Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model

Abstract

1. Introduction

Osteosarcoma (OS) is the most commonly occurring primary bone tumor and primarily occurs in children and adolescents [1,2,3,4,5]. The worldwide incidence in children and adolescents is 3–4.5 cases per 1 million people and accounts for 55% of primary bone tumor diagnoses in children in the United States [3,6,7]. Over 30 years ago, chemotherapeutics were introduced into the standard of care treatment regimen for OS patients, which greatly improved patient survival. However, survival outcomes remain at a median 5-year survival rate of 70% for patients presenting without metastatic disease at the time of diagnosis, and less than 25% for patients with metastatic or recurrent disease [1,2,4,8]. The current definitive standard of care treatment for OS consists of surgical resection of the primary bone tumor via limb salvage surgery or amputation with neoadjuvant and adjuvant chemotherapy [1,9]. Advancements in limb salvage surgeries have resulted in improving quality of life outcomes for OS patients, but these procedures are still associated with complications and risks, including mobility limitations, fracture, infection, and tumor recurrence [1,9,10]. Additionally, limb salvage surgery is an invasive procedure, and often multiple surgeries are required [11,12,13]. Consequently, there is a continued need to advance OS treatment modalities in order to improve patient quality of life and survival. A noninvasive limb salvage treatment option has the potential to immensely improve patient quality of life.

In a similar manner to many cancers, the cellular interactions that occur in the tumor microenvironment (TME) in OS result in immunosuppression, contributing to OS progression and metastatic disease development, which is the major contributing factor to OS mortality [14,15,16,17]. Thus, more recent approaches for developing novel OS treatments have focused on immunomodulation in efforts to stimulate an antitumor immune response to moderate metastatic disease development [14,18,19,20,21]. The novel focused ultrasound technique, histotripsy, has been shown to ablate targeted tumor tissue and induce immunomodulation to stimulate an antitumor immune response [22,23,24,25,26,27,28,29,30,31]. Histotripsy is thus an exciting technique that can potentially treat primary and metastatic OS to improve treatment and survival outcomes.

Histotripsy is a precise, nonthermal, noninvasive, focused ultrasound technique that utilizes high-amplitude ultrasound pulses to generate acoustic cavitation to mechanically homogenize targeted tissue [31,32,33,34,35,36]. Histotripsy has previously been employed in humans to ablate prostate tissue, heart valves, and liver tumors [36,37,38,39] and in canines for experimentally induced prostate tumors [40], spontaneously occurring primary bone tumors [25,41,42,43], and soft tissue sarcomas [43,44]. Preclinical rodent models have demonstrated effective ablation of liver tumors [45], melanoma [27], renal cell carcinoma [28], and pancreatic [24] tumors and have also reported that the resulting disruption of targeted tissue causes the release of immunogenic cellular proteins, which stimulates a proinflammatory/antitumor immune response [26,27,28,29,45,46]. The destruction of tumors with histotripsy leads to the release of tumor antigens and damage-associated molecular patterns (DAMPs), such as high mobility group box-1 (HMGB-1), orchestrating an antitumor immune response [24,26,27,28]. Furthermore, an increased infiltration of cytotoxic T-lymphocytes (CTLs) into the tumor microenvironment is observed after histotripsy treatment, as is an increase in tumor antigen recognition and tumor cell cytotoxicity [23,24,26,27,28]. In longer-term preclinical studies, the abscopal immune response associated with histotripsy was reported to decrease metastases within 20 days after histotripsy treatment [27,46,47]. A clinical trial investigating histotripsy hepatic tumor ablation observed a decrease in the growth of non-histotripsy-treated tumors in study patients [47]. Histotripsy has the potential to serve as both a primary tumor ablation modality and to stimulate an antitumor immune response. However, in the context of OS, much remains to be investigated regarding the potential of employing histotripsy ablation and the associated immunomodulatory effects.

The current study was conducted to investigate the ablative and preliminary immunological outcomes associated with histotripsy ablation of OS in the syngeneic C3H/HeN mouse and DLM8 cell line models. Ablation outcomes were evaluated histologically immediately after histotripsy and 72 h after treatment. We analyzed the expression of immune activation genes Il6, Ifnγ, Il-1β, Il10, Il13, and Pdl1 and characterized immune cell populations within the TME 72 h after treatment. We hypothesized that histotripsy ablation would result in disruption of the targeted OS tumors, yield acellular debris, and stimulate a proinflammatory immune response. Our results indicate successful targeted ablation of OS tumors and suggest that histotripsy modulates the TME, thus supporting the potential of histotripsy as a treatment modality for OS.