Induction of Th1Immune responses following laser ablation in a murine model of colorectal liver metastases
- Wen Xu Lin†1,
- Theodora Fifis†1Email author,
- Caterina Malcontenti-Wilson1,
- Mehrdad Nikfarjam1,
- Vijayaragavan Muralidharan1,
- Linh Nguyen1 and
- Christopher Christophi1
© Lin et al; licensee BioMed Central Ltd. 2011
Received: 17 September 2010
Accepted: 29 May 2011
Published: 29 May 2011
Preliminary experimental studies have suggested that the in situ destruction of tumor tissue by local laser ablation (LA) may also stimulate host immunity against cancer. We investigated local and systemic induction of immune responses after laser ablation in the setting of residual tumor.
A murine colorectal cancer (CRC) liver metastasis model was used. Selected tumors of liver CRC bearing mice and livers of mice without tumor induction were treated with LA. Liver and tumor tissues from the ablation sites and from distant sites were collected at various time points following LA and changes in CD3+ T cells and Kupffer cells (F4/80 marker) infiltration and the expression of interferon gamma (IFNγ) were investigated by immunohistochemistry and ELISpot. Base line levels of CD3+ T cells and Kupffer cells were established in untreated mice.
The presence of tumor induced significant accumulation of CD3+ T cells and Kupffer cells at the tumor-host interface, within the tumor vascular lakes and increased their baseline concentration within the liver parenchyma. LA of the liver induced accumulation of CD3+ T-cells and Kupffer cells at the site of injury and systemic induction of immune responses as discerned by the presence of IFNγ secreting splenocytes. LA of liver tumors induced significant increase of CD3+ T-cells at site of injury, within normal liver parenchyma, and the tumor-host interface of both ablated and distant tumors. In contrast Kupffer cells only accumulated in ablated tumors and the liver parenchyma but not in distant tumors. IFNγ expression increased significantly in ablated tumors and showed an increasing trend in distant tumors.
Laser ablation in addition to local tumor destruction induces local and systemic Th1 type immune responses which may play a significant role in inhibiting tumor recurrence from residual micrometastases or circulating tumor cells.
Colorectal cancer (CRC) is the most common solid organ cancer across both genders and the third most common cause of cancer related deaths . More than 50% of patients with CRC develop liver metastases (CRCLM) which is the leading cause of death in this population. Surgical resection is the only potential curative option. The spatial distribution of metastases, presence of extra hepatic disease, potential residual liver volume and function as well as the general health of the patient are the main factors that limit the surgical option to approximately 10-25% of patients [2, 3]. Advances in systemic therapies have progressively increased the potential for surgical intervention by down staging hepatic metastases in a small subset of patients . Despite successful surgery, the majority of patients develop disease recurrence most frequently in the liver.
Local thermal ablation was developed to increase the therapeutic options for patients with liver metastases [5, 6]. This involves the application of laser, radiofrequency or microwave energy to the tumor. The particular energy in each case is converted into heat that leads to tumor destruction by coagulative necrosis. The aim is to extend the necrosis into a rim of normal tissue parenchyma surrounding the tumor for total tumor destruction [7–9]. When applied as a minimally invasive technique, thermal ablation has a number of potential advantages including significantly lower morbidity, minimal destruction of normal liver tissue and transient changes in liver function enzymes, leading to a lesser regenerative response and the ability for repeated application [10–13].
Early clinical comparisons between resection and thermal ablation suggested that thermal ablation is associated with a less favourable outcome . Results from experimental animal studies however suggest that thermal ablation of metastatic liver tumors is associated with reduced incidence of tumor growth and metastasis compared to resection. Additionally a positive effect on host immune response has been reported following thermal ablation of tumors where the ablated tumor antigens appear to behave like a tumor vaccine [14–16].
This study investigated immune responses in mice with CRC liver metastases following LA of selected tumors. In particular, it focused on changes of Kupffer cells (or tumor infiltrating macrophages; TAMs) and CD3+ T cells representing innate and adaptive immunity respectively and on IFNγ expression which is associated with Th1 protective immune responses in cancer . The experimental plan was designed to investigate if protective immune responses occur in a scenario reflecting clinical application of LA, where residual micrometastases or tumor at the margins of an ablation site remain after treatment.
