Photothermal therapy improves the efficacy of a MEK inhibitor in neurofibromatosis type 1-associated malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive tumors with low survival rates and the leading cause of death in neurofibromatosis type 1 (NF1) patients under 40 years old. Surgical resection is the standard of care for MPNSTs, but is often incomplete and can generate loss of function, necessitating the development of novel treatment methods for this patient population. Here, we describe a novel combination therapy comprising MEK inhibition and nanoparticle-based photothermal therapy (PTT) for MPNSTs. MEK inhibitors block activity driven by Ras, an oncogene constitutively activated in NF1-associated MPNSTs, while PTT serves as a minimally invasive method to ablate cancer cells. Our rationale for combining these seemingly disparate techniques for MPNSTs is based on several reports demonstrating the efficacy of systemic chemotherapy with local PTT. We combine the MEK inhibitor, PD-0325901 (PD901), with Prussian blue nanoparticles (PBNPs) as PTT agents, to block MEK activity and simultaneously ablate MPNSTs. Our data demonstrate the synergistic effect of combining PD901 with PBNP-based PTT, which converge through the Ras pathway to generate apoptosis, necrosis, and decreased proliferation, thereby mitigating tumor growth and increasing survival of MPNST-bearing animals. Our results suggest the potential of this novel local-systemic combination “nanochemotherapy” for treating patients with MPNSTs.

benefits from chemotherapy, which elicits systemic effects to complement its inherently local effects. Motivated by these earlier findings, we seek to exploit these complementary effects in the context of NF1-associated MPNSTs. Specifically, we combine the MEK inhibitor, PD-0325901 (PD901), with Prussian blue nanoparticles (PBNPs) as PTT agents, to block MEK activity and simultaneously ablate MPNSTs when irradiated with a near infrared (NIR) laser. To our knowledge, our study represents the first attempt at exploiting the synergy between PTT and chemotherapy for the treatment of NF1-associated MPNSTs.
MEK inhibitors are small molecule inhibitors that target the Ras signaling pathway. NF1 and NF1-associated MPNST patients pathognomonically lack neurofibromin, a negative regulator of oncogenic Ras signaling. Without neurofibromin protein function, Ras is allowed constitutive activation 15,16 . The Ras signal transduction pathway generates a phosphorylation cascade through RAF, MEK, and ERK, which in its phosphorylated form (p-ERK) affects the transcription of genes associated with uncontrolled cell proliferation and increased cancer progression 17,18 . Research suggests the potential of using MEK inhibitors to block Ras activity in MPNSTs [19][20][21][22] , but these studies were conducted in either cell lines 19,21,22 or in animal models that yielded marginal results in treating MPNSTs 20,23 . Based on the improved efficacy of combining chemotherapy with PTT, we expect that the effects of the MEK inhibitor PD901 will be made more potent when combined with PBNP-based PTT for treating MPNSTs. PTT is a minimally invasive method for destroying tumors using light-activated nanoparticles and a low power NIR laser 24,25 . In this study, we use PBNPs [26][27][28][29] for PTT of MPNSTs, which we have previously used for ablation of subcutaneous neuroblastoma 28 . Compared to alternative nanoparticles used for PTT, PBNPs offer several advantages: they can easily be synthesized in a single, scalable step at low costs, and are already FDA-approved for human oral consumption (to treat radioactive poisoning) 30,31 suggesting their potential safety for use as PTT agents.
To determine whether PD901 combined with PBNP-based PTT results in improved treatment outcomes for MPNSTs, we use the mouse M2 MPNST cells in vitro, and a syngeneic, subcutaneous mouse model of MPNST in vivo 32 . First, we assess the efficacy of PD901 and PBNP-based PTT individually in treating MPNST cells in vitro. Specifically, we assess the effects of the individual therapies on MPNST cell viability and the mechanisms of cell death induced by the therapies. Next, we assess the efficacy of the PD901/PTT combination in treating MPNST cells in vitro and whether this combination of PD901 and PTT is synergistic (using dose reponse and drug interaction calculations). Finally, we determine the effects of the PD901/PTT combination on tumor progression and animal survival in vivo. The findings of these studies will demonstrate the feasibility of using our novel nanochemotherapy for preclinically treating MPNSTs, an important prelude to eventual clinical translation.

