M Gaillet

C Sulmont-Rossé

S Issanchou

C Chabanet

S Chambaron

Priming effects of an olfactory food cue on subsequent food-related behaviour

Keywords: Priming, Food choice, Odour, Olfactory food cue, Implicit concept activation

Studies in cognitive psychology have highlighted a link between perception and action, by revealing the non-conscious influence that a cue can have on thinking and doing. The present study aimed at exploring whether an olfactory food cue could have an impact on food choices.

We chose fruity odours as olfactory food cues, in order to examine if this kind of olfactory cue could lead individuals to choose fruit and vegetables. In the first experiment, 58 participants were assigned randomly to either a control or a melon-scent condition. In the melon-scent condition, they were unobtrusively exposed to a melon odorant in a waiting room, while in the control condition the room was non-odorized. Then, they all performed a lexical decision task and a task involving choice from a menu. The results showed that participants in the melon-scent condition answered faster only for the word 'melon' in comparison with other tested words. Moreover, participants in the melon-scent condition were more likely than control participants to choose starters with vegetables, but not main courses or desserts with fruit and vegetables. Seventy participants took part in the second experiment, in which we replicated the same protocol, but using a pear odorant as the cue. The results showed that participants in the pear-scent condition were significantly more likely than those in the control condition to choose desserts with fruits, but not starters or main courses with vegetables. These results indicate that the non-conscious perception of a fruity odour may activate a 'fruit and vegetables' concept, but also a concept of the context of consumption. Moreover, they support the idea of priming effects 'specific to a food cue'.

Introduction

In cognitive psychology, it is now well-established that the perception of a stimulus, whether consciously/attentively processed or not, can implicitly activate high-level concepts that may influence behaviour, and not only trigger direct effects (like flight or avoidance, if the stimulus corresponds to an alarm signal for example). These implicit concept activations can be explored with a paradigm called priming [START_REF] Schacter | Implicit knowledge -new perspectives on unconscious processes[END_REF]. In the first phase, participants are exposed incidentally to a stimulus called prime, which can belong to any sensory modality (visual, auditory, olfactory Etc.). Exposure to the stimulus is then inconspicuous and 'forced'.

During this exposure, mental representations related to the prime are activated (Schneider, & Shiffrin, 1977;Shiffrin, & Schneider, 1977). In the test phase, the non-conscious effects of this activation are evaluated using indirect tests. A priming effect results in an increase or a decrease in accuracy or performance during an indirect test, even though the participant has no conscious motivation to do so. Priming has been used in several fields of investigation, and is notably well described in the visual and semantic domains [START_REF] Aarts | The silence of the library: environment, situational norm, and social behavior[END_REF][START_REF] Aarts | Goal contagion: perceiving is for pursuing[END_REF][START_REF] Bargh | Automaticity of social behavior: direct effects of trait construct and stereotype activation on action[END_REF][START_REF] Bargh | The automated will: nonconscious activation and pursuit of behavioral goals[END_REF][START_REF] Baros | Automatic activation of impression formation and memorization goals: nonconscious goal priming reproduces effects of explicit task instructions[END_REF][START_REF] Custers | Positive affect as implicit motivator: on the nonconscious operation of behavioral goals[END_REF], 2007;[START_REF] Kawakami | Effect of social category priming on personal attitudes[END_REF][START_REF] Macrae | Help, I need somebody: automatic action and inaction[END_REF]. Three generalisations can be made about these studies. Firstly, priming effects occur if mental representations related to the prime and links between these representations pre-exist for participants, and if these links are strong enough. Secondly, the stronger the links, the more apparent the effects of priming should be. Thirdly, if the priming effect corresponds to the production of a particular behaviour, participants must be in a context where the expression of this behaviour of interest is relevant.

To date, very few studies have used the priming paradigm to investigate the olfactory modality, but one striking example is Holland et al.'s study [START_REF] Holland | Smells like clean spirit[END_REF]. In this experiment, participants were first asked to fill in a questionnaire in a booth with or without the odorant of a citrus-scented cleaner. Then, they performed a lexical decision task, in which they had to determine as quickly and as accurately as possible, if a letter-string appearing on a computer screen corresponded to an existing word or to a 'nonword' (i.e. a letter-string with no meaning). When exposed incidentally to the citrus odorant, participants showed shorter reaction times for cleaning-related words in the lexical decision task. Then, they moved to another room and were asked to eat a crumbly biscuit. Results showed that participants removed the biscuit crumbs more often in the scent condition even though they had not noticed the citrus odour in the booth. These findings suggest that (1) the non-conscious perception of a citrus odour creates a semantic association; (2) this semantic association activates the behavioural concept of cleaning; and (3) the odour impacts behaviour even if people are not conscious of it. This study illustrates the existence of a link between perception and action by revealing the non-conscious influence that olfactory cues can have on thinking and doing [START_REF] Dijksterhuis | The perception-behavior expressway: automatic effects of social perception on social behavior[END_REF].

In the food domain, studies using the priming paradigm have already been conducted, and have been largely devoted to the specificity of restrained eaters' responses to food cues [START_REF] Anschutz | Exposure to slim images in mass media: television commercials as reminders of restriction in restrained eaters[END_REF][START_REF] Coelho | Wake up and smell the cookies. Effects of olfactory food-cue exposure in restrained and unrestrained eaters[END_REF][START_REF] Fedoroff | The effect of pre-exposure to food cues on the eating behavior of restrained and unrestrained eaters[END_REF], 2003;[START_REF] Fishbach | Leading us not unto temptation: momentary allurements elicit overriding goal activation[END_REF][START_REF] Papies | Goal priming and eating behavior: enhancing selfregulation by environmental cues[END_REF][START_REF] Papies | Pleasure in the mind: restrained eating and spontaneous hedonic thoughts about food[END_REF], 2008[START_REF] Köster | Diversity in the determinants of food choice: a psychological perspective[END_REF][START_REF] Stroebe | Why dieters fail: testing the goal conflict model of eating[END_REF]. In one of their experiments, [START_REF] Fishbach | Leading us not unto temptation: momentary allurements elicit overriding goal activation[END_REF] assigned women concerned about their weight to a diet prime condition or a control condition. In the diet prime condition, participants were brought into a room containing nutrition, exercise and dietrelated magazines. In the control condition, participants were brought into another room that merely contained a variety of magazines about the economy and geography of the United States. All of the participants performed a lexical decision task, and then chose a gift, either a chocolate Twix® bar or an apple. Results showed that the diet-primed individuals had shorter reaction times for the target word 'diet', suggesting the neural activation of a 'diet' related concept. Furthermore, they chose an apple as a gift more frequently than did participants in the control condition.

Our study was designed in the same manner, but an olfactory prime was used. The aim was to highlight a link between the perception of a fruity odour and subsequent food-related behaviour, through the activation of the concept 'fruit and vegetables'. In the priming phase, participants were first asked to complete a 'lure' categorisation task in a room with or without a fruity odorant. Then, they performed two indirect tests. The first test was a lexical decision task, including non-words and different types of words, among which some were related to fruit or vegetables (target words), some were related to high-energy foods, and some were not related to food. The second test was a menu task that measured intentions of food choices between fruit and vegetables items and high-energy food items. We hypothesised that individuals exposed to a fruity odorant may show higher reaction speeds for fruit and/or vegetable-related words in the lexical decision task, and higher frequency of fruit and/or vegetables choices on the menu card compared with individuals in the control condition. We ran two experiments using an olfactory prime, the first with a melon odour and the second with a pear odour.

Experiment 1

For this first experiment, a melon odorant was chosen as the olfactory prime. According to [START_REF] Chrea | Semantic, typicality and odor representation: A cross-cultural study[END_REF], the odour of melon was rated as being highly typical of the fruit category which means it is a good choice as a prototypic fruity odour. A preliminary experiment run with 23 participants different from those in the present experiment confirmed the result obtained by Chrea et al., namely that the odour of melon was rated as being highly typical of the fruit category: the mean rating on a 10-cm scale was M = 8.9; SE = 0.34.

