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.

Graphical Abstract

We have developed a targeted combinatorial therapy for Ewing’s sarcoma (EwS) based on a novel drug candidate named ML111. This molecule exhibits a robust synergistic effect in combination with vincristine on EwS cells, while the same drug pair provides antagonistic effects towards nonmalignant cells. To enhance the translational potential of this combinatorial therapy regimen, we prepared ML111-loaded nanoparticles that efficiently accumulate in orthotopic EwS xenografts following systemic administration and induce marked regression of these tumors in combination with vincristine.

Introduction

Ewing’s sarcoma (EwS) is the second most common type of bone cancer in children and young adults.¹ It occurs predominately in long bones and ribs or surrounding soft tissues.^2,3 The current standard of care for EwS consists of systemic chemotherapy combined with surgery or radiation therapy, or both. Chemotherapy plays a critical role in the treatment of EwS and the most commonly used chemotherapeutic regimen, known as VDC/IE, is a combination of vincristine (V), doxorubicin (D), and cyclophosphamide (C), alternating with ifosfamide (I) and etoposide (E) ¹. Despite this aggressive chemotherapeutic strategy, the overall five-year survival rate for patients with recurrent or metastatic EwS is less than 30%. The efficacy of the VDC/IE regimen is limited by the cumulative toxicity of chemotherapeutic agents.^1,4 The development of multidrug resistance in most patients with metastatic disease is another cause of chemotherapy failure for EwS.⁵ Therefore, there is a critical need for novel strategies that can improve efficacy and decrease the side effects associated with conventional chemotherapeutic regimens in EwS patients.

Synergistic drug combinations are an active area of research, as these can be efficient strategies for combating EwS with limited adverse effects.^6,7 The aggressive cytotoxic nature of current treatments are costly to normal childhood development and novel therapeutic approaches with reduced nonspecific toxicity are especially relevant to the treatment of pediatric patients.⁸ Selective agents that synergize with conventional chemotherapeutic drugs and potentially permit dose reduction of cytotoxic agents are a high clinical priority for EwS patients. Several reports confirm that synergistic combinations of selective agents with conventional chemotherapy can potentially decrease effective doses of drugs used in the VDC/IE regimen, while providing enhanced therapeutic efficacy with minimal side effects. For example, Zollner et al. reported that administration of YK-4–279, a novel inhibitor of EWS-FLI1 protein, allowed for significantly lower doses of vincristine while maintaining its equivalent cytotoxicity.⁹ Engert et al. also revealed that some poly(ADP)-ribose polymerase (PARP) inhibitors could increase the cytotoxicity of doxorubicin, etoposide, or ifosfamide in EwS cells.¹⁰ Finally, a randomized phase 3 clinical trial is currently evaluating the ability of a targeted agent, ganitumab (IGF-1R human monoclonal antibody), to improve the therapeutic outcomes of VDC/IE regimen in patients with newly diagnosed metastatic EwS (AEWS1221/NCT02306161). These reports advocate for new therapeutic agents that are non-toxic to healthy cells and demonstrate robust synergistic effects with clinically used chemotherapeutics for EwS.

We report herein a previously uncharacterized compound, ML111, that markedly enhances the anti-EwS efficacy of some chemotherapeutic drugs used in VDC/IE regimen. Specifically, ML111 and vincristine exhibit a robust synergistic effect on EwS cells, while this drug pair exhibits antagonistic effects towards non-malignant cells, suggesting that ML111 may mitigate the toxic side effects of vincristine in patients. To enhance the translational potential of this combinatorial therapy regimen, we prepared ML111-loaded nanoparticles that efficiently accumulate in orthotopic EwS xenografts following systemic administration and induce marked regression of these tumors, alone and in combination with vincristine. Mice given the combination of ML111 and vincristine did not exhibit gross or target organ toxicities.