Results and Discussion
PD901 effectively treats MPNSTs in vitro by blocking ERK activation. In order to validate the presumed anti-MEK mechanism of action of PD901 in M2 cells, we measured its effects on the activation of ERK, which is located downstream of MEK in the Ras signaling pathway. M2 cells were treated with vehicle (DMSO) or 1 μ M PD901 for four or eight hours, and then harvested, lysed, and probed for phosphorylated ERK (p-ERK) and total ERK, using actin as a loading control. 1 μ M PD901 effectively blocked the activation of ERK (p-ERK) at both four and eight hours, demonstrated by no visible p-ERK bands on the Western blot in the PD901-treated lanes, compared with distinct p-ERK bands observed in the vehicle-treated lane (Fig. 1a). Our finding of decreased p-ERK expression in M2 cells is consistent with those in the published literature, which confirm decreased ERK activation after a few hours of PD901 treatment 33 . To determine the effect of MEK inhibition on MPNST viability, we treated M2 cells with varying doses of PD901 and measured cell viability. PD901 decreased M2 cell viability in a concentration-dependent manner, with an IC50 of approximately 1 μ M (Fig. 1b). These results are consistent with earlier studies using MEK inhibitors in other cell lines 23,34 . To determine the mechanisms causing decreased M2 viability, we analyzed the mechanisms of cell death (apoptosis versus late apoptosis/necrosis), and the effects of PD901 treatment on the M2 cell cycle. PD901 was found to decrease M2 viability by both apoptosis (Annexin V + 7AAD-) and late apoptosis/necrosis (7AAD+ ) at all concentrations tested (Fig. 1c). Further, PD901 caused a significant arrest of M2 cells in the G0/G1 phase in a concentration-dependent manner (Fig. 1d), indicating decreased proliferation after treatment. Our results with M2 cells suggest that MEK inhibition using PD901 is a feasible modality for treating MPNST in vitro, by the blockade of ERK activation, which would otherwise increase cell proliferation and division 2 .

PBNPs function as effective agents for PTT of MPNSTs in vitro.
To determine whether PBNPs function as effective agents for PTT of MPNSTs, we conducted studies in vitro using M2 cells. First, PBNPs were synthesized with monodisperse size distributions as measured by dynamic light scattering (mean hydrodynamic diameter of 68.1 nm), and with their characteristic cubic structures as observed by transmission electron microscopy (Fig. 2a). The synthesized PBNPs heated in a concentration-dependent manner when irradiated with an 808 nm NIR laser at 1.5 W/cm 2 laser power density for 10 minutes (Fig. 2b). Importantly, this incremental heating consistently resulted in decreased viability of M2 cells when the PBNPs were similarly irradiated with the NIR laser (Fig. 2c). Temperatures around 45 °C and greater appeared to decrease viability of M2 cells to a similar extent (approximately 15-25% viable). Interestingly, PBNP-based PTT triggered differing levels of apoptosis and/ or late apoptosis/necrosis in M2 cells depending on the temperature ranges to which they were heated. This temperature range was, in turn, dependent on the concentration of PBNPs used for PTT since both laser power and duration of laser irradiation were kept constant in these studies. M2 cells that were heated to the 45-50 °C temperature range triggered cell death through primarily apoptosis; M2 cells treated with 0.03 mg/mL PBNPs, which attained an average temperature of 46.8 °C after ten minutes irradiation at 1.5 W/cm 2 , resulted in apoptosis in 72.2% of cells. In contrast, M2 cells that were heated to >50 °C triggered cell death through primarily late apoptosis/necrosis; M2 cells treated with 0.05 mg/mL PBNPs, which attained an average temperature of 51.3 °C after ten minutes irradiation at 1.5 W/cm 2 , resulted in late apoptosis/necrosis in 73.2% of cells (Fig. 2d). Our finding Scientific RepoRts | 6:37035 | DOI: 10.1038/srep37035 of a temperature range preferentially triggering cell death by either apoptosis or necrosis are corroborated by an earlier study using gold nanorods to ablate tumor cells in vitro 35 . Thus, PBNPs function as effective agents for PTT of MPNSTs resulting in decreased viability of these cells in vitro. More importantly, our data suggests a potentially tunable mechanism of inducing cell death using PBNP-based PTT (apoptosis versus necrosis), which can be leveraged in subsequent studies to improve treatment outcomes for MPNSTs in vivo.