Material and methods

Participants

Fifty-eight participants were recruited and randomly assigned to either a melon-scent condition (19-43 years old; mean age = 28 years; 10 males and 19 females) or a control condition (19-47 years old; mean age = 27 years; 10 males and 19 females). To participate in the study, candidates needed to possess a normal sense of smell, normal vision and have normal body weight (Body Mass Index between 19 and 25 kg/m²). Candidates with dyslexia, food allergies or on a diet were excluded, as were those who had already taken part in a memory or priming study. In order to ensure that participants were unaware of the real purpose of the experiment (i.e. studying olfactory priming), they were told that the experiment aimed to study how people categorise environmental information. They were invited for a one-hour session during lunch-time (i.e. from 12:00 till 13:00). They were asked not to wear perfume, and not to smoke during the hour preceding the session. The experimental protocol was approved by the Comité de la Protection des Personnes Est I (Research Ethics Committee) of Dijon. In accordance with the procedures of this regulatory body, the participants received written and oral information about the study before signing a consent form. In return for their participation, they received a €10 voucher.

Olfactory stimulus

The melon food aroma sold by Meilleur du Chef© was selected on the basis of two preliminary experiments run with participants different from those in the present experiment.

In the first experiment, 24% of 22 participants identified this aroma correctly during a free identification task, and 100% identified it correctly during a 4-alternative-forced-choice identification task. In the second experiment, 23 participants rated the melon aroma on a 10cm scale as highly familiar (M = 8.1; SE = 0.52), and 48% declared that the aroma evoked the desire to consume fresh fruit and/or vegetables. The melon odorant was encapsulated, and was diffused with a scent diffuser (Scentys4 #PSIA-J-000018 v.1.00, Scentys©). Two 60-s puffs were diffused in the waiting room 30 min before the session began. Following the procedure, the odour intensity was very low in the room so that participants would not consciously notice the odour. This was checked with seven members of the laboratory staff, who were not informed about the presence of this aroma. These people were not included in the main experiment.

Procedure

All of the participants were seated for precisely 10 min in a waiting room where they performed a 'lure' categorization task. This waiting room was non-odorized in the control condition and melon-odorized in the scent condition. The 'lure' task consisted in sorting vehicles (cars, bikes, trains etc.) on the basis of their similarity. An experimenter checked that the 10 min spent in the waiting room were quiet, and that the participants did not talk to each other. Then, they were taken to the test room and seated in individual booths. First of all, they completed a lexical decision task during which they had to indicate as quickly and accurately as possible whether a letter-string appearing on a computer screen was an existing word or not. Following this, they were asked to choose a starter, a main course and a dessert from a list presented as a 'restaurant menu' (menu task). Finally, participants were asked to complete two questionnaires, an 'Investigation' Questionnaire to ascertain that they had not suspected the real goal of the study, and to check that they had not noticed or identified the prime in the waiting-room; and the Three-Factor Eating Questionnaire, to determine if there were some restrained eaters among the participants [START_REF] Stunkard | The three-factor eating questionnaire to measure dietary restraint, disinhibition and hunger[END_REF].

Lexical decision task

The task included a familiarisation phase during which participants performed six trials, and an experimentation phase during which they performed 96 experimental trials in a random order. This phase was interrupted with a break every 32 trials. Each trial proceeded as follows: participants were asked to stare at a cross in the middle of the computer screen for 2 s, then a letter-string appeared in the centre of the screen until they answered by pressing a 'yes' or 'no' key on the keyboard, and a blank screen finally appeared for a break of 1 s before the next trial. The 96 experimental trials included 48 pronounceable non-words (plour, balbu, coumete for example), and 48 words. Among these 48 real words, 24 were neutral and non-food related (e.g. French words for 'home', 'suit', 'clear'); 12 were related to high-energy foods (e.g. French words for 'cream', 'cake', 'sugar'); and 12 were related to fruit or vegetables (e.g. French words for 'salad', 'apple', 'apricot'), including the word 'melon'. The letter-strings were chosen by taking into account the number of letters (5-7), the number of syllables (as many strings with 1, 2 or 3 syllables in each categorynon-words, neutral words, high-energy-food-related words and fruit and vegetable-related words), and by respecting the frequency of the occurrence of letters in the French language for non-words.

Stimulus presentation, timing, and data collection were implemented on a PC equipped with a 14-inch LCD colour screen, using dedicated software developed by a UMR CSGA engineer.

The participants were seated in front of the screen at a typical viewing distance of about 65 cm.

Menu task.

For each course category of a typical French meal (starter, main course and dessert), 10 choices were proposed, 5 items corresponding to high-energy foods vs. 5 items corresponding to foods containing fruit and/or vegetables (Table 1). Participants were asked to select one item for each course category. The selection of the menu items had previously been validated by a preliminary experiment run with 14 subjects who did not participate in the present experiment. Hedonic ratings were recorded for each item using a 10-cm scale anchored with the statements "I am not going to like it at all"/"I am going to like it very much". Results showed no significant difference between the 'fruit and vegetables' items and the 'high energy' items (starter: F (1,117) = 0.09; p = .78; main course: F (1,117) = 0.01; p = .92; dessert:

F (1,117) = 2.46; p = .16
). It should be noted that none of the items was related to the food 'melon'. In fact, items were chosen in order to highlight an indirect link between the exposure to the melon odorant and the intentions of choice.

[Insert table 1 about here]

'Investigation' Questionnaire

Participants were asked to write down in a few words what they believed to be the goal of the study they had just taken part in, if something had surprised them during the study, and if so, if they thought that it could have influenced their performance in any of the tests.

Three-Factor Eating Questionnaire

We used the French validated version of the Three-Factor Eating Questionnaire [START_REF] Stunkard | The three-factor eating questionnaire to measure dietary restraint, disinhibition and hunger[END_REF], which includes three factors: dietary restraint, (21 items), disinhibition (16 items), and hunger (14 items).

Data analyses

With regard to the lexical decision task, incorrect responses ('no' for real words, or 'yes' for non-words) were excluded from the analyses (2.2%). Individual reaction times were transformed using a 1/Y transformation (reaction speed). This transformation improves the symmetry of the distributions [START_REF] Ratcliff | Methods for dealing with reaction-time outliers[END_REF]. Consequently, outliers disappeared after transformation, and variance homogeneity was obtained. This avoided data suppression used classically in lexical decision tasks to remove outliers. With regard to the menu task, the proportion of 'fruit and vegetables' choices was determined for each group and for each dish category (starter, main course and dessert).

Statistical analyses were conducted using SAS/STAT® statistical software package version 9.1 (SAS Institute Inc., Cary, USA). For the reaction speed, all of the analyses of variance (ANOVA) were performed with the general linear model procedure of SAS (PROC GLM).

Post-hoc comparisons of means were computed for each significant factor by using the 'leastsquare means' statement of the general linear model procedure. All of the results reported here were significant at a level of 0.05 unless otherwise stated. Means (M) are given with associated standard error (SE). As far as the proportion of fruit and vegetable choices is concerned, a logit model (generalised linear model with binomial distribution and logit link) was performed with the generalised linear model procedure of SAS (PROC GENMOD).

Results

One participant in the melon-scent condition remembered explicitly smelling an odour in the waiting room and was excluded from all the analyses.

Lexical decision task

Individual means of reaction speeds were computed for each word category (non-words, neutral words, high-energy foods related words and fruit and vegetables related words). These means were then submitted to an ANOVA with condition (control; melon-scent), word category and the interaction between the two as fixed factors, and subject (condition) as a random factor. Results showed a significant word category effect [F (3,165) = 89.37; p < .001],

with participants answering more slowly for non-words than for words (Fig. 1). No significant condition effect was observed [F (1,55) = 0.13; p = .72], nor a significant condition * word category interaction [F (3,165) = 0.65; p = .59], which ruled out a 'general' priming effect (i.e. higher reaction speed for all fruit and vegetable-related words).