Methods

Preparation and characterization of ML111-NP:

Methoxy poly(ethylene glycol)-bpoly(ε-caprolactone) (mPEG–PCL, MW: 5k-10k)-based nanoparticles loaded with ML111 (ML111-NP) were prepared via a modified solvent evaporation method (Supplementary materials) and characterized by following previously reported procedures.^11,12

Cell Viability Studies:

The cytotoxicity of ML111 alone and in combination with EwS chemotherapeutic drugs (vincristine, doxorubicin, etoposide, cyclophosphamide, and ifosfamide) was assessed in human Ewing’s sarcoma cells (SK-N-MC) by using a modified Calcein AM assay (Supplementary materials).¹³ The cytotoxicity of ML111-NP and vincristine alone and combinations of both agents at various molar ratios (vincristine: ML111 = 1:1, 1:5, 1:10, and 1:15) was also evaluated in EwS cell lines (SK-N-MC and TC-71) and non-malignant cell lines (HEK293 and NIH3T3). The drug concentrations that inhibit 50% of cell viability (IC₅₀) were determined using non-linear regression analysis (GraphPad Prism 8, GraphPad Software, Inc., San Diego, CA, USA).

The combinatorial effects (antagonistic, additive, or synergistic) of the tested drug pairs in various cell lines were analyzed based on the Chou–Talalay theory using the Compusyn software package (Version 1.0, ComboSyn Inc., Paramus, NJ, USA).^14–16 Combination index (CI) values were determined at various fractions of cells affected (F_a) based on cell viability data. CI values define the synergetic, additive, or antagonistic effects at various F_a as following: CI = 0.12013 0.5, strong synergism; 0.5–0.9, mild synergism; 0.9 – 1.1, additive effect; 1.1 – 2.0, mild antagonism; and > 2.0 strong antagonism.¹⁷

Information about cellular internalization study, cell cycle analysis, cell apoptosis analysis, and real-time cellular proliferation study can be found in Supplementary materials.

In Vivo Studies:

Animal studies were approved by the Institutional Animal Care and Use Committee of Oregon Health and Science University and the procedures followed were in accordance with institutional guidelines. Experiments were carried out on athymic nu/nu mice (4–6 weeks old, Charles River Laboratories, Wilmington, MA, USA).

Development of an intramuscular paratibial mouse model of EwS:

SK-N-MC cells (2.5 × 10⁶) were inoculated into the right gastrocnemius muscle of a mouse near the tibia and in the direction of tibial crest/tuberosity. To confirm that an intramuscular tumor mass grows in close proximity to the tibia, X-ray images of mice were obtained using an IVIS Lumina XRMS (PerkinElmer, Waltham, MA, USA). The dimensions of calves with tumors were measured with a digital caliper every other day, and the calf volume was calculated as (W² × L) × 0.5, where W is the shorter diameter, and L is the longer diameter. Mice were monitored and euthanized if one of the following signs was observed: body weight loss ≥ 20 %, calf volume ≥ 2300 mm³, and mobility difficulty.

Treatment Studies:

Mice were randomly divided into four groups. The treatment studies were initiated on day 4 or 5 after inoculation of cells when the calf volume was ~110 mm³. Mice were injected through the tail vein with the following formulations: (1) 5% dextrose (3 times/week for 3 weeks, 5 mice), (2) ML111-NP (15 mg ML111/kg, 3 times/week for 3 weeks, 10 mice), (3) vincristine (0.1 mg/kg, once a week for 4 weeks, 10 mice), and (4) combination of vincristine (0.1 mg/kg, once a week for 4 weeks) and ML111-NP (15 mg ML111/kg, 3 times/week for 3 weeks, 10 mice). When the calf volume in mice injected with 5% dextrose reached ~2300 mm³ (~27 days after cells inoculation), five mice from each group were euthanized; tumors were collected post euthanasia for analysis (Supplementary materials). The Vevo 2100 ultrasound imaging system (FUJIFILM VisualSonics, Inc., Toronto, Canada) was employed prior to euthanasia for tumor visualization and area assessment. Other non-euthanized mice in each group were monitored for survival until the calf volume reached ~2300 mm³ and median survival times were estimated from the obtained Kaplan–Meier curves. In a separate experiment, tumor growth and survival of mice were also evaluated after the following treatment regimens (1) vincristine (0.5 mg/kg, once a week for 4 weeks, 5 mice), and (2) a combination of vincristine (0.5 mg/kg, once a week for 4 weeks) and ML111-NP (15 mg ML111/kg, 3 times/week for 3 weeks, 5 mice). The body weight and calf volumes were recorded for all mice during the entire study duration. To assess potential side effects caused by the above-indicated treatments, the collected blood samples after each treatment were analyzed at IDEXX laboratory (Veterinary Diagnostic Laboratory, Portland, OR, USA).

