linked immunosorbent assay(ELISA)for IFN-P in mRNA and protain level at 1 week after infection.3.The biological effects of GMSCs,GMSCs/vector,GMSCs/IFN-P and IFN-P on TSCC cell line Cal27 cells in vitro3.1.Collection of CMTSCC cell line Cal27 cells were cultured with CM derived from GMSCs,GMSCs/vector and GMSCs/IFN-? to detect the biological effects on the cells.The normal growth media were conditioned by culturing GMSCs,GMSCs/vector or GMSCs/IFN-? for 24h.Basic medium(DMEM containing 10%FBS)served as control group,and basic medium containing 750pg/ml IFN-P served as positive control group.3.2.CCK8 proliferation assayCal27 cells were seeded in 96-well plates at a density of 1�103 cells per well.The CM or control groups medium were applied to the Cal27 that were seeded 24h before.At each time point,the culture medium was replaced with 100?1 DMEM containing 10?l CCK8 and the plates were incubated for 2h at 37?.Absorbance at 450nm was measured in a multiwell spectrophotometer.3.3.Colony formation assayCal27 cells were seeded in 6-well plates at a density of 1 � 103 cells per well and cultured for 7 days.The CM or control groups medium were applied to the Cal27 and changed every day.The colonies were stained with 1%crystal viole after fixation and counted under microscopic fields(�40).Aggregates of 50 or more cells were scored as a colony.3.4.Flow cytometry for cell cycle analysisCal27 cells were seeded in 6-well plates at a density of 2�105 cells per well.The CM or control groups medium were applied to the Cal27 that were seeded 24h before and changed every day.After 2 days culture,cells were harvested and processed for cell cycle analysis using flow cytometry.3.5.Flow cytometry for cell apoptosis analysisCal27 cells were seeded in 6-well plates at a density of 2�105 cells per well and cultured for 3 days.The CM or control groups medium were applied to the Cal27 and changed every day.After 3 days culture,cells were harvested and processed for cell apoptosis analysis using flow cytometry.3.6.Cell migration assayThe migratory effects of different groups were evaluated in 24-transwell plates.1.5�105 Cal27 cells in 100?l DMEM containing 0.I%FBS were seeded in the upper wells.The lower wells were supplemented with 500?l CM or control groups medium.The chambers were incubated for 24h at 37?.Afte removal of non-migrated cells on top of the filter,cells that had migrated through the membrane were fixed,stained with crystal violet,and counted under a phase-contrast microscope.3.7.Cell invasion assayThe diluted matrigel were added in the upper wells of 24-transwell plates for 1h to hydrate the basement membrane.The next steps were the same as cell migration assay.3.8.Western blottingTo determine the mechanism involved in the process of GMSCs/IFN-? induced Cal27 cell growth inhibition,we detected AKT signal pathway by western blotting.4.The biological effects of GMSCs,GMSCs/vector and GMSCs/IFN-? on TSCC xenografted nude mice tumor model in vivoA total of 42 male BALB/c male nude mice(5 weeks of age,15-19 g)were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd.All mice were,maintained in the animal care facility at the Second Hospital of Shandong University.The 2�106 Cal27 cells were subcutaneously inoculated on the right flank nearly axillary of each nude mouse.Before inoculation,Cal27 cells were suspended in PBS at a concentration of 1�107/ml.When tumor volume reached 50mm3,2 nude mice were used to identification of the tumor by hematoxylin-eosin(H&E)staining.The 40 nude mice were divided into 5 groups:(1)injected 200?l PBS intravenous(i.v.);(2)injected 1 �106 GMSCs in 200?l PBS i.v.;(3)injected 1 �106 GMSCs/vector in 200?l PBS i.v.;(4)injected 1� 106 GMSCs/IFN-? in 200?l PBS i.v.;(4)injected 1�106GMSCs/IFN-? in 100?l PBS intratumoral(i.t.).All of the cells were injected as described above i.v.or i.t.route twice with 2-weeks interval.After the first inoculation,tumor growth was examined every 3 days by measuring the length and width using a caliper,and tumor volumes were calculated.On day 28,all the nude mice were killed,and tumors were excised and weighed.Frozen tumor tissues were sectioned into 4 ?m slices and cell nucleus were stained by DAPI.The slices were observed under confocal laser scanning microscopy to monitor EGFP expression.Paraffin sections were made to detect the expression of IFN-? and cell proliferation antigen Ki67 in tumor tissues by immunohistochemical staining.Results:1.Isolation and