[Insert figure 1 about here]

To further check the existence of a priming effect 'specific to a food cue', a similar analysis was conducted on the two-syllable letter-strings, to compare mean reaction speeds for the word 'melon', with the mean reaction speeds for the other fruit and vegetable-related words, the high-energy-food-related words, the neutral words and the non-words (Fig. 2). The ANOVA revealed a significant condition * word category interaction [F (4,219) = 2.45; p < .05].

Post-hoc analyses showed that reaction speed for the word 'melon' was significantly higher in the melon-scent condition than in the control condition (p < .01). Furthermore, participants in the melon-scent condition answered faster for the word 'melon' than for the other fruit and vegetable-related words (p < .01). No such difference was observed in the control condition (p = .30). The same analysis performed for each of the other two-syllable fruit and vegetablerelated words did not reveal any such effect for these words.

[Insert figure 2 about here]

Choices on a restaurant menu card

To compare the proportions of 'fruit and vegetables' choices vs. 'high-energy food' choices, a logit model was performed by course category, with condition (control; melon-scent) as a factor, and unilateral tests. Results showed no significant difference for the main course (deviance = 0.14; df = 1; p = .35;) or the dessert (deviance = 0.00; df = 1; p = .47), but a tendency for the starter (deviance = 2.49; df = 1; p = .06). In fact, participants primed with the odour of melon tended to choose starters with vegetables more often than did participants in the control condition (Fig. 3).

[Insert figure 3 about here]

Three-Factor Eating Questionnaire

In accordance with previous research, dietary restraint was treated dichotomously, with female participants classified as restrained eaters if they scored 10 or above and male participants classified as restrained eaters if they scored 7 or above [START_REF] Tepper | Diet and physical activity in restrained eaters[END_REF]. A Chi-square analysis revealed no difference between the percentage of restrained eaters in the melon-scent condition (38%) and the percentage of restrained eaters in the control condition (21%;  2 = 1.83; p = .18). It should be noted that restrained eaters were not significantly more likely than unrestrained eaters to choose starters with vegetables during the menu task (71% of the restrained eaters chose a starter with vegetables vs. 58% of the unrestrained eaters;  2 = 0.73; p = .39).

Discussion

This first experiment was designed to determine whether non-conscious exposure to a melon odour might activate a 'fruit and vegetables' concept, which would then have an impact on reaction speed for fruit and vegetable-related words in a lexical decision task, and on the frequency of fruit and vegetables choices on a restaurant menu card. In fact, regarding the results of the lexical decision task, it seems that exposure to a melon odour rather activates a 'melon' concept. Participants in the melon-scent condition answered faster for the word 'melon' than did control participants, while no such effect was observed for the other fruit and vegetable-related words. However, regarding the result of the menu task, it seems that the activation of a 'melon' concept may in turn activate both the concept 'fruit and vegetables' and the concept 'starter' which corresponds to the context in which melon is mainly consumed in France [START_REF] Baros | Automatic activation of impression formation and memorization goals: nonconscious goal priming reproduces effects of explicit task instructions[END_REF]. In fact, participants in the melon-scent condition tended to choose more starters with vegetables than did participants in the control condition, while no such effect was observed for desserts. In other words, it seems that a non-consciously perceived fruity odour may activate a 'fruit and vegetables' concept, but also the concept relative to the context of consumption. To further investigate this hypothesis, we conducted a second experiment in which the odour of a fruit mainly consumed for dessert (i.e. pear odour) was used as the olfactory prime.

Experiment 2

The pear odorant was chosen as it represents a fruit mainly consumed as a dessert. According to a preliminary experiment run with 23 participants different from those in the present experiment, the odour of pear was rated as being typical of the fruit category: the mean rating on a 10-cm scale was equal to M = 7.1; SE = 0.63.

Material and methods

Participants

Seventy participants were recruited using the same criteria as in Experiment 1 and they were randomly assigned to either a pear-scent condition (18-48 years old; mean age = 28 years; 17 males and 23 females) or a control condition (18-45 years old; mean age = 29 years; 11 males and 19 females).

Olfactory stimulus

The pear food aroma sold by Meilleur du Chef© was selected on the basis of the same two preliminary experiments described in Experiment 1 for the selection of the melon food aroma.

According to these preliminary experiments, 24% of 22 participants identified correctly this aroma through a free identification task, and 76% identified it correctly through a 4alternative-forced-choice identification task. Twenty-three participants rated the pear aroma on a 10-cm scale as familiar (M = 6.4; SE = 0.63), and 17% declared that this aroma evoked the desire to consume fresh fruit. The odorization procedure was the same as in Experiment 1, except that three 30-s puffs were diffused 30 min before the session began.

Procedure

The priming procedure was identical to that in Experiment 1. During the test phase, participants completed first the lexical decision task and then the menu task. They were then asked to choose between an apple compote or a slice of brownie as a gift for their participation (choice of a snack). They finally completed the 'Investigation' Questionnaire.

The lexical decision task was the same as in Experiment 1, with 48 non-words, 24 neutral words, 12 words related to high-energy foods and 12 words related to fruit or vegetables, including the word 'pear'. Concerning the menu task, a preliminary experiment run with 20 subjects who did not participate in the present experiment enabled to select new dessert items unrelated to the fruit 'pear' (Table 2.). Hedonic ratings were recorded for each item using 10cm scales. No significant differences for hedonic ratings were found between the 'fruit and vegetables' items and the 'high-energy' items (starter: F (1,189) = 0.05; p = .83; main course: F (9,189) = 1.54; p = .25; dessert: F (1,189) = 0.49; p = .50). Concerning the choice of a snack, two snacks were proposed, one unrelated (slice of brownie) and one related (apple compote) to the 'fruit and vegetables' concept. Participants were asked to select one of these snacks as a gift for their participation.

[Insert table 2 about here]

Data analyses

The analyses conducted were the same as in Experiment 1. With regard to the choice of a snack, the proportion of 'fruity' choices was determined for each group, and a similar logit model as for the menu task was used to analyse these data.

Results

No participant in the pear-scent condition remembered explicitly smelling an odour in the waiting room.

Lexical decision task

Mean reaction speeds for each word category (non-words, neutral words, high-energy-foodrelated words and fruit and vegetable-related words) were submitted to an ANOVA with condition (control; pear-scent), word category and the interaction between the two as fixed factors, and subject (condition) as a random factor. Results showed a significant word category effect [F (3,203) = 130.15; p < .001], with participants answering more slowly for nonwords than for words. No significant condition effect was observed [F (1,203) = 0.43; p = .51], nor a significant condition * word category interaction [F (3,203) = 0.18; p = .91]. As in Experiment 1, this result ruled out a 'general' priming effect. The similar analysis, conducted on the two-syllable letter-strings was performed to compare mean reaction speeds for the word 'pear', with the mean reaction speeds for the other fruit and vegetable-related words, the high-energy-foods-related words, the neutral words and the non-words. The ANOVA did not reveal a significant condition * word category interaction [F (4,268) = 1.39; p = .24]. Participants in the control condition answered as fast as participants in the pear-scent condition for the word 'pear'.

Choices on a restaurant menu card

A logit model was performed by course category, with condition (control; pear-scent) as a factor, and unilateral tests. Results showed no significant difference for the starter (deviance = 0.23; df = 1; p = 1.26) or the main course (deviance = 0.61; df = 1; p = .86). But the analyses revealed that participants primed with the odour of pear chose desserts with fruits more often (deviance = 3.02; df = 1; p < .05) (Figure 4).

[Insert figure 4 about here]

Choice of a snack

A logit model was performed with condition (control; pear-scent) as a factor, and unilateral tests. The analyses revealed a tendency (deviance = 2.55; df = 1; p = .06). In fact, participants primed with the odour of pear tended to choose desserts with fruit more often than did participants in the control condition (Figure 4).

General discussion

The results of the second experiment did not reveal any priming effect of the odour of pear on the lexical decision task. Nevertheless, exposure to the odour of pear had a clear impact on food choices. In fact, participants in the pear-scent condition chose a dessert with fruits on a menu significantly more frequently, and they tended to choose compote as a gift more often than did participants in the control condition. In line with the results of Experiment 1, these results seem to indicate that a non-consciously perceived odour of pear may activate not only a 'fruit and vegetables' concept, but also a concept of 'dessert' which corresponds to the context in which pear is mainly consumed.