Statistical Analysis:

All experiments were performed in triplicate, and data are presented as the mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). Significance for tumor growth data was determined using two-way ANOVA at 5% significance level with Tukey’s Multiple Comparison post-test with groups compared to each other at 5% significance level. Significance for all other data was determined using one-way ANOVA at 5% significance level with Tukey’s Multiple Comparison post-test with groups compared to each other at 5% significance level.

Results

In vitro evaluation of ML111, VDC/IE chemotherapeutic drugs and their combinations

ML111 belongs to the 2-amino-4-phenyl-4H-benzo[h]chromenes chemical family (Figure 1A). In the first step, we compared in vitro anticancer efficacy of ML111 with five chemotherapeutic drugs that are currently used in the clinical VDC/IE regimen for EwS treatment. The obtained dose–response curves (Figure 1B) and calculated IC₅₀ values (Figure 1C) reveal that ML111 is at least eight times more potent against SK-N-MC EwS cells than doxorubicin, etoposide, cyclophosphamide, and ifosfamide alone. In contrast, vincristine was very potent in disrupting SK-N-MC cell viability (Figure 1C).

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.

In the second step, we examined the anticancer activity of each VDC/IE chemotherapeutic drug with ML111 in SK-N-MC cells to determine if any drug pair exhibited a synergistic effect (Figure S1). To account for the potential molar ratio dependence,¹⁸ these experiments were conducted at two molar ratios of chemotherapeutic drug to ML111. These analyses revealed that both vincristine and ifosfamide exhibited synergistic effects in combination with ML111 at both molar ratios (Table 1). CI values also demonstrate that between these two drug combinations, the strongest synergism was observed for vincristine with ML111. Therefore, this drug pair (ML111 and vincristine) was selected for further studies. ML111 also substantially decreases IC₅₀ values of doxorubicin, cyclophosphamide, and etoposide, and tested drug pairs demonstrate synergistic effects at a certain molar ratio, although less consistently (Table 1 and Figure S1).

Table 1.

Summary of IC₅₀ and CI values for chemotherapeutic drugs in combinations with ML111

Drug name	IC50 (nM)			CI values
	Drug alone	Drug:ML111 1:1	Drug:ML111 1:5	Drug:ML111 1:1	Drug:ML111 1:5
Vincristine	2.5 ± 0.3	0.12 ± 0.04	0.07 ± 0.05	0.035	0.024
Doxorubicin	179.2 ± 7.9	45.5 ± 6.2	4.9 ± 1.6	3.560	0.794
Etoposide	1705.0 ± 6.5	613.9 ± 8.1	2.7 ± 2.4	>5	0.764
Ifosfamide	>5000	4.5 ± 3.8	3.8 ± 1.4	0.473	0.254
Cyclophosphamide	>5000	2461.0 ± 10.8	4.5 ± 3.4	>5	0.604
ML111	22.1 ± 2.8
Strong synergism CI = 0.1– 0.5	Mild synergism CI = 0.5– 0.9	Additive CI = 0.9– 1.1	Mild antagonism CI = 1.1– 2.0	Strong antagonism CI > 2.0

