Time was the most frequently used indicator, given in 92% of the recipes, with 15% using only time. Other indicators included: set (89), browned (76), clean toothpick/knife (60), puffed (27), and jiggled (13). Only two recipes indicated final endpoint temperatures. Three recipes  were chosen and prepared in triplicate to see if they would reach recommended temperatures. The pie and quiche were still liquid at 71 °C, and were well over the recommended temperature when cooked according to instructions, but the egg casserole was not consistently above 71 °C, when the recipe instructions indicated it was done and the center was light brown and “jiggled” This research indicates that consumers are not receiving information on endpoint temperatures in egg recipes, but the likelihood of foodborne illness is low since most dishes probably be cooked past the recommended temperature before the consumer considers them done unless there are many inclusions that may absorb liquid and reduce the appearance of liquid in the dish.Many consumers do not follow recommended food safety practices for cooking egg dishes, such as pies, quiches, and casseroles, potentially leading to foodborne illnesses such as Salmonellosis. The United States Department of Agriculture (USDA) recommends cooking egg mixtures until the center reaches 71 °C (160 °F). The objectives of this study were to determine what endpoint temperature information consumers receive from egg dish recipes, and if recipes would lead to safe temperatures when followed. Egg dish recipes ( Salmonella. Although there have been decreases in many other foodborne illnesses, Salmonella rates have remained steady [Salmonella [Salmonella [Salmonella [Foodborne illness continues to be a public concern, with large social and economic tolls due to hospitalizations, loss of productivity, and death . Many ofd steady . The U.Slmonella , making lmonella ,6. Europlmonella  and a Brlmonella .Temperature recommendations for eggs when cooked by themselves vary in the US, with some guidelines stating only that they should be cooked until the yolk and white are firm, not runny  and otheSalmonella in grossly contaminated eggs [Salmonella to a potentially safe level, other methods such as sunny-side-up, over-easy, and scrambling egg cooking do not adequately reduce bacteria levels [Salmonella survival rates in eggs have typically been done with either whole or liquid eggs, and have not looked at more complicated dishes, such as casseroles and pies that might be the source of illness in the home [Common methods of egg cooking do not completely eliminate ted eggs . Althouga levels ,14. Studthe home .One potential source of information on consumer food safety is recipes. Consumers have expressed that one of their preferred sources of food safety information was cookbooks and other print recipes . A studyThe objective of this study was to determine what recommendations concerning determination of doneness consumers receive when using egg dish recipes found in cookbooks and internet sources, and if those recommendations are valid. A comparison of the information to the recommendations given by regulatory agencies, such as the FDA and USDA, will demonstrate gaps between what information consumers are being exposed to through recipes, and what information can potentially be added to recipes to improve food safety information for consumers. In addition, three recipes were selected from those surveyed and prepared in triplicate to determine if the recommendations given would lead to dishes that were safely cooked.Recipes were selected from multiple sources used by consumers, both online and in print. Sources included retail locations (including supermarkets and kitchen supply stores that sell direct to consumers), blogs , nonprofit , magazines, cooking shows, traditional cookbooks, and local cookbooks . The number and types of recipes used from each source are shown in In total, 226 egg recipes were analyzed for this study. Recipes were chosen where eggs were one of the critical components, and where they could potentially be undercooked. Both those that used the whole egg and those that used either the yolk or whites only were included in the analysis, though the focus was predominantly on whole eggs. These recipes came from 65 websites, 50 cookbooks, and nine magazine titles (with multiple issues of each). Recipe types included for analysis were (in decreasing order of frequency): pie, quiche, casserole, frittata, custard, strata, soufflé, omelet, cheesecake, pudding, torte, and other.Pies included in these recipes were mostly custard and cream type pies, such as pumpkin, lemon, custard, and coconut. When recipes fell into two categories, such as a custard put into a pie, the category where the final cooking of the eggs happened was selected. Recipes that were unique and did not fall into any category were placed in the “other” category.The directions of each recipe were categorized by what method was recommended to the consumer for determining when the dish was finished cooking. The majority of the recipes gave more than one method for determining the doneness of the product, so an overall count of indicator type was used. Where recipes used different wording to describe the same method, they were combined into a single category; for instance, “browned,” “golden brown,” and “light brown” were all combined into one category, as were the variations of utensils, including using a fork, knife, or toothpick. Other directions that were only seen once, and therefore not placed in a category, included instructions such as “cooked