immune system
Duijnhoven, Frederieke van
Citation
Duijnhoven, F. van. (2005, June 22). Local ablative therapies for colorectal liver metastases
and the immune system. Dept. of Surgery, Leiden University Medical Center, Leiden
University. Retrieved from https://hdl.handle.net/1887/2706
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Chapter 5
Local therapy of colorectal liver m etastases and form ation of
Local therapy of colorectal liver m etastases and form ation of
Local therapy of colorectal liver m etastases and form ation of
Local therapy of colorectal liver m etastases and form ation of
antibodies
antibodies
antibodies
antibodies
Introduction IntroductionIntroduction Introduction
Several studies have shown the presence of tumour specific T cells in patients with colorectal cancer1-4.
The presence of T cells in the tumour epithelium is associated with a better survival5-8. Besides a cellular
immune response, a humoral response may be detected in colorectal cancer patients. Several studies have demonstrated presence of antibodies directed against tumour associated antigens (TAA) in serum from colorectal cancer patients9-11. Vaccination with tumour-derived antigens12-14 or autologous tumour cells15
resulted in induction or enhancement of an anti-tumour immune response, but the clinical relevance of these immunotherapeutic regimens is still uncertain2,14,16-20.
W hen colorectal liver metastases are treated with local ablative techniques (radiofrequency ablation (RFA), photodynamic therapy (PDT) or locoregional chemotherapy (isolated hepatic perfusion, IHP)), the necrotic tumour remains in situ after treatment, contrary to the situation following resection. And as the therapy is not systemic, the immune system is not compromised but may contribute to the anti-tumour efficacy. W e hypothesized that the presence of dead tumour cells in situ may induce or enhance a tumour specific immune response, as antigen exposure is facilitated by the local destruction of the tumour structure. Experimental animal studies have already shown that local tumour ablation can indeed result in increased anti-tumour immune response21-23.
To investigate this hypothesis, we evaluated the immune status of colorectal cancer patients with liver metastases using serum samples taken before and after treatment with local ablative therapies or loco-regional chemotherapy. A panel of six randomly selected human colon carcinoma cell lines represented a wide array of possible cell surface TAAs and presence of serum antibodies against these TAAs was assessed using flow cytometry. In addition, the cytokines interferon-gamma (IFN-γ) and interleukine-10 (IL-10) that may indicate a systemic cellular immune response were measured in the sera, using Enzyme-Linked Immunosorbent Assay (ELISA).
Materials and methods Materials and methodsMaterials and methods Materials and methods
Patient sera
Blood samples were centrifuged for 20 minutes at 2060 G (Beckman GS-6R centrifuge, Beckman Coulter, Fullerton, CA, USA); serum samples were collected and stored at -70°C until use.
Human colon carcinoma cell lines
To expose the patient sera to a broad panel of human tumour cell surface antigens, six human colon carcinoma cell lines (SW480, SW116, HCT-81, HT29, CaCo2 and Co115) were randomly selected. All cell lines were cultured in RPMI 1640 supplemented with 2mM L-glutamine, 10% heat inactivated calf serum, 50 U/ml penicillin and 50 Gg/ml streptomycin sulphate (complete medium) (all from Gibco, Grand Island, NY, USA). Cells were maintained by serial passage.
