Investigation of HIFU-induced anti-tumor immunity in a murine tumor model

Zhenlin Hu 1 Xiao Yi Yang 0 Yunbo Liu 1 Georgy N Sankin 1 Eric C Pua 1 Michael A Morse 0 H Kim Lyerly 0 Timothy M Clay 0 Pei Zhong 1 0 Program in Molecular Therapeutics, Departments of Surgery, Medicine, Pathology, and Immunology, and Duke Comprehensive Cancer Center, Duke University , Durham, NC 27708 , USA 1 Department of Mechanical Engineering and Materials Science, Duke University , Durham, NC 27708 , USA Background: High intensity focused ultrasound (HIFU) is an emerging non-invasive treatment modality for localized treatment of cancers. While current clinical strategies employ HIFU exclusively for thermal ablation of the target sites, biological responses associated with both thermal and mechanical damage from focused ultrasound have not been thoroughly investigated. In particular, endogenous danger signals from HIFU-damaged tumor cells may trigger the activation of dendritic cells. This response may play a critical role in a HIFU-elicited anti-tumor immune response which can be harnessed for more effective treatment. Methods: Mice bearing MC-38 colon adenocarcinoma tumors were treated with thermal and mechanical HIFU exposure settings in order to independently observe HIFU-induced effects on the host's immunological response. In vivo dendritic cell activity was assessed along with the host's response to challenge tumor growth. Results: Thermal and mechanical HIFU were found to increase CD11c+ cells 3.1-fold and 4-fold, respectively, as compared to 1.5-fold observed for DC injection alone. In addition, thermal and mechanical HIFU increased CFSE+ DC accumulation in draining lymph nodes 5-fold and 10-fold, respectively. Moreover, focused ultrasound treatments not only caused a reduction in the growth of primary tumors, with tumor volume decreasing by 85% for thermal HIFU and 43% for mechanical HIFU, but they also provided protection against subcutaneous tumor re-challenge. Further immunological assays confirmed an enhanced CTL activity and increased tumor-specific IFN--secreting cells in the mice treated by focused ultrasound, with cytotoxicity induced by mechanical HIFU reaching as high as 27% at a 10:1 effector:target ratio. Conclusion: These studies present initial encouraging results confirming that focused ultrasound treatment can elicit a systemic anti-tumor immune response, and they suggest that this immunity is closely related to dendritic cell activation. Because DC activation was more pronounced when tumor cells were mechanically lysed by focused ultrasound treatment, mechanical HIFU in particular may be employed as a potential strategy in combination with subsequent thermal ablations for increasing the efficacy of HIFU cancer treatment by enhancing the host's anti-tumor immunity. - Background High-Intensity Focused Ultrasound (HIFU) has recently emerged as a promising non-invasive treatment modality for localized solid malignancies [1]. The fundamental principle of HIFU is to focus an acoustic beam to a small, well-defined target region. For current clinical treatment, lesion formation occurs primarily through the accumulation of heat and the subsequent coagulative necrosis at the focus, with temperatures exceeding 65C as the target tissue absorbs the focused acoustic energy. With a typical HIFU system, the size of the induced lesion is approximately 10 mm 1 mm. Thus, complete ablation of a tumor site is performed through progressive scanning of the tumor volume with the assistance of image guidance, such as magnetic resonance imaging or B-mode ultrasound. This treatment can be performed external to the body, provided a path devoid of air or other gaseous regions between the HIFU transducer and the target is available. In addition to the thermal mechanism, HIFUinduced tissue damage can also occur through mechanical means. With longer exposures at high pressures, HIFU can induce cavitation, the formation of microbubbles under high tensile pressure, with the resultant secondary shock wave generation and jet formation upon inertial bubble collapse [2,3]. This mechanism can cause mechanical lysis of tumor cells. Because the onset of cavitation in vivo is unpredictable, this method has been generally avoided in early clinical applications [2]; however, recent studies suggest that cavitation can be used to enhance HIFU-induced thermal ablation, as well as in other potential therapeutic applications such as ultrasound-mediated gene transfer and drug delivery [4-6]. Compared to conventional cancer therapy modalities, HIFU has the advantages of being noninvasive and generally well-tolerated by the patient, enabling it to be administered repetitively. Despite this advantage, several limitations in the current form of HIFU cancer therapy still exist. First, incomplete tissue necrosis, especially in large tumors, may lead to local recurrence of the tumor post-treatment. For example, about 20% local recurrence of soft tissue sarcoma has been reported [7]. This phenomenon presumably occurs due to inhomogeneities in tissue properties and heat conduction. Second, HIFU cannot be used to kill metastatic cancer cells outside the primary tumor site. In fact, distant metastasis, especially in the air-rich lung tissue, is a major cause of mortality following clinical HIFU therapy [8]. Clearly, the quality and effectiveness of HIFU cancer therapy need further improvement. Historically, research in HIFU has been focused almost exclusively on enhancing thermal ablation efficiency with more precise control of targeting and monitoring of lesion formation, while largely ignoring the diverse range of biological responses that may be induced by HIFU. One of the most important biological consequences of HIFU treatment is the creation of a large amount of tumor antigens in the form of necrotic cells and the local release of a diverse array of endogenous danger signals from HIFUdamaged tumor cells. This biological response has the potential to stimulate an anti-tumor immune response [9,10]. However, little is known about how such significant HIFU-induced changes in the tumor microenvironment may influence the host's anti-tumor immune response. It is nevertheless interesting to note that several clinical studies have provided preliminary evidence that suggest HIFU treatment could affect the patient's immune status. For example, a marked increase in CD3+ and CD4+ subsets and in the CD4+/CD8+ ratio in peripheral blood of cancer patients has been observed following HIFU treatment [8,11,12]. In addition, several animal studies have revealed encouraging evidence that suggests HIFU treatment may induce systemic anti-tumor immunity. For example, Yang et al. reported that HIFU treatment of subcutaneous murine C1300 neuroblastoma could cause a significant reduction in subsequent challenged tumor growth [13]. Despite these previous efforts, the specific immune response elicited by HIFU has not been determined using well-established immunologic assays, and the underlying mechanism is l (...truncated)