Systemic siRNA Nanoparticle-Based Drugs Combined with Radiofrequency Ablation for Cancer Therapy

Abstract

Purpose: Radiofrequency thermal ablation (RFA) of hepatic and renal tumors can be accompanied by non-desired tumorigenesis in residual, untreated tumor. Here, we studied the use of micelle-encapsulated siRNA to suppress IL-6-mediated local and systemic secondary effects of RFA.

Methods: We compared standardized hepatic or renal RFA (laparotomy, 1 cm active tip at 70 ± 2 °C for 5 min) and sham procedures without and with administration of 150 nm micelle-like nanoparticle (MNP) anti-IL6 siRNA (DOPE-PEI conjugates, single IP dose 15 min post-RFA, C57Bl mouse:3.5 ug/100ml, Fisher 344 rat: 20 ug/200 ul), RFA/scrambled siRNA, and RFA/empty MNPs. Outcome measures included: local periablational cellular infiltration (α-SMA+ stellate cells), regional hepatocyte proliferation, serum/tissue IL-6 and VEGF levels at 6-72 hr, and distant tumor growth, tumor proliferation (Ki-67) and microvascular density (MVD, CD34) in subcutaneous R3230 and MATBIII breast adenocarcinoma models at 7 days.

Results: For liver RFA, adjuvant MNP anti-IL6 siRNA reduced RFA-induced increases in tissue IL-6 levels, α-SMA+ stellate cell infiltration, and regional hepatocyte proliferation to baseline (p < 0.04, all comparisons). Moreover, adjuvant MNP anti-IL6- siRNA suppressed increased distant tumor growth and Ki-67 observed in R3230 and MATBIII tumors post hepatic RFA (p<0.01). Anti-IL6 siRNA also reduced RFA-induced elevation in VEGF and tumor MVD (p < 0.01). Likewise, renal RFA-induced increases in serum IL-6 levels and distant R3230 tumor growth was suppressed with anti-IL6 siRNA (p < 0.01).

Conclusions: Adjuvant nanoparticle-encapsulated siRNA against IL-6 can be used to modulate local and regional effects of hepatic RFA to block potential unwanted pro-oncogenic effects of hepatic or renal RFA on distant tumor.

Fig 1. Hepatic thermal ablation increases liver IL-6 levels at 12hr post-treatment and periablational α-SMA+ activated myofibroblasts infiltration—which are suppressed with single dose adjuvant nanoparticle anti-IL6 siRNA.

(A) Liver ELISA quantification of IL-6 levels 12hr after treatment (mean ± standard deviation). C57Bl mice were randomized to receive sham treatment, liver RF ablation, liver RFA / IP MNP anti-IL6 siRNA, MNP anti-IL6 siRNA alone, RFA / MNP scrambled siRNA, or RFA / empty carrier (n = 5–6 per group). Liver RFA increased local 12hr liver IL-6 levels (1,463±108 pg/ml) which was suppressed with adjuvant IP MNP anti-IL6 siRNA (1,139±159 pg/ml, p = 0.04). Additionally, adjuvant MNP anti-IL6 siRNA given 15 minutes after hepatic thermal ablation (D, E) in C57Bl mice suppressed periablational infiltration of α-SMA positive myofibroblasts compared to hepatic thermal ablation alone (B) or RFA combined with scrambled siRNA (C) (F, mean ± standard deviation, p<0.01).

Fig 2. Adjuvant nanoparticle anti-IL6 siRNA suppresses thermal ablation-induced hepatocyte proliferation in the untreated, distant hepatic lobe.

Adjuvant MNP anti-IL6 siRNA given 15 minutes after hepatic thermal ablation (C) in C57Bl mice (n = 5–6 animals/arm) suppressed hepatocyte proliferation in the distant, untreated liver lobe (with CDC47 staining, mean ± standard deviation) compared to hepatic thermal ablation alone (A, D, p<0.01). Hepatic thermal ablation combined with MNP scrambled siRNA was not significantly different from either thermal ablation alone or ablation combined with MNP anti-IL6 siRNA (B, D).

Fig 3. Hepatic thermal ablation-induced distant subcutaneous R3230 tumor growth is suppressed with adjuvant nanoparticle anti-IL6 siRNA.