Six to eight week old male CBA mice (Laboratory Animal services, University of Adelaide, South Australia) were used in all experiments. Mice were maintained in standard cages with access to irradiated food and water ad libitum, and exposed to a twelve hour light/dark cycle. All procedures were implemented in accordance with the guidelines of the Austin Health Animal Ethics Committee.
Experimental model of CRC liver metastases
The primary cell line MoCR was derived from a dimethyl hydrazine (DMH)-induced primary colon carcinoma in the CBA mouse and maintained in vivo by serial passage in the flanks of CBA mice . For passage and experimentation, tumors grown subcutaneously were teased, passed through a filter, treated with EDTA and washed in PBS to make a single cell suspension. Liver metastases were induced by an intrasplenic injection of 5 × 104 tumor cells prior to splenectomy as reported previously . In this model, liver metastases are fully established by 21 days following tumor induction.
Laser Ablation Treatment
Twenty-one days after tumor induction animals were used for LA study. Similarly located intra-parenchymal tumors of 7 mm diameter were chosen for sub-total laser ablation and was performed as described previously . Briefly a Neodymium Yttrium-Aluminium-Garnet (Nd:YAG-wavelength of 1064 nm) laser (Dornier medilas fibertom 4100 Medizintechnik GmbH, Munchen) was used. Animals were anaesthetized and a bilateral sub-costal incision was performed to allow full exposure of the liver. A 400 μm bare tip optical quartz fibre delivered laser energy, applying 100J of power per tumor (50 seconds at 2 Watts). The treatment parameters were chosen based on our previous extensive studies where the nature and extent of injury including temperature profiles were examined [19–21]. Average tissue temperatures reach 65°C adjacent to the fibre site without causing tissue charring. Higher power settings in this animal model generally produce charring. This setting in tumor tissue produces incomplete necrosis that does not extend into the liver. For endpoints other than 0 the treated tumors were marked with special dye (Davidson Tissue Marking System, Bradley Products, Grale Scientific, Melbourne, Australia), the abdomen was closed and animals recovered.
Tissue Sample Collection
At each endpoint after LA treatment, mice were anesthetized and their liver was excised. The two ablated or sham treated (no activation of the probe) tumors were identified and then immediately dissected from the liver together with surrounding liver tissue. Samples of liver tissue and untreated tumors were also collected. All specimens were fixed in formalin for 48 hours and processed for immunohistochemistry.
Three study groups were used: The first study aimed to establish the baseline distribution of T cells and Kupffer cells in tumor bearing livers and consisted of two groups of mice. The experimental group was induced with metastatic tumor cells 21 days prior to tissue collection and controls consisted of a group of mice from the same cohort but not induced with tumor. The second study investigated temporal changes in the distribution of T cells, Kupffer cells and IFNγ expression, when non tumor bearing animals were treated with TA in the liver tissue and compared to baseline controls (shams: liver not treated with TA). The third study investigated temporal changes in the distribution of T cells, Kupffer cells and IFNγ expression in liver and metastatic tumor tissues following TA treatment of two selected tumors. The results were compared to baseline controls (day 21 post tumor induction and day 0 post TA treatment).
Formalin fixed paraffin embedded 4-μm-thick sections of the tissues were deparaffinized and rehydrated using standard techniques. Endogenous peroxidases were blocked by incubation in 3% peroxide in methanol for 10 minutes. Antigen retrieval was achieved by incubation in Proteinase K in a 37°C oven for 20 minutes, followed by a cooling down period of 10 minutes at room temperature (RT). Normal goat serum (20%) was used to block non specific binding. Commercially available primary antibodies used for staining (CD3; rabbit anti-human CD3+ polyclonal A0452, Dakocytomation, Denmark at 0.6 μg/ml, IFNγ; rat anti-mouse IFNγ monoclonal 3321-3-1000, Mabtech, Australia, at 1 μg/ml, Kupffer cell staining; rat anti mouse F4/80 monoclonal antibody, ATCC no. HB-198, culture supernatant at 1:50 dilution). Negative controls were incubated with the respective non immune antibody isotypes at the same concentration as the primary antibody. Sections were incubated with primary antibodies overnight at 4°C. Sections treated with the rat antibodies were treated with a rabbit anti-rat linker antibody before treatment with a polymer based detection kit containing goat anti-rabbit immunoglobulins (IgG) linked to horseradish peroxidase (HRP) (Envision Plus, Dako, Australia). Each incubation step was followed by two five minute washes with PBS + 0.05% Tween 20. Positive staining was visualized using diaminobenzidine (DAB) as a substrate.
