Between September 2014 and January 2015, 58 patients with cancer, including solid tumors and hematological malignancies, were treated using immunotherapy (Table I). Criteria for inclusion in the present study were: Histopathologically confirmed cancers; expected survival duration >3 months; Karnofsky performance status (KPS) >40%; aged >65 years; free of congestive heart failure, severe coronary artery disease, cardiac arrhythmias, organ transplant, serious infection, severe autoimmune disease or central nervous system disease; and without chemotherapy or immunomodulatory treatment during the previous 4 weeks. Informed consent was obtained from all patients prior to therapy.

The DCs and CIKs were generated following Good Manufacturing Practice guidelines (17). Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; Peprotech, London, UK) was injected (150 µg) 24 h prior to blood collection to mobilize white blood cells. PBMCs were collected from patients or healthy donors using a COBE Spectra continuous flow blood cell separator (Caridian BCT, Lakewood, CO, USA). The concentrated PBMCs were suspended immediately in CIK medium [X–VIVO 20 serum-free medium (Lonza, Cologne, Germany), 50 ng/ml anti-CD3 antibody (BD Biosciences, San Jose, CA, USA), 1,000 U/ml recombinant human IL-2 (rhIL-2; Peprotech) and 1,000 U/ml recombinant human IFN-γ (Peprotech)], at 37°C with 5% CO₂ and fed every 5 days in fresh complete medium with various types of cytokines, as aforementioned.

For DC culture, PBMCs were cultured in X-VIVO 20 medium containing 1,000 U/ml IL-4 and 500 U/ml rhGM-CSF. Autologous tumor lysate (100 µg/ml) was added at day 6 and co-cultured with DCs for 24 h. For CIK activation, CIKs were co-cultured with DCs loaded with tumor antigen for 7 days. The DC-CIK cells were harvested and analyzed for phenotype, then suspended in 100 ml saline for intravenous injection. The final cell products were assessed for viability by the dye-exclusion test and checked twice for possible contamination by bacteria, fungi and endotoxins.

Approximately 5×10⁵ CIKs, with or without activation by DCs, were resuspended in 20 µl 2% fetal bovine serum and 1% sodium azide in phosphate buffered saline (PBS) and incubated with 10 µl antibody against CD3-fluorescein isothiocyanate (FITC; clone, UCHT1; dilution, 1:2.5; catalog no., 11-0038-80; eBioscience, San Diego, CA, USA), CD4/FITC and CD8-RPE (clones, MT310 and DK25; dilution, 1:50; catalog no., FR86850), and CD3-FITC/CD56-RPE (clones, UCHT1 and C5.9; dilution, 1:50; catalog no., FR91250; Dako, Carpentaria, CA, USA) for 30 min at 4°C. Following incubation, cells were washed twice with PBS and resuspended in 1 ml staining buffer (BD Pharmingen, San Diego, CA, USA). The cell population was analyzed using flow cytometry (FCM; BD Biosciences).

Patients received 1–4 cycles of DC-activated CIK treatment at intervals of 1 month after chemotherapy/radiotherapy. For each cycle, patients were treated with a median of (8.3±0.61)x10⁹ DC-activated CIKs (range, 8.0–12.6×10⁹) at 1-day intervals for two intravenous infusions (Fig. 1). Additional care was provided whenever required. Clinical examinations of these patients were performed by oncology specialists weekly or biweekly, including a complete blood count, liver and renal function tests, computed tomography (CT) or magnetic resonance imaging (MRI) scan.

Efficacy evaluation was performed prior to and subsequent to 2 cycles of DC-CIK treatment. The evaluation indexes of therapeutic efficiency included the objective remission rate of measurable focus region, which was performed according to the World Health Organization criterion, and comparing the tumor size prior to and subsequent to 2 cycles of treatment by CT/MRI. The curative effect was further divided into complete remission (CR), partial remission (PR), stable disease (SD) and progressive disease (PD). The ratio of CR and PR was analyzed. The clinical benefit rate (CBR) was also analyzed. The quality of life and physical improvement were measured by KPS, and the assessment of pain grade was evaluated by numeric rating scale (NRS). The tumor markers carcinoembryonic antigen (CEA), α fetoprotein (AFP), carbohydrate antigen-125 (CA125), carbohydrate antigen-199 (CA199), carbohydrate antigen-724 (CA724), prostate special antigen (PSA) and neuron-specific enolase (NSE) were also detected by Immunofluorescence Analysis system (Zeiss AG, Thornwood, NY, USA). The lymphocyte subpopulation, consisting of the CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ populations of T lymphocytes, was measured by FCM, and the ratio of CD4:CD8 cells was also analyzed.

The measurement data was compared using Student's t-test and analyzed by SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The median count of untreated PBMCs of all patients was 3.16×10⁹ (range, 2.67×10⁹-3.59×10⁹) per cycle. The median count of CIK cells after 14 days of amplification could reach 9.64×10⁹ (range, 8.57×10⁹-12.41×10⁹) per cycle. On the basis of trypan blue staining, the cellular vitality was >95%.

To determine the immunological activation of DCs on CIKs, the phenotype of CIKs prior to and subsequent to induction by DCs was analyzed using FCM. Fig. 2 demonstrated that the population of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD3⁺CD56⁺CIK cells were significantly increased between 47.94±7.02, 27.56±4.05, 18.13±4.26 and 4.28±1.18 (prior to co-culture) to 80.78±8.11, 40.31±6.94, 37.02±5.78 and 18.33±4.60% subsequent to co-culture, respectively (P=0.044).

In total, 26 out of 58 patients had a measurable focus region: 1 achieved CR and 25 achieved PR, with an objective remission rate of 44.83%. Among the remaining patients, 30 achieved SD (51.72%) and 2 demonstrated PD (3.45%). There were 42 of 58 patients who underwent CBR evaluation: 36 had both KPS that was increased >20% and pain relief of >50%, with an overall CBR of 85.71% (36/42). Additionally, following treatment with DC-activated CIKs, the KPS was 87.28±5.46, which was significantly higher than prior to treatment (69.02±7.45). Subsequently, the level of tumor markers including CEA, AFP, CA125, CA199, CA724, PSA and NSE was monitored. The results demonstrated that there were 15, 8, 4, 2 and 2 patients with high CEA, CA199, NSE, CA724 and AFP expression, respectively. Subsequent to DC-CIK mediated immunotherapy for 2 cycles, 9 patients attained almost normal tumor marker expression levels. The remaining patients did not demonstrate a decline to the normal range, but CEA expression decreased (Fig. 3). Referring to the lymphocyte subpopulation, the present test results indicated that 75% of patients showed an increase in CD3⁺CD4⁺ lymphocytes (P=0.017) and 50% of patients showed an increase in CD3⁺CD8⁺ lymphocytes (P=0.023). The population of CD4/CD8 cells was significantly increased as well (P=0.024) (Fig. 4).

The distributions of side effects in the patients are shown in Table II. No serious side effects that may present a safety hazard to life were observed. No patient failed to complete the DC-CIK immunotherapy. There were no grade III–IV cell-associated toxicities, and common grade I–II toxicities consisted of transient chills, fever, fatigue, headache and anemia.