Radiosensitizing Effect Of Adipose Tissue-derived Mesenchymal Stem Cells On Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is the most common primary liver tumor,ranking fourth in leading cause of tumor-related death.Radiation therapy(RT)is one of the main methods for treating malignant tumors,which produces ionizing radiation damage to biologically active molecules such as DNA in cancer cells.However,due to the high degree of HCC malignancy and poor radiotherapy tolerance,the clinical benefit of RT is relatively low,and the prognosis of HCC patients is still poor.It is of great clinical significance to seek a radiosensitizer that can increase the radiosensitivity of tumors and in the meanwhile has little or no side effects on normal tissues.Adipose tissue-derived mesenchymal stem cell(AD-MSC)is a kind of easily assessable and abundant mesenchymal stem cell.Many studies have found that it regulates signal transduction,cell cycle progression,apoptosis induction and inhibition values in tumor cells.The purpose of this study was to investigate the radiosensitization effect of AD-MSC on HCC cells and explore its underlying mechanism.Section I Study on radiosensitization of AD-MSC in treatment ofHCC in vitroObjectiveThe in vitro experimental model of combined radiation therapy(RT)and adipose-tissue derived mesenchymal stem cell(AD-MSC)treatment was explored to clarify the changes in cell proliferation,colony formation,and tumor stemness of hepatocellular carcinoma(HCC)cell lines after RT combined with stem cell therapy.This section is to investigate whether stem cell therapy has radiosensitization effect on HCC cells and to find the best combination therapy model.MethodsThe liposuction aspirates of healthy patients in the department of plastic surgery,the First Affiliated Hospital of Zhejiang University Medical College,were collected.Adipose tissue was separated,and AD-MSC was extracted and cultured.The stem cell surface specific markers CD44,CD73,CD90,and CD105 were identified by flow cytometry,and their adipogenic and osteogenic cell lines were divided into 4 groups,namely the control group,the radiation treatment group,the AD-MSC treatment group and the combination treatment group.The cells of each group were given different irradiation doses and different irradiation time in combination with stem cell therapy,and the cell proliferation was detected by the CCK-8 method;the colony formation experiment was used to detect the cell colony forming ability;Scratch test was used to detect cell movement repair ability;Transwell test was used to detect cell migration and invasion ability;cell flow was used to detect specific stemness markers expression.ResultsUsing normal liver cell line LO2 as a control,the results of CCK-8 experiments showed that HCC cell lines Huh7 and Hep G2 cells in the combined treatment group significantly inhibited proliferation.At the same time,compared with the control group,RT and AD-MSC treatment groups,the combined treatment significantly inhibited the ability of cell colony formation,move ability,migration and invasion,and significantly reduced tumor stemness.But the above changes were not significant in the control LO2cell line.ConclusionsIn vitro experiments show that for different types of HCC cell lines,MSC can increase the growth inhibition effect of RT on HCC,and the combined treatment results show that it is closely related to the irradiation dose and time.Significant tumor suppressive effect was seen compared to monotherapy.Section II Study on radiosensitization of AD-MSC in treatmentof HCC in vitroObjectiveSeveral preclinical studies have shown that mesenchymal stem cells(MSCs)have antitumor effects.The above in vitro studies combining MSC and RT have shown that stem cells may have a radiosensitizing effect,and the therapeutic effect is closely related to time and irradiation dose.In this section,in vivo experiments in animals are used to further determine the inhibitory effect of MSC combined with RT on HCC.MethodsAccording to the conclusions of the first section of the study,a model of human HCC cell line-derived nude mouse xenograft tumor was established.Female nude mice of Balb/c strain were purchased with same characteristics,and the same amount of Huh7 cells were injected into right flank of each mice,and the tumor formation was observed every 3 days.After the tumor volume reached?100 mm~3 in volume at day0after implantation,nude mice were randomly divided into 4 groups:the control group was injected intravenously with saline;the radiation treatment group was treated with irradiation,delivered by a Varian trilogy linear accelerator;the AD-MSC treatment group was administrated 1×10~6 AD-MSC suspended in 100?L saline via the tail vein;and the combined treatment group was treated with 1×10~6 AT-MSC immediately after irradiation.Observe and record the tumor growth as well as the general situation of mice,and draw the tumor irradiation dose-response curve.Nude mice were sacrificed at the end of the experiment,and tumor tissue samples were taken for subsequent immunohistochemistry,PCR and other experiments.ResultsOn the basis of the constructed the HCC xenograft tumor model,4 groups of nude mice were treated accordingly.Experiments showed that the combined treatment of AD-MSC and RT significantly delayed tumor growth time,and through immunohistochemisty experiments found that the tumor proliferation coefficient of the combined treatment group was significantly improved,and TUNEL staining showed a significant decrease according to the number of apoptosis.ConclusionsThe combination treatment of AD-MSC and RT can produce a significant radiosensitization effect in the xenograft model of HCC in nude mice.AD-MSC may have a potential therapeutic effect in synergistic RT to inhibit tumor proliferation and promote apoptosis,but its internal mechanism remains to be further explored.Section III The mechanism of radiosensitization of AD-MSC intreatment of HCCObjectiveBased on the conclusions of the previous two sections,the tumor tissue samples of each group were analyzed by transcriptome sequencing.Gain and loss studies including sh RNA lentiviral transduction,cell function experiments,Western blot,rt-pcr,etc.were verified to explore the potential mechanism of the radiotherapy sensitization of HCC cells by stem cell therapy.MethodsAnalysis of high-throughput transcription sequencing differences showed abnormal expression of 76 genes in the combined therapy group compared to other groups'data.By comparing heatmap analysis,Reactome FIViz network-based analysis and previous literatures,we believe that interferon-induced transmembrane protein 1(IFITM1)may be the main functional gene.Through sh RNA lentiviral transduction we obtained gene knockdown group sh IF Huh7 cell,overexpression group IF-OE Huh7 cell,and transfection control group IF-NC Huh7 cell.Cell functions such as colony formation and sphere-forming experiments were used to in sh IF and IF-OE Huh7 cells after treatments respectively.Expression changes such as STAT-3,P53,MMP family,Caspase family were detected by Western Blot,RT-PCR experiments in sh IF and IF-OE Huh7 cells after treatments respectively.ResultsProliferation of sh IF Huh7 cell line was inhibited.Also compared with IF-NC Huh7 cells,sh-IF Huh7 cells in treatment groups were significantly reduced in cell proliferation,clone formation,invasion and migration,while IF-OE group tumor reduction was significantly inhibited.Similar to the transcriptional changes detected by RT-PCR experiment,Western Blot analysis showed that the expression of STAT3,MMP2 and MMP9 was inhibited in sh IF Huh7 cells after combined treatment,while the expression of Caspase family,P21 and P53 was increased compared with that of if-NC cells.The expressions of STAT3,MMP2 and MMP9 in if-OE Huh7 cells were increased after combined treatment,while the expressions of Caspase family,P21 and P53 were significantly inhibited.ConclusionsThrough mechanisms such as overexpression and knockdown IFITM1 gene,it was found that IFITM1 function in the combination therapy of AD-MSC and RT may be attributed to changes in its downstream STAT3,MMP family and Caspase family.