PD901 and PTT synergistically combine to yield improved treatment outcomes for MPNSTs in vitro.
After determining the individual efficacies of both PD901 and PBNP-based PTT in treating M2 cells in vitro, we next sought to examine the potential benefit of combining the treatments. Cell viability studies demonstrated that combining the two treatments in vitro was able to significantly decrease the viability of M2 cells more than either treatment administered alone (Fig. 3a). At both low doses (0.125 μ M PD901, "LOW PD901;" 0.005 mg/mL PBNP-based PTT, "LOW PTT") and high doses (1 μ M PD901, "HIGH PD901;" 0.05 mg/mL PBNP-based PTT, "HIGH PTT"), the combination treatment resulted in significantly decreased cell viability over the individual treatments administered under identical conditions. Additionally, PD901 + PTT ("HIGH Combo") functioned to effectively block p-ERK expression in M2 cells, mirroring the effects of PD901 treatment alone (Fig. 3b). It is important to note that while both PD901 alone and the HIGH Combo treatment resulted in similar Western blot readouts, further studies are needed to elucidate whether these effects are sustained in the two treatment groups over time. Earlier studies have demonstrated a rescue of p-ERK expression over time after MEK inhibition 23 . Therefore, therapies that yield sustained decrease of p-ERK expression may be important for improved treatment outcomes in MPNSTs. Interestingly, PTT alone was able to decrease p-ERK expression (Fig. 3b), signifying its potential impact on Ras signaling at a point along its signal transduction pathway. This finding suggests that although PD901 and PTT function differently to decrease M2 cell viability (cell cycle arrest vs. ablation, respectively), these effects converge along the Ras signaling pathway. To our knowledge, using PTT to alter a prominent signal transduction pathway in cancer has not been previously explored. Deeper mechanistic studies to determine  (Fig. 3c). The coefficients of drug interaction (CDI) 36 for three of the doses tested (doses 2, 3, and 4) were calculated to be less than 0.7, indicating significant drug synergy between PD901 and PTT.

PD901 and PTT combine to decrease MPNST progression and increase survival in vivo.
Motivated by our in vitro findings, we generated a syngeneic mouse model of MPNST using the M2 cell line in B6129SF1/J mice based on previous literature 32 to test the in vivo efficacy of our novel combination therapy. Mice were subcutaneously injected with M2 cells. When the mice exhibited established tumors (~10 mm in diameter), the MPNST-bearing mice were divided into four groups and were: 1) untreated (n = 5), 2) treated with PD901 (n = 5), 3) treated with PTT (n = 5), or 4) treated with both PD901 plus PTT (n = 5). Tumor progression was measured daily and mouse survival was monitored post-treatment. Tumors in the untreated group progressed at the fastest rate (Fig. 4a, black lines), and mice in this group succumbed to high tumor burden after 14 days or less (median survival = 13 days; Fig. 4b). In contrast, mice treated with PD901 plus PTT exhibited slower tumor growth compared to all other groups, as demonstrated by the decreased slopes of their tumor progression curves (Fig. 4a, purple lines). This slower tumor growth translated to significantly increased survival in mice treated PBNPs exhibit their characteristic cubic morphology when visualized using transmission electron microscopy (scale bar = 100 nm). (b) PBNPs heat to higher temperatures with increasing concentrations of PBNPs when irradiated with an 808 nm NIR laser at 1.5 W/cm 2 for ten minutes; measured at one-minute intervals using a thermocouple. *p < 0.05; ***p < 0.001; compared to adjacent lower concentration's temperature at 10 minutes. (c) The viability of M2 cells decrease when subject to varying doses of PTT (PBNP concentrations ranging from 0.01 mg/mL to 0.05 mg/mL, with or without 808 nm laser irradiation at 1.5 W/cm 2 for ten minutes), measured after 24 h. *p < 0.05; ***p < 0.001; compared to matched controls. (d) M2 cells treated with various doses of PTT can trigger cell death via apoptosis (light blue, Annexin V + 7AAD-population) and/or late apoptosis/necrosis (dark blue, 7AAD + population) depending on the resulting temperature range to which they are heated; quantified by flow cytometry. Data in all plots expressed as mean ± standard deviation (n ≥ 3/group). *p < 0.05; **p < 0.01; compared to all other samples in group.  Figure S1). It should be noted that the improved treatment outcomes were observed using a single local administration of PBNP-based PTT and daily oral dosing of PD901. Further optimization of this combination therapy that leverages the convergent biological effects of these two therapies observed in vitro may result in further improvement in treatment outcomes for MPNSTs over those currently observed, in addition to conferring potential benefits such as less frequent doses of the MEK inhibitor. Taken together, our findings demonstrate the feasibility of using PD901 in combination with PBNP-based PTT for achieving improved treatment outcomes for MPNSTs in vivo.
In summary, we have presented a novel combination therapy comprising two distinct but synergistic treatments: MEK inhibition using the small molecule inhibitor PD901 and PBNP-based PTT. We demonstrated a convergence of the two treatment strategies on the Ras signal transduction pathway, which resulted in decreased ERK activation. These effects, when combined with the effects of photothermal ablation via PTT, resulted in decreased growth of MPNSTs both in vitro and in vivo. Ongoing studies in our group will build upon these data to further mechanistically describe the combined effect of PD901 and PTT so as to better exploit the synergy of this combination nanochemotherapy in clinically treating NF1-associated MPNSTs.

