Nanoparticles target early-stage breast cancer metastasis in vivo

Despite advances in cancer therapy, treating cancer after it has metastasized remains an unmet clinical challenge. In this study we demonstrate that 100 nm liposomes target triple-negative murine breast-cancer metastases post intravenous administration. Metastatic breast cancer was induced in BALB/c mice either experimentally, by a tail vein injection of 4T1 cells, or spontaneously, after implanting a primary tumor xenograft. To track their biodistribution in vivo the liposomes were labeled with multi-modal diagnostic agents, including indocyanine green and rhodamine for whole-animal fluorescent imaging, gadolinium for magnetic resonance imaging (MRI), and europium for a quantitative biodistribution analysis. The accumulation of liposomes in the metastases peaked at 24 h post the intravenous administration, similar to the time they peaked in the primary tumor. The efficiency of liposomal targeting to the metastatic tissue exceeded that of a non-liposomal agent by 4.5-fold. Liposomes were detected at very early stages in the metastatic progression, including metastatic lesions smaller than 2 mm in diameter. Surprisingly, while nanoparticles target breast cancer metastasis, they may also be found in elevated levels in the pre-metastatic niche, several days before metastases are visualized by MRI or histologically in the tissue. This study highlights the promise of diagnostic and therapeutic nanoparticles for treating metastatic cancer, possibly even for preventing the onset of the metastatic dissemination by targeting the pre-metastatic niche.


Introduction
Breast cancer is the most prevalent cancer among women [1]. The stage at which the disease is diagnosed is an important predictor of prognosis. Up to 99% of patients diagnosed and treated for a confined primary breast cancer tumor will live beyond five years [2]. The prognosis of those diagnosed with a metastatic disease worsens dramatically, with only 23% of patients surviving beyond five years [2,3].
Metastasis is defined as one, or multiple, cancer lesions that spread to a tissue that is distant to the primary tumor.
The primary tumor sheds nearly one-million cancer cells per gram tumor every day into circulation [4][5][6]. A wellestablished theory is that these circulating cells colonize at distant sites, seeding metastases [6,7]. The main sites of breast-cancer metastases are the lungs, liver and bone [8]. As the metastases progress, they impair the function of these vital organs.
In this study, we assessed the ability of nanoparticles to target triple-negative breast cancer (TNBC) metastases in vivo . TNBC is characterized by the lack of expression of two hormone receptors (estrogen and progesterone) and a deficiency of human epidermal growth factor receptor 2 (HER2). These cell-surface receptors are utilized for targeting medicines to the cancerous lesions. In their absence, TNBC has limited treatment options and a poor prognosis [27].
Targeting nanoparticles to cancerous tissues can exploit physical or biological mechanisms [28][29][30][31][32][33][34], one of which is targeting the leaky vasculature in cancerous tissue [35,36]. In contrast to normal tissues, solid tumors undergo unregulated angiogenesis, providing the tumor with vasculature to supply the metabolic needs of the rapidly dividing cancerous cells [37]. The resultant blood vessels exhibit a discontinuous endothelial-cell lining, with gaps ranging from 200 to 2000 nm between individual endothelial cells [38][39][40]. The poor architecture of the tumor vasculature is an access point for therapeutic nanoparticles circulating in the blood [41]. In order to penetrate the tumor, the nano dimensions of the drug carriers must be smaller than the size of the pores in the vasculature. Experimental studies show that particles smaller than 150 nm are advantageous for tumor penetration [8,42]. Once the nanoparticles penetrate the tumor vasculature, they are taken up by cells [43], or are retained in the extracellular matrix. Nanoparticles are usually not trafficked out of the tumor region, because of the impaired lymphatic drainage in tumors [44][45][46]. This phenomenon, in which nanoparticles accumulate in cancerous tissues post intravenous administration, has been coined the enhanced permeability and retention (EPR) effect, or passive targeting [47][48][49].
In total, nearly 250 nanotechnologies have either been approved or are in advanced approval stages by the FDA and EMA for treating a wide range of diseases, including breast cancer [53][54][55][56][57][58][59]. For example, Doxil is a 100 nm PEGylated liposome formulation loaded with the chemotherapeutic agent doxorubicin, and is used in first-line breast cancer management [60]. PEG (polyethylene glycol) on the surface of the liposomes increases their half-life in the circulation and reduces the uptake of the nanoparticle by the mononuclear phagocyte system [48,61]. These liposomes were shown to target tumors in patients [48], while reducing life-threatening side-effects, such as cardiomyopathy [62]. Another clinical nanotechnology used for breast cancer treatment is Abraxane -130 nm albumin nanoparticles loaded with the microtubule inhibitor paclitaxel [63].
In this study, we examined whether intravenously injected PEGylated liposomes can target breast cancer metastases. We studied the effect of several disease conditions on nanoparticle accumulation at the metastatic site, including the size of the metastases, the presence or absence of a primary tumor alongside the metastases, and the size of the metastatic lesion.