Mouse spleens were collected from LA treated and sham LA treated mice and the spleen cells from each group were pooled. Spleen cells (106 per well in 100 μL RPMI complete medium) were incubated without stimulation for 18 h in 96-well plates (MAIPS; Millipore, Australia) pre-coated with host species anti-murine IFNγ (clone R4, American Type Culture Collection, Manassas, VA). Triplicate wells were set up for each condition. After washing wells with PBS, secreted cytokine was detected with biotinylated anti-murine IFNγ (MAb XMG.21-biotin; Pharmingen, Australia) followed by extravidin-alkaline phosphatase at 100 μg/mL (Sigma). Spots of activity were detected with a colorimetric alkaline phosphatase kit (Bio-Rad, Hercules, California, USA) and counted using a plate reader (AID GmbH, Germany) with AID ELISpot software Version 3.0. Data are presented as mean spot-forming units (SFU) per million cells ± standard error of the mean (SEM).
Quantification of CD3+ T cell and Kupffer cell staining
All sections were examined using a digital microscope system (Coolscope, Nikon Corporation, Chiyokd-ku, Tokyo, Japan). Areas of interest were identified and photomicrographs for each region were captured for enumeration of lymphocytes within (1) tumor-host interface (treated and untreated distant tumors), (2) LA injury front and (3) distant normal liver away from the ablation sites. Images were coded and analyzed using an image analysis program in a blinded manner (Image-Pro Plus Version 4.5.1, Media Cybernetics, USA). Counts were expressed as the number of positive cells per mm2 of tissue. Alternatively positive stained areas were calculated using Image-Pro Plus software and expressed as arbitrary units.
Semi-quantitative analysis of IFNγ
Areas of interest were identified using a light microscope (Olympus BH2, Japan) at a magnification of 125x. The entire margin of treated normal liver, tumor host interface of treated/untreated tumor and normal liver tissues were examined. Scoring criteria was used to estimate the amount and intensity of staining seen in each sample. The grading system used was: as: 0: no staining 1: faint staining; 2: small amount or weak staining; 3: moderate staining; 4: abundant or strong staining; 5: Abundant or very strong staining. Means for each group were determined using the individual scores from each sample.
Statistical analyses were performed using SPSS program (Statistical Package for the Social Sciences™, version 10, Chicago, Illinois, USA). All data was expressed as the mean ± standard error of the mean unless otherwise specified. Data was tested for normality using detrended Q-Q plots, descriptive statistics such as skewness and kurtosis and the Kolgomorov-Smironov test prior to statistical analysis. Differences between groups were assessed by non parametric Kruskall Wallis followed by Mann Whitney U tests or parametric ANOVA followed by Tukey post hoc analysis as appropriate. A P value of 0.05 or less was regarded as statistically significant.
Tumor induces accumulation of CD3+ T cells and Kupffer cells in liver and tumor tissues
Laser ablation of liver tissue induces local and systemic immune responses
Laser ablation of selected tumors induces concentration and distribution changes of CD3+ T cells in tumor and liver tissues
Temporal biphasic changes of CD3+ T cell numbers were seen within the liver parenchyma distant from the ablation sites Figure 4b. These changes showed an immediate peak which was then followed by a second peak, with significant differences seen in CD3+ T cell numbers between the treated and sham groups (P values: Immediate: 0.050, Day 1: 0.086; Day 2: 0.086; Day 3: 0.043; Day 5: 0.008; Day 7: 0.433, t tests) after LA treatment, suggesting systemic trafficking of these cells.
Laser ablation of specific tumors induces increased IFNγ expression in tumor tissues
Changes in concentration and distribution of Kupffer cells in tumor and liver tissues following laser ablation of tumors
Thermal ablation has evolved as a significant minimally invasive treatment for unresectable CRC liver metastases as well as an adjunct to liver resection . Accumulating evidence suggests that in situ tumor destruction by thermal ablation may also stimulate local and systemic anti-tumor immunity, with the potential to eliminate not only treated tumors, but also residual micrometastases which normally give rise to tumor recurrence; reviewed by Gravante et al . In previous studies we have shown that LA destroys tumor or liver tissue by generating immediate focal necrosis followed by a marked inflammatory response and progressive increase in the area of injury . We have also demonstrated significant accumulation of Kupffer cells and increased expression of HSP70 at the injury front persisting for a number of days following LA treatment . In the present study we demonstrate T-cell accumulation not only at the LA injury site, but also within liver parenchyma and tumor/host interface of both ablated tumors and residual tumors distant from the site of ablation. In contrast Kupffer cells only accumulated in ablated tumors and the liver parenchyma but not in distant tumors. IFNγ expression increased significantly in ablated tumors and showed an increasing trend in tumors distant from the ablation site. In addition significantly more splenocytes from liver ablated animals secreted IFNγ compared to controls.