Development and characterization of ML111-loaded polymeric nanoparticles

ML111 is characterized by poor aqueous solubility (0.0057 mg/mL, evaluated by Percepta 2012, ACD/Labs, Toronto, Ontario, Canada), and therefore, DMSO-based formulation was used in the above described in vitro studies. Although DMSO, as a co-solvent, is widely used for in vitro testing of small drug molecules, it is not well suited for commercial intravenous formulations due to toxicological concerns.¹⁹ To overcome this limitation, eight formulations for parenteral administration of ML111 were developed and characterized (Table S1). Although many of the developed formulations (e.g., PEG-PLA and Pluronic F-127 micelles) were able to substantially enhance the apparent aqueous solubility of ML111 (> 17 times), these preparations demonstrated limited stability upon dilution with corresponding vehicles (e.g., 0.9% NaCl and 5% dextrose) and therefore, none was suitable for in vivo application. In contrast, the formulation prepared by encapsulation of ML111 into the hydrophobic core of PEG-PCL-based polymeric nanoparticles (ML111-NP, Figure S2) was highly stable under the same experimental conditions and increased the apparent aqueous solubility of ML111 up to 350-fold (Table S1). The nanoparticles with a drug loading capacity of 2.4% have an average hydrodynamic size of 32 nm (Figure 2A), zeta potential of 0.12 mV, polydispersity index of 0.07 and display spherical morphology (Figure 2B). The developed nanoparticles are stable in solution for at least 3 weeks at 4 °C because no noticeable changes in a nanoparticle size distribution (Figure 2A), and ML111 concentration (2.0 mg/mL) occurred over the tested period.

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.

In vitro evaluation of ML111-NP alone and in combination with vincristine

In vitro studies demonstrate that PEG-PCL nanoparticles are efficiently internalized into EwS cells (Figure 2C) and enhance anticancer efficacy of ML111 (IC₅₀ = 9.2 nM) when compared to DMSO-based formulation (IC₅₀ = 22.1 nM, Figure 2D). Synergistic anticancer effect of ML111-NP in combination with vincristine at four molar ratios was validated in two EwS cell lines. Cell viability measurements revealed a significant reduction in the IC₅₀ values of vincristine from 2.5 nM to 0.1 nM in SK-N-MC cells and from 4.1 nM to 0.9 nM in TC-71 cells when ML111-NP were added at 1:1 molar ratio (Table 2 and Figure S3). The results further suggest that by increasing the dose of ML111-NP in the combinatorial treatment (vincristine: ML111-NP =1:5, 1:10, and 1:15), the IC₅₀ values of vincristine can be further decreased. Finally, the combination of vincristine and ML111-NP at four molar ratios revealed strong synergistic effects with CI values from 0.008 to 0.11 in both cell lines (Table 2).

Table 2.

Summary of IC₅₀ and CI values for vincristine in combinations with ML111-NP

Vincristine/ML111 ratio	IC50 of Vincristine (nM)		CI values
	SK-N-MC cells	TC-71 cells	SK-N-MC cells	TC-71 cells
Vincristine alone	2.5 ± 0.3	4.1 ± 0.6
ML111-NP alone	9.2 ± 2.4	12.7 ± 3.1
1:1	0.1 ± 0.03	0.9 ± 0.2	0.06	0.11
1:5	0.03 ± 0.01	0.11 ± 0.02	0.02	0.04
1:10	0.02 ± 0.01	0.02 ± 0.01	0.013	0.02
1:15	0.003 ± 0.001	0.003 ± 0.001	0.008	0.02
Strong synergism CI = 0.1– 0.5	Mild synergism CI = 0.5– 0.9	Additive CI = 0.9– 1.1	Mild antagonism CI = 1.1– 2.0	Strong antagonism CI > 2.0

Importantly, ML111-NP did not inhibit viability of non-malignant human embryonic kidney cells 293 (HEK293) and murine fibroblasts (NIH3T3) (Figure 3A). Moreover, ML111-NP antagonized the cytotoxic effect of vincristine in both HEK 293 (CI > 2.5) and NIH 3T3 (CI > 1.5) cells (Figure 3B and C and Figure S4).

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.

We also evaluated the effect of ML111-NP/vincristine-based combinatorial treatment on EwS cell cycle arrest, proliferation, and apoptosis when compared with either single-drug treatment. Both vincristine and ML111-NP caused the arrest of the EwS cell cycle at G₂/M in a time-dependent manner (Figure 4A). Moreover, combinatorial treatment markedly increased the fraction of cells arrested in G₂/M phase, compared with either vincristine or ML111-NP alone at all time points (Figure 4B).