Study design
This study compared the biodistribution of 100 nm liposomes to the primary tumor and metastasis, post intravenous injection. Three disease models were compared to mirror the clinical scenario: (1) tumor alone, mimicking the existence of a primary tumor; (2) metastasis alone, mimicking metastatic development after resection of the primary tumor; and (3) tumor plus induced lung metastasis, mimicking an advanced stage of the disease (figure 1).
The lipids were dissolved in chloroform. The solvent was then evaporated under reduced pressure at 55°C, 100 mbar, using a R-210 Rotorvaporator (Buchi, Switzerland). The dry lipid film was then hydrated with 5% w/v dextrose or phosphate buffered saline (PBS), depending on the entrapped molecule (see below), to reach a final lipid concentration of 50 mM.
To produce nanoscale vesicles, the liposomes were extruded (Lipex Extruder, Northern Lipids, Vancouver, Canada) in a stepwise manner at 60°C through polycarbonate membrane filters (Whatman, Newton, USA) with 400, 200, and 100 nm pores. Four extrusion steps were applied per filter type. After extrusion, the liposome suspension was dialyzed against 5% w/v dextrose or PBS solution using a 12-14 kDa dialysis tube to remove non-entrapped materials.
Liposome size distribution was measured by dynamic light scattering using a Zetasizer Nano ZSP (Malvern Instruments, UK). Samples were diluted 1-100 in the appropriate buffer. Sizing measurements were conducted at room temperature and the back-scattered light was detected at an angle of 173°. loaded with rare earths and contrast agents, and their biodistribution to triple-negative breast cancer (TNBC) metastasis, primary TNBC tumors, or both, were evaluated quantitatively and qualitatively in mice. Liposomes were loaded with traces of rare-earths and clinical contrast agents, including indocyanine green (ICG), rhodamine, gadolinium (Gd) and europium (Eu). The liposomal biodistribution to the cancerous tissues was evaluated quantitatively, using ICP, and qualitatively, by MRI and whole-animal fluorescent imaging. Liposomes were sized around 100 nm using dynamic light scattering (B) and cryoTEM microscopy (C). ICG-liposomes have a far-red fluorescent emission that can be detected in the cancerous tissue (D). Metastatic levels of Eu and Gd were measured analytically by ICP (E) or using MRI. Experimental TNBC lung metastases occur in BALB/c mice after injecting 4T1 cells intravenously (F). The metastatic lesions have a white, blister-like, appearance within the pink lung tissue.
CryoTEM imaging of the liposomes was performed as follows: lipid dispersions at concentration of 5 mM are prepared in a controlled-environment vitrification system at 25°C and 100% relative humidity and examined in a Philips CM120 cryo-electron microscope operated at 120 kV. Specimens were equilibrated in the microscope below -178°C, then examined in the low-dose imaging mode to minimize electron beam radiation damage, and recorded at a nominal under-focus of 4-7 nm to enhance phase contrast an Oxford CT-3500 cooling holder was used. Images were recorded digitally by a Gatan MultiScan 791 CCD camera using the Digital Micrograph 3.1 software package (Gatan).
ICG entrapment was determined using an Infinite 200PRO multimode plate reader (Tecan, Switzerland), excitation/emission 780/810 nm. For this, the liposomes were dissociated using the Bligh and Dyer method [67]. Briefly, liposomes were diluted in the volume ratio of liposome: chloroform:methanol:water of 0.8:2:2:1 in order to obtain two separate phases; (1) an organic phase that contained all the lipids and (2) an aqueous phase that contained all the watersoluble materials. The absorbance of the ICG in water phase was measured.
Eu/Gd entrapment was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES, 5100 system, Agilent Technologies, US). The amount of metal was calculated using a calibration curve according to Europium/Gadolinium ICP standards (Sigma-Aldrich, Rehovot, Israel).