In clinical studies, thermal ablation of tumors has been shown to result in early systemic inflammation, the induction and systemic trafficking of specific anti-tumor T-cell responses involving CD4+ and CD8+ T cells [23, 24] and a generalized adjuvant effect that also involved the activation of natural killer cells [25, 26].
In experimental studies, total tumor destruction by thermal ablation protected animals from further tumor challenge  while partial tumor removal by thermal ablation resulted in significant residual tumor inhibition and systemic tumor specific CD4+ and CD8+ T-cell induction compared to resection . The mechanisms by which thermal ablation activates the immune system are not clear at this stage, accumulating evidence however suggest the involvement of both the innate and the adaptive immune systems and their cytokines .
Our results indicate that presence of tumor alters the molecular environment of the liver in ways that attract accumulation of immune cells (CD3 T cells and Kupffer cells). Infiltration of Kupffer cells (or TAMs) into tumors has been reported in many other studies. Macrophages activated in the classical pathway (M1) favoring a Th1 immune response (IFNγ, NO, TNFα, IL-1 and IL-12 secretion) are associated with tumoricidal functions. Macrophages infiltrating the tumor microenvironment however are usually activated along the M2 pathway promoting Th2 type immune responses  and tumor progression by releasing proangiogenic cytokines and growth factors (VEGF, IL-8, b-FGF) and matrix metalloproteases (MMPs) that digest the tumor basement membrane, facilitating tumor metastasis .
Infiltration and accumulation of CD3+ T cells within colorectal and other tumors has also been reported in several other studies. The significance of this infiltration is controversial. Early studies associate it with a favourable outcome [31, 32]. More recent studies however indicate that T cell infiltration in solid tumors are at best ineffectual in controlling tumor growth and most often contribute to tumor progression by enabling the neutralisation of immune responses . The tumor microenvironment subverts the immune response in a variety of ways to support tumor growth. All CD3+ T cell subtypes have been shown to be capable of promoting tumor progression, either through altered cytokine production such as IL-1, IL-4, TGF-β and IL-10 [34–36] or through cell-cell contact after being converted into FoxP3 regulatory T cells by the influence of tumor stroma derived immunosuppressive factors such as PGE2, TGF-β or IDO by-products [37, 38]. Thermal ablation studies suggest that the treatment induces protective Th1 immune responses to counteract the immunosuppressive tumor microenvironment. Antigens from thermally ablated hepatocellular carcinoma induced superior stimulation of in vitro immune responses than untreated tumor antigens  and vaccination with antigens from thermally treated tumors prior to thermal ablation enhanced the treatment outcome . This is most likely achieved by the upregulation of HSP proteins including HSP70 that we and others have shown to occur after LA treatment . HSP70, a stress induced molecular chaperone, has a dual role in inducing a Th1 anti-tumor response. HSP70 acts as a general adjuvant, signaling through toll-like receptor 4 (TLR-4)  resulting in the maturation and activation of dendritic cells (DCs). Maturation of DCs is required to efficiently present antigenic peptides for protective immune responses. HSP70 also forms complexes with all the tumor antigens so it also induces tumor specific immune responses by delivering specific antigens to DCs . Maturation of dendritic cells and efficient antigen presentation results in a Th1 immune response capable of overcoming the tumor immunosuppressive environment. It was shown that a Th1 response and upregulation of IFNγ is required for the prevention of tumor establishment or the elimination of already established tumors . The presence of Th1 activated T cells is an independent prognostic marker for patient survival .
