Modifying RF ablation-induced upregulation of Interleukin-6 using nanocarrier-based anti-IL6 siRNA
Project Investigator: Dr. Muneeb Ahmed
Nanotechnology innovation: Traditionally, anti-tumoral studies with siRNA have focused on enhancing the effects of other pharmaceutical agents. Here, we propose to extend the siRNA paradigm beyond these conventional approaches by combining liposomally-delivered siRNA with an entirely different modality, a thermal energy source that can be rapidly clinically implemented such as percutaneous tumor ablation, to modulate treatment induced tissue changes and improve treatment efficacy. The successful completion of these studies will set the stage for expansion of traditional siRNA applications into a new direction to include adjuvant use with non-pharmacologic treatments (such as ablation, surgery, or radiation).
Specific Aims: Radiofrequency (RF) tumor ablation is an image-guided minimally-invasive technique in widespread clinical use that generates high-temperature focal tissue heating around percutaneously placed needle-like electrodes to kill focal tumors in the liver, lung, kidney, bone and other organs (1). As RF ablation has demonstrated greatest reproducible efficacy for treating small tumors, we have performed extensive animal and early clinical studies combining it with nanoparticle platforms such as liposome-encapsulated doxorubicin, paclitaxel, and quercetin, to create large and more complete tumor ablation zones. These combination paradigms have taken advantage of our previous work demonstrating that RF ablation, when combined with nanocarrier delivery platforms, can provide very specific and targeted delivery of the designated biopharmaceutical to the affected area.
From these studies, we have shown that RF ablation-induced tissue effects occur in successive zones around the ablation zone, and include increases in free radical formation, apoptosis, and heat shock protein expression (2). Most recently, our preliminary studies have also identified RF-induced increases in cytokines, such as interleukin-6, that leads to the recruitment of infiltrating cells into the red “inflammatory” zone in tissue/tumor at the margins of the zone in which we have seen therapeutic benefit combining RF ablation with the above mentioned nanoparticles. We have further demonstrated that these cytokines have more global effects including increasing cellular proliferation in distant liver tissue (such as in the untreated liver lobe) and increased tumor growth in distant sites such as the mammary fat pad [See Preliminary Data]. These effects are likely to be deleterious to overall patient survival in many cases. Accordingly, identifying successful strategies to modulate these secondary effects of RF ablation could potentially lead to significant improvements in treatment efficacy.
We have further performed comparative studies using IL-6 knock-out mice models to demonstrate that increased cell recruitment and liver growth can be largely reversed. Thus, SiRNA, with its ability to interfere with protein production of specific molecular targets, has the unique potential to target and modulate specific cellular pathways to improve RF ablation efficacy. Indeed, our preliminary studies demonstrate a single properly timed adjuvant dose of liposomal siRNA can successfully suppress RF-induced increases in IL-6 and associated secondary effects of cellular infiltration. Having demonstrated that nanocarrier based anti-IL6 siRNA can successfully be combined with RF ablation to reduce periablational cellular infiltration; we now propose to further develop this concept by completing the following Specific Aims:
- To determine whether combination liposomal anti-IL6 siRNA and RF ablation can be used to modulate global tissue effects such as hepatic parenchymal growth (as measured by proliferative markers such as CDC47 and Ki-67) and,
- To determine whether adjuvant liposomal anti-IL6 siRNA be used to suppress RF-induced stimulation of tumor growth in remote tumors.
One of the Preliminary Data:
In this preliminary proof- of-concept study, RF ablation of normal liver in C57Bl mice, was combined with intraperitoneal liposomal anti-IL6 siRNA. Treatment arms included 1) RFA (21g electrode, 1 cm active tip, 70°C x 5 min) of normal liver followed 15 min later by a single dose IP dose of liposomal anti-IL6 siRNA (150 nm carrier size, 3.5 mcg per dose), 2) RFA followed 15 min later by a single IP dose of liposomal scrambled siRNA, 3) RFA followed 15 min later by a single IP dose of empty liposomes, and 4) RFA alone (control) [n=5 animals/group]. Animals were sacrificed at 4 and 7d post- treatment, and tissue was harvested from both the treated and remote (non-treated) hepatic lobes. Outcome measures included immunohistochemical staining for aSMA cells and macrophages (4 and 7d) and cell mitosis (7d). Preliminary results of these studies demonstrated that liposomal anti-IL6 siRNA reduces the number of aSMA positive cells in the periablational rim after RF ablation on immunohistochemistry (compared to RFA alone and scrambled siRNA). This was confirmed on subsequent quantification of aSMA marker cell positivity and border zone thickness, as combined RFA+antiIL6 siRNA significantly reduced the number of aSMA cells observed compared to control groups (p<0.03). This exciting proof-of-concept data builds specifically on our experience using nanocarrier platforms to deliver pharmaceutical agents directly to tissues surrounding the RF ablation zone. Thus, here we demonstrate the feasibility of our approach by specifically showing that this synergy can be further extrapolated using siRNA to specifically target RF-induced tissue cellular responses (and associated cell infiltration).