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Cancer Therapy: Preclinical |
Authors' Affiliations: Laboratories of 1 Biomedical Magnetic Resonance, 2 Medicinal Chemistry and Radiopharmacy, 3 Pharmacology and Therapeutics, and 4 Molecular Imaging and Experimental Radiotherapy; 5 Pharmacokinetic, Metabolism, Nutrition and Toxicology Unit, Université Catholique de Louvain, Brussels, Belgium
Requests for reprints: Bernard Gallez, CMFA/REMA Units, Université Catholique de Louvain, Avenue E. Mounier 73.40, B-1200 Brussels, Belgium. Phone: 32-2-7642792; Fax: 32-2-7642790; E-mail: Gallez{at}cmfa.ucl.ac.be.
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Abstract |
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Experimental Design: To test this hypothesis, BoNT-A was injected locally into mouse tumors (fibrosarcoma FSaII, hepatocarcinoma transplantable liver tumor), and electron paramagnetic resonance oximetry was used to monitor pO2 in vivo repeatedly for 4 days. Additionally, contrast-enhanced magnetic resonance imaging was used to measure tumor perfusion in vivo. Finally, isolated arteries were mounted in wire myograph to monitor specifically the neurogenic tone developed by arterioles that were co-opted by the surrounding growing tumor cells.
Results: Using these tumor models, we showed that local administration of BoNT-A (two sites; dose, 29 units/kg) substantially increases tumor oxygenation and perfusion, leading to a substantial improvement in the tumor response to radiotherapy (20 Gy of 250-kV radiation) and chemotherapy (cyclophosphamide, 50 mg/kg). This observed therapeutic gain results from an opening of the tumor vascular bed by BoNT-A because we showed that BoNT-A could inhibit neurogenic tone in the tumor vasculature.
Conclusions: The opening of the vascular bed induced by BoNT-A offers a way to significantly increase the response of tumors to radiotherapy and chemotherapy.
BoNT-A has a wide variety of clinical indications, particularly for treating intractable muscle hyperactivity associated with strabismus, spasticity, hemifacial spasm, and blepharospasm (10). BoNT-A is largely known by the public for its cosmetic uses to rejuvenate the aging face. The Botulinum toxin is a neurotoxin composed of two polypeptide chains held together by a single disulfide bridge. The toxin works locally at the injection site and acts on neurotransmitter-mediated processes by blocking their release. Its mechanism consists of a metalloprotease activity against the synaptosomal-associated protein of 25 kDa (SNAP-25), which prevents the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex involved in the fusion of vesicles with presynaptic membranes and consequently impedes the release of neurotransmitters, such as acetylcholine and noradrenaline, at the neuromuscular junction (10–12). Thus far, there has been no investigation into the possible usefulness of BoNT-A as an adjuvant to cancer therapies. Our data present a paradigm according to which BoNT-A could interfere with neurotransmitter release at the perivascular sympathetic varicosities, leading to inhibition of neurogenic contractions of tumor vessels and therefore improving tumor perfusion and oxygenation. To characterize the changes in the tumor microenvironment induced by BoNT-A, we used several powerful tools: contrast-enhanced magnetic resonance imaging (MRI) has been used to measure tumor perfusion noninvasively (13); electron paramagnetic resonance (EPR) oximetry has made it now possible to monitor pO2 in vivo repeatedly and relatively noninvasively (14, 15); and myograph setup has been used to study the particular reactivity of arterioles co-opted by the tumor. We showed a substantial increase in tumor perfusion and oxygenation after BoNT-A administration, which led to a substantial improvement in the response of tumors to radiotherapy and chemotherapy.
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Material and Methods |
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BoNT-A preparation and injection procedure
BoNT-A solution in saline (Botox, Allergan, Antwerp, Belgium) or saline solution (control) was injected when tumors reached a diameter of 6.7 ± 0.2 mm. BoNT-A was administered in the tumor at two different sites (two injections of 20 µL, corresponding to a total dose of 29 units/kg).