Upregulation of the Th1 pathway cytokines IL-12 and/or IFNγ within the tumor resulted in tumor killing  and directly inhibited tumor angiogenesis . In the current study we demonstrated local and systemic upregulation of IFNγ and the accumulation of CD3+ T cells at the site of LA injury and at distant tumors. These findings suggest that LA treatment induces a Th1 immune response. Retention of CD3+ cells at the site of injury could be due to the presence of antigen presenting cells activated by HSP70 and displaying antigens from necrotic cells after LA treatment. While both Kupffer cells and CD3+ T accumulated at the tumor host interface and the injury site of the ablated tumor, only CD3+ T cells showed significant accumulation at the tumor host interface of distant tumors. This finding implies that a large proportion of CD3+ cells must recognise tumor specific signals, whereas KCs respond to a general inflammation response and specifically accumulate in the ablated tissues. Accumulation of CD3+ T cells within tumor margins of untreated tumors following thermal ablation treatment have also been reported in other studies using different tumor models [14, 27].
In addition to local upregulation of IFNγ, significantly more splenocytes in LA treated animals produced IFNγ compared to sham treated animals indicating induction of a systemic Th1 immune response. IFNγ is produced by activated T cells and other cells of the immune system such as NK cells. The temporal kinetic pattern of IFNγ expression in this study was similar to that of CD3+ cell infiltration following LA treatment. This finding suggests that the infiltrating CD3+ T cells would also be activated along the Th1 pathway and may provide an effective mechanism for control of CRC liver metastases.
We have shown LA treatment induces significant innate and adaptive immune responses, including IFNγ upregulation locally and systemically, indicating these responses to be Th1 and therefore tumor inhibiting. The accumulation of CD3+ T cells and the increase of IFNγ in distant unablated tumors suggest that the response could be beneficial in suppressing outgrowth of residual micrometastases in the clinic. Future work will identify the composition and activation status of the CD3+ T cell population after LA therapy and will validate their protective roles as their modulation may further enhance treatment outcomes.
This work was supported by the Cancer Council of Victoria and Austin Health Medical Research Foundation.
- Landis SH, Murray T, Bolden S, Wingo PA: Cancer statistics, 1999. CA Cancer J Clin. 1999, 49 (1): 8-31. 10.3322/canjclin.49.1.8. 31View ArticlePubMedGoogle Scholar
- Cromheecke M, de Jong KP, Hoekstra HJ: Current treatment for colorectal cancer metastatic to the liver. Eur J Surg Oncol. 1999, 25 (5): 451-463. 10.1053/ejso.1999.0679.View ArticlePubMedGoogle Scholar
- Adam R, Avisar E, Ariche A, Giachetti S, Azoulay D, Castaing D, Kunstlinger F, Levi F, Bismuth F: Five-year survival following hepatic resection after neoadjuvant therapy for nonresectable colorectal. Ann Surg Oncol. 2001, 8 (4): 347-353. 10.1007/s10434-001-0347-3.View ArticlePubMedGoogle Scholar
- Ruers T, Bleichrodt RP: Treatment of liver metastases, an update on the possibilities and results. Eur J Cancer. 2002, 38 (7): 1023-1033. 10.1016/S0959-8049(02)00059-X.View ArticlePubMedGoogle Scholar
- Abdalla EK, Vauthey JN, Ellis LM, Ellis V, Pollock R, Broglio KR, Hess K, Curley SA: Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Ann Surg. 2004, 239 (6): 818-825. 10.1097/01.sla.0000128305.90650.71. discussion 825-817PubMed CentralView ArticlePubMedGoogle Scholar
- Iannitti DA, Martin RC, Simon CJ, Hope WW, Newcomb WL, McMasters KM, Dupuy D: Hepatic tumor ablation with clustered microwave antennae: the US Phase II Trial. HPB (Oxford). 2007, 9 (2): 120-124.View ArticleGoogle Scholar
