Isolation And Characterization Of Gastric Cancer Cells With The Different Digress Of Differentiation And Roles In Adoptive Cell Therapy

BackgroundGlobally, Gastric cancer is the second leading cause of cancer deaths. The mian cause of death is recurrence of gastric cancer in patients after therapy. The principal focus of research work has been on this problem.Recently, emerging of cancer stem cells (CSC) hypothesis provides researchers a new way to treatment gastric cancer. According to the cancer stem cells hypothesis, only a small quantity of cancer cells in tumors possess the ability to self-renewal and infinite proliferation, these cells can gives rise to a number of regular cancer cells (RCC) CSC hypothesis supporters think the only effective way to treat cancer is to target the CSC which are the root of tumor metastasis and recurrence. But so far, lack of an effective method for CSC sorting cause limit the further researchIt is generally known that progenitor cells have some low degree of "stemness" and also express stem cell markers. By that analogy, cancer progenitor cells (CPC) should possess this characteristic.Because isolation of CSC cannot be removing CPC, some researchers use the term "CSC/CPC" to describes this subpopulationg. However, the biological characteristics between CSC and CPC may have a large difference and cannot reflecting the real biological characteristics. In addition, suggesting theories of CSC is based on stem cells (SC) hypothesis, it means that tumor should conclude the process of differentiation:SC that produce progenitor cells that differentiate into different types of cells.However, most of the previous researches simply separate the cancer cells into CSC or no-CSC. So, found the way to isolate CSC,CPC and RCC is important for CSC hypothesis.Aldehyde dehydrogenase(ALDH) is a detoxifying enzyme that oxidizes intracellular aldehydes. ALDH involved in the oxidation of aldehydes and thought to participate in cellular differentiation and SC self-protection. SC were shown to contain higher levels of ALDH activity than their more differentiated progeny. High ALDH activities have been reported to sort CSC in many cancers. Because of this property, it maybe suitable for isolate different differentiated level of cells in cancer.Adoptive cellular immunotherapy (ACI) have good application prospects,especially based on used of dendritic cells(DC).One ACI strategy uses DC-based vaccination to initiate cytotoxic t lymphocyte (CTL) antitumor immunity have many advantages. Adoptive transfer of antitumor CTL has shown to be effective in ACI of many types of CSC, but it has not been reported in GCSC and GCPC.Objective1. Isolate different degree of differentiated subpopulation in gastric cancer based on ALDH activity.2.To study the biological characteristics of different degree of differentiated subpopulation in gastric cancer.3.Measure the efficiency of inhibiting tumor between different CTL therapy groups.MethodsLAldefluor assay of MFC performed by fluorescence activated cell sorting. Aldefluor flow cytometry-based assay to identify cells based on ALDH activity:highest positive activity (ALDHhigh), weakly positive activity(ALDHlow), negative activity (ALDHneg).2.The expression of CD44,CD133 and cell cycle were determined by flow cytometry analysis.3.RT-PCR was used to detect the expression of OCT-4 and SOX-2.4.Spheroid colony formation assay was used to detect the ability of self-renew.5.CCK8 detect the ability of proliferation and chemoresistance.6.Transwell assays detect the ability of invasion.7.Plate clone formation assay detect the ability of clone.8.SPSS17.0 statistical software was used for statistical analysis.Result1.ALDH activity closely related to the sternness of MFC(1)The percent of positive activity ALDH was 5%±0.91% in MFC, choose the 1% of highest positive activity as ALDHhigh, the 1% of weakly positive activity as ALDHlow, the 1% of weakly negative activity as ALDHneg. (2)The positive expression rates of CD44 was too high to use. The positive expression rates of CD133 was (44.07%± 3.97%、34.33%±3.06%、1.60%±0.66%) in ALDHhigh,ALDHlow and ALDHneg respectively. The positive expression rates of CD133 was higher in ALDHhigh and ALDHlow, with no significant difference in both group; (3) The expression of OCT-4 was (0.99±0.10; 0.43±0.04; 0.28±0.03, P<0.05) in ALDHhigh,ALDHlow and ALDHneg respectively; The expression of SOX-2 was (1.00±0.08; 0.63±0.05; 0.14 ±0.03, P<0.05) in ALDHhigh,ALDHlow and ALDHneg respectively. (4)Both ALDHhigh and ALDHlow have the ability of spheroid colony formation,The expression rates of ALDH+(70.1%±7.1%; 30.5%±5.7%, P<0.01) was in ALDHhigh and ALDH"eg respectively;(5)Both ALDHhigh and ALDHneg have the different ability of tumor igenicity.2.The different biological characteristics in ALDH subpopulation(1)The ALDHlow degree of proliferation was highest,while the ALDHhigh degree of proliferation was lowest. (2)The percent of S/G2/M phase was (40.6%±4.6%; 91.1% ±1.3%; 60.5%±3.4%, P<0.05) in ALDHhigh,ALDHlow and ALDHneg respectively; (3)The ability of chemoresistance decline with ALDH activity reducing; (4)The numbers of clone was (166.3±14.9) in ALDHlow that higher than ALDHhigh and ALDHeg(P<0.01).(5)The numbers of invasion was (51.7±9.1) in ALDHhigh that higher than ALDHlow and ALDHneg(P<0.05).3.The efficiency of inhibiting tumor between different CTL therapy groupsThe efficiency of inhibiting tumor was (88.2%±6.3%,68.7%±11.3％；43.2%± 29.6%,P<0.05)in CTL-ALDHlow,CTI-ALDHhigh and CTL-ALDHneg respectively； The rate of periheral blood CD3+T cell was(53.4%±2.7%；52.2%±3.8%；52.3%± 6.1%;44.1%±3.2%) in CTL-ALDHhigh,CTL-ALDHlow,CTL-ALDHneg and control group respectively;and the rate of peripheral blood CD3+CD8+T cell was (24.4%± 3.2%；26.3%±1.7%；21.9%±2.5%；11.5%±1.8%；P<0.01) in CTL-ALDHhigh, CTL-ALDHklow,CTL-ALDHneg and control grouo;The splenic lymphocyte CD3+T cell was(20.3%±2.3%；19.06%±1.4%；19.4%±4.7%；10.2%±2.1%) in CTL-ALDHhigh, CTL-ALDHlow,CTL-ALDHneg and control group respectively and treatment groups higher than control group(P<0.05);The splenic lymphocyte CD3+CD8+T cell was (6.7%±2.0%;5.4%±0.9%;5.8%±O.5%:1.8%±0.5%)in CTL-ALDHhigh, CTL-ALDHlow,CTL-ALDHneg and control group respectively,and treatment groups higher than control group(P<0.05).Conclusions1.Aldefluor assay as a new way for isolate different differentiated level of cells in MFC.2.CPC have some low degree pf "stemness" and high degree ofproliferation,should getting more attention.3.Specifically killing targeted CPC maybe a better way for cancer treatment.