characterization of human GMSCsPrimary cultures of single cell suspensions from human gingival tissues exhibited a spindle-shaped fibroblast-like morphology.All trials of single cell-derived colony forming efficiency were successfully performed.14 days after seeding,CFU-Fs were(21.4�2.8)%.Human GMSCs exhibited long-term proliferation capacity exceeded 12 passages in culture and the total population doublings of GMSCs was 40.95�4.19.By flow cytometry analyses,human GMSCs were positive for mesenchymal stem cell(MSC)associated makers CD44,CD73,CD90,CD 105,CD 166 and STRO-1,and were negative for the haematopoietic stem cell markers CD 14,CD34 and CD45.After 28 days of culture in osteogenic induction medium,GMSCs showed formation of mineralized nodules or aggregates.Calcium mineralization was confirmed by Alizarin Red S staining.After 28 days of culture in adipogenic induction medium,GMSCs showed formation of lipid droplets that were positively stained with Oil Red O.After 28 days of culture in chondrogenic induction medium,cell pellets of GMSCs exhibited synthesis of collagen type II by immunohistochemistry.GMSCs demonstrated in vitro osteogenic,adipogenic and chondrogenic differentiation capacities,mRNA expression levels were significantly increased for osteogenic-associated markers(RUNX2,OPN and BSP2),adipogenic-associated markers(leptin,adipsin and PPARy2),and chondrogenic-associated markers(aggrecan,COL-? and SOX9)compared with control groups by qRT-PCR analyses after induction.2.High-level expression of IFN-? produced by GMSCs/IFN-?There was no change for IFN-? transduced GMSCs from the cell shape.The GMSCs/IFN-? and GMSCs/vector appeared similar to fibroblasts,with a characteristic spindle-shaped morphology.Both GMSCs/IFN-? and GMSCs/vector had strong green fluorescence expression 48h after transfection under fluorescence microscope,QRT-PCR showed that IFN-? mRNA expression level in GMSCs/IFN-?group was significantly higher than that in GMSCs and GMSCs/vector group(P<0.01).IFN-? concentration was 756.7�19.914pg/ml for GMSCs/IFN-? group,which was significantly higher than that in GMSCs and GMSCs/vector group detected by ELISA(P<0.01).The expression level of IFN-P displayed no significant difference between GMSCs and GMSCs/vector group.The results indicated a stable expression of IFN-? in GMSCs/IFN-?.3.GMSCs,GMSCs/vector,GMSCs/IFN-? and IFN-? significantly attenuated Cal27 cell growth in vitroCCK8 assay showed a significant reduction of Cal27 cell proliferation in GMSCs,GMSCs/vector,GMSCs/IFN-P and IFN-P group compared with control group after the second day(P<0.01).The results displayed no significant difference between GMSCs and GMSCs/vector group.GMSCs/IFN-? and IFN-? group drastically reduced Cal27 cell proliferation compared with GMSCs and GMSCs/vector group(P<0.01).GMSCs/IFN-? showed similar inhibition effect compared with IFN-? group.Such a proliferation inhibitory activity of GMSCs/IFN-? was further demonstrated with the colony formation assay.The number of colonies detected in GMSCs,GMSCs/vector,GMSCs/IFN-? and IFN-? group was decreased compared with the control group(P<0.01).Moreover,fewer colonies were seen in GMSCs/IFN-? and IFN-? group than that in GMSCs and GMSCs/vector group(P<0.01).The number of colonies displayed no significant difference between GMSCs and GMSCs/vector group.There was also no significant difference in colony numbers between GMSCs/IFN-? and IFN-? group.To investigate the mechanism that mediates the proliferation inhibition function of GMSCs,GMSCs/vector,GMSCs/IFN-? and IFN-?,flow cytometry analyses of cell cycle and cell apoptosis were performed.There was no significant difference in the percentage of Cal27 cells in the S peak among all the groups,which indicated that GMSCs,GMSCs/vector,GMSCs/IFN-P and 1FN-? had no significant effects in Cal27 cell cycle compared with control group.Flow cytometry analyses showed that GMSCs,GMSCs/vector,GMSCs/IFN-P and IFN-?significantly promoted cell apoptosis compared with control group(P<0.01).GMSCs/IFN-? and IFN-? group drastically increased Cal27 cell apoptosis rate compared with GMSCs and GMSCs/vector group(P<0.01).The results indicated that the proliferation ability of Cal27 cells was significantly inhibited by GMSCs/IFN-P.We found that GMSCs/IFN-p could inactivate the AKT pathway through decreasing phosphorylation of AKT(p-AKT).The result suggested that GMSCs/IFN-P effectively inhibiting the proliferation of Cal27 cells was