Cell culture
A TNBC cell line, 4T1 (ATCC), was used to induce primary tumors and metastasis. Cells were cultured at 37°C in a humidified atmosphere and 5% CO 2 in air. RPMI 1640 medium, supplemented with 10% heat inactivated Fetal Bovine Serum, 1% v/v Penicillin-Streptomycin solution (10 000 U ml −1 of Penicillin G Sodium Salt and 10 mg ml −1 of Streptomycin Sulfate), and 1% v/v L-Glutamine (all from Biological Industries, Beit Haemek, Israel). The cells were washed thoroughly with PBS at 4°C before injection.

In vivo
All animal studies were approved and performed according to the guidelines of the Institutional Animal Research Ethical Committee at the Technion-Israel Institute of Technology. Animal welfare was monitored daily both by the researchers and staff veterinarians.
Eight to ten-week-old (∼20 gr) BALB/c female mice (Harlan laboratories, Jerusalem, Israel) were used for all animal models.
Combined primary tumor and metastasis. 2×10 5 4T1 cells in 100 μl of PBS were injected IV, to form metastases; and an additional 7×10 5 4T1 cells in 100 μl of PBS were injected SC, to form the primary tumor. Both the primary tumor and metastasis formed ∼14 d post injection.
Spontaneous metastasis and the pre-metastatic niche. 5×10 5 4T1 cells in 100 μl of PBS were injected SC to the rear right flank to induce the formation of a primary tumor, metastasis evolved spontaneously in the lungs 3-4 weeks later. During the time between the induction of the primary tumor and the histological detection of metastases, a premetastatic niche is formed in the lung, conditioning this tissue to harbor the disseminated metastatic cells.

Quantitative biodistribution analysis
Once the metastases evolved mice were injected intravenously with 300 μl Gd-loaded liposomes (100 nm, 50 mM lipid). Twenty-four hours after the injection, the mice were sacrificed and the metastatic lesions were excised and analyzed for the liposome presence using Gd ICP analysis.

MR imaging
MRI scans were acquired using a 1 T micro-MRI (Aspect M2 MRI, Aspect Imaging, Modi'in, Israel) equipped with a cylindrical radiofrequency volume coil (35 mm inner diameter) for signal excitation and reception. During imaging the animal was placed in a coronal position on a holder and kept anaesthetized with 0.5%-1.5% Isoflurane, supplemented with oxygen (0.8 l min −1 ) via a facial mask. The respiratory rate of the mice was monitored using a pressure pad (Aspect M2, Aspect Imaging, Israel) located on the abdomen.
Image processing was performed using MRI image analysis and a MATLAB based software (MRItool). For each image, regions of interest in the lungs were manually segmented to measure the total signal intensity, average signal, and area.

Fluorescent imaging
100 μl ICG liposomes (100 nm, 50 mM lipid) or the same amount of free (non-liposomal) ICG was injected IV via the mouse tail vein. Animals were sacrificed at time intervals of 0, 3, 6, 12, 24, and 48 h post injection, and the metastatic lungs and or tumors were excised and imaged using a MaestroEX in vivo fluorescent imaging system equipped with an excitation filter at 690 nm. A 750 nm long-pass emission filter was applied to prevent the interference of excitation light with the charge coupled device (CCD) camera. In vivo spectral imaging from 780 to 820 nm (in 10 nm steps) was carried out with an exposure time of 5000 ms for each image frame. Background was removed by using the spectral unmixing software (Maestro 2p20).
Gd-rhodamine liposomes (enabling dual imaging modalities; Gd-MRI; and rhodamine-fluorescent) were injected IV (300 μl); 24 and 48 h after the injection mice were scanned by MRI, then the lungs were resected and analyzed histologically to visualize the exact tissue deposition of the liposomes [68].

Quantification of Eu and Gd delivery to tumor and metastasis
After imaging, organ samples were washed thoroughly with PBS, dried and weighed. Europium (Eu) or Gadolinium (Gd) content in the organs was quantified using ICP. For this purpose samples were carbonized at 500°C for 5 h and their ash was dissolved in nitric acid 1% v/v (Bio Labs, Israel).