The results of these two experiments were obtained in conditions in which the odorants were diffused so that their intensity was so low that participants would not consciously perceive it.

Among the 69 participants in the scent conditions, only one (in Experiment 1) remembered explicitly having smelt an odour in the waiting room and was excluded from all the statistical analyses. All of the others were neither conscious of the presence of an odorant in the waiting room, nor of the impact it could have had on their performances to the experimental tests.

Moreover, it should be noted that we obtained these results while the odour exposure was not concomitant with our experimental tests. In fact, in Holland et al.'s study (2005), participants performed the lexical decision task while the citrus odorant was diffused in the test room, whereas they were no longer exposed to the citrus odour when asked to eat a biscuit. In the present study, the participants were exposed to the melon or the pear odour for 10 min, and then performed the different tasks in a non-odorized test room. All these findings lead us to assume that the processes involved in the priming effects we observed, operate at an implicit level.

While participants primed with a melon odour showed higher reaction speeds for the word 'melon' in the lexical decision task, participants exposed to a pear odour did not answer faster than control participants for the word 'pear'. This result could be explained by the fact that in the French language, the word 'pear' is twice as frequent as the word 'melon' [START_REF] New | Lexique 2: a new French lexical database[END_REF]. In fact, it is well established that the frequency of occurrence is a strong determinant of performance in word recognition tasks such as lexical decisionmaking, with high frequency words recognised more quickly than lower frequency words [START_REF] Frederiksen | Spelling and sound -approaches to internal lexicon[END_REF][START_REF] Monsell | The nature and locus of word frequency effects in reading[END_REF]. Concerning the results of Experiment 2, mean reaction speeds for the words 'melon' and 'pear' were submitted to an ANOVA with condition and word as fixed factors, and subject (condition) as a random factor. The results showed a significant word effect [F (1,66) = 4.61; p < .05], with all of the participants answering more slowly for the word 'melon' (M = 579.56 ms; SE = 15.51) than for the word 'pear' (M = 556.38 ms; SE = 17.68). Consequently, it can be hypothesised that reaction speed for the word 'pear' could be related to a 'ceiling level', which left no room for a possible increase in reaction speed in the pear-scent condition compared with the control condition.

Regarding the results for the menu task in the two experiments, two explanations may be proposed. The first is that the non-conscious perception of a fruity odour led to the activation of a 'fruit and vegetables' concept. This is evidenced by the fact that exposure to the odour of melon, which is typical of the fruit category, tended to induce more choices of starters with vegetables. This finding does not seem so surprising because fruit and vegetables are always associated in nutritional recommendations through French public health campaigns ("Eat five fruit and vegetables per day") [START_REF] Hercberg | The French National Nutrition and Health Program: 2001-2006-2010[END_REF]. Furthermore, fruit and vegetables are almost always gathered in the same section in markets (fruit and vegetable section), and share many common points, from both a culinary and a botanic points of view.

The second explanation is that the priming effects observed in our experiments seem to be rather 'specific to the food cue'. In fact, exposure to the odour of melon, which is mainly consumed as a starter, tended to modify choices of starters, but not of other courses, among participants in the melon-scent condition. Similarly, exposure to the odour of pear, which is mainly consumed as a dessert, had a significant impact on the choices of desserts, among participants in the pear-scent condition. In accordance with these results, several arguments in the literature suggest that these priming effects 'specific to the food cue' may be an inherent characteristic of olfactory priming in the food domain. In [START_REF] Fedoroff | The specificity of restrained versus unrestrained eaters' responses to food cues: general desire to eat, or craving for the cued food?[END_REF], exposure to a pizza odour induced greater pizza intake than that following the exposure to a cookie odour.

In [START_REF] Coelho | Wake up and smell the cookies. Effects of olfactory food-cue exposure in restrained and unrestrained eaters[END_REF], we could even talk about priming effects ultra specific to the food cue. In fact, an odour of chocolate-chip cookies made restrained participants eat a smaller amount of (the cued) chocolate-chip cookies than restrained participants in a control condition, but had no effect on total cookie intake (cued cookies plus oatmeal-raisin, and double-chocolate cookies). As mentioned in the introduction, the stronger the link between a prime and a related mental representation, the stronger the priming effects should be. In the present experiments, the melon and the pear odours were perceived as familiar and were explicitly well-recognised and identified (see Sections 2.1.2 and 3.1.2). It can therefore be expected that the link between these primes and the subsequently activated concepts were quite strong and should produce even more specific priming effects. This raises the question of what kind of effect could be observed if a not well-recognised food odour or a food odour not typical of the corresponding food category was to be used. Further studies should clarify what characteristics of a food odour may be able to activate more general concepts, and have an impact not only on one course of a menu.

Conclusions

Based on the results of these two experiments, we can conclude that non-consciously perceived odours seem to have an impact on food choices, in this study, guiding participants towards more fruit and/or vegetables (Fig. 5). The results of the lexical decision task provided evidence that the non-consciously perceived odour of melon can first activate the corresponding food concept. Concerning the food-choice tasks, the results indicated that incidental exposure to the odour of melon or pear could activate a concept of 'fruit and vegetables' together with the more specific concept of the context of consumption.

[Insert figure 5 about here] Although it was often considered in the past that "people were aware of their attitudes, beliefs, and values that guided their behaviour, and that they would be willing to reveal them if asked appropriately" [START_REF] Kihlstrom | Implicit methods in social psychology[END_REF], current psychological research suggest that a significant part of human feelings, behaviour and decision-making are influenced by implicit, unconscious processes [START_REF] Bargh | The unbearable automaticity of being[END_REF][START_REF] Bargh | Beyond behaviorism: on the automaticity of higher mental processes[END_REF][START_REF] Dijksterhuis | On making the right choice: the deliberation-without-attention effect[END_REF][START_REF] Dijksterhuis | The unconscious consumer: effects of environment on consumer behavior[END_REF][START_REF] Greenwald | Implicit social cognition: attitudes, self-esteem, and stereotypes[END_REF]. As Köster pointed out in his review published in 2009 [START_REF] Köster | Diversity in the determinants of food choice: a psychological perspective[END_REF], "intuitive reasoning and the clear demonstration of the unconscious nature of most of our decision making […] probably play a more important role in food-related behaviour than anywhere else". In line with this thought, our study provides for the first time scientific evidence that a non-consciously perceived fruity odour can indirectly influence food choices.

Figure 1 Fig. 1 .Figure 2 Fig. 2 .Figure 3 Fig. 3 .Figure 4 Fig. 4 .

 11223344 Figure 1

Acknowledgments

The Burgundy region funded the present work. The authors thank Jacques Maratray for the development of the software used in the lexical decision task; Fabienne Bouillot, Christophe Martin (Chemosens) and Valérie Feyen for their precious logistic help for the preparation of the experiment; The society Scentys for its work on the odorant encapsulation; Nicholas Winning and Didier Carnet for providing language help. The authors have no conflict of interest to disclose.

Table 1

Means of ratings on declared hedonicity for each item selected for the Menu task in Experiment 1. For each course category of a typical French meal (starter, main course and dessert), 10 choices were proposed, 5 unrelated and 5 related to the 'fruit and vegetables' concept. No item contained or referred to the fruit melon.


Spiros Kotopoulis
email: spiros.kotopoulis@uib.no

Odd Georg Dimcevski

Helge Gilja

Dag Hoem

Michiel Postema

Odd Helge Gilja

Treatment of human pancreatic cancer using combined ultrasound, microbubbles and gemcitabine: a clinical case study

published or not. The documents may come

Treatment of human pancreatic cancer using combined ultrasound, microbubbles, and gemcitabine: a clinical case study

comparing to a historical control group of 80 patients. In two out of five patients treated, the maximum tumour diameter was temporally decreased to 80±5% and permanently to 70±5% of their original size, whilst the other patients showed reduced growth.

