Antitumor Effects Of Oncolytic Herpes Simplex Virus Type 2 Against Colorectal Cancer In Vitro And In Vivo

ObjectiveColorectal cancer(CRC)is a common malignant tumor of digestive tract,and its morbidity and mortality rate increased year by year.Furthermore,latestage diagnoses and limited efficacious treatment options make it complex clinical challenge.Therefore,the new therapeutic regimen with completely novel therapeutic mechanism is necessary for colorectal cancer.Biological treatment of cancer therapy,as the fourth model in a new cancer treatment is unique.Oncolytic viruses(OVs)are either naturally occurring or genetically engineered viruses that selectively damage malignant cells.They have broad prospects in the treatment of many kinds of solid tumors.oHSV2,a conditionally replication-competent herpes simplex type 2 which had been insertion of granulocyte-macrophage colony stimulating factor(GM-CSF)gene.GM-CSF is a pleiotropic cytokine which can recruitment and activation of antigen presenting cells to produce specific anti-tumor immunity.In present study,it was the first time to investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor in colorectal cancer in vitro and vivo.MethodsIn vitro,we investigated the cytopathic effects of oHSV2 in colorectal cancer cell lines by MTT.Then the cell cycle progression and the apoptosis of oHSV2 were examined by flow cytometry,respectively.In vivo,we generated a model of colorectal cancer with mouse colorectal cancer cell CT26 in BALB/c mice.After intratumoral administration of oHSV2,the GM-CSF expression levels in blood of CT26 xenograft model were determined by ELISA method.The antitumor effects and adaptive immune response of oHSV2 were assessed on tumor-bearing mice.In our study,the therapeutic efficacy of oHSV2 had been compared with the traditional chemotherapeutic agent 5-fluorouracil(5-FU).ResultsIn vitro: 1)The human LoVo,HT29,HCT116 cell lines and murine CT26 cell line were sensitive to oHSV2-induced cytotoxicity.After infection,oHSV2 caused all cancer cell lines to form a typical syncytium.2)MTT analysis revealed that the percentage of dead cells was increased at a time and dose dependent manner.3)Flow type result display that treatment with 5-FU had a marked effect on S phase frequency compared with the control group(all P<0.05).However,treatment with oHSV2 had no any effect on G0/G1 phase,G2/M phase or S phase compared with the control group(all P>0.05).The Annexin-V/PI assay showed that infected with oHSV2 caused a greater proportion of necrosis cells compare with control group(all P<0.05)and presents dose-dependent manner.In vivo: 1)In CT26 xenograft model,the level of GM-CSF was continuously elevated and the peak value(3150±327.1 pg/m L)was found on day 8 in the blood.Then GM-CSF expression gradually reduced as time process.2)In vivo study showed that both oHSV2 and 5-FU had an antitumor effect.The median survival time was prolonged compared with PBS-treated mice(Median survival: 36 days for the control vs 51 days for 5-FU alone,50 days for oHSV2 alone;p < 0.01 for both 5-FU,oHSV2,log-rank test).However,the group treated with 5-FU had a continuous reduction in mice body weight.At day 28,the average body weight of the mice treated with 5-FU was(16.61±0.74)g and presented the extremely significant difference with the other groups(P<0.01).The body weight of mice treat with oHSV2 showed increase slowly and there was no statistically significant change compare with control groups(21.16±0.68 g vs 22.07±0.54 g,P>0.05).3)Flow cytometry showed that treatment with either 5-FU alone or oHSV2 alone result in reductions on the MDSC frequency in the spleen(7.84% and 2.50%,respectively)compared with the control group(14.60%,p<0.01),but oHSV2 more effective than 5-FU in depleting MDSC.The mean frequency of Tregs was higher in 5-FU group compared with the control group(14.50% vs 8.94%,P<0.01),but oHSV2-treated mice showed a slightly reduction on Tregs frequency(4.60%,P<0.05).In the TDLN,the frequency of DC was increased when treated with oHSV2 compared with the control group(6.49% vs 3.73%,P<0.01).However,a significantly lower frequency of DC was found in 5-FU treatment(2.04%,P<0.05).Therefore,oHSV2 induced down Results regulation of MDSC,Tregs and up regulation DC.Correspondingly,Flow cytometric analysis of the frequency of T lymphocytes in tumor tissue showed that the percentage of both CD4+ T and CD8+ T cells from oHSV2-treated group was significantly higher than that from mock-treated tumors(15% vs 8.57%,P<0.01;8.19% vs 5.15%,P<0.01).However,5-FU treatment appeared to reduce the proportion of CD4+T cells and CD8+T cells levels(4.17% and 2.98%,all P<0.05)compared with the control group.ConclusionOur study provides the first evidence that oHSV2 could induce cell death in colorectal cancer in vitro and in vivo.These findings underline the oHSV2 as an effective therapeutic cancer candidate that cause oncolysis effect and recruite adaptive immune responses for enhanced therapeutic impact,providing a potential therapeutic tool for treatment of colorectal cancer.