- Nikfarjam M, Muralidharan V, Malcontenti-Wilson C, Christophi C: Progressive microvascular injury in liver and colorectal liver metastases following laser induced focal hyperthermia therapy. Lasers Surg Med. 2005, 37 (1): 64-73. 10.1002/lsm.20194.View ArticlePubMedGoogle Scholar
- Christophi C, Winkworth A, Muralihdaran V, Evans P: The treatment of malignancy by hyperthermia. Surgical Oncology. 1998, 7 (1-2): 83-90. 10.1016/S0960-7404(99)00007-9.View ArticlePubMedGoogle Scholar
- Gravante G, Ong S, Metcalfe M, Strickland A, Dennison A, Lloyd D: Hepatic microwave ablation: a review of the histological changes following thermal damage. Liver international. 2008, 28 (7): 911-921. 10.1111/j.1478-3231.2008.01810.x.View ArticlePubMedGoogle Scholar
- Solbiati L, Ierace T, Tonolini M, Osti V, Cova L: Radiofrequency thermal ablation of hepatic metastases. Eur J Ultrasound. 2001, 13 (2): 149-158. 10.1016/S0929-8266(01)00127-6.View ArticlePubMedGoogle Scholar
- Vogl TJ, Straub R, Zangos S, Mack MG, Eichler K: MR-guided laser-induced thermotherapy (LITT) of liver tumours: experimental and clinical data. Int J Hyperthermia. 2004, 20 (7): 713-724. 10.1080/02656730400007212.View ArticlePubMedGoogle Scholar
- Berber E, Siperstein AE: Perioperative outcome after laparoscopic radiofrequency ablation of liver tumors: an analysis of 521 cases. Surgical endoscopy. 2007, 21 (4): 613-10.1007/s00464-006-9139-y.View ArticlePubMedGoogle Scholar
- Dobbins C, Brennan C, Wemyss-Holden S, Cockburn J, Maddern G: Bimodal electric tissue ablation-long term studies of morbidity and pathological change. The Journal of surgical research. 2008, 148 (2): 251-259. 10.1016/j.jss.2007.09.008.View ArticlePubMedGoogle Scholar
- Isbert C, Ritz JP, Roggan A, Schuppan D, Ruhl M, Buhr HJ, Germer CT: Enhancement of the immune response to residual intrahepatic tumor tissue by laser-induced thermotherapy (LITT) compared to hepatic resection. Lasers Surg Med. 2004, 35 (4): 284-292. 10.1002/lsm.20097.View ArticlePubMedGoogle Scholar
- Hu Z, Yang XY, Liu Y, Sankin GN, Pua EC, Morse MA, Lyerly HK, Clay TM, Zhong P: Investigation of HIFU-induced anti-tumor immunity in a murine tumor model. J Transl Med. 2007, 5: 34-10.1186/1479-5876-5-34.PubMed CentralView ArticlePubMedGoogle Scholar
- Gravante G, Sconocchia G, Ong S, Dennison A, Lloyd D: Immunoregulatory effects of liver ablation therapies for the treatment of primary and metastatic liver malignancies. Liver international. 2009, 29 (1): 18-10.1111/j.1478-3231.2008.01915.x.View ArticlePubMedGoogle Scholar
- Ikeda H, Old L, Schreiber R: The roles of IFN gamma in protection against tumor development and cancer immunoediting. Cytokine & growth factor reviews. 2002, 13 (2): 95-10.1016/S1359-6101(01)00038-7.View ArticleGoogle Scholar
- Kuruppu D, Christophi C, Bertram JF, O'Brien PE: Characterization of an animal model of hepatic metastasis. J Gastroenterol Hepatol. 1996, 11 (1): 26-32. 10.1111/j.1440-1746.1996.tb00006.x.View ArticlePubMedGoogle Scholar
- Nikfarjam M, Muralidharan V, Su K, Malcontenti-Wilson C, Christophi C: Patterns of heat shock protein (HSP70) expression and Kupffer cell activity following thermal ablation of liver and colorectal liver metastases. Int J Hyperthermia. 2005, 21 (4): 319-332. 10.1080/02656730500133736.View ArticlePubMedGoogle Scholar
- Muralidharan V, Nikfarjam M, Malcontenti-Wilson C, Christophi C: Effect of interstitial laser hyperthermia in a murine model of colorectal liver metastases: scanning electron microscopic study. World J Surg. 2004, 28 (1): 33-37. 10.1007/s00268-003-6973-0.View ArticlePubMedGoogle Scholar
