| Background and Objective:Breast cancer is the leading cause of cancer death in women.According to the national cancer registration center,about 269,000 cases of breast cancer and about 70,000 deaths were reported in 2015.Patients with TNBC typically have a relatively poor outcome compared with those with other breast cancer subtypes owing to an inherently aggressive clinical behavior.In particular,about 20 to 30%of women worldwide will develop invasive breast cancer during their lifetime,leading to more than 500,000 deaths a year due to metastasis from the breast to other organs,with a median survival of only 2 to 3 years.However,there are currently no effective and selective treatments that directly target metastatic cancer.Surgical resection of wide spread metastases is generally not feasible,whereas various classes of chemotherapeutic drugs are ineffective at treating disseminated cancer and often have severe side effects.With the rapid development of immunotherapy,various therapeutic schemes such as CAR-T,TCR-T,tumor vaccine,bispecific antibody,and checkpoint inhibitor were applied to the treatment of malignant tumors,and achieved promising clinical effects,especially the PD-1/PD-L1 blockage in the treatment of solid tumor.The studies on the tumor microenvironment of TNBC have shown that TNBC is a type of tumor with vast heterogeneity and high tumor mutant burden,which displays a full array of different levels of lymphocyte and monocyte infiltration and activation of PD-1/PD-L1 inhibitor.With the expansion of clinical indications,the adverse events of anti-PD-1 antibodies have gradually increased,and the low response rate to some patients has always been a problem.In situ immunomodulation is a promising avenue of investigation.Concentrating the therapy at the site of disease allows one to break local immune tolerance,thereby enabling generation of systemic antitumor immunity in the absence of systemic exposure that can evoke severe side effects.Mesenchymal stem cells(MSCs)are a population of adult stem cells that exist in various of organs and tissues with the capacity of low immunogenicity and multi-lineage potential.MSCs have been served as promising delivery for gene therapy owing to the characteristics of tumor tropism.In this study,we investigated a multi-targeting therapeutic system based on the homing of MSCs,which were modified by E1A to serve as vehicles of AdCD3-HAC.Subsequently,the bi-functional fusion protein,CD3-HAC,induced the interaction between lymphocytes and PD-L1 positive tumor cells in metastatic niche.Method:The methods of PCR,overlap PCR,restriction enzyme cleavage and linkage were used to construct the adenoviral vector pAd-hTERT-CD3-HAC,pAd-hTERT-CD3scFv,pAd-hTERT-HAC,eukaryotic expression vector pcDNA3.1(+)-CD3-HAC,pcDNA3.1(+)-CD3scFv,pcDNA3.1(+)-HAC,and the control vectors pLentiR and pAdTrack.The correctness of constructs was verified by DNA sequencing.The supernatants of 293T cells transiently transfected with the corresponding lentiviral expression vectors were collected,and the recombinant protein was purified by affinity chromatography with His-tag.In addition,the purified or un-purified protein was detected by western blot analysis.Western blot,flow cytometry and immunofluorescence analysis were performed to confirm the expression and cell surface binding of CD3-HAC.The dynamic interaction between PBMC and AdCD-HAC infected MDA-MB-231 target cells were monitored using living cells work station to confirm the membrane-bound CD3-HAC were functional.Then,cytotoxicity detection was performed by Lactic dehydrogenase(LDH)release assay to quantify the lysis efficiency of PBMC against different virus infected target cells.The expression of surface markers CD69 and CD25 of T cells was detected by FACS.And the supernatants in the co-culture system were collected for the assay of cytokines produced by T cells.The proliferation of PBMC was detected by CellTrace Far Red assay.ELISA and apoptosis detection were performed to ascertain the function of HAC in blocking the PD-1/PD-L1 axis.Meanwhile,we combined with low-dose of 5-FU,and detected its effect of improving the immunotherapy.In vitro,the exactly copy number of adenovirus in the intracellular and supernatant of MSC.Adtrack.E1A at indicated time points were examined by QX200 Droplet Digital PCR system;then,the existence of adenovirus particles in MSC.Adtrack.E1A was demonstrated by transmission electron microscopy.Then MSC.AdLuc.LentiR or MSC.AdLuc.E1A was injected intravenously into MDA-MB-231 tumor-bearing mice and then monitored by bioluminescence imaging(BLI).Immunofluorescence analysis was performed to confirm the activated of lymphocyte and interaction between tumor cells and PBMC mediated by CD3-HAC after systematic infusion of MSC.Adtrack.LentiR or MSC.AdCD3-HAC.E1 A.To evaluate the antitumor effect of the CD3-HAC combined with low dose 5-FU,MSC.AdCD3-HAC.E1 A were injected intravenously into tumor-bearing mice,followed by PBMC infusion two days later.Besides,5-FU was given(ip,20 mg/kg)every other day after MSCs injection for three times.In vivo luciferase signal was monitored via Xenogen IVIS imaging as indicated time point and survival curve was established.Results:We successfully constructed the adenoviral vector pAd-hTERT-CD3-HAC,pAd-hTERT-CD3scFv,pAd-hTERT-HAC,eukaryotic expression vector pcDNA3.1(+)-CD3-HAC,pcDNA3.1(+)-CD3scFv,pcDNA3.1(+)-HAC,and the control vectors pLentiR and pAdTrack.Immunofluorescence and flow analysis showed that CD3-HAC fusion protein was specifically expressed in breast cancer cells and successfully bind to the surface of cells.Moreover,typical cell interaction,including binding and then lysis in 15 hours,was examined by living cell work station.Results from LDH release assays demonstrated that CD3-HAC secreted from virus infected tumor cells could induce specific cytotoxicity of T cells to PD-L1-positive cells.PD-1/PD-L1 blockage of HAC was confirmed.In addition,5-FU sensitizes adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity.The exactly copy number of hexon gene(late adenoviral gene)in the intracellular and supernatant of co-transfected MSCs(MSC.Adtrack.E1A)was measured at indicated time.Furthermore,the intracellular viral particles in MSC.AdTrack.E1A were identified by electron microscopy 48 hours after infection.To examine the re-infection ability of adenoviruses assembled by E1A-modified MSCs,flow cytometry was applied to measure the infection efficiency of adenoviruses released from MSC.Adtrack.E1A to MDA-MB-231 cells in a co-culture system.The number of GFP-positive cells increased along with the addition of MSCs.Besides,pretreatment with 5-FU significantly improved infection efficiency at all MDA-MB-231:MSCs ratios.In 231-Luc lung metastatic model the gene modified MSCs selectively colocalized with cancer metastasis of tumor-bearing(but not tumor free)mice 1 day after injection intravenously,then new generated adenoviruses infected and modified the tumor cells by expressing CD3-HAC proteins,which inducing the lymphocytes cytotoxicity.Results from anti-tumor experiment indicated that MSC.AdCD3-HAC.E1A+PBMC with or without 5-FU significantly prolonged the survival of mice.Conclusion:This work is an exploration of a promising targeted therapeutic strategy using gene modified MSCs to deliver bi-functional fusion protein CD3-HAC to tumor sites against metastatic TNBC.MCSs based local immunotherapy can boost more effective antitumor immune response and reverse cancer immunotolerance,while leading less autoimmune toxicity.In a word,this targeted therapeutic strategy should be explored further,as it represents a potential treatment alternative for tumor patients with metastatic diseases. |
PDF Full Text Request