through,” “edges pull away,” and “clumped.” Categorization of the recipes was done by two separate researchers, and then compared for accuracy and consistency. Any differences were resolved by a third researcher.Recipes gave one (22%), two (46%), three (27%), or four (5%) indicators of doneness. Only two recipes gave no indicator of doneness. Of those recipes that gave only one indicator, the most common (78%) was time. Indicators used by the recipes in order of frequency included cooking time (92%), being “set” (39%), brownness (34%) , having a probe or utensil come out clean (27%), puffiness (12%), reduced jiggling (6%), other (5%), thickening (3%), bubbling (3%), final temperature (1%), and raw eggs (1 recipe). Time (or a range of time) was by far the most common indicator given, found in 92% of the recipes. Those recipes that gave no other method of determining doneness other than time could be especially difficult for consumers who were not familiar with preparing the product and knowing when to stop cooking the items. This could result in undercooked items that carry an increased risk of leading to foodborne illness.Approximately 40% of recipes used the word “set” as a doneness indicator with over half (54%) stating only “until set,” and others specifying what part of the dish should be set, whether sides (27%), top (9%), center (6%), or bottom (4%). The bottom being set was used in recipes such as frittatas where the eggs were undisturbed during the cooking process. It is important to note that those that gave a location other the center could have resulted in a center that was not set or not cooked completely.Only two of the recipes, both from retail sources, gave an endpoint temperature for the egg dish and both recommendations met USDA minimum temperature guidelines. One, a strata with ham, recommended that the thermometer inserted in the center read 170 °F (76.7 °C), while the other, a custard pie, recommended 160 °F (71 °C) as an endpoint temperature for the custard before baking the pie. Both still used time as the primary method for determining doneness, with temperature as a secondary means of checking the product if a thermometer was used.Almost all of the indicators of doneness found in these recipes were based on methods that were either visual  or time based. Almost none of the recipes gave a temperature to reference during the cooking procedure. This shows that consumers are not receiving the information on endpoint temperatures recommended by the USDA in the recipes that they likely use for preparing egg dishes.Based on the findings of this study that almost no recipes contain temperature information, a second study was conducted to determine if the directions given would give lead to undercooked egg dishes that could increase the risk of foodborne illness. The temperature recommendations given by USDA and other groups are based on reducing the risk of foodborne illness, rather than on consumer measures such as doneness of the dish . It was Three recipes were chosen for testing the instructions, a lemon chess pie, a quiche, and an egg based casserole. Recipes were chosen to represent different types of recipes with varying difficulty and inclusions. The chess pie was the simplest with no inclusions, the quiche had some inclusions as well as cheese which could change the texture and browning appearance, and the casserole had large inclusions including chunks of bread. Ingredients for each recipe were purchased at a local supermarket. A test item from each recipe was baked to test proper placement of instrumentation for each phase of the experiment, and recipe measurements were modified slightly for consistency. Household measures were changed to standard metric weights and volumes for cooking. Where applicable, standard sizes were obtained from the National Nutrient Database-Foods list . For exaOven temperature was monitored using a calibrated oven thermometer . An oven safe cooking thermometer and a digital thermometer with probe  that had been previously checked for consistency and calibrated for accuracy were used to monitor the temperature of the dishes. Thermometers were affixed to a ring stand with clamps for support and stability, and to ensure that the thermometer placement and depth were consistent. Thermometers were placed in the center of the dishes at a 45° angle, not touching the bottom of the pan or the crust. When a dish reached the recommended safe temperature (71 °C) the cooking time that had passed was noted, and a spoon was placed into the center of the dish to check for consistency. The dish was then allowed to continue the cooking process until the time stated on the recipe, at which point it was removed from the oven, placed on a wire cooling rack, and temperature readings were taken both immediately and after the item was allowed to rest before cutting, to mimic typical consumer behavior as listed in the recipes or in similar recipes . Subjective measurements of set, coloring, jiggling, clean toothpick, and clean knife tests were assessed by two observers and recorded by video and photography. An additional factor of “puff” was assessed in the quiche recipe due to doneness instructions. Definitions for the measurements were agreed upon before assessment began. Consistency was assessed at the time that the item reached 71 °C by dipping a spoon into the center of the item, and was defined as the holding together or retention of form, and was assessed as to being liquid, semi-formed, or firm. All other evaluations were done immediately upon removal from the oven once the cooking time was