Tumor oxygenation
EPR Oximetry, using charcoal (CX0670-1, EM Science, Gibbstown, NJ) as the oxygen sensitive probe, was used to evaluate the tumor oxygenation (14). EPR spectra were recorded using an EPR spectrometer (Magnettech, Berlin, Germany) with a low-frequency microwave bridge operating at 1.2 GHz and extended loop resonator. Mice were injected in the center of the tumor 1 day before measurement using the suspension of charcoal (suspension in saline containing 3% Arabic gum, 100 mg/mL, 50 µL injected, 1-25 µmol/L particle size). The localized EPR measurements correspond to an average of pO2 values in a volume of 
10 mm3 (14). To avoid any acute effect of the treatment, data acquisition was made before the injection of BoNT-A or saline and then on a daily basis.
Flow and permeability measurements
Patent Blue staining. Patent Blue (Sigma-Aldrich, Bornem, Belgium) was used to obtain a rough estimate of the tumor perfusion (8) 3 days after administration of BoNT-A or vehicle. This technique involves the injection of 200 µL of Patent Blue (1.25%) solution into the tail vein of the mice. After 1 minute, a uniform distribution of the staining through the body was obtained, and mice were sacrificed. Tumors were carefully excised and cut in two size-matched halves. Pictures of each tumor cross-section were taken with a digital camera. To compare the stained versus unstained area, an in house program running on IDL (Interactive Data Language, RSI, Boulder, CO) was developed. For each tumor, a region of interest (stained area) was defined on the two pictures, and the percentage of stained area of the whole cross-section was determined. The mean of the percentage of the two pictures was then calculated and was used as an indicator of tumor perfusion.
Dynamic contrast enhanced MRI. The perfusion was monitored on day 3 after injection of BoNT-A (n = 3) or saline (n = 4) via single-slice dynamic contrast-enhanced MRI at 4.7 T using the rapid clearance blood pool agent P792 (Vistarem, Guerbet, Roissy, France; ref. 19). High-resolution multislice T2-weighed spin echo anatomic imaging was done just before dynamic contrast-enhanced imaging. Pixel-by-pixel values for Ktrans (influx volume transfer constant, from plasma into the interstitial space, units of min–1), Vp (blood plasma volume per unit volume of tissue, unitless), and Kep (fractional rate of efflux from the interstitial space back to blood, units of min–1) in tumor were calculated via tracer kinetic modeling of the dynamic contrast-enhanced data (19), and the resulting parametric maps for Ktrans, Vp, and Kep, were generated. Statistical significance for Vp or Ktrans identified "perfused" pixels (i.e., pixels to which the contrast agent P792 had access; refs. 19, 20).
Miles assay. The Evans blue technique was used to measure the tumor vasopermeability to macromolecules. This technique provides a good estimate of the extravasation and interstitial accumulation of albumin (21) as Evans blue dye makes a complex with the negatively charged intravascular albumin by electrostatic combination. Briefly, Evans blue (2.5 mg/mL in NaCl 0.9%) was administered i.v. (10 mg/kg) in control and treated mice. The dye was allowed to circulate for 1 hour before sacrifice of the animals. Tumors were then excised, weighed, and put in formamide (2 mL/tumor) for 24 hours at 55°C for Evans blue extraction. The Evans blue concentrations were determined by spectrophotometry at 620 nm and expressed in terms of concentration per gram of tumor tissue.
Irradiation and tumor regrowth delay assay
The FSaII tumor-bearing leg was locally irradiated with 20 Gy of 250-kV X-rays (RT 250; Philips Medical Systems, Andover, MA). The tumor was centered in a 3-cm diameter circular irradiation field. A single dose irradiation of 20 Gy, 3 days after the i.t. injection of BoNT-A, was done. After treatment, the tumor growth was determined daily by measuring transversal and anteroposterior tumor diameters until they doubled in size, at which time the mice were sacrificed. A linear fit was done between 8 and 16 mm, which allowed determination of the time to reach a particular size for each mouse.