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.

Real-time proliferation studies revealed that either vincristine or ML111-NP significantly reduced proliferation of EwS cells. However, the combination of two drugs inhibited proliferation of the tested cells completely (Figure 5A).

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).

Our in vitro studies demonstrated that vincristine, in combination with ML111-NP, induces apoptosis in EwS cells earlier and to a greater extent than either agent alone. The percentage of early and late apoptotic cells was higher at various time points after combinatorial treatment when compared to single treatments with vincristine and ML111-NP at the same doses, respectively (Figure 5B).

Orthotopic xenograft model of EwS

An orthotopic xenograft model of EwS was established by injecting SK-N-MC cells into the right gastrocnemius muscles of nude mice. This approach generates an intramuscular tumor mass that grows in close proximity to the tibia (Figure 6A).

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).

Biodistribution of the ML111-NP

The tumor-targeting ability and biodistribution of the NIR dye-labeled ML111-NP following systemic administration were evaluated by fluorescence imaging. A strong NIR fluorescent signal was primarily localized in the tumor-containing hindlimb at 24 h post-injection (Figure 6B), indicating efficient accumulation and retention of ML111-NP in cancer tissue via passive targeting. Ex vivo analyses of both hindlimbs and other organs further validated these results (Figure 6C).

In vivo antitumor efficacy of the ML111-NP

Prior to the evaluation of the combinatorial therapy, toxicity and anticancer efficacy of ML111-NP as a single treatment modality were. Five days following inoculation of SK-N-MC cells, mice were treated with ML111-NP (15 mg ML111/kg) three times per week (every other day) for three weeks (Figure S5). As shown in Figure 6A, the mouse calf significantly increased in size at day 4 post-injection of cells due to tumor growth, validating that a cancer xenograft was well established prior to treatment initiation. It is challenging to measure the size of a xenograft in the hindlimbs of a mouse accurately due the tight integration of the tumor into adjacent tissue (Figure 6A). Therefore, the cancer growth was quantitatively monitored by measuring the medial-lateral and anterior-posterior lengths of a calf with digital calipers, and the calf volume calculated at any given point in time during the treatment was compared to the calf volume obtained prior to cells injection (~ 80 mm³, day 0).²⁰ These measurements suggested that ML111-NP significantly inhibited tumor growth in comparison with a control group (Figure 7A). By day 25 of the study, the average calf volume in mice treated with ML111-NP was 2.4 times smaller (827 ± 129 mm³) than that of control mice injected with 5% dextrose (1970 ± 179 mm³). Analysis of two-dimensional (2D) ultrasound images of tumors recorded prior to euthanasia, revealed that the average tumor area after the ML-111-NP treatment (162 ± 11 mm²) was 1.4 times lower than the area of tumors in the control group (226 ± 9 mm², Figure 7D and E), validating the trend in results obtained by the measurements of the calf volume with a caliper (Figure 7A). Animals from both groups were euthanized at the defined end point (day 27) when the calf volume in control mice reached ~2300 mm³, and tumors were excised. The average tumor weight of the ML-111-NP treatment group (2.3 ± 0.5 g) was 1.6 times lower than the control group (3.7 ± 0.7 g) (Figure 7B).

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.

TUNEL staining of the collected tumor sections suggests that ML111-NP inhibited tumor growth by inducing apoptosis in cancer cells. The number of apoptotic-positive cells in the ML111-NP-treated cancer tissues was 6.7 times higher than that in the control group (Figure 8).

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.

Survival studies were also performed to further investigate the anticancer efficacy and tolerability of ML111-NP-based treatment. Kaplan–Meier survival curves demonstrate that ML111-NP treated mice have a median survival of 36 days, whereas control mice reached a predetermined tumor limit of ~2300 mm³ at 27 days (Figure 7C). Importantly, none of the ML111NP-treated mice died, exhibited altered serum biomarker levels (Figure S6), lost more than 10% of their body weight, (Figure S6), or exhibited any signs of toxicity (e.g. walks hunched or slowly) during the course of therapy.