complete, with the exception of the clean slicing, which was done after the resting period. Set was determined to mean if the item had a fixed or rigid appearing center. Jiggle was used to describe whether an item moved lightly and quickly from side to side or up and down when given a quick jerk, i.e., like gelatin. Color change was noted as no change, golden, light brown, or brown when assessing the outer top of the finished product. The clean toothpick and knife tests were defined as the amount of adherents on the instrument after it was inserted into the center of the item and removed.2 cups sugar (454 g)1 tablespoon all-purpose flour (7 g)1 tablespoon cornmeal (7 g)¼ teaspoon salt (0.2 g)¼ cup butter or margarine, melted (57 g)2 teaspoons grated lemon rind (15 g)¼ cup lemon juice, fresh (60 mL)¼ cup whole milk (60 mL)4 large eggs, grade A (200 g)1 unbaked 9-inch pastry shell, frozenThe first recipe tested was a lemon chess pie, modified as follows, with the SI measurements in parenthesis .2 cups Preparation: Combine sugar, flour, cornmeal, and salt. Add butter, lemon rind, lemon juice, and milk: mix well. Add eggs, one at a time, beating well with a wire whisk after each addition. Pour into pastry shell. Bake at 350 °F (177 °C) for 50 min. Yield: one 9-inch pieThe uncooked pie crust was set out on the counter (between 23 °C and 26 °C) to defrost 1 h before cooking time along with the butter, which was allowed to soften before mixing. Dry ingredients were weighed and stirred together before wet ingredients were added to the mixing bowl. Eggs were combined, beaten, and then added to the mixture in 50 g increments, with beating in between each addition. The mixture was poured into the pie crust and placed in a preheated Kenmore 24 inch oven, with a timer set for 50 min. Time and consistency were noted once the dish reached 71 °C, and then it was allowed to continue cooking until the time stated in the recipe.After the 50 min of cooking time was completed and temperature and consistency measures were noted, the pie was placed on a wire rack and allowed to cool for 2 h, at which point the internal temperature was again taken and recorded.1 unbaked deep dish 9-inch pastry shell, frozen3 cups shredded cheddar cheese (170 g)4 eggs (200 g)1 cup whole milk (236 mL)1/2 cup heavy cream (118 mL)3 tablespoons flour (19 g)1–2 teaspoons dried chives (0.1 g)1/4 teaspoon salt (1.2 g)1/8 teaspoon pepper (0.2 g)The quiche recipe was adapted as follows, with standardized measurements in parenthesis .1 unbakPreparation: Preheat oven to 375 °F (191 °C). Let frozen crust stand at room temperature for 1 h to thaw. Sprinkle cheese into the bottom of the pie crust; set aside. In medium bowl, combine remaining ingredients and beat with a wire whisk until smooth and incorporated. Pour carefully over cheeses in crust. Bake for 40–50 min until quiche is puffed, golden brown, and set. Let stand 5 min, cut into wedges to serve.The pie crust was set out on the counter (23 to 26 °C) 1 h before cooking time to thaw. Dry ingredients were weighed and mixed together, followed by wet ingredients as stated in the recipe. A wire egg whisk was used to beat ingredients thoroughly. The mixture was then poured over the cheese-filled pie crust and placed in the preheated oven. The temperature of the quiche was monitored for an internal temperature of 71 °C, at which point the time was recorded and the consistency of the quiche was checked by placing a spoon into the center of the quiche.The item was allowed to continue cooking for the set 40 min and the temperature was taken, at which point it was allowed to cook the entire 50 min stated in the recipe. The quiche was then removed from the oven and placed on a wire rack on the counter. Final temperatures were taken and the quiche was assessed for consistency, set, color, jiggling, and whether a toothpick and knife came out clean. Temperatures were recorded from the center of the dish, but various spots around the quiche were checked to ensure uniformed heating. The quiche was allowed to set for 10 min and again assessed for temperature and sliced.1 pound sausage (454 g)9 eggs (450 g)1.5 cups whole milk (355 mL)1 cup grated cheese (113 g)1 teaspoon salt (4.8 g)1/2 teaspoon pepper (1.1 g)1 teaspoon dry mustard (2 g)4 slices bread, cubed (94 g)The breakfast casserole was adapted as follows, with standardized measurements in parenthesis .1 poundPreparation: Brown sausage; drain. Mix eggs, milk, salt, pepper, and mustard together. Add meat and cheese. Mix well and pour over bread; toss lightly. Pour into buttered baking dish. Refrigerate overnight. Bake at 350 °F (177 °C) for 45 min. Let stand for about 10 min before serving.The sausage was fried and drained, and had a final resting weight of 219 g on average. Other ingredients were mixed and added together as instructed. An 8″ × 8″ pan was buttered with a 5 g pat of butter prior to adding the mixture. Plastic wrap was placed over the dish, which was refrigerated overnight for 14 h. The casserole was then removed from the refrigerator and allowed to wait while the oven preheated, and then was placed in the oven with a thermometer inserted into the center and the timer set for 45 min. The casserole was checked at 45 min, at which point none of the repetitions had reached the recommended temperature and were still in a semi-liquid state, and so were allowed to continue cooking. When the product reached 71 °C, a second thermometer was used to check the internal endpoint temperature, and consistency was checked. The dish was then removed from the oven and placed on a wire rack to