Histology
After sacrificing the mice, the lungs were removed and kept in 10% neutral buffered formalin (Sigma) at room temperature. Later, the tissues were paraffin embedded and sectioned into 5 μm slides. Slides were stained with hematoxylin and eosin (H&E) to evaluate the general morphology and further stained with 4′,6-diamidino-2-phenylindole (DAPI) for fluorescence imaging.
All data groups we analyzed statistically using an unpaired, two-sided Student's t-test. Confidence level was taken to be P=0.05.

Results and discussion
Breast cancer is a global epidemic [69], of which the metastatic condition is the most lethal [70]. Nearly 250 000 patients are diagnosed annually with metastatic breast cancer in the US alone, and more than 40 000 die of this condition [71]. In developing countries too, numbers of breast cancer patients are increasing as life span, awareness to cancer and medical infrastructures are improving [72,73]. Diagnosing and treating metastatic TNBC, a subtype of breast cancer, is especially challenging, due to the lack of biological drugs that can target this condition [74].
In this study, we investigated whether 100 nm liposomes can target TNBC metastases in vivo.
Three murine disease models that mirror relevant clinical scenarios were studied: (1) Targeting a primary tumor alone -modeling the early stages of the disease; (2) Targeting metastasis alone-modeling the metastatic disease after a primary tumor has been resected; and, (3) Targeting both the metastases and a primary tumor simultaneously-modeling a metastatic disease with a non-resected primary tumor ( figure 1(A)). In addition, two complementary experimental approaches were employed to induce metastases. The first is 'experimental metastasis', in which triple-negative (4T1) cancer cells are injected intravenously to induce lung metastases in BALB/c mice [75][76][77]. Experimental metastasis is commonly used because of its experimental reproducibility [75,78]. Moreover, we studied nanoparticle biodistribution to a 'spontaneous metastatic model', in which a primary 4T1 tumor xenograft was implanted in mice, spontaneously seeding distant metastases [75].
We first studied the ability of 100 nm liposomes to accumulate in primary breast cancer tumors. For this, we compared the biodistribution of liposomes loaded with ICG (a clinical contrast agent) to an equal amount of free ICG injected intravenously ( figure 2(A)). Animals injected with the free dye had a maximal fluorescent signal in the primary tumor 3 h post intravenous administration, after which the signal decayed ( figure 2(B)). In comparison, the accumulation of liposomes peaked in the tumor after 24 h. The intensity of the fluorescent signal in tumors of mice injected with the ICG-liposomes was 4.5-fold greater than the maximal signal in tumors of mice injected with the free dye [79]. These data confirm previous findings that nanoparticulate systems have an advantage over the free small molecules in accumulating in primary tumor [35,48,80]. The mechanism governing nanoparticle biodistribution to primary tumors has been shown to be dependent on the nanoparticles' half-life in circulation as well as their ability to extravasate through the compromised tumor vasculature [81][82][83][84][85]. PEG conjugated to the corona of the liposomes extends the liposomes' circulation time in vivo [86], and their nano-dimensions facilitates extravasation through the compromised tumor vasculature [87,88].
After confirming the liposomes were detected in the primary tumor, we tested whether 100 nm liposomes can also be detected in the metastasis post intravenous administration. For this, experimental TNBC metastasis was induced in the lungs of BALB/c mice. Liposomes containing ICG or free ICG were injected intravenously, and the mice were imaged over 48 h. A fluorescent signal was detected in the metastatic lungs after the injection ( figure 2(A)). Similar to the primary tumor, the free dye biodistribution in the metastases peaked after 3 h and the nanoparticles peaked at 24 h post intravenous injection ( figure 2(C)). The similar kinetics in the primary tumor and metastases, suggest that a similar mechanism governs accumulation in both, namely, extravasation of the nanoparticles from the circulation into the metastatic lesions [89,90]. If the patient is diagnosed after cancer has already metastasized, the primary tumor is usually not resected [91][92][93][94][95][96]. To this end, we studied the liposome accumulation in metastasis in the presence, or absence, of a primary tumor.