In vivo antitumor efficacy of the ML111-NP in combination with vincristine

The observed efficacy and safety of the ML111-NP-based treatment were equivalent to that of vincristine alone (Figures 7, 8 and S6) injected at a dose of 0.1 mg/kg once per week for four weeks (Figure S5). The combination of ML111-NP (15 mg/kg) and vincristine (0.1 mg/kg), administered according to the schedule outlined in Figure S5, provided superior therapeutic outcomes compared with the individual treatments. The measurements obtained on day 25 of the study (after the last injection of vincristine) revealed that the average volume of a calf with a tumor was 258 ± 101 mm³ in mice exposed to the combinatorial therapy, while these values were 2.8 and 3.2 times higher in mice treated vincristine (733 ± 172 mm³) and ML111-NP (827 ± 129 mm³) alone, respectively (Figure 7A). Ultrasound measurements, performed on day 27 of the study, further demonstrated that the area of tumors after the combinatorial therapy (70 ± 23 mm²) was 2.0 and 2.3 times lower than tumor areas treated with vincristine (140 ± 26 mm²) and ML111-NP (162 ± 11 mm²) alone (Figure 7E–G), respectively. The average weight of tumors after the combinatorial therapy (1.0 ± 0.1 g) was less than half that of tumors from ML111-NP (2.3 ± 0.6 g) and vincristine (2.0 ± 0.2 g) groups; there was no statistically significant difference between the latter two groups (Figure 7B). Histological analysis of tumor tissues revealed that the combinatorial therapy induced a significantly higher level of apoptosis than individual treatments (Figure 8). Finally, survival studies confirmed that the combinatorial therapy increased the median survival time of mice by 14 and 17 days compared to vincristine and ML111-NP treatments, respectively (Figure 7C).

We further demonstrated that EwS xenografts can be significantly regressed or eradicated by increasing the dose of vincristine from 0.1 mg/kg to 0.5 mg/kg in the above-described combinatorial treatment regimen (Figure S7). For example, on day 25 of the study (after the last dose of vincristine), the average calf volume in the combinatorial treatment group was not statistically different from the average calf volume measured at the beginning of the experiment (day 0), prior to injection of cells (75 ± 14 mm³ vs. 81 ± 11 mm³, respectively). In contrast, the average calf volume in mice treated with vincristine alone (0.5 mg/kg) was 107 ± 21 mm³. Although cancer regrowth was detected in two out of five mice in each treatment group after therapy discontinuation, tumor regrowth rate was significantly lower in mice exposed to the combinatorial therapy (Figure S7). Volumes of the calf in two mice treated with the combinatorial therapy increased from 80 mm³ and 96 mm³ to 108 mm³ and 163 mm³, respectively, 31 days after cessation of therapy (Figure S7). In contrast, these values in two mice treated with vincristine alone changed from 97 mm³ and 121 mm³ to 340 mm³ and 546 mm³, respectively, within the same period (Figure S7). Tumor weights measured post-euthanasia on day 50 after therapy discontinuation were 1.0 g and 1.9 g in mice treated with the combinatorial therapy and 2.3 g and 2.5 g in vincristine-treated mice. Cancer tissues were not visually detected in other mice from both treatment groups (VIC and VIC + ML111-NP) euthanized 66 days after therapy discontinuation.

The above combinatorial treatment regimens were well tolerated by mice and no overt changes in bodyweight and concentrations of serum biomarkers were observed between control and treatment groups (Figures S6 and S8).

Discussion

Conventional combinatorial chemotherapy regimens for EwS and other types of cancer remain limited by acute toxicity and long-term side effects.^1,21,22 Synergistic combinations of novel selective anticancer agents with clinically used chemotherapeutic drugs can potentially overcome these issues by using lower doses of the combination constituents to maintain the required therapeutic efficacy. Therefore, it is important to identify drug combinations that demonstrate synergistic therapeutic efficacy against cancer cells, while simultaneously exhibiting antagonistic effects in non-malignant cells ¹⁸. It is also well documented that synergism for a drug combination strongly depends on the ratio of doses, and failure to achieve the optimal drug ratio in cancer tissues following systemic administration can lead to antagonistic effects.^18,23,24 Drug combinations that act synergistically at multiple dose ratios are required to overcome this challenge.

