The structural effect of high intensity ultrasound on peritoneal tissue: a potential vehicle for targeting peritoneal metastases

High intensity ultrasound (HIUS) has been increasingly investigated as a possible tool in the treatment of many different tumor entities, e.g. cancers of prostate, kidney, liver, pancreaticobiliary and other intrabdominal malignancies [1‐3]. While HIUS is still under clinical evaluation, previous studies indicate its potential to improve overall antitumoral activity regardless of chemotherapeutic applications [4]. Despite encouraging first clinical results [5], no studies have been conducted yet to assess HIUS’ possible application in the treatment of peritoneal metastases (PM). In all previous clinical applications, the HIUS beam was “focused” on a single spot in the body (High intensity focused ultrasound, HIFU). However, while PM usually covers a large surface, its depth is only minimal. This might be one reason as to why HIFU has not been considered for peritoneal applications. This study aims to modify the conventional HIFU to a “non-focused” HIUS approach and assess its potential in PM treatment. While HIUS is assumed to impact the peritoneum when used in the treatment of liver cancer, the validity of this assumption remains unclear [6]. Considering that any interaction with the peritoneum may be used in a therapeutic capacity, and inaccessibility remains one of the main difficulties in PM treatment, it seems astonishing that HIUS has never been investigated as a potential tool in PM treatment. PM is a common manifestation of advanced gastrointestinal and gynecological cancers, and affected patients usually have a very poor prognosis with median survival rates of only a few months [7]. Recent studies indicate that the combination of HIUS with intraperitoneal chemotherapy (IPC) could significantly increase drug penetration depths and therefore enhance the overall antitumoral effect, especially when applied with liposomal doxorubicin [8, 9]. While this effect has mostly been attributed to the rupture of liposomal doxorubicin [10], the results partially exceeded penetration levels observed in conventional chemotherapeutic solutions [8]. At the same time, no structural damage to the peritoneum was detected. Still, some authors have suggested that HIUS might affect the peritoneal surface when accidently applied during hepatocellular carcinoma treatment (HCC) [6]. To our knowledge, neither the application of HIUS and its potential, nor its possible side effects on the peritoneum have ever been systematically studied in the context of PM. In general surgery, HIUS is an established procedure predominantly used in the treatment of HCC [11, 12]. In a previous study, HIUS was assumed to cause local heat on the peritoneum, which could possibly induce peritoneal tissue destruction [6]. However, recent clinical evaluations indicate that HIUS might be safe for intraperitoneal use [13]. Knowing the antitumoral properties demonstrated by HIUS in HCC, it seems reasonable to assume similar effects in PM. Thus, with respect to its low invasiveness and absence of radiation, HIUS may potentially play an important role in future PM treatment. To evaluate the structural effects of HIUS on the peritoneum, we studied a well-established ex-vivo model in which we investigated peritoneal samples following HIUS application using light, fluorescence and electron microscopy.

No approval of the local board on animal welfare was required as the experiments were performed using commercially available tissue samples. A local animal supplier (Zerniki Wielkie, 55–020 Wroclaw, Poland) provided the fresh post-mortem swine peritoneum. This peritoneum was cut into proportional samples and placed into petri dishes. Then, the samples were covered with NaCl 0.9% until a layer of 6 mm covering the samples was attained (Fig. 1). HIUS (Sonopuls HD 2070, Bandelin, Berlin, Germany) at 70 W and 20 kHz was applied on the peritoneal tissue using a metal pen. The applied HIUS beam was not focused with high intensity, but rather spread from the tip of the metal pin to the periphery with continuously decreasing intensity. The tip of the pen was as close as 3 mm to the tissue. Each sample group included 3 peritoneal tissue samples and received either 30, 60, 120 or 300 s of HIUS treatment. The control group did not receive any HIUS exposure and was only placed in a petri dish for 300 s and covered by NaCl 0.9%. One sample of each group was subject to further analyses by means of light, fluorescence, or electron microscopy. Experiments were independently performed three times.

While in the past few years, many improvements have been observed in chemotherapeutic regimens and new drug compositions, a significant amount of PM patients fail to respond to systemic and local treatments. This circumstance is mostly attributed to molecular mechanisms and limited drug distribution into the tumor [14]. Similar limitations have been observed in IPC during PM treatments [15, 16]. However, higher local drug disposition and increased tissue drug penetration is reported to enhance the anti-tumoral effect [17‐19]. Attempts to improve tissue penetration rates by treating the peritoneal surface prior to chemotherapeutic application were mostly unsuccessful. For example, concepts like using an energy beam via radiation to prepare the peritoneal tissue for IPC have unfortunately not shown any improved penetration effects [20‐22]. However, our data suggests that HIUS might be an easy, feasible additional feature in the treatment of PM. While our data is limited, and the study is preliminary in nature, our findings present the potential effects of HIUS on the peritoneum. In the future, these effects can be used in various applications. By creating very small tissue disruptions within the peritoneal surface, the transport of various particles through this main barrier is facilitated, resulting in significantly improved penetration rates of chemotherapeutic drugs. Some previous studies suggest these possible HIUS effects in combination with chemotherapy [8, 23]. However, these studies were primarily investigating drug tissue penetrations without emphasizing structural tissue changes, thus giving little explanation for this effect. A very recent study has, for the first time, analyzed drug penetration on the peritoneum following HIUS application, and the findings of this study indicate that penetration rates can be enhanced by more than threefold depending on the duration of the HIUS beam [24]. Since the effect of HIUS seems to show limitations in depth, it could be used for PM treatment during cytoreductive surgery to possibly disrupt the vascular network of single nodules. This concept is quite interesting since tumor nodules in PM are assumed to have a reduced blood supply compared to regular peritoneal tissue [25]. Other HIUS aspects. e.g. its role in the enhanced apoptosis of cancer cells has been recently discovered and requires further analysis [26]. Thus, HIUS potential for PM must be further investigated and warrants more studies to thoroughly investigate its potential. However, this present study offers important first insight of potential HIUS application to treat PM.

Our data indicate that HIUS creates disruptions in the peritoneal surface and its underlying tissues. In the subperitoneal tissue, HIUS application results in microbubble formation. Beside its direct effects on the peritoneum, these structural surface changes might also result in increased drug permeability.

To adequately assess HIUS’ efficacy as well as its therapeutic possibilities on the peritoneum, further studies are required.