Function Of Dendritic Cells In LSCC Patients And Induction Of LSCC Specific Anti-tumor Effects Using DCs Transfected With Hep-2 Cells Total RNA Marked With EGFP In Vitro

Laryngeal squamous cell carcinoma(LSCC)is one of the most common malignancies, with an increasing occurrence of new cases and deaths annually.Despite all the progresses made in surgical,radiotherapy and chemotherapy techniques,long-term survival of LSCC patients has not been improved significantly over the past three decades,which poses a formidable challenge for physicians in ENT department.Thus, studies of certain key issues pertinent to LSCC,such as tumor immunology,are of great importance in getting insight into the mechanisms underlying the pathogenesis of LSCC and,in turn,resulting in effective therapeutics eventually.It has been suggested that anti-tumor immunity plays a pivotal role in the initiation and progression of malignancy.Induction of an effective anti-tumor immune response requires the activation of effector cells by antigen-presenting cells(APCs) that are responsible for the presentation of tumor-specific antigens.Of particular interest is the role of dendritic cells(DCs),the key APCs which are specialized in taking up,processing,and presenting tumor antigen to stimulate na(?)ve T cells and to initiate strong tumor antigen-specific immune responses.Because of the important role in initiating,modulating and keeping immune response,DC-based immune therapy becomes the focus of present antitumor researches.It has been demonstrated that DCs could induce in present of combination cytokines.DCs pulsed with tumor antigens administered in different forms,such as peptides,cell lysates,proteins,apoptotic cells,DNA or RNA,could induce specific antitumor immune response.DC-based anti laryngeal carcinoma immune therapy is at its very beginning.Because of the fail in recognizing tumor specific antigens of laryngeal squamous cell carcinoma,DC pulsed with whole tumor-derived material present as a main strategy.It has been reported that DCs loaded with tumor lysates and fusions of DCs with tumor cells could induce effective anti-tumor effects in LSCC.Recently,it has been demonstrated that DCs transfected with specific or whole cell ribonucleic acid(RNA)is more effective and safe than DNA vector constructs. However,little is known about the antitumor effects of RNA pulsed DCs in LSCC.However,patients with advanced cancer including squamouse cell carcinoma of the head and neck(SCCHN)are known to be immunologically compromised,which allows malignant cells to evade the host's anti-tumor defenses.The MHC restriction made it necessary to use autologous DCs in immune therapy.On the other hand,it would be ideal to use DC-based immunotherapy as a first choice,but in fact,it is more likely to be of use as an adjuvant treatment together with the conventional therapies.In concerning of laryngeal SCC,surgery and radiotherapy is still regarded as the most efficient treatment strategy world wide.Therefore,it is important to understanding changes of DCs in malignant tumor patients not only caused by the disease per se,but also by other aspects,such as different therapeutic regimens or other clinical features.In this study,we firstly investigate the effects of surgery and adjunctive radiotherapy on both circulating dendritic cells(DCs)and MoDCs of LSCC patients. Then we investigate the influence of Hep-2 cells total RNA transfection on monocyte derived dendritic cells(DCs)and the possibility of induction of antigen specific anti-tumor effects,as well as the feasibility of EGFP as a marker during RNA transfection.The aims of this current research are providing insight into the immune system status of LSCC patients,meanwhile assisting us in developing the proper DC-based anti-cancer immunotherapeutic strategies. PARTâ… Circulating Dendritic Cell Subsets detection by FACS and Induction of DC from Human Peripheral Blood MonoeyteObjective To investigate the convenient and reliable assay for circulating dendritic cell subsets detection,induct DC from peripheral blood monocyte of healthy donors.Methods Peripheral blood samples were collected from 15 healthy donors.Using multicolor flow cytometry,the percentages of lineage negative(LIN^-)and HLA-DR⁺ DC precursors,as well as their HLA⁺LIN^-CD11c+(myeloid)and HLA⁺LIN^-CD123⁺ (lymphoid)subsets,were determined in whole blood.Moreover,monocyte-derived DCs were generated utilizing granulocyte macrophage colony-stimulating factor (GM-CSF),interleukin-4(IL-4)and tumor necrosis factor-α(TNF-α).The morphous of cultured cells were observed using inverted microscope and electron microscope, then the phonotype of MoDCs was measured by flow cytometry and the ability to stimulate autologous T cells were tested in mixed leukocyte reaction(MLR).Results The percentages of HLA-DR⁺ DC precursors,HLA⁺LIN^-CD11c+ DCs and HLA⁺LIN^-CD123⁺ DCs in peripheral WBCs were 0.414±0.13%,0.284±0.13%, 0.068±0.032%,respectively.The absolute numbers of these cells were 22.75±5.22/μl, 15.12±4.66/μl and 3.78±1.40/μl,respectively.Mature monocyte derived DCs of healthy donors displayed typical characteristics of morphology,which were non-adherent,displayed many fine spines on surface,rich chondriosome and poor