cool. The casserole was then checked for set, color, jiggling, and the clean knife and toothpick test. Final temperatures were taken after 10 min, and the casserole was then sliced and plated.The quiches reached 71 °C at an average of 34 min and were all still liquid in consistency. They reached an average temperature of 79 °C at 40 min and 81 °C at 15 min. The quiches were fairly consistent as finished products. They were all set, did not puff or jiggle, and had clean knife and toothpick tests. All of the quiches had a light brown top coloring and reached a resting temperature of 79 °C at 10 min. When plated, all were semi clean slices.The casserole had the only problematic results of any of the dishes. At the cooking time given in the recipe (45 min), all of the replicate dishes were still under the recommended temperature, and all of them had some liquid remaining in the center and on the top of the dish and did not look done. Would a consumer use the time as the definite endpoint or cook longer to get a casserole that was set and not runny? Once the casseroles reached 71 °C, all of them were considered set, though some still had some jiggle. After a 10 min resting period, the average temperature of the casseroles was 67 °C, and all of them sliced cleanly.Salmonella would be destroyed through the cooking process. Some studies have shown that the ability of Salmonella to survive cooking in eggs can be dependent on factors such as pH, heat resistance, initial bacterial load, and previous exposure to environmental stress [Limitations to this study included the limited number of recipes that were prepared and observed. Also, this study did not attempt to inoculate recipe preparations and measure bacterial loads to determine what level of l stress . For thiIn all of the trials of the pies and quiches, the products achieved the target temperature of 71 °C well before the indicated cooking time had been reached. Upon reaching 71 °C, considered to be safe in terms of reduced risk for harboring a foodborne pathogen, recipes were still in a liquid phase and typically would not have been eaten or considered finished by consumers. Once the products were cooked for the recommended time in the recipes, they were all well above the recommended safe temperature, and all had a final set consistency, clean toothpick, and clean knife test.The breakfast casseroles was unique in that the replications took longer than the recommended time to reach the appropriate temperature. The observers also noted that it was more difficult to determine if the products were “set” compared to other dishes due to not being able to determine if the liquid on top was from uncooked eggs or from oils from the cheese or sausage. At the 45 min mark, there was some light browning despite the liquid on top, meaning some consumers may have chosen to remove the dish at that point, though it is likely that those familiar with the type of dish would have continued cooking it, especially given that the dish still jiggled markedly and appeared undercooked.Salmonella. The thermal death rate of these bacteria can depend on multiple factors, including pH and additives such as salt. Some studies have shown that the thermal death rates of Salmonella spp. Do not always follow a log-linear curve, making it difficult to predict when a product has reached a specific death rate [60D = 0.17 min [One of the main reasons for the recommendation of cooking egg dishes to a temperature of 71 °C is to ensure thermal inactivation of bacteria that cause foodborne illness such as strains of ath rate . From re0.17 min ). AlthouThe results of these trials indicate that consumers are at minimal risk for undercooking simple egg dishes such as pies and quiches, since they would continue cooking them past the liquid stage that was seen at 71 °C, though some caution should still be exercised with more complex egg dishes such as casseroles.Salmonella spp. in more complex dishes such as casseroles as well, though there may be some risk based on their interpretation of when a dish is considered set. Following the directions found in egg dish recipes led to safely cooked dishes in terms of endpoint temperature, with the possible exception of the casserole. Microbial testing for more complex egg dishes may need to be done to ensure that they are indeed safe based on the given directions, and additional trials of other egg dishes could be done to determine if this applies to other egg products as well. Food safety agencies and educators can use this information to focus on other products or aspects besides egg dish temperature where there is a greater risk for foodborne illness.Efforts to educate consumers on egg safety should prioritize the safe handling of raw eggs and the reduction of cross contamination, rather than endpoint temperatures. Almost no recipes contain endpoint temperatures for egg dishes, despite the recommendations of various food safety organizations. Although this does seem like a potential concern in theory, the risk appears to be minimal in practice based on our results. We found that simple egg dishes did not solidify until well after they reached the recommended temperature of 71 °C, at which point the risk for foodborne illness would be minimal. Consumers would likely also reach a high enough temperature for thermal death of


The funding section in the original publication of this article  omitted “KPR (grant number T32 AI007433) and RPW (grant number R37 AI093269) are funded by the National Institutes of Health (NIH). The report is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.”