Chemotherapy and tumor regrowth delay assay
BoNT-A–treated mice received a single dose (50 mg/kg via 100 µL i.p.) of cyclophosphamide, an alkylating agent. Regrowth delay experiments in transplantable liver tumor have shown that this dose of 50 mg/kg is just below the efficacy threshold for this product (experiments done with dose range of 250-10 mg/kg).
Clonogenicity assay
FSAII tumors in mice were dissected in a sterile environment and gently pieced in McCoy's medium. The cell suspension was filtered (100-µm-sized pore nylon filter, Millipore, Brussels, Belgium) and centrifuged (5 minutes, 450 x g, 4°C), and cells were set to culture in DMEM containing 10% fetal bovine serum. Confluent cells were treated with BoNT-A (0.73 units/108 cells) 4 hours before being irradiated at 2 Gy. To assess the cell radiosensitivity, trypan blue exclusion dye method and a clonogenic cell survival assay were done. For the former, the cells were counted for viability 24 hours after irradiation. For the latter, the cells were washed and reincubated in the conditioned medium without drug 24 hours after irradiation. After a 7-day incubation in a humidified 5% CO2 atmosphere at 37°C, the dishes were stained with crystal violet, and colonies with >50 cells were counted. The experiments were carried out in triplicate.
Apoptotic activity
The apoptotic activity inside tumors was assessed by measuring the activity of caspase-3, a well-known effector involved in the apoptosis induced by chemotherapeutic or radiotherapeutic treatments. The activation of caspase-3 was measured by immunoblotting, and the results were confirmed by measuring the cleavage of poly(ADP-ribose) polymerase, a polypeptide cleaved during apoptosis process. Tumors were dissected, minced, put in an extraction buffer solution [10 mmol/L Tris, 1 mmol/L EDTA, 250 mmol/L sucrose, 0.1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L NaF, 1 mmol/L Na3VO4, supplemented with protease inhibitor cocktail (Complete Mini, Roche Applied Science, Mannheim, Germany; pH 7.4] and homogenized with a Potter-Elvehjem tissue grinder. The tumor homogenates were centrifuged at 10,000 x g for 20 minutes, and the supernatant fraction was saved for analysis. From these homogenates, protein concentration was determined by the Bradford protein assay (22). Equal amounts of proteins (50 µg) were subjected to SDS-PAGE [6% and 15% separating gels, respectively, for poly(ADP-ribose) polymerase and caspase-3 detection] followed by electroblot to nitrocellulose membranes. The membranes were blocked 1 hour in TBS buffer (pH 7.4) containing 5% powdered milk protein followed by an incubation of 2 hours with diluted antibodies in a fresh solution of powdered milk protein (1% w/v in TBS buffer). The membranes were washed and incubated for 60 minutes with a dilution of secondary antibody coupled with horseradish peroxidase. Anti-poly(ADP-ribose) polymerase and anti-caspase-3 rabbit polyclonal antibodies were diluted by 1:200 and goat anti-rabbit polyclonal antibody by 1:10 000. They were purchased respectively from Santa Cruz Biotechnology (Santa Cruz, CA) and Chemicon, Inc. (Temecula, CA). The quantification of the Western blot bands was done with by densitometry (Image Master V1.1, Pharmacia Biotech, Piscataway, NJ).
