Chemicals, cell lines, and animals
The human cell lines AGS and NCI-N87 (subsequently referred to as N87) are Lauren intestinal-type gastric adenocarcinoma cell lines, and MKN-45 is a Lauren diffuse-type gastric adenocarcinoma cell line. AGS, N87, and MKN-45 cells were obtained from the America Type Culture Collection and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and L-glutamine 2 mmol/L (“regular media”). The cancer cell lines were passaged within six months from the time that they were received. The United Kingdom Coordinating Committee on Cancer Research guidelines was followed [21].
Patients
Sixty-three patients who had histological or cytologically confirmed stage III–IV gastric cancer participated in the study. The Ethical Review Board of the Medical Ethics Committee of the National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College approved the study protocol. All subjects gave written informed consent in accordance with the Declaration of Helsinki. After enrollment, all patients provided a complete medical history and underwent a physical examination. The demographics and baseline characteristics of the patients are summarized in Table 1. The criteria for patient selection were 1) age 18–80 years; 2) expected survival of ≥ 3 months; 3) Karnofsky performance status score > 40%; 4) peripheral blood white blood cell count ≥ 4 × 109/L; 5) platelet count ≥ 10 × 1010/L; 6) serum aspartate aminotransferase/alanine aminotransferase values below the normal upper limit; 7) no cardiac arrhythmias, congestive heart failure, or severe coronary artery diseases; and 8) not pregnant or lactating.
Table 1
Demographics and baseline characteristics of gastric patients with cancer.
Demographic variable
|
Combination therapy (n = 34)
|
Chemotherapy (n = 29)
|
P
|
|
Age (years)
|
55.7 ± 10.89
|
56.2 ± 9.88
|
0.36
|
|
Sex, n (%)
|
Female
|
9 (26.5%)
|
7 (24.1%)
|
0.78
|
Male
|
25 (73.5%)
|
22 (75.9%)
|
ECOG Performance status
|
|
0
|
11
|
7
|
0.63
|
|
1
|
23
|
22
|
|
Histology
|
|
Poorly differentiated
|
27
|
17
|
0.58
|
|
Well/Moderately differentiated
|
7
|
12
|
|
Number of organs with metastasis
|
≤2
|
24
|
18
|
0.42
|
|
≤3
|
10
|
11
|
|
ECOG, Eastern Cooperative Oncology Group.
ACT cell generation
Peripheral blood mononuclear cells were obtained using Ficoll density centrifugation[22]. The cells were resuspended at a density of 3 × 106 cells/mL in X-VIVO 15 medium (Lonza, Switzerland), supplemented with 10% heat-inactivated autologous plasma and primed by adding 1,000 U/mL IFN-γ on day 0, and 100 ng/mL anti-CD3 antibody (MACS GMP CD3 pure, Miltenyi Biotech, Bergisch Gladbach, Germany) and 500 U/mL IL-2 was provided on day 1. On day four of culture, the cell density was adjusted to 1 × 106 cells/mL. Fresh medium with IL-2 (500 U/mL) or IL-15 (50 ng/mL) was added every three days. On day 14 of culture, ACT cells were harvested and used for analysis. Cells cultured in IL-15 and IL-2 were designated as ACTIL-15 and ACTIL-2 cells, respectively.
In vitro cytolytic assays and cytokine release
Europium release assay was used for in vitro cytotoxicity analysis as described[22]. Target cells (MKN-45, AGS, or N87) were co-cultured with ACT cells in triplicate at an effector-to-target cell ratio of 50:1, 10:1, 5:1, and 1:1 in U-bottom 96-well culture plates (NUNC, Roskilde, Denmark). The supernatant from each well was collected and co-incubated with europium solution (Europium, Perkin Elmer, Turku, Finland) in flat-bottom 96-well plates (NUNC, Roskilde, Denmark). Fluorescence was measured with a time-resolved fluorometer (1420–018 Victor, Perkin Elmer, Waltham, MA, USA). The percentage of specific cytolysis was calculated for each well as described [22], and the mean ± SD was calculated for each duplicate or triplicate.
Flow cytometry assay
Expression of surface markers in the cell was analyzed by flow cytometry. All antibodies (FITC-conjugated anti-CD3, PECy7-conjugated anti-CD56, APC-conjugated anti-CD25, PE-conjugated anti-CD45RO, PerCP-conjugated anti-CD62L, APC-conjugated anti-CCR7) were purchased from BD Biosciences. Samples were analyzed using a FACS Calibur flow cytometer and CellQuest software (Becton Dickinson).