associated with suppression of AKT signal pathway.4.GMSCs,GMSCs/vector,GMSCs/IFN-? and IFN-? significantly attenuated Cal27 cell migration and invasion in vitroThe number of cells migrated to the lower surface of the transwells in GMSCs,GMSCs/vector,GMSCs/IFN-P and IFN-? groups were decreased compared with control group(P<0.01).Moreover,less migrated cells were seen in GMSCs/IFN-?and IFN-? groups than that in GMSCs and GMSCs/vector group(P<0.01).The number of migrated cells displayed no significant difference between GMSCs and GMSCs/vector group.There was also no significant difference in migrated cell numbers between GMSCs/IFN-? and IFN-P group.Such invasion inhibitory activity of GMSCs/IFN-P was further demonstrated with the cell invasion assay,which indicated that the migration and invasion ability of the Cal27 cells was significantly inhibited by GMSCs/IFN-p.5.GMSCs,GMSCs/vector and GMSCs/IFN-? significantly attenuated TSCC xenografted nude mouse tumor model growth in vivo5.1.Systemic administration of GMSCs,GMSCs/vector and GMSCs/IFN-?migrated to TSCC in vivoIn this study,the H&E staining showed that the TSCC xenografted nude mouse tumor model was established and each experimental mouse bearing TSCC treated with PBS,GMSCs,GMSCs/vector or GMSCs/IFN-? respectively.Green fluorescence was observed under confocal laser scanning microscopy in TSCC which received GMSCs/vector or GMSCs/IFN-? injection by different routes.Immunohistochemical staining results demonstrated that the tumor tissues expressed IFN-? in GMSCs,GMSCs/vector,GMSCs/IFN-P(i.v.)and GMSCs/IFN-?(i.t.)group.The number of IFN-? positive cells in GMSCs/IFN-?(i.v.)and GMSCs/IFN-p(i.t.)group was larger than that in GMSCs and GMSCs/vector groups(P<0.01).The observation suggested that exogenous GMSCs can migrate and engraft to TSCC in vivo.5.2.GMSCs,GMSCs/vector and GMSCs/IFN-? significantly attenuated tumor growth in vivoThe tumor volumes of GMSCs,GMSCs/vector,GMSCs/IFN-?(i.v.)and GMSCs/IFN-?(i.t.)group were decreased compared with control group from day 6(P<0.01).GMSCs/IFN-?(i.v.)and GMSCs/IFN-?(i.t.)group drastically reduced TSCC growth compared with GMSCs and GMSCs/vector group(P<0.05).GMSCs/IFN-?(i.v.)group exhibited the most potent antitumor activities compared with other groups(P<0.05).The trend was confirmed by the volumes and weights of dissected tumors,suggesting markedly decelerated proliferation of tumors by GMSCs/IFN-?.The results suggested that the local production of IFN-? in TSCC tissues might exert anti-tumor effects for GMSCs/IFN-?-based cancer therapy.Results of immunohistochemistry staining of Ki67 indicated the number of proliferating tumor cells was significantly higher in control group compared with the other groups(P<0.05).The number of Ki67 positive cells in GMSCs/IFN-?(i.v.)and GMSCs/IFN-?(i.t.)group was less than that in GMSCs or GMSCs/vector groups(P<0.05).GMSCs/IFN-?(i.v.)group exhibited the the strongest inhibition of tumor cell proliferation compared with other groups(P<0.05).The result suggested that GMSCs/IFN-? effectively inhibited the growth of TSCC,which maybe associated with the suppression of tumor cells proliferation.Conclusions:1.Human GMSCs were successfully isolated from normal gingival tissues by limiting dilution method.2.GMSCs/IFN-P was successfully constructed,which can stably secrete high level of IFN-?.3.Human GMSCs can inhibit TSCC cell line Cal27 cells proliferation,migration and invasion,and promote cell apoptosis.The results indicated human GMSCs can act as biological vehicles for TSCC gene delivery effectively and safely.4.GMSCs/IFN-? achieved the same biological effects as the IFN-P,and exhibited more significant effects on inhibiting Cal27 cell growth,invasion and migration,and promoting cell apoptosis than GMSCs/vector.The result suggested that GMSCs/IFN-? effectively inhibited the proliferation of Cal27 cells,which was associated with suppression of Akt signal pathway.5.Systemic administration of GMSCs,GMSCs/vector or GMSCs/IFN-? migrated to TSCC tissues and significantly attenuated tumor growth in vivo.Intravenous injection of GMSCs/IFN-P induced the most potent antitumor activities compared with other groups,while the local production of IFN-? in TSCC tissues might exert anti-tumor effects for GMSCs/IFN-?-based cancer therapy.The results provided evidence that delivery of IFN-? by GMSCs may be an excellent and promising approach to develop an effective treatment protocol for TSCC therapy.