Aggressive behaviour was very rare and no effect of the backpacks was found. In line with our hypothesis, backpacks increased preening on the day of fitting, both when isolated (No backpack: 0% (IQR: 0–1) vs. Backpack: 22% (IQR: 1–43), P < 0.01) and when back in the group (No backpack: 0% (IQR: 0–27) vs. Backpack: 43% (IQR: 5–77), P < 0.001). However, no effect on preening was observed 2–7 days afterwards. Other behavioural changes suggested that on the day of fitting hens prioritized attempts to (re)move the backpack and were less attentive to their surroundings. However, only equipment pecking  was still affected 2–7 days after fitting (No backpack: 0 pecks/hen/minute (IQR: 0–0), vs. Backpack: 0 (IQR: 0–0.07), P < 0.05). We found no effect of our backpacks on bodyweight. In conclusion, our backpacks seem suitable to attach monitoring equipment to hens with only a very minor effect on their behaviour after a short acclimation period (≤2 days).Automated monitoring of behaviour can offer a wealth of information in circumstances where observing behaviour is difficult or time consuming. However, this often requires attaching monitoring devices to the animal which can alter behaviour, potentially invalidating any data collected. Birds often show increased preening and energy expenditure when wearing devices and, especially in laying hens, there is a risk that individuals wearing devices will attract aggression from conspecifics. We studied the behavioural and physiological response of 20 laying hens to backpacks containing monitoring devices fastened with elastic loops around the wing base. We hypothesised that backpacks would lead to a stress-induced decrease in peripheral temperature, increased preening, more aggression from conspecifics, and reduced bodyweights. This was evaluated by thermography of the eye and comb (when isolated after fitting backpacks), direct observations of behaviour , and weighing . Each hen wore a backpack during one of the two experimental periods only and was used as her own control. Contrary to our hypothesis, eye temperature was higher when hens wore a backpack (No backpack: 30.2 °C (IQR: 29.0–30.6) vs. Backpack: 30.9 °C (IQR: 30.0–32.0), P < 0.001). Eye temperature of hens wearing a backpack was strongly correlated to the time spent preening (r It alloNo previous studies have assessed whether adult chickens adapt their behaviour when fitted with devices for automated behavioural monitoring, and only two have assessed this in sub-adults. In our pilot studies we used ‘vests’ of stretchy fabric or plastic cases in contrasting colours to fit devices and observed immediate marked responses including sidestepping/reversing (interpreted as attempts to escape from underneath the equipment), running away in apparent panic, and simply lying down during the first 15 min after fitting. Several days later hens still pecked or pulled the devices frequently, were often attacked and chased by conspecifics, and were seen to isolate themselves in nest boxes or on perches. We therefore developed a less visible and obtrusive attachment system. This consisted of a ‘backpack’ only slightly larger than the devices contained, with smooth angles and in the same colour as the hen, which was attached by elastic loops around the base of the wings. In a small scale trial on a commercial farm , these bWe hypothesised that if our backpacks would not be well tolerated, hens would increase the time they spent preening, sidestepping/reversing, sitting/lying, and the frequency of equipment pecking . We also hypothesised that hens wearing a backpack would be pecked and attacked more often, leading to increased plumage damage and attempts to withdraw by fleeing, perching, or hiding in the nest box. This was predicted to reduce foraging and eating/drinking resulting in lower body weights.Physiological responses shortly after fitting the backpacks were analysed by infrared thermography, a non-invasive indicator of arousal. Acute stress leads to an initial decrease in peripheral temperature due to vasoconstriction . Mild st2The study was carried out following ethical approval by the University of Bristol (license number UB/17/002).2.12 floor pen covered with wood shavings. Hens had continuous access to commercial layer mash, water, a three-tier perch, nest boxes, a slatted ramp and environmental enrichment , except when put in the holding pen . Room temperature was maintained between 16 and 19 °C throughout the study.Twenty 18-week-old British Blacktail laying hens were obtained from a commercial rearing farm and transported to the test facility after weighing and fitting leg rings for individual identification. All hens were housed together throughout, in a 13.8 mIn the week before data collection the hens were habituated to human presence. In addition to the normal exposure to humans during routine husbandry procedures (replacing feed and water and egg collection), at least one person was present in the house during most of the light period. During the first two days of habituation she moved around the pen freely, but did not enter the pen. On the third and fourth day she entered the pen, but did not actively approach any of the hens. Hens that approached her calmly were picked up briefly and placed back on the floor immediately and carefully. All hens had allowed this by the end of the fourth day and none showed clear avoidance behaviour after being picked up. Two days before the experiment started all hens were picked up, handled, weighed, mite-treated, and put back in the pen.2.2Each hen was fitted with an approximately 50 g backpack when 23–24 weeks old. Each backpack contained three monitoring devices intended for later studies: a light sensor , tri-axial accelerometer , and a location device . The equipment was wrapped in brown electrical tape and attached to the back of the hen using elastic loops around the wing base. This meant that the larger part of the package was covered by