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Cancer is one of the leading causes of death in industrialized nations. Nowadays, cancer therapy mainly consists of surgery, radiation and chemotherapy. Thanks to intensive research alternative treatment strategies like gene therapy and especially immunotherapies are on the rise. Immunotherapies base on the idea of stimulating and supporting the patients immune system to generate an effective anti-tumor immune response. Dendritic Cells are perfect targets for this purpose, since these potent antigen-presenting immune cells influence the balance of the immune system by defining the route of action. Stimulation of these cells by activation of cellular signaling pathways results in maturation, upregulation of surface molecules and secretion of cytokines. A20 has been identified as a regulator of dendritic cell maturation and attenuator of their immune stimulating properties. Hence, the blockade of that natural inhibitor reveals an elegant way to activate cellular pathways of DCs. A siRNA against A20 obtains a functional blockade via RNA interference if it can be delivered into the cytoplasm of the target cells. CpG oligodeoxynucleotides can be used for this intracellular transport. CpGs contain DNA motifs similar to those found in bacteria. Innate immune cells can detect this DNA via the toll-like receptor 9 getting activated and stimulated. CpG oligodeoxynucleotides are already in clinical use as adjuvants in vaccines and in cancer therapy approaches. Linking A20-specific siRNA to CpG enables A20 regulation and cell stimulation selectively in toll-like receptor 9 expressing cells, like dendritic cells. Aim of this study was to investigate if these constructs trigger immune cell activation and if they are able to break immune-suppression in the tumor environment to enhance anti-tumor immunity. A long-term growth factor dependent bone marrow-derived dendritic cell culture has been established in order to analyze the CpG-siRNA A20 effects on murine dendritic cells. The constructs were internalized shortly after administration (1 hour) and led to cell stimulation/activation. The intraperitoneal treatment with the constructs induced local cellular activation and systemic IL-6, TNF-α cytokine production in healthy mice. Subcutaneous growing B16 melanoma tumors were treated peritumorally to analyse whether the observed immune-stimulation has effects on established tumors. The silencing of A20 enhances CpG-induced activation of NF-κB followed by elevated expression of IL-6, TNF-α and IL-12 in this tumor model. These changes led to enhanced anti-tumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. New constructs were designed and tested on dendritic cells isolated from healthy donors in order to test whether the obtained results for the murine system are applicable to the human system. CpG-siRNA A20 constructs induced cell activation and cytokine expression (IL-6, TNF-α) significantly more than CpG alone. Even though responds of the donor DCs were variable, there are promising similarities to the results of the mouse experiments. The significant role of A20 in controlling the immune-stimulatory activity of DCs has been confirmed in this study. The novel CpG-siRNA A20 constructs provide a strategy for simultaneous A20 silencing and CpG-mediated cell stimulation directly in vivo. This therapeutic approach induces potent adaptive and innate immune responses against established tumors in mouse melanoma model leading to prolongation of survival. CpG-targeted A20 blockade is a new immune-stimulatory approach, which could be suitable for supplementation or optimization of clinical tumor treatments.
Introduction
The concept of thermal ablation has proven a minimally invasive alternative or accompaniment to conventional tumour therapy. Patients with hepatic primary tumours or metastases are able to profit from it. Several modalities of thermal ablation exist, including radiofrequency ablation, microwave ablation and laser ablation. They differ in regards to their indications and their physical backgrounds, yet they all share the same aim: the hyperthermic ablation of tumorous target tissue.
At this point in time the maximum ablation diameter attained in a singular session using a singular applicator is about 30 mm. The maximum attainable volume is about 23 cm3. However, the mean and median of hepatic lesions exceed that number with about 50 mm. Most hepatic tumours therefore cannot be easily ablated in toto.
One of the main limitations of thermal ablation is the periprocedural transformation of vital tissue into a boundary layer. This boundary layer prevents efficient energy transmission into peripheral tissue and thus limits the potential of thermal ablation. The boundary layer is usually located centrally around the ablation applicator. In laser ablation the formation of this boundary layer is called carbonisation.
A technically simple, yet potentially effective approach to delay or prevent the formation of this boundary layer is the usage of a spacer. This perfused spacer cools the central zone surrounding the applicator. Therefore central temperatures remain beyond the point of carbonisation.
Methods
The development of two spacer prototypes took place in cooperation with the AG “Experimentelle Radiologie” of the University Clinic Charité. The first fully closed prototype featured an internal circulation of cooling fluid without tissue perfusion. The second open prototype perfused into tissue through an opened tip.
The conduct of this study included ex vivo experiments on bovine livers (n = 15) by means of laser ablation. Ablation diameter and ablation volume were recorded through MR-guided volumetry and manual displacement volumetry. The mean values of diameter and volume that were recorded when the stand-alone applicator system was used were then compared to the mean values recorded when using the closed spacer-supported applicator system and the open spacer-supported applicator system. The difference in values between the three applicator types were then examined for statistical significance using SPSS.
To exclude covariates a preliminary experiment was conducted which aimed to maximise power input of the laser and time interval while minimising the chance of carbonisation. For that, one of the variables was increased in intervals and the ablation diameter of all three applicator types was measured until carbonisation occurred.
Results
In the preliminary experiment it was found that following the increase of the pre-set power input of the laser a proportional increase of ablation diameter followed. However when increasing power input above 25 Watt almost instantaneous carbonisation of the central tissue occurred. This was the same for all three applicator types.
When increasing the time interval > 10 minutes the stand-alone applicator system showed central carbonisation, which was not the case when using the closed spacer-supported applicator system or the open spacer-supported applicator system. The two spacer prototypes only experienced carbonisation when a time interval of > 25 minutes was set. Thus the comparison of all three applicator types was conducted at 25 Watt and 10 minutes, whereas the comparison between the closed spacer-supported applicator system and the open spacer-supported applicator system was conducted at 25 Watt and 25 minutes.
During the first experiment the stand-alone applicator system achieved mean values of 37.50 mm ablation diameter and 23.61 cm3 ablation volume. This was a statistically significant (p < 0.001) increase to the values either spacer was able to attain: the closed spacer-supported applicator system recorded a mean value of 28.67 mm ablation diameter and 18.12 cm3 ablation volume, whereas the open spacer-supported applicator system recorded a mean value of 31.00 mm ablation diameter and 18.49 cm3 ablation volume. However, setting a longer time interval was not possible when the stand-alone applicator system was used for ablation. Due to this, a second experiment comparing mean ablation diameter and volume between the two spacer prototypes followed.
During the second experiment with a time interval of 25 minutes the closed spacer-supported applicator system attained a mean value of 52.07 mm ablation diameter and 75.25 cm3 ablation volume. These values showed a statistically significant (p < 0.001) difference in comparison to the open spacer-supported applicator system with mean values of 47.60 mm ablation diameter und 72.20 cm3 ablation volume.
Discussion
Within the framework of this study it was proven that the presence of a spacer between laser applicator and hepatic tissue was able to achieve a significant increase in ablation diameter and ablation volume. By using a closed spacer an increase in volume by a 3.19 factor of change was possible. The open spacer obtained an increase in volume by a 3.06 factor of change. The concept of using a spacer in thermal ablation as a proof of concept study is therefore valid and suitable for further pre-clinical studies.