We also explain and characterise the settings and acoustic output obtained from a commercial clinical scanner used for combined ultrasound microbubble and chemotherapy treatment.

Conclusion: It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available clinical ultrasound scanners to increase the number of treatment cycles, prolonging the quality of life in patients with pancreatic adenocarcinoma compared to chemotherapy alone.

Purpose

Cancer is the world's second largest cause of death with over 7.6 million deaths a year (21% of NCD deaths) [1]. There are over 217 000 new cases of pancreatic cancer worldwide every year [START_REF] Hariharan | Analysis of mortality rates for pancreatic cancer across the world[END_REF]. Pancreatic cancer is very difficult to treat due to its aggressive biology, late diagnosis, the encasement of large blood vessels, and the presence of metastasis. Hence, surgery is rarely an option. Chemotherapy produces modest responses but is not curative in this setting, mainly because its use is severely hampered by toxic effects to vital organs. As a result, the survival is very low. The mortality of the inoperable patients is 50% within 3 months and 90% within 12 months [START_REF] Neoptolemos | Adjuvant chemoradiotherapy and chemotherapy in resectable pancreatic cancer: a randomised controlled trial[END_REF][START_REF] Neoptolemos | A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer[END_REF].

Sonoporation is a novel method for non-invasive targeted drug and gene delivery [START_REF] Bao | Transfection of a reporter plasmid into cultured cells by sonoporation in vitro[END_REF][START_REF] Van Wamel | Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation[END_REF][START_REF] Delalande | Sonoporation at a low mechanical index[END_REF][START_REF] Postema | Sonoporation: Why microbubbles create pores[END_REF]. Sonoporation is defined as the transient formation of pores in cell membranes owing to ultrasound or a combination of ultrasound and microbubbles. These pores range in size from several nanometres to several micrometres [START_REF] Lee | Sonoporation enhances chemotherapeutic efficacy in retinoblastoma cells in vitro[END_REF][START_REF] Deng | Ultrasound-induced cell membrane porosity[END_REF][START_REF] Schlicher | Mechanism of intracellular delivery by acoustic cavitation[END_REF][START_REF] Zhao | Phospholipids-based microbubbles sonoporation pore size and reseal of cell membrane cultured in vitro[END_REF], allowing for increased drug uptake in highly targeted regions [START_REF] Postema | Ultrasound-directed drug delivery[END_REF][START_REF] Postema | CEUS and sonoporation[END_REF][START_REF] Postema | Ultrasound-guided delivery and sonoporation[END_REF].

The acoustic parameters used for sonoporation showing increased cellular uptake of chemotherapeutics and genes vary from low-intensity diagnostic ultrasound (Mechanical Index (MI) < 0.3) [START_REF] Escoffre | Microbubble attenuation and destruction: are they involved in sonoporation efficiency?[END_REF][START_REF] Miller | Membrane damage thresholds for 1-to 10-MHz pulsed ultrasound exposure of phagocytic cells loaded with contrast agent gas bodies in vitro[END_REF][START_REF] Miller | Membrane damage thresholds for pulsed or continuous ultrasound in phagocytic cells loaded with contrast agent gas bodies[END_REF][START_REF] Miller | DNA transfer and cell killing in epidermoid cells by diagnostic ultrasound activation of contrast agent gas bodies in vitro[END_REF][START_REF] Miller | Sonoporation of monolayer cells by diagnostic ultrasound activation of contrast-agent gas bodies[END_REF][START_REF] Miller | Gene transfection and drug delivery[END_REF][START_REF] Kooiman | Sonoporation of endothelial cells by vibrating targeted microbubbles[END_REF][START_REF] Delalande | Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype[END_REF][START_REF] Nejad | Optical observation of cell sonoporation with low intensity ultrasound[END_REF][START_REF] Yang | Experimental study on cell self-sealing during sonoporation[END_REF][START_REF] Qiu | Microbubble-induced sonoporation involved in ultrasound-mediated DNA transfection in vitro at low acoustic pressures[END_REF][START_REF] Matsuo | Synergistic inhibition of malignant melanoma proliferation by melphalan combined with ultrasound and microbubbles[END_REF][START_REF] Lamanauskas | Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue® or BR14® microbubbles[END_REF][START_REF] Watanabe | Low-intensity ultrasound and microbubbles enhance the antitumor effect of cisplatin[END_REF] to high-intensity diagnostic ultrasound (MI > 1.0) [START_REF] Lee | Sonoporation enhances chemotherapeutic efficacy in retinoblastoma cells in vitro[END_REF][START_REF] Wu | Sonoporation, anti-cancer drug and antibody delivery using ultrasound[END_REF][START_REF] Lai | Quantitative relations of acoustic inertial cavitation with sonoporation and cell viability[END_REF][START_REF] Hallow | Measurement and correlation of acoustic cavitation with cellular bioeffects[END_REF][START_REF] Forbes | Examination of Inertial Cavitation of Optison in Producing Sonoporation of Chinese Hamster Ovary Cells[END_REF][START_REF] Tlaxca | Analysis of in vitro Transfection by Sonoporation Using Cationic and Neutral Microbubbles[END_REF]. Throughout literature, the acoustic settings used to induce sonoporation vary drastically, with a broad range of these settings showing improved drug and gene delivery. Several studies also show the effect of clinical diagnostic ultrasound in standard colour-Doppler and B-mode imaging on cellular uptake [START_REF] Miller | DNA transfer and cell killing in epidermoid cells by diagnostic ultrasound activation of contrast agent gas bodies in vitro[END_REF][START_REF] Miller | Sonoporation of monolayer cells by diagnostic ultrasound activation of contrast-agent gas bodies[END_REF]. These studies, which made use of clinical diagnostic scanners, concluded that a larger duty cycle was necessary to increase the effect of sonoporation. It has been shown that the ideal settings to induce sonoporation are when shock-waves were not present, in order to sustain the microbubbles, and when the duty cycle is long enough, to excite the microbubbles in the targeted area without heating the surrounding tissue [START_REF] Escoffre | Microbubble attenuation and destruction: are they involved in sonoporation efficiency?[END_REF]. Furthermore, higher intensities correlating to cavitation and jetting result in increased cell death due to mechanical damage instead of (transient) sonoporation [START_REF] Forbes | Examination of Inertial Cavitation of Optison in Producing Sonoporation of Chinese Hamster Ovary Cells[END_REF][START_REF] Ohl | Detachment and sonoporation of adherent HeLacells by shock wave-induced cavitation[END_REF][START_REF] Miller | Induction of Apoptosis in Sonoporation and Ultrasonic Gene Transfer[END_REF][START_REF] Postema | Jetting does not cause sonoporation[END_REF]. As a result, there is no consensus on the exact ultrasound settings to be used for sonoporation [START_REF] Geers | Crucial factors and emerging concepts in ultrasound-triggered drug delivery[END_REF]. For this reason we aimed to use settings that matched our previous in-vitro and in-vivo work as much as possible, i.e., an in-situ MI=0.2, maximum duty cycle, and minimum shockwave generation in order to preserve the microbubbles [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF][START_REF] Postema | Sonoporation: Why microbubbles create pores[END_REF][START_REF] Delalande | Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype[END_REF].

To date, all sonoporation experiments have been done either in vitro or in animal models, hence the effect of sonoporation in humans is not truly known yet.

Ultrasound has been used as a tool in the clinic for many years, especially in transabdominal imaging. Specifically, the pancreas can easily be imaged ultrasonically [START_REF] Erchinger | Transabdominal ultrasonography of the pancreas: basic and new aspects[END_REF]. In clinical ultrasonic imaging, ultrasound is combined with socalled ultrasound contrast agents to locate tumours [START_REF] Postema | Contrast-enchanced and targeted ultrasound[END_REF][START_REF] Piscaglia | The EFSUMB Guidelines and Recommendations on the Clinical Practice of Contrast Enhanced Ultrasound (CEUS): update 2011 on non-hepatic applications[END_REF]. These agents consist of gas microbubbles encapsulated by elastic shells [START_REF] Postema | Fundamentals of medical ultrasonics[END_REF]. Using a clinical diagnostic scanner for combined imaging and treatment allows for precise acoustic field alignment ensuring that the correct ultrasound intensity reaches the target area.