The presence of a primary tumor decreased liposome accumulation at the metastatic site ( figure 2(D)). The accumulation of the liposomes in metastases, in the absence of a primary tumor reached 3.4% ±0.3% of the injected dose per gram tissue. In comparison, when the primary tumor coexisted with the metastasis, the accumulation of the liposomes in the metastasis declined to 1.6%±0.6% ( figure 2(D)). This is explained by a sink effect the primary tumor has on liposomal biodistribution, in which liposomes are deterred from the circulation to the primary tumor reducing their accumulation in the metastasis [5,38,[97][98][99][100][101]. Furthermore, previous studies have demonstrated that the primary tumor suppresses vascular progression in the disseminated metastasis [102][103][104][105][106], thereby possibly reducing the ability of liposomes to extravasate from the capillaries into the metastasis [107]. While in the clinical scenario, there is controversy whether resection of the primary tumor in patients with metastatic breast cancer improves prognosis [91][92][93][94][95]. Our data suggest that nanoparticles target metastases more effectively in the absence of a primary tumor.
While fluorescent imaging is used mainly for pre-clinical cancer research, MRI is a common imaging modality for diagnosing cancer clinically. We tested whether nanoparticles can also be detected in the metastatic lesions by MRI and their Differences between two means were tested using an unpaired, two-sided student's t-test.  Before the metastases can be detected histologically or by imaging, the pre-metastatic lungs are conditioned to harbor the metastatic cells. We examined the ability of liposomes to target the pre-metastatic niche in the lungs, i.e. the lung tissues before metastasis could be detected in them by imaging. Mice were scanned throughout the study using MRI: before inducing the primary tumor (B), on day 8 (C), and day 15 (D). None of the mice presented detectable metastases by MRI (B)-(D). Gd-loaded liposomes were administered intravenously after the MRI scans. Twenty-four hours post injection the mice were sacrificed, lungs were excised and evaluated histologically for the presence of micro-metastases that were not detected by MRI, and by ICP for a quantitative analysis of the presence of Gd-liposomes. ICP analysis indicated that the accumulation of Gd-liposomes increased in the lungs as the time since induction of the primary tumor increased (F). In some mice, micro-metastases were visualized histologically (G), even though they were undetectable by MRI (E). Lungs diagnosed histologically with micro-metastases (G) had greater amounts of Gd-liposomes compared to pre-metastatic lungs (F). Data are shown as the mean ± SDE of n=2 (control), n=6 (day 8), n=4 (day 15), n=2 (mets initiating); * p=0.074. Differences between two means were tested using an unpaired two-sided Student's t-test. presence in the metastasis subsequently confirmed histologically by fluorescent-histology of the tissue. For this, we replaced the fluorescent ICG inside the liposomes with an MRI contrast agent-gadolinium (Gd) [68]. In addition, we doped the liposome membrane with sulforhodamine-lipids, generating a multi-modal (fluorescence and MRI) diagnostic liposome. A bright co-localization signal of the Gd-liposomes inside the metastatic lesions was recorded by MRI 24 h after the intravenous injection (figures 3(A)-(D)). The same experiment carried out in healthy mice showed no Gd-liposomes signal in the lungs (figures 3(E)-(H)). The accumulation of Gd-liposomes in the metastatic lesions was confirmed histologically. For this, the metastatic lungs were resected and stained with H&E. The liposomal rhodamine signal coincided with metastatic lesions (figure 3(J)). A fluorescent signal was not found in the lungs of healthy animals ( figure 3(L)). These data confirmed our initial whole-animal fluorescent imaging findings that 100 nm liposomes injected intravenously accumulate in the metastatic tissue (figure 1), and indicated that liposomes can be used as MRI contrast agents for imaging metastasis.
One major requirement from nanoparticles is the ability to target small metastatic lesions [101,108]. For this, we quantified the accumulation of nanoparticles in metastases of different sizes: smaller than 2 mm, between 2 and 4 mm, and larger than 4 mm. Metastases were induced experimentally in the lungs of BALB/c mice by an intravenous injection of 4T1 cells. Once the metastases were detected by MR imaging, mice were injected 100 nm Gd-loaded liposomes intravenously. Twenty-four hours later, the mice were sacrificed, lungs were excised, and the individual metastatic lesions were resected and grouped according to size (figures 3(M), (N)). All three metastasis groups (<2 mm, 2-4 mm, and >4 mm) had significant Gd-liposomal presence. Higher levels of nanoparticle accumulation were detected in the smallest metastasis, reaching 3.1%±0.2% of the injected dose per gram tumor in lesions smaller than 2 mm in diameter, compared to 1.5%±0.02% and 1.0%±0.01% in the 2-4 mm and >4 mm groups, respectively (figure 3(M)). The higher accumulation of nanoparticles in the smaller lesions is explained by higher vascular density and permeability in the smaller lesions [109][110][111]. Similar tendencies, in which nanoparticle' accumulation per gram tumor increases as the lesion size decreases, was reported also in primary tumors [112]. It should also be noted that 2 mm lesions are below the limit-of-detection of most clinical imaging systems, thereby targeting imaging agents to such small lesions can facilitate early metastatic detection [113].
