A Clinical Pilot Study On Comparison Of Cancer Patients Treated With Autologous And Healthy Allogeneic CIK

Background and Objects of the Study：Cancer is one of the most difficulty disease to overcome nowadays. In recent years,with the in-depth research on the immune system and its function, adoptive cellular transferimmunotherapy (ACT) has become the fourth effective strategy in succession to surgicalresection, chemotherapy and radiotherapy. ACT refers to infusion of immune cells withanti-tumor activity in order to arouse anti-tumor immune responses or kill tumor cellsdirectly and finally achieve the desired therapeutic effect.Cytokine-induced killer cells (CIK) were reported by Schmidt-Wolf in1991andrefered to a group of heterogeneous cells generated from peripheral blood mononuclearcells (PBMC) or umbilical cord blood mononuclear cells, among which CD3+CD56+cellshold major cytotoxicity against tumor cells. In contrast with those earlier adoptive cells,such as lympokine activated killer cells (LAK), tumor infiltrating lymphocytes (TIL),cytotoxic T lymphocytes (CTL) and anti-CD3monoclonal antibody activated killer cells(CD3AK), CIK have higher multiplication efficiency, more powerful anti-tumorcytotoxicity, less side effects and equal effects to tumor cells of multidrug-resistance.Therefore, CIK cells were applied to treating several hematic or solid malignant tumors andwhich have become current research focus because of the good curative effect.CIK were mainly derived from patients in clinic, however, researchers reported thatthe overall efficiency against solid tumors of autologous CIK from patients was onlyaround30%. Beyond autologous CIK, allogeneic CIK from healthy donors were also usedfor treatment of old and infirm patients because of the cells’ convenience to be cultured andminor side effects, but there haven’t been any complete reports about the differences of invitro proliferation activity, anti-tumor cytotoxicity and clinical curative effect betweenautologous CIK and healthy allogeneic CIK.Moreover, survival of CIK after infusion was a key factor in achieving good curative effects, but there also haven’t been reports about survival time of CIK. In this study, wecompared in vitro proliferation activity, anti-tumor cytotoxicity and curative effect betweenautologous CIK and allogeneic CIK, and preliminarily analyzed survival time of allogeneicCIK after infusion with the purpose to provide a basis and experience for application ofCIK in clinic.Methods:Part I: Isolation and culture of CIK derived from cancer patients and healthydonors.1. PBMC derived from cancer patients (n=12) and healthy donors (n=12) were isolatedby Ficoll-Hypaque densi ty gradient centrifugation and stimulated in RPMI media(1.0×106/mL) with interferon-γ (IFN-γ), OKT3monoclonal antibody (OKT3), recombinanthuman interleukin-1(rhIL-1) and recombinant human interleukin-2(rhIL-2) for14d untilharvested.2. CIK derived from patients or healthy donors cultured for3d,7d and14d wereresuspended in phosphate buffer solution (PBS) and stained with0.04%Trypan blue, thenthe multiplication capacity was evaluated according to viable cell count.3. CIK derived from patients or healthy donors cultured for3d,7d and14d wereresuspended in PBS and stained with CD3-FITC/CD56-PE/IgG control, then the changes ofCD3+CD56+phenotype were analyze by flow cytometry.Part II: Comparison between CIK derived from cancer patients and healthydonors on cytotoxicity against human colon cancer cell line HCT116or humanhepatocellular cancer cell line HepG2and expression of Bcl-2/Bax in cancer cells.1. HCT116and HepG2cells in exponential growth phase were collected as target cells,autologous or allogeneic CIK cultures for14d and healthy PBMC were collected as effectorcells and added at the indicated ratio of effector cells to target cells (E/T). Afterco-incubation for24h at37°C, the specific lysis of target cells was analyzed by cellcounting kit-8(CCK-8).2. HCT116and HepG2cells in exponential growth phase were collected as target cells,autologous or allogeneic CIK cultures for14d and healthy PBMC were collected as effectorcells and added at E/T=40:1. After co-incubation for48h at37°C, the total protein of targetcells was extracted then the B-cell lymphoma-2(Bcl-2) protein and Bcl-2associated X (Bax) protein were determined by Western blotting and the relative ratio of Bcl-2to Baxwas counted.Part III: Comparison between autologous with allogeneic CIK on the short-termcurative effects of patients and CIK cell survival time in vivo.1. Lymphocyte subsets of