- Nikfarjam M, Muralidharan V, Malcontenti-Wilson C, Christophi C: The apoptotic response of liver and colorectal liver metastases to focal hyperthermic injury. Anticancer Res. 2005, 25 (2B): 1413-1419.PubMedGoogle Scholar
- Clasen S, Boss A, Schmidt D, Schraml C, Fritz J, Schick F, Claussen C, Pereira P: MR-guided radiofrequency ablation in a 0.2-T open MR system: technical success and technique effectiveness in 100 liver tumors. Journal of magnetic resonance imaging. 2007, 26 (4): 1043-10.1002/jmri.21120.View ArticlePubMedGoogle Scholar
- Hansler J, Wissniowski TT, Schuppan D, Witte A, Bernatik T, Hahn EG, Strobel D: Activation and dramatically increased cytolytic activity of tumor specific T lymphocytes after radio-frequency ablation in patients with hepatocellular carcinoma and colorectal liver metastases. World J Gastroenterol. 2006, 12 (23): 3716-3721.PubMed CentralPubMedGoogle Scholar
- Napoletano C, Taurino F, Biffoni M, De Majo A, Coscarella G, Bellati F, Rahimi H, Pauselli S, Pellicciotta I, Burchell J, Gaspari L, Ercoli L, Rossi P, Rughetti A: RFA strongly modulates the immune system and anti-tumor immune responses in metastatic liver patients. International journal of oncology. 2008, 32 (2): 481-PubMedGoogle Scholar
- Zerbini A, Pilli M, Penna A, Pelosi G, Schianchi C, Molinari A, Schivazappa S, Zibera C, Fagnoni F, Ferrari C, Missale G: Radiofrequency thermal ablation of hepatocellular carcinoma liver nodules can activate and enhance tumor-specific T-cell responses. Cancer research. 2006, 66 (2): 1139-10.1158/0008-5472.CAN-05-2244.View ArticlePubMedGoogle Scholar
- Zerbini A, Pilli M, Laccabue D, Pelosi G, Molinari A, Negri E, Cerioni S, Fagnoni F, Soliani P, Ferrari C, Missale G: Radiofrequency thermal ablation for hepatocellular carcinoma stimulates autologous NK-cell response. Gastroenterology. 2010, 138 (5): 1931-10.1053/j.gastro.2009.12.051.View ArticlePubMedGoogle Scholar
- Ivarsson K, Myllymäki L, Jansner K, Stenram U, Tranberg KG: Resistance to tumour challenge after tumour laser thermotherapy is associated with a cellular immune response. British Journal of Cancer. 2005, 93 (4): 435-440. 10.1038/sj.bjc.6602718.PubMed CentralView ArticlePubMedGoogle Scholar
- Dromi SA, Walsh MP, Herby S, Traughber B, Xie JW, Sharma KV, Sekhar KP, Luk A, Liewehr DJ, Dreher MR, Fry TJ, Wood BJ: Radiofrequency Ablation Induces Antigen-presenting Cell Infiltration and Amplification of Weak Tumor-induced Immunity. Radiology. 2009, 251 (1): 58-66. 10.1148/radiol.2511072175.PubMed CentralView ArticlePubMedGoogle Scholar
- Allavena P, Sica A, Garlanda C, Mantovani A: The Yin-Yang of tumor-associated macrophages in neoplastic progression and immune surveillance. Immunological reviews. 2008, 222: 155-161. 10.1111/j.1600-065X.2008.00607.x.View ArticlePubMedGoogle Scholar
- Hagemann T, Robinson S, Schulz M, TrÃ¼mper L, Balkwill F, Binder C: Enhanced invasiveness of breast cancer cell lines upon co-cultivation with macrophages is due to TNF-alpha dependent up-regulation of matrix metalloproteases. Carcinogenesis. 2004, 25 (8): 1543-10.1093/carcin/bgh146.View ArticlePubMedGoogle Scholar
- Menon AG, Janssen CM, Rhijn CM, Morreau H, Putter H, Tollenaar R, van de Velde CJH, Fleuren GJ, Kuppen PJK: Immune system and prognosis in colorectal cancer: a detailed immunohistochemical analysis. Laboratory Investigation. 2004, 84 (4): 493-501. 10.1038/labinvest.3700055.View ArticlePubMedGoogle Scholar
- Ohtani H: Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human colorectal cancer. Cancer immunity. 2007, 7: 4-PubMed CentralPubMedGoogle Scholar
- Ruffell B, DeNardo D, Affara N, Coussens L: Lymphocytes in cancer development: polarization towards pro-tumor immunity. Cytokine & growth factor reviews. 2010, 21 (1): 3-10.1016/j.cytogfr.2009.11.002.View ArticleGoogle Scholar