In this study, we worked towards optimising the ultrasonic settings for invoking sonoporation in the target region of a pancreatic tumour using a common commercial clinical ultrasound scanner without physical modifications.

Methods

A clinical scanner was calibrated in a degassed water bath in order to map the beam profile and optimise the acoustic settings. After the chemotherapeutic dose was delivered, the clinical probe was positioned aiming directly at the pancreatic tumour and locked in place for 31.5 minutes. The probe was attached to a ball joint and was positioned near the upper abdomen. Stomach and intestine were avoided in all cases to ensure propagation only through soft tissue, to ensure delivery of the aimed ultrasound intensity at the desired area. Once the tumour was located the probe orientation was fine-tuned in order to locate the largest slice of the tumour and as much vasculature as possible, i.e., the feeding vessels. The probe was then locked in position until the completion of the treatment. The natural breathing motion aided the treatment as the ultrasound slice gently oscillated through the tumour. By visualising the vasculature and tumour it could be ensured that the microbubbles were being sonicated at the target. These vessels were then used as a reference point for future treatments. Nine doses of ultrasound contrast agent were intravenously injected over this time period to enhance the sonoporation effect. To evaluate the efficacy of the combined treatment we compared the amount of chemotherapy cycles the patient was able to receive. Furthermore, the tumour size was measured over the course of the treatment cycles to monitor and compare the tumour growth.

Ultrasound scanner configuration

A GE LOGIQ 9 ultrasound scanner (GE Healthcare, Waukesha, WI) combined with a 4C curvilinear probe (GE Healthcare) was used for both diagnosis and therapy.

To calibrate and program the diagnostic scanner for the optimised therapeutic settings the probe was locked in position in a custom-made 250-L 3D scanning tank, containing degassed water. A calibrated HGL-200 bullet-type hydrophone (Onda, Sunnyvale, CA) connected to a WaveJet 354a oscilloscope (Teledyne LeCroy SA, Geneva, Switzerland) was used to measure the acoustic signal. The scanning tank had a spatial resolution of 0.4 µm. For the calibration a 200-µm resolution was used.

AQUASONIC® ultrasound transmission gel (Parker Laboratories, Fairfield, NJ) was placed on the transducer transmission surface and the probe was subsequently covered using a latex ultrasound probe cover (Sheathing Technologies, Inc., Morgan Hill, CA) prior to submersion. The diagnostic scanner settings were modified in order to achieve a maximum duty cycle without completely degrading the image quality, in addition to having a linear acoustic signal. We aimed for minimal acoustic shockwaves and harmonics minimising potential cavitation. The absence of nonlinear content was verified by visualising the temporal extent of the pulses and performing a Fast Fourier Transform (FFT) [START_REF] Gerold | Laser-nucleated acoustic cavitation in focused ultrasound[END_REF]. Multiple focal depths (from 2.8 cm to 8.4 cm) and different settings (varying gain, changing window size, etc.) were evaluated to ensure similar acoustic conditions in all cases. To calculate the in-situ acoustic pressures and intensities, the in-water values were derated by 0.3 dB/MHz/cm, an approximation of soft tissue attenuation in accordance to FDA and IEC guidelines [START_REF]Information for Manufacturers Seeking Marketing Clearance of Diagnostic Ultrasound Systems and Transducers[END_REF][START_REF]Ultrasonics -Hydrophones -Part 2: Calibration for ultrasonic fields up to 40 MHz[END_REF]. The attenuation factor of 0.3 dB/MHz/cm is only valid for soft tissue. Hence, this calibration was representative for our clinical positioning for targeting the pancreas.

Table 1 shows the ultrasound scanner settings used to perform the simultaneous observation and treatment of the pancreatic tumours. Skilled clinical sonographers were called upon to judge the image quality. As there are variations between patients, such as tumour depth and tissue attenuation, certain settings had to be adjusted to ensure the correct ultrasound intensity reached the required area whilst maintaining the image quality. The settings that were varied are labelled as Patient-depending.

The three settings that were adjusted prior to treatment were: the focal depth, image depth, and gain. The focal and image depths were adjusted in order to visualise and position the acoustic focus directly in the middle of the tumour. By doing so we could ensure that the acoustic conditions the tumour received was as similar as possible in all patients. The gain is only applied after the received signal, hence it did not affect the acoustic output. The gain simply allowed for a brighter image.

Once the probe was locked in position and the tumour was "targeted", no changes to the ultrasonic conditions were made. [START_REF]Information for Manufacturers Seeking Marketing Clearance of Diagnostic Ultrasound Systems and Transducers[END_REF][START_REF]Ultrasonics -Hydrophones -Part 2: Calibration for ultrasonic fields up to 40 MHz[END_REF]. The beam profile showed formation of multiple foci in close proximity along the lateral direction merging to form a quasi-continuous focus (Figure 1E). In the elevation direction side lobes can be clearly seen (Figure 1A and1D). Using the full width half maximum (FWHM) to define the beam size, the active or treatment area can be defined as a volume of 69 × >100 × 1.0 (mm) 3 (l×w×h). It is assumed that this is the region were sonoporation occurred most efficiently. Figure 2 shows the pulse repetition pattern generated by these settings. The pulse was amplitude-modulated, consisting of 5 cycles (2.1 µs) every 210 µs corresponding to a 1% duty cycle (repetition rate optimised). The duty cycle is defined as the percentage of time that ultrasound is being generated. This was measured during the spatial calibration process, in the acoustic focus with the hydrophone, for the duration of the inverse of the frame rate. Due to synthetic aperture and contrast enhanced imaging the pulse pattern at the focus was amplitude-modulated [46] [47]. This can be seen in the upper panel of Fig. 2. The lower panel of Fig. 2 shows the time signal of a single pulse. The pulse is still relatively sine-shaped, thus the transfer function of the propagation path is linear. Minor nonlinear effects can be seen after the 4 th cycle. This indicates that shockwave occurrence and therefore microbubble destruction is negligible. A Fast Fourier Transform (FFT) of the acoustic signal is shown in Fig. 3. The centre frequency is 1.9 MHz. Using a -3-dB or FWHM cut-off the bandwidth was measured to be 1.1 MHz; from 1.3 -2.4 MHz. A second harmonic peak can be seen at 3.6 MHz due to the minor non-linear effects. This peak was 11 dB lower than the primary peak.

B-mode Contrast mode

These settings complied with current safety guidelines for clinical diagnostic imaging [START_REF]Information for Manufacturers Seeking Marketing Clearance of Diagnostic Ultrasound Systems and Transducers[END_REF][START_REF]Guidelines for the safe use of diagnostic ultrasound equipment[END_REF][START_REF] Barnett | International recommendations and guidelines for the safe use of diagnostic ultrasound in medicine[END_REF]. Figure 4 shows two images of pancreatic cancer in two separate patients captured using the sonoporation treatment settings.

Chemotherapeutic and Microbubble dosage

The recommended chemotherapeutic protocol was followed [START_REF] Lilly | Highlights of prescribing information -Gemzar[END_REF]. This protocol dictates which patients are eligible for chemotherapy and the dosages that can be administered. It includes dosage reduction values depending on platelet and absolute granulocyte count. The chemotherapeutic used, gemcitabine (Gemzar ® , Eli Lilly and Company, Indianapolis, IN) was administered once weekly for up to 7 weeks (or until toxicity necessitates reducing or holding a dose), followed by a week of rest from treatment. Subsequent cycles consisted of infusions once weekly for 3 consecutive weeks out of every 4 weeks. Our protocol used the Eastern Cooperative Oncology Group (ECOG) performance status as a measure of the clinical condition [START_REF] Oken | Toxicity and Response Criteria of the Eastern-Cooperative-Oncology-Group[END_REF]. The ECOG performance status ranges from 0-5, where 0 denotes a "fully active patient able to carry on all pre-disease performance without restriction", and 5 denotes a "dead" patient. Chemotherapy was halted if the patient exceeded a grade of 2 that states the patient is "ambulatory and capable of all self-care but unable to carry out any works activities. Up and about more than 50% of waking hours." The ECOG guidelines can be considered as a measure of how "healthy" a patient is. We used the ECOG guidelines to monitor the effectiveness of the combined treatment i.e., the longer a patient stays below an ECOG grade of 3, the more effective the treatment is considered.