Before metastases colonize in a healthy organ, biochemical signals are sent from the primary tumor, to enable the metastatic cells to adhere and progress at the distant metastatic site [7,114]. During this process the vasculature at the destination organ becomes leaky [115]. Since lungs are a primary site of metastatic dissemination, we tested whether nanoparticles can be detected in the pre-metastatic lungs [8]. For this, a primary tumor was implanted in the rear flank of BALB/c mice ( figure 4(A)). One and two weeks postimplantation of the primary tumor the mice were scanned using MRI. No metastatic malignancy was detected by MRI in the lungs (figures 4(B)-(E)). At these time points, mice were injected with 100 nm Gd-liposomes intravenously. Twenty four hours later (day 9 or day 16), mice were sacrificed and the lungs were excised and fixated. All the histological slides were evaluated as normal (no evidence of metastasis), except for two slides that showed foci of isolated tumor cells (micro-metastasis, <0.2 mm, figure 4(G)). After the histological analysis, the tissue blocks were digested and analyzed for the presence of Gd-liposomes using ICP. A gradual elevation in the levels of Gd was detected in the premetastatic lungs compared to the healthy lungs over time ( figure 4(F)). Lungs that were evaluated histologically with micro-metastasis had greater concentrations of Gd-liposomes, reaching 0.059% of the injected dose. Having this said, using an unpaired two-sided student t-test, we did not reach the statistical level of significance we hoped for nanoparticle detection in the pre-metastatic niche (p=0.074 versus p<0.05). This is explained by the inefficiency of the metastatic process, and the limit-of-detection of the existing analytical tools. In metastasis, only 0.01% of the circulating tumor cells colonizes at the metastatic site and form viable metastases [4-6, 9, 116-118]. Respective to the injected nanoparticles, 0.01% of the injected dose is 1.2 nM Gd, which is near the limit of detection of Gd by ICP (0.3 ppb). This explains the lower level of confidence of detection in the premetastatic niche, rendering the development of more accurate systems for studying nanoparticle distribution to the premetastatic niche.
These results suggest that nanoparticles accumulate preferentially in the pre-metastatic lungs. This is explained by biological conditioning of the lung tissue for the emergence of metastases [7,9,114,115,[119][120][121]. During this process the vasculature at the target organ becomes hyper-permeable [115], enabling nanoparticle accumulation in the pre-metastatic lungs [120,122,123].
Ultimately, when a patient is diagnosed with a primary tumor, the main concern is preventing future metastatic dissemination. Our data suggest that even before having imaging evidence of the existence of metastatic lesions in an organ, the pre-metastatic niche may be targeted and treated with nanoparticles; thereby, possibly, preventing future metastatic progression at this site.
In summary, this study demonstrates that 100 nm liposomes target triple negative murine breast cancer metastasis. Our findings support the use of nanotechnology for imaging and targeting medicines to metastases and ultimately to the pre-metastatic niche.
Ministry of Science Technology and Space-Office of the Chief Scientist (3-11878); the Israel Science Foundation (1778/13); the Israel Cancer Association (2015-0116); the German-Israeli Foundation for Scientific Research and Development for a GIF Young grant (I-2328-1139.10/2012); the European Union FP-7 IRG Program for a Career Integration Grant (908049); a Mallat Family Foundation Grant; AS acknowledges Alon and Taub Fellowships.
We wish to thank the dedicated staff of the Technion Pre-Clinical Centers for their great help in conducting the in vivo experiments.
Ms Goldman wishes to thank the Technion Interdepartmental Biotechnology Program for its generous support.
Finally, we would like to thank Integremed Medical Communications and Mrs Rebekah Roiter for editing this manuscript.