(A) Subcutaneous R3230 tumors implanted in Fisher 344 rats with similar growth rates were randomized at Day 0 to one of six different treatment arms (n = 6–7 animals/arm). Hepatic thermal ablation alone or combined with either empty carrier or MNP scrambled siRNA resulted in significantly greater tumor growth and change in diameter (5d before to 7d after treatment) compared to sham treatment (p<0.01 for all comparisons, mean ± standard deviation for all numbers presented). Adjuvant MNP anti-IL6 siRNA combined with thermal ablation resulted in distant tumor growth rate and end diameter that was the lowest of all treatment arms (p<0.01 for all comparisons). (B) Adjuvant MNP anti-IL6 siRNA combined with hepatic thermal ablation also reduced distant tumor proliferation (Ki-67) to sham levels compared to hepatic thermal ablation alone or combined with empty carrier or MNP scrambled siRNA (p<0.01 for relevant comparisons). (C) Hepatic thermal ablation alone or with MNP scrambled siRNA increased serum IL-6 levels at 6hr compared to the sham procedure (n = 3–4 animals/arm, p<0.02). This effect was suppressed with adjuvant MNP anti-IL6 siRNA (p = 0.03 vs. RF liver alone). (D) Comparison of the effect of siRNA administration timing showed that adjuvant MNP anti-IL6 siRNA administered at Day 0 resulted in the lowest post-treatment tumor growth rate and endpoint diameter (n = 3–4 animals/arm, p<0.05 for all comparisons). Adjuvant MNP anti-IL6 siRNA administered 3d post-ablation reduced the endpoint diameter compared to hepatic ablation alone, but was still significantly greater that either sham or combined-Day 0 treatment (p<0.05 for all comparisons).

Fig 4. Adjuvant nanoparticle anti-IL6 siRNA suppresses hepatic ablation-induced distant subcutaneous R3230 tumor growth via reduction in VEGF-mediated angiogenesis.

(A) Tissue levels of VEGF (Y-axis², dark gray columns) in the periablational tissue surrounding the ablation zone were quantified at 72hr after different treatments (mean ± standard deviation, n = 3–4 animals/arm). Hepatic thermal ablation increased periablational VEGF levels, which were then suppressed with adjuvant MNP anti-IL6 siRNA (p<0.05 for all comparisons). (B-E) Similarly, hepatic thermal ablation led to increased microvascular density/angiogenesis (immunohistochemistry for CD34, A: Y-axis¹, light gray columns) in distant subcutaneous R3230 tumor that was also suppressed with adjuvant anti-IL6 siRNA (n = 6–7 animals/arm).

Fig 5. Thermal ablation of a second primary organ (kidney) leads to increased distant subcutaneous R3230 tumor growth that is suppressed with adjuvant nanoparticle anti-IL6 siRNA.

(A) Subcutaneous R3230 tumors implanted in Fisher 344 rats with similar growth rates were randomized at Day 0 to one of four different treatment arms (n = 6–7 animals/arm). Thermal ablation of normal kidney alone resulted in significantly greater tumor growth and change in diameter (5d before to 7d after treatment) compared to sham treatment or MNP anti-IL6 siRNA alone (p<0.01 for all comparisons, mean ± standard deviation for all data). MNP anti-IL6 siRNA combined with thermal ablation reduced distant tumor growth rate and endpoint diameter to baseline sham levels. (B) Adjuvant MNP anti-IL6 siRNA combined with kidney thermal ablation also reduced distant tumor proliferation (Ki-67) and microvascular density (CD34) to baseline levels compared to kidney thermal ablation alone (p<0.01 for relevant comparisons). (C) Adjuvant MNP anti-IL6 siRNA suppressed kidney thermal ablation-induced elevations in serum IL-6 levels at 6hr post-treatment (n = 3–4 animals/arm, p = 0.006).

Fig 6. Hepatic thermal ablation-induced distant tumor growth is suppressed with adjuvant nanoparticle anti-IL6 siRNA in a second breast adenocarcinoma tumor model (MATBIII).

Subcutaneous MATBIII tumors implanted in Fisher 344 rats with similar growth rates were randomized at Day 0 to one of three different treatment arms (sham treatment and hepatic RF ablation without and with adjuvant IP MNP anti-IL6 siRNA; n = 6–7 animals/arm). Given the rapid growth rate of this line, tumors were measured twice daily 2.5d pre- to 3.5d post-treatment (mean ± standard deviation). Hepatic thermal ablation resulted in a significantly larger endpoint tumor diameter compared to the sham group (p<0.05). Adjuvant MNP anti-IL6 siRNA suppressed the effects of hepatic thermal ablation, such that endpoint tumor diameter was equivalent to the sham group.