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10% human AB serum). “Catch” reagent (20 μL) containing Lonza, Cologne, Germany). Supernatant (20 μL) was incubated
a bivalent CD45 capture and interferon gamma (IFNγ) with AK-detection reagent (Lonza, Cologne, Germany)
binding antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, for 5 min at room temperature. Bioluminescence was
Germany) were added; the suspension was kept for 5 min measured by a luminometer (BD Monolight 3096 Microplate
on ice and 5 mL medium was added before incubation Luminometer, BD Biosciences, Heidelberg, Germany) and
at 37 °C in closed roller tubes for 45 min. Thereafter, expressed as relative luminescence units (RLU).
20 μL phycoerythrin-conjugated antibody against IFNγ
(1:5 dilution ratio) as well as 10 μL fluoresceine-thiocyanate Statistical analysis
+
conjugated anti-CD8 antibody (1:400 dilution ratio) The stimulation index (SI) of CD8 T cells was determined
−
+
+
+
(both from Miltenyi Biotec GmbH, Bergisch Gladbach, by the ratio of IFNγ CD8 vs. IFNγ CD8 T cells stimulated
Germany) were added to the cooled and washed cell with tumor tissue lysate vs. stimulation with normal liver
®
suspension and incubated for 10 min on ice. Erythrocyte tissue, respectively, and calculated with Excel 2003
lysis buffer (5 mL), containing 0.155 mol/L NH Cl, 10 mmol/L software (Microsoft, Seattle, USA). Results were analyzed
4
®
KHCO and 0.1 mmol/L EDTA diluted 1:10, was added for statistically by the SPSS software package (Version 14,
3
10 min and the cell suspension centrifuged at 300 g for SPSS GmbH Software, Munich, Germany). SI values were
10 min. The cell pellet was resuspended in 500 μL ice divided into two groups according to the histological origin
cold washing buffer and immediately analyzed by flow of the tumors. SI values were calculated numerically and are
cytometry (FACS Calibur, BD Biosciences, Heidelberg, presented as columns. RLU were expressed numerically and
Germany) after addition of 0.25 mg propidium iodide in 5 μL are shown as columns. The significance of the enhanced SI
distilled water. FACS data were analyzed using software Win after RFA treatment and augmented cytotoxic activity was
MDI Version 2.8 (Scripps Research Institute, La Jolla, CA, USA). tested with Fisher’s test for dependent samples. P < 0.05
were considered significant.
Cytotoxicity assay
Cytolytic activity of T cells was measured by adenylate RESULTS
kinase (AK) release assay. Cells (10 ) were incubated with
4
1,000 effector cells in a final volume of 200 μL growth Of 3 patients suffering from CRC, 2 had a recurrence-free
medium with fetal calf serum in round-bottom 96-well survival and 1 developed secondary metastases. One patient
microtiter plates. After incubation for 4 h at 37 °C, 100 μL of with HCC developed a local recurrence, and the other one
supernatant was harvested and stored at -20 °C for further had a disease-free survival after 12 months [Table 1].
analysis.
All patients had a significant activation of tumor-specific
The human HCC cell line HepG2 and the human CRC cell line T cells (SI baseline = 2.02, SD ± 0.2; SI CRC at
CaCO served as target cells for T cells isolated from patients 12 months = 12.3, SD ± 0.14; SI HCC at 12 months = 11.8,
2
with HCC and CRC metastases, respectively. All cell lines SD ± 0.23) (P < 0.05) [Figure 1].
were human leukocyte antigen matched (ABO-system) and
tested previously. HepG2 (ACC-180) and CaCO (ACC169) cells Disease-free patients showed a significantly risen cytolytic
2
were purchased from DSMZ (Leibniz Institute DSMZ-German activity of PBMC against matched tumor cells even 12 months
Collection of Microorganisms and Cell Cultures, Braunschweig after treatment (P < 0.05). The cytolytic activity after
Germany) and were cultured with RPMI1640 and DMEM 12 months was comparable to baseline data (RLU before
supplemented with 10% fetal bovine serum, penicillin (107 U/L) RFA = 10.4, SD ± 1.3; RLU 4 weeks = 354.7, SD ± 42.1;
and streptomycin (10 mg/L) (Biochrom AG, Berlin, Germany) RLU CRC 12 months = 298.4, SD ± 23.1; RLU HCC
as previously described. [9,10] 12 months = 317.4). In contrast, patients with recurrence
of malignancy showed a significantly decreased cytolytic
Maximum AK release was obtained by incubating target and activity (RLU for CRC at 12 months = 76.2; RLU for HCC at
effector cells with Total-Lysis Reagent™ (Lonza, Cologne, 12 months = 102.5) (P < 0.05) [Figure 2].
Germany), and baseline AK release was obtained by incubating
cells with medium alone. Baseline release from T cells and DISCUSSION
tumor cells were < 10% of maximum release in all experiments,
and baseline value was subtracted from each value. In the last few years, local ablative therapies such as laser
induced or radiofrequency induced thermal ablation have
The activity of AK was determined by detection of become more attractive as therapeutic options for patients
auto-luminescence using a luciferase assay (ToxiLight Kit, with unresectable solid tumors. The results of local ablative

94 Hepatoma Research | Volume 1 | Issue 2 | July 15, 2015