the neck feathers when the head was up . On day Hens that belonged to the group that was not fitted with backpacks on a certain day were instead held by an experimenter for 30 s (the approximate amount of time it took to put on a backpack) before the start of data collection . To avoid confounding between our treatment and possible circadian patterns, hens that were to be equipped on that day and those that were not were collected from the pen alternatingly. As hens would be used as their own control  individuals were handled in the same order on day 8 as on day 0.2.3Immediately after being equipped or held, each hen was held and photographed (in profile orientation) at a standardized location 1 m from the FLIR E50bx thermal camera (emissivity: 0.96). One minute later a second thermal image was taken after which the hen was placed in a holding pen (91 × 62 × 62 cm length × width × height) which permitted visual, but not physical, contact with conspecifics. Whilst in the pen five thermal images were taken at approximate 1 min intervals from a standardized distance, attempting to photograph the hen in profile and when not moving. Hens were photographed away from metal surfaces and air currents . Because acquiring reliable images  was not possible in the home pen, no thermal images were taken before collecting the hen, after placing the hen back, or on subsequent days.Thermal images were analysed using the Thermacam Reporter Professional 2000 software package , acquiring the temperature of the midpoint of the eye and an average temperature within the traced area of the comb.2.4Behaviour was assessed using direct continuous focal observation. Each hen was observed during the five minutes she spent in the holding pen and during the first five minutes after release from the holding pen back into the home pen. In addition, all hens were observed 2, 3, 5, 7 days after each day of backpack fitting for 10 min per hen per day. Hens were always observed in the same order in which they had been handled during backpack fitting.All observations were made by a single observer using Obansys software  on a handheld device. The percentage of observed time spent sitting/lying, standing, walking, sidestepping/reversing, foraging, preening, eating/drinking, dustbathing and performing and receiving gentle feather pecks was recorded, simultaneously scoring the percentage of time spent in different locations . In addition, the frequency of agonistic behaviour , body pecks, severe feather pecks , wing flaps and wall pecks was simultaneously recorded, as was the frequency of equipment pecks (to the backpack or leg rings if hens were wearing a backpack and to the leg rings only when hens were not wearing a backpack). We did not distinguish between ring pecks and backpack pecks, as we would not consider a shift from pecking one type of equipment to another as a reason not to recommend the use of backpacks in further research.2.5Any faeces produced whilst the hen was in the holding pen or whilst being weighed seven days later were noted. Faeces were collected and stored with the intention to analyse glucocorticoid metabolites.2.6Individual bodyweights and plumage damage were assessed one day before and 7 days after fitting the backpacks (and handling the hens that did not receive a backpack in that week). These were recalculated into weight gain (or loss) and increase in plumage damage.2, 2: bare skin visible ≥5 cm2). Plumage damage to the back was not scored as the backpack covered part of this area.Plumage damage was assessed separately in three areas  on a 3-point scale  and in the second (day 8–15) experimental period) were analysed using Wilcoxon rank sum tests. Correlations between behaviour and eye temperature in the holding pen were analysed using Spearman rank correlation tests. These analyses were done separately for hens with and without a backpack.33.1Throughout the experiment, agonistic behaviour other than head pecking was extremely rare. Focal hens were never observed to flee or claw, engaged in only one fight and two stand-offs, and were clawed by a conspecific only twice. Similarly, performing and receiving severe feather pecking was very rare . Because of their low occurrence none of these behaviours were analysed statistically.Newly backpacked hens placed in the holding pen spent significantly more time preening, sitting/lying and sidestepping/reversing, but less time standing and walking than when they were placed into the holding pen without a backpack . When neTwo days after fitting the backpacks, hens jumped or flew less and pecked their equipment more when they wore a backpack, although these effects were minor and displayed by a minority of the hens (jump/fly – Backpack: 0 (IQR: 0–0) vs. No backpack: 0 (IQR: 0-0.1), P < 0.05, Z = 2.2, peck equipment – Backpack: 0 (IQR: 0–0.12) vs. No backpack: 0 (IQR: 0–0), P < 0.05, Z = −2.4). Three days after fitting, hens received more feather pecks when they wore a backpack (Backpack: 0 (IQR: 0–0) vs. No backpack: 0 (IQR: 0–0), P < 0.05, Z = −2.0). Seven days after fitting hens pecked their equipment more when wearing a backpack (Backpack: 0 (IQR: 0–0.1) vs. No backpack: 0 (IQR: 0–0), P < 0.05, Z = −2.0). No other significant effects were detected on days 2, 3, 5 and 7 (Supplementary Tables 1 and 2). Because the effect of the backpacks thus seemed far subtler on the days after (rather than immediately after) fitting, data from these four days were averaged to acquire a more robust estimate of behaviour per hen. Analysis of this averaged data showed that when wearing a backpack, hens pecked their equipment significantly more often .Behaviour whilst wearing a backpack was also influenced by whether the backpack was worn in the first (day 0–7) or the second (day 8-15) experimental period . CompareThe hens had usually all laid their egg before the observations started, and no clear signs of pre-lay behaviour were observed. In line with this, nestbox