Methods
Chemicals and PBNP synthesis. PD0325901 (#PZ0162; PD901) was purchased from Sigma-Aldrich (St. Louis, MO). PBNPs were synthesized from their constituent salts purchased from Sigma-Aldrich as previously described 28 . 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, complete protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitor (Roche, Basel, Switzerland)]. Samples were analyzed by SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). The blots were then blocked in 5% non-fat milk in TBST, followed by incubation of primary antibodies at 4 °C overnight. After washing, the blots were incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 hour. Signals were detected using ECL or ECL plus (GE healthcare, Little Chalfont, United Kingdom) followed by film development.  Photothermal therapy. An 808 nm NIR laser (Laserglow Technologies, Toronto, Ontario, Canada) at 1.5 W/cm 2 was used for all PTT studies. PBNPs were added to 96-well plates at varied concentrations and irradiated for ten minutes. A thermocouple (Omega Engineering, Stamford, CT) was used to measure the temperature of the wells at one-minute intervals.

Analysis of PD901 and PBNP-based PTT interaction in vitro.
The coefficient of drug interaction (CDI) was used to analyze the interaction between PD901 and PBNP-based PTT in treating M2 cells in vitro, as previously described 36 . Briefly, M2 cells were subject to varying doses of PD901, PBNP-based PTT, or combination PD901/PTT. The doses of PD901 used were 0.125-100 μ M; the doses of PBNP-based PTT were 0.005-0.1 mg/mL irradiated for 10 min with the NIR laser. The doses for the PD901 + PTT combination were the combined doses used for each individual treatment. The viability of the M2 cells at each dose was measured using the ATP-based CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). The CDI for a given dose of PD901, PTT, or the PD901/PTT combination was calculated using the formula: CDI = (M2 viability in response to PD901/PTT)/(M2 viability in response to PD901 × M2 viability in response to PTT). A value of CDI < 1 indicated drug synergy, CDI < 0.7 indicated significant drug synergy, CDI = 1 indicated additivity, and CDI > 1 indicated drug antagonism.

Animals.
All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Children's National Health System, Washington, DC (Protocol #00030571). The studies were conducted in accordance with the approved IACUC guidelines. 4-6 week old female B6129SF1/J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were monitored daily for symptoms of toxicity (e.g. sick mouse posturing, infection, impaired mobility, weight loss, self-mutilation, bleeding). No mice were euthanized due to toxicity symptoms, and no measurable weight loss was recorded. The animals were acclimated for 3-4 days prior to tumor inoculation.
In vivo studies. 1 million M2 cells were injected into the backs of B6129SF1/J mice in 100 μ L 50% phosphate buffered saline (PBS, Life Technologies, Carlsbad, CA)/50% Matrigel (Corning, Corning, NY). Treatment commenced when tumors reached 10 mm in diameter. Animals were treated in four groups: (1) Untreated (n = 5): animals received no treatment, (2) PTT-treated (n = 5): animals were intratumorally injected with 50 μ L of 1 mg/mL PBNPs and irradiated for 10 minutes with 1.5 W/cm 2 NIR laser, (3) PD901-treated (n = 5): animals were given 5 mg/kg PD901 daily by oral gavage in 0.5% hydroxypropyl methylcellulose and 0.2% Tween80, and (4) PD901 plus PTT combo-treated (n = 5): animals were given both PTT and PD901 treatments as listed above. Animals were euthanized if tumors reached >20 mm in diameter, if their tumors became ulcerated, or if they exhibited signs of distress. Tumors were measured daily by calipers. For histological analysis of the effects of the treatments, tumors were harvested eight hours post treatment (treatments described above; n = 2-3 per group), processed, and stained with hematoxylin and eosin (H&E) for subsequent microscopy. Statistical analysis. Statistical analysis was conducted using Prism V5.00 (GraphPad Software, San Diego, CA). Statistical significance between groups in the plots was determined using a two-tailed Student's t-test and values with p < 0.05 qualified as statistically significant and were marked with an asterisk (*) to indicate comparison between two specified groups. ** indicates p-values < 0.01, while *** indicates p-values < 0.001. p-values > 0.05 qualified as not statistically significant. The samples sizes (n) for each group were either ≥3/group or explicitly mentioned for each study. For the animal survival study, a log-rank test was used to determine statistically significant differences in survival between the various groups, (α = 0.05, rejecting the null hypothesis if x 2 > critical value for the test). Survival results were analyzed by generating Kaplan-Meier plots. A p-value < 0.05 was considered statistically significant in this analysis. All relevant p-values are listed in Supplementary Table 1.