Flow cytometry
Tumour cells were harvested with a solution of 0,1% (w/v) EDTA and 0.25% (w/v) trypsine in HBSS (Sigma, St. Louis, MO, USA) and washed twice using 0.9% (w/v) NaCl solution buffered with 1.4 mM phosphate buffered saline (PBS) supplemented with 0,5% (w/v) bovine serum albumin (BSA, Boehringer Mannheim, Germany) Serum samples were diluted to 1:100 with PBS with 0.5% BSA w/v (PBS/BSA) and 100 Gl of test serum was added to 0.5 x 106 viable tumour cells and incubated for 30 minutes at 4°C. Cells were washed
twice with PBS/BSA, resuspended and incubated with a saturating amount of FITC-conjugated goat-anti-human IgG (PickCell Laboratories B.V., Amsterdam, The Netherlands) for 30 minutes at 4 ºC. As positive control for each tumour cell line known positive sera or specified human-α-human HLA sera were used (kindly provided by Dr A. Mulder, Leiden University Medical Center, The Netherlands). After washing once with PBS/BSA, 300 Gl of a 1.0 GM solution of propidium iodide in PBS/BSA was added in order to discri-minate between dead and vital tumour cells. Cells were then analysed in a flow cytometer (FACScalibur, Becton Dickinson Immuno-cytometry, San Jose, CA, USA). A total of 10,000 gated vital cells was analysed and the relative immuno-fluorescence was taken as measure for the fluorescence intensity. Level of anti-bodies was expressed in units of fluorescence intensity (FU). All samples from each patient were analysed within the same flow cytometry experiment.
Statistical analysis
Statistical differences between groups were calculated using two-tailed Pearson's Chi-Square test for qualitative analysis, with a p value of < 0.05 considered statistically significant.
Results ResultsResults Results
Sera were tested for the presence of antibodies directed against cell surface antigens on a panel of six different human colorectal carcinoma cell lines by flow cytometry, as described in the materials and methods section. In none of the serum samples antibodies directed against C0115 cell surface antigens were detec-ted. We therefore considered this cell-line non-informative and excluded this cell-line from further analysis. Results for all patients obtained with five remaining cell lines and in 6 IHP patients for four cell lines are shown in table 1.
group n CaCo2 HCT81 HT29 SW480 SW1116 qualification 1 0 0 + + 0 + PDT 2 0 − − − 0 − 3 0 0 0 0 0 0 4 0 0 + + 0 + 5 − 0 0 0 0 − 1 0 0 0 0 + + RFA 2 − + + + + + 3 0 0 0 0 0 0 4 0 + + 0 + + 5 0 0 0 + 0 + 1 0 0 − − − − resection 2 − − + − 0 − 3 0 0 + + − + 4 − − − − 0 − 5 0 0 0 0 − − 6 0 0 − − 0 − 7 0 0 0 0 0 0 1 0 0 + + 0 + IHP 2 0 0 + + 0 + 3 0 0 + + 0 + 4 0 0 + + 0 + 5 0 + + + 0 + 6 + + + + 0 + 7 0 + + + + 8 + 0 + 0 + 9 0 + + + + 10 0 + + + + 11 0 0 0 0 0 12 0 + 0 − +/−
Figure 1. Number of tests in separate cell lines showing unchanged, decreased or increased serum levels of antibodies directed against 5 different human colon carcinoma cell lines following isolated hepatic perfusion (IHP), radiofrequency ablation (RFA), photodynamic therapy (PDT) or hepatic resection, with * p = 0.001 and ** p = 0.003. Italic numbers represent the number of patients from which test results depicted in the column were derived
After analysis for each cell line separately and taking them all together for each treatment group, we qualified patients as "increase", "no change" or "decrease" in serum antibody levels. Qualification was based on the most frequently occurring change in antibody levels in each patient. In only one patient, decrease in antibody levels occurred as often as increased antibody levels. This patient was graded as +/− and excluded from statistical Chi-Square test analysis. In accordance with results from separate cell line analysis,
increased antibodies were most often seen in patients that were treated with IHP (10 out of 11 patients) or RFA (4 out of 5 patients)(figure 2). Of 7 patients treated with resection, 5 were qualified as "decrease" and only one was qualified as "increase". Antibody response following PDT was not as pronounced, with 2 patients qualified as "increase", two patients showing no change and one patient qualified as "decrease". As in cell line analysis, differences between RFA and resection groups as well as between IHP and resection groups were significant.