use was rare .3.2Tracing the outline of the comb on the thermal images taken when the hens were in the holding pen proved difficult, as the comb was often partly obscured by the bars of the pen. Therefore, the comb images when in the holding pen were discarded. In addition, 11 out of 40 comb images and 29 out of 120 eye images had to be discarded due to poor image quality.Comb and eye temperature taken directly and one minute after handling did not differ when hens had been equipped vs. when they were held only . However, average eye temperature whilst in the holding pen was significantly higher when hens were wearing a backpack (No backpack: 30.2 °C (29.0–30.6) vs. Backpack: 30.9 °C (30.0–32.0), P < 0.001, Z = −3.7).More detailed analysis of the pattern of eye temperature over time showed that eye temperature was significantly higher (or tended to be) when the hen was wearing a backpack than when she was not, for all minutes in the holding pen (The hens that did not receive a backpack during the first period (day 0–7) of the experiment had a lower eye temperature 30 s after retrieval from the pen than those that did not receive a backpack in the second period (day 8–15) of the experiment . No batc3.3s = 0.78, P < 0.001). A similar association was found when not wearing the backpack, although in this case it was less expressed .When hens were wearing a backpack, their average eye temperature whilst in the holding pen was strongly correlated to the percentage of time they spent preening  and negatively associated with time spent sidestepping/reversing  and sitting/lying . These behaviours were rare to absent in hens not wearing a backpack though, and no association was found in this subset.In addition, eye temperature of hens wearing a backpack was positively associated with the number of equipment pecks . The two hens that did not defecate when not wearing a backpack did not do so when wearing the backpack either. Defaecation rate was 3 out of 10 for backpacked hens and 9 out of 10 for non-backpacked hens in both halves of the experiment.Only two hens showed any plumage damage (one when wearing a backpack and one when without) and therefore plumage damage was not analysed statistically.3.5Bodyweight increased during the experiment. More specifically, hens gained weight in the first period of the experiment rather than in the second period, regardless of the half of the experiment during which they wore a backpack .Median body weight gain during the 7-day period when wearing the backpack was not found to differ significantly from weight gain during the 7-day period when not wearing the backpack (No backpack: 110 g (IQR: 28–190) vs. Backpack: 70 g (IQR: 10–195), P = 0.82, Z = 0.2).4We aimed to assess how laying hens responded  to being fitted with a backpack containing measuring devices, as studies using such hen-mounted devices are gaining popularity.Agonistic behaviour was very rare throughout the study, which was an important finding as in our pilot studies  hens with more discernible devices had frequently been attacked and chased, which distorted their behaviour. In the current study the only elements of agonistic behaviour that occurred frequently enough for analysis (receiving and performing head pecks) did not increase significantly when hens wore backpacks, in line with previous observations in pullets . FeatherAlthough agonistic behaviour was very rare, the hens did show other changes in behaviour when wearing our backpacks. In line with our expectations, newly equipped hens spent more time performing behaviours that likely reflect attempts to remove the backpack or to move it to a more comfortable position  when placed in the holding pen. Conversely, hens seemed less interested in exploring or escaping the holding pen when wearing a backpack, as they spent less time walking and pecked the pen less. Most of these differences were also observed in hens that had been placed back in the home pen, where hens also ate/drank and foraged less and received more equipment pecks. These equipment pecks were mainly received by hens wearing the backpacks during the first period of the experiment (day 0–7), suggesting that hens became less interested in the backpacks upon repeated exposure. A quarter of the hens spent most of their time sitting or lying down in the holding pen when wearing a backpack, whilst no hens sat or lay down in the holding pen when not wearing a backpack. In the context of the open-field test, more time spent sitting still is interpreted as a sign of increased fear . Our holIf the backpack or the fitting procedure induced fear, a lower eye temperature would be expected, as hens are reported to show an immediate decrease in peripheral temperature as an initial response to stress . However5The effects of our backpacks on hen behaviour were relatively mild, even nearly absent from 2 days after equipping on. This enables application providing a short period of acclimation is permitted. There was no clear evidence of an overall stress response when studying peripheral temperature and weight gain. This method of attachment thus seems appropriate when fitting technology to monitor laying hen behaviour shortly, though perhaps not immediately, following fitting.


Copyright: Alameda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Present: The grant support information is incomplete.Correct: The complete grant support is as follows.29531-29547. https://doi.org/10.18632/oncotarget.8792Original article: Oncotarget. 2016; 7:FUNDINGThis research was supported partially by funds from Fondo Europeo de Desarrollo Regional (FEDER) and by grants from the Ministry of Economy and Competitiveness of Spain  and by grants 1.010.511 (Fundacion Banco de Santander-Universidad Alfonso X el Sabio) to M.J Fernandez-Acenero; Comunidad Autonoma de Madrid grant S2010/BMD-2470 (Oncocycle Program) and CIEM13-4E-1944 to JMP; AES grants ISCIII-RETIC RD06/0020/0029 and RD12/0036/0009 to JMP.