A single treatment cycle is defined as a single infusion of chemotherapeutic followed by ultrasound and microbubble treatment. The week pause was not counted as a treatment cycle. Once the granulocyte or platelet count was permanently too low, or the patient surpassed an ECOG performance status grade of 2, no more treatment was administered.

Gemcitabine was administered by intravenous infusion at a dose of 1000 mg/m 2 over 30 minutes. The start of the chemotherapeutic delivery is defined as .

During the last 10 minutes ( ) of chemotherapeutic delivery, diagnostic imaging was performed in standard abdominal imaging mode and the tumour was located. Here the tumour dimensions were measured with ultrasonography. Once the tumour was located, a custom made clamp was used to lock the probe in position and the clinical scanner was switched to therapeutic settings (Fig 5). As the maximum systemic concentration of the chemotherapeutic starts at the finish of delivery ( ) this was chosen as the initiation point for the ultrasound treatment.

Clinically approved SonoVue ® (Bracco Imaging Scandinavia AB, Oslo, Norway)

ultrasound contrast agent was used as the microbubble for sonoporation. To ensure microbubbles were present throughout the whole treatment 0.5 mL of contrast agent followed by 5 mL saline were injected every 3.5 min, i.e., at . A single vial (4.5 mL) was used throughout each treatment. Treatment was stopped at The total cumulated ultrasound treatment time was only 18.9 s. This time frame can be seen in Fig. 5A.

Measurement of disease and tumour progression

The primary measure for evaluating the effectiveness of the treatment was the amount of cycles the patient could undergo. The more treatment cycles the patient underwent, the longer the patient was considered healthy [START_REF] Lilly | Highlights of prescribing information -Gemzar[END_REF][START_REF] Oken | Toxicity and Response Criteria of the Eastern-Cooperative-Oncology-Group[END_REF]. Furthermore, if the tumour size was reduced substantially in accordance to the Response Evaluation Criteria in Solid Tumours (RECIST) [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)[END_REF], the treatment modality was re-evaluated, e.g. transfer to radiation therapy or surgery. This was considered a successful treatment.

Diagnostic ultrasound imaging was performed weekly assessing the tumour size. As

Computerised Tomography CT scans are considered the golden standard for following tumour growth [START_REF] Sohaib | CT assessment of tumour response to treatment: comparison of linear, cross-sectional and volumetric measures of tumour size[END_REF], every 8 weeks a CT scan was also performed to validate the tumour size. This value was used to follow the tumour progression.

Positron Emission Tomography (PET) imaging was also performed at the start of the treatment to assess the presence of metastasis.

Figure 6 shows the pancreatic adenocarcinoma in patient 5 prior to ultrasound and microbubble treatment as seen by CT and PET imaging modalities. show PET scans in transverse and coronal plane respectively. The location of the tumour can be clearly identified by the brighter colour in the middle of the abdomen.

In Panels B and C, the tumour and pancreas are respectively indicated by the blue and green dashed lines. The pancreas tail is behind the large colon in panel C.

Treatment group

Patients with inoperable pancreatic cancer and fulfilled the inclusion criteria at the Haukeland University Hospital, Bergen, Norway, who have volunteered to participate, were included. The inclusion critera primarily stated that the patients must be > 18 years of age, a diagnosis of inoperable pancreatic cancer, histologically verified, locally advanced (stage II/III) or metastatic (stage IV) adenocarcinoma of the pancreas, and must be ambulatory with an ECOG performance status between 0 and 2.

For this case report a total of five patients were recruited. Table 3 shows the characteristics of the five patients enrolled in this pilot study prior to treatment in addition to the start and end dates of the treatment for every patient.

Control group

Taking into account the guidelines for gemcitabine treatment, it can be deduced that the more treatment cycles the patient can undergo, the longer the patient can be considered healthy; hence the more effective the treatment. Once the patient surpasses a Level 2 in the ECOG performance status guidelines, they would no longer receive treatment; this would accordingly define the end of the healthy and ambulatory period. Our control group consisted of 80 patients from 2009-2011 with histology showing pancreatic adenocarcinoma (matching the same criteria as our patients).

These patients received the identical chemotherapy treatment (in accordance to

Gemzar guidelines [START_REF] Lilly | Highlights of prescribing information -Gemzar[END_REF]) at Haukeland University Hospital, Bergen, Norway. The control treatments were also discontinued once they surpassed and ECOG performance grade of 2 or their blood counts dropped below the chemotherapy guidelines. Patients who received a different treatment were excluded from the control group. The data was accessed through the internal hospital medical system. The same anonymous data will be available on the Norwegian national cancer registry.

Ethical Considerations

All experiments were performed with approval from the regional ethics committee under reference number 2011/1601/REK vest.

Results and discussion

The beam characterisation showed that the clinical scanner took into account the attenuation of soft tissue when varying the focal depth. This allowed for a good prediction of the ultrasound profile in-situ and easy manipulation of the ultrasound intensity and positioning. The "active" area that we assume enhances the chemotherapy effect was long and wide in all cases independent of depth, surpassing the tumour size, allowing a maximum flexibility on treatment area. It has be assumed that there are some fluctuations in the sound field pressures due to tissue property variations, but this should not drastically change the sound field in our case, as acoustic propagation was only though soft tissue. Taking into account the vast range of ultrasound intensities used to induce sonoporation, as seen in literature, we assume that sonoporation may be occurring at lower or higher acoustic pressures independent of the varying attenuation of tissue. A benefit of using a clinical probe is also that due to the synthetic aperture, objects obscuring the field of view do not affect the beam formation in other areas; hence we can predict the ultrasound dose delivered to our target area.

The image generated using our customised treatment settings allowed easy identification of both microbubbles and tumours. Figure 4A shows clear signs of microbubble presence in the tumour vasculature and surrounding tissue. Figure 4B shows the dimensions of a pancreatic tumour indicating the ease of detecting and aligning the probe to the tumour using the modified settings. Figure 8 and Table 3 show the effect of our combined treatment on the tumour size.

After 8 weeks two patients showed a tumour diameter reduction. Patient 1 had a temporary tumour reduction from 4.0 cm to 3.1 cm. The next CT image was taken 24 weeks later and showed a growth to 4.6 cm; an increase of 15% from the original tumour size after 32 weeks of treatment. In patient 2, the treatment resulted in a continuous tumour reduction over 16 weeks, a very rare response from chemotherapy alone. As a result of his increased health, after 10 treatment cycles, he was removed from the clinical trial to undergo radiation therapy. As this patient was removed from the trial due to the success of the treatment, a lower number of total and average treatments was seen, reducing the apparent effectiveness of the treatment as a whole.

It should be noted that none of the patients in the control group stopped treatment due to its success but on the contrary, due to their deterioration.

Two patients showed slow tumour growth from the 8 th week onwards (patient 3 and patient 4). Patient 5 also had a biopsy verified primary tumour in the pancreas. This was surgically removed but re-occurred with a small tumour in the operation sight and a large metastasis. This indicated that the tumour was at a late stage of development hence a limited response could be expected from the chemotherapeutic. Nevertheless, this patient was also able to receive 11 cycles of treatment.