Figure 2. Number of patients showing increase, no change or decrease in serum levels of antibodies directed against 5 human colon carcinoma cell lines following isolated hepatic perfusion (IHP), radiofrequency ablation (RFA), photodynamic therapy (PDT) or hepatic resection, with * p = 0.036 and ** p = 0.002
Levels of IFN-γ and IL-10 were measured in all serum samples by ELISA. In all but two samples, values were below negative control levels for IFN-γ. Positive samples were seen in RFA group (7.33 pg/ml, before treatment) and IHP group (4.54 pg/ml, after treatment). For IL-10, only three samples were positive. One positive sample was taken after resection of liver metastases (19.59 pg/ml). The other two positive samples were from an IHP treated patient, taken before (59.86 pg/ml) and after (85.35 pg/ml) treatment.
Discussion DiscussionDiscussion Discussion
Research into the involvement of the immune system in efficacy of local ablative therapies is scarce. A few experimental studies report inflammation and immunological parameters to be influenced or increased by RFA or by a similar technique, laser induced thermotherapy (LITT). In LITT, local heat is generated by laser light instead of by ionic oscillation. Treatment of experimental liver metastases in rats by LITT resulted not only in local tumour destruction, but also inhibited growth of nearby, untreated liver tumours and decreased
0 2 4 6 8 10 12
PDT RFA resection IHP
treatment modality n p a ti e n ts no change decrease increase * ** 0 2 4 6 8 10 12
PDT RFA resection IHP
peritoneal tumour spread21,24. Wissniowski et al. showed the presence of circulating tumour specific T cells
after RFA treatment of VX2 hepatoma in 11 rabbits as well as increased T cell infiltration in tumour margins after RFA25. Another experimental study showed that upon induction of focal brain injury by RFA in mice,
macrophages and monocytes infiltrated the RFA lesion26. Immunohistochemical analysis indicated
expres-sion of TNF-α in these infiltrating macrophages to be increased at four days after RFA, remaining at this high level until 8 days after RFA. A recent study by Scheller et al. did not confirm these results in serum from patients whose liver tumours were treated with RFA, as they did not find a significant increase in cytokines (IL-1, IL-6, IL-8, IL-10 and TNF-α)27. In our study, we also did not find increased levels of IFN-γ or IL-10
following RFA, PDT or IHP. Although tumour specific T cells may be induced and local production of cyto-kines increased upon local tumour ablation, this is not necessarily reflected in serum levels of cytocyto-kines, as systemic cytokine levels may mostly be too low for detection by ELISA techniques.
The inducement of a specific immune response upon PDT of cancer is well documented in experimental models, with several studies indicating the necessary presence of a functioning immune system for PDT to exert its optimal anti-tumour effects23,28. Not only does the immune system play a pivotal part in the initial
PDT efficacy, it is also responsible for the long-term tumour resistance that is found after PDT29. In addition,
an experimental study comparing surgical treatment of tumour with PDT showed that there was a systemic anti tumour effect upon PDT as growth of untreated tumours and development of metastases was inhibi-ted21. This enhancing effect of PDT on the host antitumour immune response is possibly due to the
stimu-lated expression of proinflammatory cytokines23,30, but a recent study by Gollnick et al. indicated that PDT
may also directly influence tumour cell immunogenicity31. Vaccines were generated by PDT of cultured
tumour cells and inoculated intradermally for 4 weeks in mice, before intradermal inoculation with tumour cells. Vaccination with PDT-generated lysates resulted in substantial protection against tumour growth and was associated with higher survival when compared to non-vaccinated rats and vaccination with ultraviolet generated vaccines. These results indicate that PDT-vaccines elicit a tumour specific immune response.
In our study, no increased amount of antibodies was found following PDT, whereas antibody levels were increased upon IHP and RFA. Possibly, the immune response is not affected by PDT or is mainly of a cellular nature. The pronounced increase in antibody levels following IHP may be explained by its mode of action. Upon IHP, cell death occurs through apoptosis followed by secondary necrosis, whereas initial effects of RFA and PDT are due to primary necrosis. The less acute destruction that is induced by IHP could result in longer exposure of TAA and immunogenicity of tumour cells may be better preserved.
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