Myogenic tone measurements
Segments of the co-opted saphenous arteries (±2 mm in length) were carefully dissected. For each tumor, two adjacent segments were mounted in a multiwire myograph (610M, DMT, Aarhus, Denmark). Briefly, two 40-µm wires were threaded through the lumen of the vessel segment. One wire was attached to a stationary support driven by a micrometer, whereas the other was attached to an isometric force transducer. The bath of the myograph was filled with physiologic salt solution [120 mmol/L NaCl, 5.9 mmol/L KCl, 25 mmol/L NaHCO3, 17.5 mmol/L dextrose, 2.5 mmol/L CaCl2, 1.2 mmol/L MgCl2, 1.2 mmol/L NaH2PO4 (pH 7.4)], gassed, and maintained at 37°C. After mounting, vessels were maintained under zero force for 45 to 60 minutes. A passive diameter-tension curve was constructed. The vessel was set at a tension equivalent to that generated at 90% of the diameter of the vessel under a transmural pressure of 100 mm Hg. The viability of the vessels was assessed by measuring the contractile response to a depolarizing solution (physiologic salt solution where 100 mmol/L KCl replaced NaCl stoichiometrically). After washing, the vessels (n = 4) were incubated in the presence of BoNT-A (0.12 unit/mL) for 2 hours, whereas the matched controls (adjacent segments) were kept in physiologic salt solution + solvent. All vessels were then challenged with a high KCl solution (40 mmol/L KCl) to depolarize smooth muscle cells of the media and nerve endings, thereby activating the Ca2+-dependent release of neurotransmitter. The amplitude of the neurotransmitter release was estimated by measuring the relaxation to an 
-adrenoceptor blocker (phentolamine) or to a cholinergic antagonist (atropine) for noradrenaline and acetylcholine, respectively.
Statistical analysis
Results are given as means ± SE from n animals. Comparisons between groups were made with Student's two-tailed t test or two-way ANOVA where appropriate, and P < 0.05 was considered significant.
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-adrenoreceptor blocker (phentolamine) or to a cholinergic antagonist (atropine) for noradrenaline and acetycholine, respectively. We observed that in four vessels treated with BoNT-A, the relaxation to phentolamine was significantly smaller (52.4 ± 12.8% of the control) confirming that BoNT-A could interfere with the release of neurotransmitters (e.g., noradrenaline) and neurogenic vasoconstriction (Fig. 9). A similar experimental protocol using atropine failed to reveal a neurogenic acetylcholine-evoked contraction.
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Discussion |
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For the first time, using an experimental model dedicated to the study of the functionality of isolated vessels, we have shown that BoNT-A can modulate the vasoreactivity of vessels feeding tumors. This unique information should be evaluated in the context of the vascular architecture of tumors. As emphasized in the introduction, hastily formed immature tumor microvessels lack smooth muscle layers and are less sensitive to vasomodulations. However, it is not uncommon to find mature blood vessels with smooth muscle layers and neural junctions inside slowly growing tumors (1). It was previously confirmed that these arterioles remain sensitive to vasomodulation under tumor co-option but also are subject to major adaptations to the tumor microenvironment (26, 27). Here, we found that BoNT-A inhibits the release of noradrenaline, a major endogenous vasomodulator that is responsible for the maintained sympathetic vascular tone through activation of vascular smooth muscle -adrenoceptors. This is consistent with the opening of the tumor vascular bed that we observed in vivo. Even if we did not show an effect on the release of acetylcholine, we certainly cannot exclude such an effect. The lack of observation of this effect could result from the sparse cholinergic vascular innervation, the relatively short life of acetylcholine (rapidly destroyed by acetylcholine-esterase on its way from the synapse to the smooth muscle media) and/or from the relatively small number of muscarinic receptors present on the media. Overall, the effect on the release of neurotransmitters was sufficient to produce significant changes in the hemodynamics of the tumors.