Our results validate that ML111, in combination with vincristine, exhibits robust synergistic effects against EwS cell lines at various molar ratios. Vincristine is widely used in combination with various chemotherapeutic drugs to treat EwS, but its clinical dosage is limited by neurotoxicity.²⁵ The obtained results demonstrate that, depending on the ratio, synergy with ML111 can lead to a decrease in the IC₅₀ value of vincristine by ten to several hundred fold in different EwS cell lines.

To increase the aqueous solubility of ML111 and improve its accumulation in cancer tumors, PEG-PCL-based nanoformulation was prepared and validated both in vitro and in vivo. The obtained in vitro data suggested that ML111 encapsulated into nanoparticles was 2.4 times more potent than ML111 dissolved in DMSO-containing aqueous solution, as evidenced by the IC₅₀ values. Our results are consistent with numerous reports indicating that nanoparticles can substantially improve the anticancer activity of hydrophobic agents by increasing their aqueous solubility and improving cellular uptake.^26,27 In vitro studies further revealed that ML111-NP can behave as a selective anticancer agent for EwS, produce a strong synergistic anticancer effect with vincristine at multiple molar ratio and potentially reduce the adverse effects of vincristine on non-malignant cells. To assess potential side effects of ML111-NP alone and in combination with vincristine on various healthy cells, fibroblasts (NIH3T3) and kidney cells (HEK293) were employed in our studies. To demonstrate the rigor of our approach, we used both non-malignant murine (NIH3T3) and human (HEK293) cells.

Safety and anticancer efficacy of ML111-NP in combination with vincristine were further validated in an intramuscular paratibial mouse model of EwS. This animal model was previously used for the evaluation of various therapeutic modalities for the treatment of osteosarcoma and EwS.^20,28–30 Crenn et al. even demonstrated that the intramuscular model of osteosarcoma exhibits lower therapeutic response to certain chemotherapeutic agents (e.g., doxorubicin) when compared to the intra-osseous model.²⁸ To our knowledge, this is one of the first studies demonstrating the targeting and therapeutic efficacy of a nanoparticle-based drug formulation in an orthotopic xenograft model of EwS.^31–33 Our studies suggest that the PEG-PCL nanoparticles can provide efficient delivery and retention of ML111 in orthotopic xenografts of EwS following systemic administration. Animal studies validated in vitro results demonstrating that ML111-NP combined with vincristine at two different ratios provides superior anticancer effects than single treatments. Notably, ML111-NP administered three times a week for three weeks at a dose of 15 mg/kg do not cause any measurable toxicity in mice as a single treatment or in combination with different doses of vincristine. This remarkable safety profile of ML111-NP suggests that ML111 doses higher than 15 mg/kg can be tested to enhance the anticancer efficacy of vincristine further.

Overall, our data suggest that the identified molecule, ML111, encapsulated in the polymeric nanoparticles, has a significant potential to become an effective agent for treatment of EwS. Moreover, the ML111-NP exhibited a robust, synergistic effect with vincristine, and this novel synergistic drug combination can provide an efficient therapeutic approach for EwS with the possibility of diminishing severe side effects associated with vincristine. The current data provide the rationale for further assessing this novel combinatorial therapy in advanced animal models of metastatic EwS.

Abbreviations:

EwS

Ewing’s sarcoma

ML111

2-amino-4-(3-methoxyphenyl)-4Hbenzo[h]chromene-3-carbonitrile

ML111-NP

nanoparticles loaded with ML111

VDC/IE

a combination of vincristine (V), doxorubicin (D), and cyclophosphamide (C), ifosfamide (I) and etoposide (E)

EWS/FLI

1 is a chimeric protein found in Ewing’s sarcoma tumors

mPEG–PCL

methoxy poly(ethylene glycol)-b-poly(ε-caprolactone)

CI

combination index

SiNc

Silicon 2,3-naphthalocyanine bis(trihexylsilyloxide)

VIC

vincristine