patients at different times after infusion of autologous CIK(n=12) or allogeneic CIK (n=12) were tested by flow cytometry, taking healthy donors ascontrol.2. Clinical benefit rate (CBR) of patients treated with autologous CIK (n=12) orallogeneic CIK (n=12) was evaluated by taking karnofsky performance status (KPS),numerical rating scale (NRS) and weight as the main evaluation parameters.3. Side effects, such as fever, erythra and other discomforts during treatment withautologous or healthy allogeneic CIK were recorded and compared.4. Sry derived from male CIK cells in female patients’ peripheral blood wasqualitatively analyzed by PCR to evaluate survival time of CIK.Results:Part I: Isolation and culture of CIK derived from cancer patients and healthydonors.1. Trypan blue staining showed that PBMC derived from healthy donors (n=12)cultured for3d,7d and14d had a higher proliferation activity than cells from cancerpatients (n=12) at the same time.2. Result of flow cytometry showed that rate of CD3+CD56+cells in CIK from healthydonors (n=12) cultured for3d,7d and14d was higher than cells from cancer patients (n=12)at the same time..Part II: Comparison between CIK derived from cancer patients and healthydonors on cytotoxicity against human colon cancer cell line HCT116or humanhepatocellular cancer cell line HepG2and expression of Bcl-2/Bax in cancer cells.1. The specific lysis of HCT116and HepG2was analyzed by CCK-8, result showedthat three kinds of effector cells could inhibit proliferation of target cells and the anti-tumorcytotoxicity was proportional to E/T. In addition, at the same E/T, CIK from healthy donorshad a stronger anti-tumor cytotoxicity than that of CIK from patients, and the cytotoxicityof CIK from patients was stronger than healthy PBMC. 2. Western blotting showed that three kinds of effector cells could increase expressionof Bax in HCT116and HepG2, meanwhile, expression of Bcl-2was down-regulated, andthe ratio of Bcl-2to Bax was decreased. Among the three effector cells, CIK from healthydonors had the greatest effect on target cells, and effects of both CIKs from patients andhealthy donors were stronger than that of healthy PBMC, too.Part III: Comparison between autologous with allogeneic CIK on the short-termcurative effects of patients and CIK cell survival time in vivo.1. Result of flow cytometry showed that immune function of all24patients improvedin varying degrees after treated with CIK, and CIK from healthy donors would have morebeneficial and lasting effect on improving of CD3+T lymphocyte and the ratio of CD4+Tlymphocyte to CD8+T lymphocyte than CIK from patients.2. Short-term curative effect was evaluated on60d after ACT with CIK, result showedthat there were few differences of KPS, NRS, changes of weight and total CBRbetween patients treated with autologous CIK (n=12) or allogeneic CIK (n=12).3. There were few side effects after infusion of either autologous or healthy allogeneicCIK during our observation period.4. Sry gene could be detected in these8female patients’ peripheral blood after treatedwith allogeneic male CIK by PCR, the intensity of which diminished over time and theaverage duration was (7.0±0.93) w.Conclusion：In this study, we preliminarily compared differences of proliferation activity,cytotoxicity and clinical curative effect between autologous CIK and allogeneic CIK, andanalyzed survival time of allogeneic CIK after infusion by detecting Sry gene in8femalepatients’ peripheral blood treated with male CIK.1. CIK derived from healthy donors had a higher proliferation activity and a higherCD3+CD56+rate than cells from patients in vitro culture process.2. Like PBMC, both CIK derived from healthy donors and CIK from patients showedin vitro anti-tumor cytotoxicity against HCT116and HepG2, besides, these three kinds ofeffector cells could increase expression of Bax and down-regulate expression of Bcl-2inHCT116and HepG2. Our further study showed that CIK derived from healthy donors had astronger effect of anti-tumor and down-regulating Bcl-2/Bax than CIK from cancer patients. 3. Immune function of all patients improved in varying degrees after treated with CIK,and CIK from healthy donors would have more beneficial and lasting effect on improvingof CD3+T lymphocyte and the ratio of CD4+T lymphocyte to CD8+T lymphocyte thanCIK from patients. There were few differences of KPS, NRS, changes of weight and thetotal CBR between patients treated with autologous CIK or allogeneic CIK. After infusion,Sry gene could be detected and the average duration was (7.0±0.93) w.