lysosome in endoplasm.They expressed high level of CDS0,CD83,CD86 and HLA-DR,the percentages of positive expression of these surface marks were 46.54±7.1%,62.64±10.3%,51.14±9.6%and 73.44±8.1%.Few MoDCs could induce the proliferation of autologous T cells at the ratio of 1:10.Conclusion Multicolor FACS is convenient and reliable for circulating dendritic cell subsets detection but need proper gating strategy.We could successful isolate and culture mature DCs from peripheral blood of healthy donor in presence of GM-CSF, IL-4 and TNF-α.Those cells maintained the typical morphological characteristics and had immune activities. PARTâ…¡Therapeutic Influence on Circulating and Monocyte-derived Dendritic Cells in Laryngeal Squamous Cell Carcinoma PatientsObjective To investigate the effects of surgery and adjunctive radiotherapy on both circulating dendritic cells(DCs)and MoDCs of LSCC patients.Methods Forty-six laryngeal SCC patients and 15 age-matched healthy control subjects were enrolled in this study.Both in patients underwent surgery only(n=18) and those accepted adjunctive radiotherapy after tumor removal(n=28),blood samples were taken before,during and after conventional treatment.Three-color flow cytometry was used for circulating DC subsets determination.Moreover, monocyte-derived DCs were generated utilizing granulocyte macrophage colony-stimulating factor(GM-CSF)and interleukin-4(IL-4),then the phonotype of MoDCs was measured by flow cytometry and the ability to stimulate autologous T cells were tested in mixed leukocyte reaction(MLR).Results The pre-operative mDC counts,MoDC surface molecular expression and stimulatory capacity were impaired in patients in comparison with controls.The number of mDC and the expression of CDS0,CD83,HLA-DR on MoDC were significantly increased as compared to those pre-treatment both in patients underwent surgery only and surgery followed with adjunctive radiotherapy.However,the recovery of CD86 expression and allostimulatory activity was only observed in patients accepted surgery only.Conclusion Surgical resection of laryngeal squamous cell carcinoma(LSCC) could be associated with improved circulating myeloid dendritic cell(mDC)number and monocyte-derived dentritic cell(MoDC)function.Although adjunctive radiotherapy after surgery did not effect the normalization of mDC number,it may have an impact on MoDC function. PARTâ…¢Induction of Laryngeal Squamous Cell Carcinoma Specific Anti-tumor Effects Using Dendritic Ceils Transfected with Hep-2 Cells Total RNA Marked with EGFP in VitroObjective To investigate the influence of Hep-2 cells total RNA transfection on monocyte derived dendritic cells(DCs)and the possibility of induction of laryngeal squamous cell carcinoma specific anti-tumor effects,as well as the feasibility of EGFP as a marker during RNA transfection.Methods Immature dendritic cells were generated from peripheral blood monocyte of healthy donor in the present of granulocyte macrophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4).Hep-2 cells were transfected with plasmid pEGFP-N1 using liposome and stably selected by G418.Total Hep-2-EGFP cellular RNA was generated using Trizol following the manufacturer's protocol,the integrity and purity of total RNA were detected.Immature DCs were transfected with total RNA of Hep-2-EGFP cells by simple co-incubation and electroporation.Transfection efficiency was detected by fluorescence microscope. After transfection,DCs were incubated with TNF-αfor maturation.The phonotype of matured MoDCs transfected with total RNA was measured by flow cytometry and the ability to stimulate autologous T cells was tested in mixed leukocyte reaction(MLR). For CTL induction,RNA transfected DCs were incubated with allogeneic T cells for 7 days,and the cytolytic activity of induced CTLs was analyzed by a standard ⁵¹Cr-release assay.Results EGFP was stably expressed in Hep-2 cells,the green fluorescence lasted for at least 20 generations.To attest the intactness of total Hep-2-EGFP cellular RNA, all RNA preparations were subjected to electrophoresis in gel under denaturing conditions with clear visualization of intact 18S and 28S ribosomal bands.Simple co-incubation and electroporation yield transfection efficiency of 7～9%and 21～23% respectively.The expression of maturation marks,such as CD83 and HLA-DR,on DCs transfected with total Hep-2-EGFP cellular RNA was significant higher than on those simply matured.Allogeneic T cell proliferation induced by transfected DCs was significantly higher than untransfected DCs.DCs transfected with total Hep-2 cellular RNA were capable of stimulating CTLs that specific recognized and lysed Hep-2 ceils, but not HT-29 cells(colon cancer)and OVCAR3 cells(ovarian cancer).Conclusion EGFP could be used as a marker to observe the effect of transfection of DCs with total Hep-2 tumor cellular RNA.Transfection with total Hep-2 cellular RNA could up-regulation the expression of DC maturation marks,promote the stimulatory capacity to allogenic T cells,and induce specific CTL against Hep-2 cells. Thus,toal tumor RNA-transfected DCs may represent a broadly applicable vaccine strategy to induce potentially therapeutic T cell responses in laryngeal squamous cell carcinoma patients.