- Aspord C, Pedroza-Gonzalez A, Gallegos M, Tindle S, Burton E, Su D, Marches F, Banchereau J, Palucka AK: Breast cancer instructs dendritic cells to prime interleukin 13-secreting CD4+ T cells that facilitate tumor development. The Journal of experimental medicine. 2007, 204 (5): 1037-10.1084/jem.20061120.PubMed CentralView ArticlePubMedGoogle Scholar
- DeNardo D, Barreto J, Andreu P, Vasquez L, Tawfik D, Kolhatkar N, Coussens L: CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer cell. 2009, 16 (2): 91-102. 10.1016/j.ccr.2009.06.018.PubMed CentralView ArticlePubMedGoogle Scholar
- Jarnicki A, Lysaght J, Todryk S, Mills KHG: Suppression of antitumor immunity by IL-10 and TGF-beta-producing T cells infiltrating the growing tumor: influence of tumor environment on the induction of CD4+ and CD8+ regulatory T cells. The journal of immunology. 2006, 177 (2): 896-904.View ArticlePubMedGoogle Scholar
- Sharma S, Yang SC, Zhu L, Reckamp K, Gardner B, Baratelli F, Huang M, Batra R, Dubinett S: Tumor cyclooxygenase-2/prostaglandin E2-dependent promotion of FOXP3 expression and CD4+ CD25+ T regulatory cell activities in lung cancer. Cancer research. 2005, 65 (12): 5211-10.1158/0008-5472.CAN-05-0141.View ArticlePubMedGoogle Scholar
- Curti A, Pandolfi S, Valzasina B, Aluigi M, Isidori A, Ferri E, Salvestrini V, Bonanno G, Rutella S, Durelli I, Horenstein A, Fiore F, Massaia M, Colombo M, Baccarani M, Lemoli R: Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25- into CD25+ T regulatory cells. Blood. 2007, 109 (7): 2871-2877.PubMedGoogle Scholar
- Zerbini A, Pilli M, Fagnoni F, Pelosi G, Pizzi M, Schivazappa S, Laccabue D, Cavallo C, Schianchi C, Ferrari C, Missale G: Increased immunostimulatory activity conferred to antigen-presenting cells by exposure to antigen extract from hepatocellular carcinoma after radiofrequency thermal ablation. Journal of immunotherapy. 2008, 31 (3): 271-282. 10.1097/CJI.0b013e318160ff1c.View ArticlePubMedGoogle Scholar
- Liu Q, Zhai B, Yang W, Yu LX, Dong W, He YQ, Chen L, Tang L, Lin Y, Huang DD, Wu HP, Wu MC, Yan HX, Wang HY: Abrogation of Local Cancer Recurrence After Radiofrequency Ablation by Dendritic Cell-based Hyperthermic Tumor Vaccine. Mol Ther. 2009Google Scholar
- Chen T, Guo J, Han C, Yang M, Cao X: Heat shock protein 70, released from heat-stressed tumor cells, initiates antitumor immunity by inducing tumor cell chemokine production and activating dendritic cells via TLR4 pathway. The journal of immunology. 2009, 182 (3): 1449-View ArticlePubMedGoogle Scholar
- Castelli C, Rivoltini L, Rini F, Belli F, Testori A, Maio M, Mazzaferro V, Coppa J, Srivastava P, Parmiani G: Heat shock proteins: biological functions and clinical application as personalized vaccines for human cancer. Cancer immunology and immunotherapy. 2004, 53 (3): 227-10.1007/s00262-003-0481-9.View ArticlePubMedGoogle Scholar
- Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc P-H, Trajanoski Z, Fridman W-H, Pages F: Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006, 313 (5795): 1960-1964. 10.1126/science.1129139.View ArticlePubMedGoogle Scholar
- Kilinc M, Rowswell-Turner R, Gu T, Virtuoso L, Egilmez N: Activated CD8+ T-effector/memory cells eliminate CD4+ CD25+ Foxp3+ T-suppressor cells from tumors via FasL mediated apoptosis. The journal of immunology. 2009, 183 (12): 7656-7660. 10.4049/jimmunol.0902625.View ArticlePubMedGoogle Scholar
- Sorensen E, Gerber S, Frelinger J, Lord E: IL-12 suppresses vascular endothelial growth factor receptor 3 expression on tumor vessels by two distinct IFN-gamma-dependent mechanisms. The journal of immunology. 2010, 184 (4): 1858-10.4049/jimmunol.0903210.PubMed CentralView ArticlePubMedGoogle Scholar
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