The consequences of the modulations in the vasoreactivity of tumor vessels were remarkable in two ways. First, the accessibility of xenobiotics to the tumor was increased, as shown by the dynamic contrast-enhanced MRI study as well as the penetration of dyes in the tumor. Second, the oxygenation of these hypoxic tumor models was substantially increased after the treatment. Interestingly, the opening of the tumor vascular bed was observed over the whole tumor, as shown by both perfusion studies. This observation is consistent with the diffusion of BoNT-A in tissues. It was previously shown, using radiolabeled BoNT-A in animal models, that the toxin remains in the tissue injected, diffusing over a depth of 7 mm from the injection site (28). This diffusion is even larger in humans when using larger volumes for the injection (29). The practical consequence was that the injection in two discrete sites was sufficient to affect the whole vascular bed of this tumor model, whose volume was <1 cm3 at the time of the treatment. From the oximetry study, we also learned that the opening of the vascular bed was sufficient to improve the oxygen supply of the tumor, enabling these hypoxic tumor models to increase their pO2 from a radioresistant level to a radiosensitive level (below and above 5 mm Hg, respectively; refs. 18, 30). Moreover, this increase in tumor oxygenation was prolonged because the effect was already observed 1 day after the injection of BoNT-A and increased up to 3 days after the treatment.
To show the relevance of these findings for the treatment outcome, we carried out radiotherapy and chemotherapy experiments using suboptimal doses of the cytotoxic treatments. The response of the tumors was remarkably higher when the tumors received BoNT-A 3 days before the cytotoxic anticancer treatments. At this stage, our study consists of a proof-of-principle that the modulation of the vasoreactivity of the tumor vessels is a powerful strategy to increase the response to common anticancer treatments. Moreover, because BoNT-A is widely used in the clinic with a long-term established absence of toxicity (absence of systemic effect) when used appropriately (31), it is certainly valid to envision clinical trials with local administration of BoNT-A in easily accessible tumors to assess the relevance of the present study in human tumors. It should be emphasized that the dosage used in the present study is well within the range of dosage used in humans (1-100 units/kg). Interestingly, defining the issue of therapeutic gain using BoNT-A should not be a major source of concern. A potential clinical advantage of the combination of a sensitizing agent and radiation can only be foreseen if the potentiation of radiation response is higher in tumors than in normal tissues in the radiation field, indicating that the therapeutic ratio of the combined treatment is above unity. Classically, several normal tissue models are used to determine the whether an agent is responsible for toxicity on early responding tissue (e.g., intestinal regenerated crypt assay) or late responding tissues (e.g., leg contracture assay). However, in the case of BoNT-A, these assays are unlikely necessary as BoNT-A is administered directly inside the tumor with a very limited diffusion in the surrounding normal tissues. Even in this case, a radiosensitizing property on the normal tissue is unlikely as we showed that BoNT-A acts via an oxygen effect. Consequently, the radiosensitizing property will more than likely be higher for hypoxic tumor regions than for well-oxygenated tissues.
In summary, we have established that local administration of BoNT-A in the tumor significantly increases the tumor perfusion and oxygenation, most probably through an inhibition of neurotransmitter release and neurogenic contraction. We further showed that the opening of the vascular bed induced by BoNT-A offers a way to substantially increase the response of tumors to radiotherapy and chemotherapy. Further work is required to assess the usefulness in humans of this novel strategy to alleviate the resistance of tumors to cytotoxic treatments.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: R. Ansiaux is a Belgian National Fund for Scientific Research-Televie fellow. C. Baudelet is Scientific Research worker of the Belgian National Fund for Scientific Research. C. Dessy and O. Feron are Research Associates of the Belgian National Fund for Scientific Research.
6 P. Martinive, submitted for publication. 
Received 6/ 7/05; revised 10/ 5/05; accepted 12/14/05.
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, control group (n = 8 and 4 for FSAII and transplantable liver tumor, respectively). 
, treated group (n = 10 and 4 for FSAII and transplantable liver tumor, respectively). Note the significant increase in pO2 24 hours after the injection with a maximum on day 3. Points, mean; bars, SE. **, P < 0.01.
; n = 6), treated with vehicle (
; n = 5), treated with 20 Gy of radiation 3 days after the injection of the vehicle (
; n = 5), or treated with 20 Gy of radiation 3 days after the injection of BoNT-A (