Tumor xenograft model
For establishing human gastric cancer xenograft model in mice, 6–8-week old NCG mice (Institute of Zoology, Chinese Academy of Sciences, Shanghai, China) were housed under specific pathogen-free conditions. The mice were irradiated with 200 cGy for 24 h. MKN-45 cells (1 × 106 cells/mouse) were subcutaneously injected into the flanks. When tumor nodules became palpable (seven days after injection), mice were randomized into three groups: blank control (injected with physiological saline via the tail vein), ACTIL-2 (injected with ACTIL-2 cells via the tail vein), and ACTIL-15 (injected with ACTIL-15 cells with the tail vein). Animals were euthanized on day 32 after transplantation of malignant cells, or they were monitored for survival. Mice that showed signs of physical abnormalities or poor health were sacrificed by carbon dioxide asphyxiation followed by cervical dislocation. Toxicity was defined as 20% or more bodyweight loss or toxic death. Body weight was measured once a week, and survival was calculated from the time of tumor-cell injection until death. Animals were euthanized when they exhibited a lack of a righting reflex or lack of response to stimuli to avoid suffering. Tumor volume was measured every 2–3 days in a blind fashion and calculated according to the following formula: tumor volume = length × width2 /2.
Immunohistochemistry
Tumors were harvested and fixed for 24 h with 10% buffered formalin before being embedded in paraffin. Serial sections 5 μm thick were cut for histologic analysis and were stained with hematoxylin-eosin according to standard procedures. For immunohistochemistry, sections were incubated with anti-CD3 (1:200), then incubated with Dako ChemMateTM EnVision System (Dako, Glostrup, Denmark) for 30 min. Staining was visualized using diaminobenzidine and sections were counterstained with hematoxylin.
Enzyme-linked immunosorbent assay (ELISA)
Single-cell suspensions from tumors were cultured in the medium at 37°C for 6 h. The secretion of IFN-g, TNF-a, IL-4, and IL-10 by ACT cells was measured in the supernatant of the culture media with ELISA kits (R&D Systems) according to the manufacturer instructions.
Statistical analysis of animal studies
The differences in the mean relative tumor volume (RTV) between the treated and control groups on day 25 were analyzed with the two-tailed Student’s t-test. All statistical analyses were performed by SPSS 20.0 software. P < 0.05 was considered statistically significant.
Study design
Subjects were randomly assigned to receive S-1 plus oxaliplatin plus ACTIL-15 cells (a combination therapy group) or S-1 plus oxaliplatin alone (chemotherapy group). To avoid bias, randomization was performed (Bellsystem24, Tokyo, Japan). Assignment to the treatment groups was balanced according to stratified factors, including Eastern Cooperative Oncology Group performance status and whether the cancer was unresectable or recurrent. A unique random sequence generated by an independent statistician was sequentially applied to each patient allocation by the biased coin method. The two groups were matched for gender, age at onset of disease, pathology, tumor size, metastases, and stage at the first visit.
All patients in the chemotherapy group and the combination therapy group received the same chemotherapy component of the treatment, including doses and cycles. S-1 was orally administered at 80 mg/m2 divided into two daily doses for 12 days, followed by nine days off. Oxaliplatin was administered intravenously at 130 mg/m2 over 1–3 h on day 1, then every 21 days until the disease had progressed or unacceptable toxic effects had developed. The dose of S-1 was calculated according to the patient’s body surface area. Patients in the combination therapy group received ACTIL-15 cell therapy on day 14 and again after the second chemotherapy treatment. The mean lymphocytes count in the ACTIL-15 cell agent was 5.9×109 cells.
Assessments during treatment and follow-up
Toxicity assessments, compliance with S-1, and blood test results were recorded after each cycle of treatment. Tumors were assessed after every other cycle. Computed tomography was performed every eight weeks until cancer progressed. The therapy was discontinued if the disease progressed, the patient refused, unacceptable toxicity occurred, or the patient died. Tumor status was assessed by imaging approximately every two months or until death.
Toxicity was evaluated according to the National Cancer Institute Common Toxicity Criteria for Adverse Events version 3.0. Response to treatment was evaluated according to the Response Evaluation Criteria in Solid Tumors (www.cancer.gov/). Radiographic evidence of response to treatment was independently reviewed. An independent data-monitoring committee oversaw the safety and efficacy of the trial and other aspects of the study conduct.
Statistical analysis of human studies
Statistical analyses were conducted with SPSS 20.0 and the GraphPad program (version Prism 5). Differences in demographic and clinical variables between the two groups were evaluated with the Pearson χ2 test, while Fisher’s exact test was used for categorical variables. The Kaplan–Meier method was used to analyze the progression-free survival (PFS) and overall survival (OS). PFS was calculated from the time of the first treatment to the time of the first disease progression or the last follow-up. OS was calculated from the date of the first treatment to the date of death resulting from any cause or the date of the last follow-up. All P values were two-tailed, and significance was set at P < 0.05.