As pancreatic cancer is such an aggressive form of cancer it is very uncommon to see any decrease in tumour growth from chemotherapy. Our aim was to improve quality of life, to extend the healthy period of life, and conclusively extend the patients survival. If the patient was "healthy" enough (well-defined state in both groups, ECOG performance status 0-2 [START_REF] Oken | Toxicity and Response Criteria of the Eastern-Cooperative-Oncology-Group[END_REF]), they would be able to receive treatment for a longer period. In fact, as long they are ambulatory and capable of all self-care they are able to receive the treatment. Seeing a decrease in the primary tumour size was an added benefit to the increased number of treatment cycles and thereby the anticipated survival. The addition of the sonoporation procedure following the standard chemotherapeutic protocol did not add any discomfort to the patients. All patients were very relaxed during the treatment to a state where they could comfortably sleep throughout the whole treatment.

Patient

In this study we also aimed to show that it is possible to induce sonoporation in the clinic using existing commercial equipment, whilst fitting in the current safety regulations for the use of diagnostic ultrasound. In our previous work we showed that a duty cycle of 40% was ideal for sonoporation [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF][START_REF] Delalande | Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype[END_REF]. Here we are using a duty cycle of 1%; hence expecting a small effect of sonoporation. There are many ways to improve this method of therapy such as by increasing the duty cycle from 1% to 40% and introducing targeted microbubbles that could attach to specific cancer cells [START_REF] Postema | Contrast-enchanced and targeted ultrasound[END_REF].

The efficacy of our combined treatment should be compared to the efficacy of the current golden standard, the chemotherapeutic gemcitabine alone, where the viability of the patient has been extended by approximately 1 month [START_REF] Neoptolemos | Adjuvant chemoradiotherapy and chemotherapy in resectable pancreatic cancer: a randomised controlled trial[END_REF][START_REF] Neoptolemos | A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer[END_REF].

Conclusion

Using a clinical diagnostic scanner for therapeutic purposes allows accurate acoustic field alignment ensuring that the desired ultrasound dose reaches the target area. This configuration allows simultaneous visualisation of the microbubbles present whilst treating the pancreatic tumour. In this pilot study, we saw an extended treatment period when comparing to the control group. Furthermore, we did not notice any adverse side effects. Combined ultrasound, microbubble and chemotherapeutic treatment could pave the way for a novel enhanced drug delivery pathways.

Figure 1 :

 1 Figure 1: 1D and 2D beam profiles at sonoporation settings using the 4C probe at two

Figure 2 :

 2 Figure 2: Ultrasonic pulse generated by the clinical scanner. The top panel shows the

Figure 3 :

 3 Figure 3: Fast Fourier transform of ultrasonic signal. The centre frequency of the

Figure 4 :

 4 Figure 4: Images captured using customised sonoporation settings using a clinical

Figure 5 :

 5 Figure 5: Time frame of each chemotherapy cycle (Panel A) and photograph of probe

Figure 6 .

 6 Figure 6. CT (Panel A) and PET (Panels B and C) images of patient 5 showing

Figure 7

 7 Figure 7 shows the normalised perfusion curve where the arrows indicate the contrast

Figure 7 :

 7 Figure 7: Normalised microbubble presence in tumour locality during the first 800 s

Figure 8 :

 8 Figure 8: Change in tumour diameter over time measured from CT images in patients

Table 1 :

 1 Parameters as indicated on a GE LOGIQ 9 clinical ultrasound scanner.

	Parameter		Value	Unit	Description	Variability
	MI	0.4	0.4		Mechanical Index	None
	TIs	0.0	0.0		Thermal Index of soft	None
					tissue
	Freq	4.0	4.0	MHz	Centre Receive	None
					frequency
	AO	1	36	%	Normalised acoustic	None
					output
	FR	4	4	fps	Frame rate	None
	Gn	30-45	30-45	dB	Gain	Patient-depending
	S/A	3/3	2/0		Synthetic Aperture	None
	Map	F/0	2/0		Colour map	None
	F	5.2-6.8	5.2-6.8	cm	Focal depth	Patient-depending
	D	10-15	10-15	cm	Image Depth	Patient-depending
	DR	66	66	dB	Dynamic Range	None
	SRI HD	3	3		Image smoothing	None
	Grey Map	F/0	H		Image colour maps	None

Table 2 :

 2 Acoustic

conditions generated by the 4C probe for sonoporation in-water and derated for in-situ values

Table 3 :

 3 Patient characteristics prior to treatment. ND denotes non-discernable values. Start and end date of treatment are also stated.

						Patient
				Patient 1	Patient 2	3	Patient 4	Patient 5
		Age		66	55	70	68	51
		Sex		Male	Male	Female	Female	Female
	Pathology Findings		Pancreatic ductal adenocarcinoma
	ECOG Performance	0	1	1	0	1
		ALAT	IU/L	20	55	138	23	66
	Biochemistry	LD Leuk	IU/L x10 9 U/L	121 6.8	146 3.8	153 6.9	117 6.1	176 11.1
		Neutr	x10 9 U/L	4.3	5.8	3.8	3.5	7.1
	Tumour Markers	Ca 125 Ca 19-9		ND 59	54.1 ND	102 ND	ND 4608	136.6 ND
	Treatment dates	Start date		06/01/2012 04/04/2012 07/03/2012	22/02/2012 15/02/2012
	(dd/mm/yyyy)	End date		26/092012 01/08/2012 11/07/2012	11/05/2012 08/06/2012

Maximum tumour diameter (cm) Total number of cycles Inclusion day Week 8 Week 16 Week 24 Week 32

	1	4.0	3.1	-	-	4.6	27
	2	4.9	3.9	3.6	-	-	10
	3	1.5	3.0	3.5	-	-	11
	4	2.2	-	2.3	2.9	3.4	16
	5	4.0	-	5.5	6.2	-	16

Table 4 :

 4 Maximum tumour diameter as measured from CT images. Empty values denote skipped CT scans.

Acknowledgements

This study has been supported by funds from the Norwegian Cancer Society (NCS) and MedViz (http://medviz.uib.no/), an interdisciplinary research cluster from Haukeland University Hospital, University of Bergen and Christian Michelsen Research AS. We would like to thank Dr. Martin Biermann, Dr. Tormod Bjårnes, Dr.

Bjørn Tore Gjertsen, Dr. Anders Molven and Dr. Halfdan Sørbye for their support throughout this project.


Spiros Kotopoulis

Ragnhild Haugse

Michiel Postema

Sonoporation: The hurdles that need to be surpassed

Sonoporation, the transient formation of pores in cellular membranes is being investigated for multiple purposes such as enhanced localised drug or gene delivery.

The concept of sonoporation dates back to the early 80's [START_REF] Fechheimer | Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading[END_REF] but has yet to see the light in common day clinical practice. This is partially due to the lack of understanding of how sonoporation truly occurs and in consequence how we can improve its efficacy. There have been numerous studies [START_REF] Postema | High-speed photography during ultrasound illustrates potential therapeutic applications of microbubbles[END_REF][START_REF] Prentice | Membrane disruption by optically controlled microbubble cavitation[END_REF][START_REF] Delalande | Sonoporation at a low mechanical index[END_REF][START_REF] Krasovitski | Subcellular sonophores: ultrasound induced intramembrane cavitation[END_REF][START_REF] Marmottant | Controlled vesicle deformation and lysis by single oscillating bubbles[END_REF] investigating the mechanisms behind sonoporation but there is still a lot to be understood. These mechanisms are shown in Figure 1.

A large majority of studies simply investigate if sonoporation is possible and rarely try to understand the underlying mechanisms and the variance of efficacy when translating into a clinical setting. We hypothesis that different acoustic conditions and microbubbles are necessary depending on the cell line that we desire to sonoporate. Our studies aim at understanding the ideal acoustic settings for a variety of cell lines, translating this into preclinical tests and finally moving into clinical trials. In order to investigate such a broad range of conditions we have designed a modular sonication chamber that allows bulk evaluation cell sonoporation. This design is shown in Figure 2. A further setup will be to design custom microbubbles designed to enhance sonoporation. To be able to accurately manufacture monodispersed microbubbles with a narrow band property distribution we will attempt to manufacture flow focusing microbubble generation chambers using E-beam technology with nanometre accuracy.

Figure 1 -

 1 Figure 1-Sonoporation mechanisms.

Figure 2 -

 2 Figure 2 -Modular custom-made sonoporation evaluation chamber.