A targeted combinatorial therapy for Ewing's sarcoma

Abstract

Ewing's sarcoma (EwS) is the second most common bone cancer in children and adolescents. Current chemotherapy regimens are mainly ineffective in patients with relapsed disease and cause long-term effects in survivors. Therefore, we have developed a combinatorial therapy based on a novel drug candidate named ML111 that exhibits selective activity against EwS cells and synergizes with vincristine. To increase the aqueous solubility of hydrophobic ML111, polymeric nanoparticles (ML111-NP) were developed. In vitro data revealed that ML111-NP compromise viability of EwS cells without affecting non-malignant cells. Furthermore, ML111-NP exhibit strong synergistic effects in a combination with vincristine on EwS cells, while this drug pair exhibits antagonistic effects towards normal cells. Finally, animal studies validated that ML111-NP efficiently accumulate in orthotopic EwS xenografts after intravenous injection and provide superior therapeutic outcomes in a combination with vincristine without evident toxicity. These results support the potential of the ML111-based combinatorial therapy for EwS.

Keywords: Chemotherapy; Combinatorial therapy; Ewing's sarcoma; ML111; Nanoparticle.

Figure 1.

The anticancer activity of ML111 and VDC/IE chemotherapeutic agents against EwS cells. (A) Chemical structure of ML111, (B) Dose-response curves of SK-N-MC cells treated with various concentrations of ML111 and VDC/IE chemotherapeutic drugs for 48 h, (C) IC₅₀ values of the tested agents in SK-N-MC cells.

Figure 2.

PEG-PCL nanoparticles improve the cellular uptake and anticancer activity of hydrophobic ML111 in vitro. (A) Representative size distribution profiles of ML111-NP measured by dynamic light scattering before and after storing for 21 days at 4 °C. (B) Representative TEM image of ML111-NP. (C) Flow cytometry analysis of cellular uptake of Nile Red-labeled ML111-NP by SK-N-MC cells after 24 h (red curve). The black curve represents non-treated cells (control). (D) Viability of SK-N-MC cells treated with various concentrations of ML111 in DMSO and ML111-NP for 48 h.

Figure 3.

ML111-NP selectively compromised viability of EwS cells and exhibited antagonistic effects in a combination with vincristine on non-malignant cells. (A) Dose-response curves of non-malignant (HEK293 and NIH3T3) and EwS (SK-N-MC and TC-71) cell lines treated for 48 h with ML111-NP. (B) Dose-response curves and (C) CI vs. fraction affected plots of HEK 293 cells treated for 48 h with various concentrations of vincristine (VIC) alone and in combination with ML111-NP at 1:1 and 1:5 molar ratio.

Figure 4.

Combinatorial treatment induces cell cycle arrest at the G₂/M phase in EwS cells. (A) Flow cytometry analysis of cell cycle alterations in unsynchronized SK-N-MC cells treated for as indicated. The final concentrations of vincristine and ML111 (in context of ML111-NP) were 2 nM and 100 nM, respectively. (B) The percentage of SK-N-MC cells in the G₂/M phase following the indicated treatments. The symbols represent *p < 0.05, **p < 0.01, and ^nsp > 0.05.

Figure 5.

Combinatorial treatment induces cell cycle arrest at the G₂/M phase in EwS cells. (A) Flow cytometry analysis of cell cycle alterations in unsynchronized SK-N-MC cells treated for as indicated. The final concentrations of vincristine and ML111 (in context of ML111-NP) were 2 nM and 100 nM, respectively. (B) The percentage of SK-N-MC cells in the G₂/M phase following the indicated treatments. Data are represented as mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, and ^nsp > 0.05).

Figure 6.

Biodistribution of ML111-NP in an orthotopic xenograft model of EwS. (A) Representative X-ray image of a mouse (ventral) recorded with an IVIS Lumina XRMS live animal imaging system 4 days after intramuscular injection of SK-N-MC cells into the right hind limb. (B and C) Representative NIR fluorescence images of a mouse body (without skin) (B) and resected tissues (C) 24 h after intravenous injection of ML111-NP loaded with a NIR dye, silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (SiNc).

Figure 7.

ML111-NP, in combination with vincristine (0.1 mg/kg), significantly inhibit tumor growth and improve survival of mice with orthotopic SK-N-MC xenografts. (A) The mean change in a calf volume of right hindlimbs with orthotopic SK-N-MC xenografts over time in mice treated with 5% dextrose (control), ML111-NP (15 mg/kg), vincristine (VIC, 0.1 mg/kg), and combination of both vincristine (0.1 mg/kg) and ML111-NP (15 mg/kg) according to schedules provided in Figure S4. (B) Average weight of resected tumors in each treatment group at day 27 after the beginning of studies. (C) Kaplan–Meier survival curves of mice after the above-indicated treatments. The symbols represent *p < 0.05, **p < 0.01, ***p < 0.001 and ^nsp > 0.05. (D – E) Representative 2D ultrasound images of SK-N-MC xenografts in the hindlimb of mice treated as indicated. Mice were imaged prior to euthanasia on day 27 after the beginning of studies. Tumors are indicated with circles.

Figure 8.

Combinatorial therapy induces a significantly higher level of apoptosis in EwS tumors than individual treatments. (A-D) Representative TUNEL-stained images of orthotopic xenografts after treatment with (A) 5% Dextrose (control), (B) ML111-NP (15 mg/kg), (C) vincristine (0.1 mg/kg) and (D) combination of both vincristine (0.1 mg/kg) and ML111-NP (15 mg/kg). Apoptotic cells are represented by a dark brown stain. Scale bars, 50 μm. (E) The graph represents an average percentage of TUNEL-positive cells per field (five fields were analyzed)) in each treatment group. Analysis performed at 40× magnification. The symbols represent *p < 0.05, **p < 0.01, ***p < 0.001 and ^nsp > 0.05.