The preclinical development of anti-sarcoma drugs has been primarily based on the subcutaneous transplantation of xenografts. Transplant survival remains an obstacle of current models which has been attributed to the period of hypoxia after transplantation. We hypothesized that primary soft tissue sarcoma models with an intrinsic tissue engineered vascular supply would be easily reproducible. The aim of this study was to establish a model of primary human soft tissue sarcoma with an intrinsic vascular supply.Primary soft tissue sarcoma cells from resected human liposarcomas isolated and divided into tumour fragments were transplanted into a silicon chamber, placed around the superficial epigastric vessels in mice. Sarcoma xenograft samples were analysed histomorphologically (light/electron microscopy and immunohistochemistry).All primary soft tissue sarcoma transplants engrafted, leading to solid tumours within 3 weeks. Histological and immunohistochemical staining confirmed the mouse xenografts as identical high grade liposarcomas compared to original tumour tissue.This study established a reproducible xenograft model of primary human liposarcoma. This animal model could be of high value for studying human soft tissue sarcomas and their therapy. Liposarcomas are tumors derived from primitive mesenchymal cells that undergo adipose differentiation and constitute approximately 10% of all soft tissue sarcomas. Liposarcomas are predominantly a disease of adulthood with peak incidence around the 5th to 6th decade of life and with a slight predominance toward males. The extremities are the most common primary site, accounting for about 40% of cases. Nearly one-third of cases arise in visceral spaces, and there is an association between site of primary tumor and histologic subtypes -5. Complin vivo biological and preclinical studies [in vivo [Subcutaneous and orthotopic xenografts of human tumours in nude mice are an accepted model for  studies -19. Xeno[in vivo . The sta[in vivo , 19, 21 [in vivo , 19, 21.Six weeks old, sexually mature NMR nude mice  weighing about 20 - 25 g were used in the present study. Animals were housed in ventilated racks with controlled humidity and temperature (20 ± 2 °C) under pathogen-free conditions and a 12 h light-dark photoperiodicity and. Food and water as well as boxes, bedding, were sterilised. The surgical procedures used for the inoculation of the cells to give rise to solid tumours and for subsequent removal and transplantation of tumours was conducted under general anaesthesia and steril conditions. All experimental procedures were carried out under the guidelines and with the permission of the ethics committee of the Ruhr University Bochum. Animal care and manipulation adhered to institutional guidelines and the Guide for the Care and Use of Laboratory Animals . Writtenin vivo experiment. The tumour tissue was cut into 1 - 2 mm fragments prior to transplantation.On the day of surgery soft tissue sarcoma samples were directly transferred from the operating theatres to the laboratory. Following the tumour ablation tumour samples were taken under sterile conditions at a representative area of the original tumour mass. The sample was then divided into two adjacent parts, one for further histology  and the other for the Tissue engineering chambers (n = 6) consisting of 5 mm of length of silicone laboratory tubing  were inserted into the groins of the mouse as previously described . The aniThe tissue engineering chamber was then placed around the vessels. The chamber was secured to the underlying muscle with a 10-0 nylon microsuture. The longitudinal slid and proximal aperture of the chamber was sealed with bone wax . Care was taken to avoid occlusion of the vascular pedicle. Tumour fragments suspended in Matrigel®  were inserted into the chamber. At 37 °C the matrix solidifies and thus ensures that the cells remain in situ. Then the distal aperture of the chamber was also sealed, creating a semiseald chamber again taking care to not compromise the vascular patency. The construct was carefully returned to the dissected plan within the groin. The wound was closed in layers. The anaesthesia protocol was uneventful in all cases. All animals survived the surgical procedure and recovered well. After recovery animal behaviour returned to normal and remained unsuspicious. No wound complications were noted.in vivo in which the animals were allowed to move freely and were fed a standard mouse chew ad libitum the animals were again anaesthetised. The wounds were explored and the chamber (tissue engineering chamber) was exposed. In the chamber the vascular patency was recorded. The vascular pedicle was cut outside the chamber and the chamber retrieved from the implantation site. The outside of the chamber was freed from surrounding fibrous capsular and the chamber reopened. The newly formed tissue was then carefully removed. Tissue specimens were photographed and the tissue was divided into two sections one for histological assessment the other one for electron microscopy. Tumour pathology was analyzed by histological and immunohistochemical staining.After an incubation of 3 weeks Tumour samples were placed in 10% formaldehyde. A minimum of five individual sections of the primary tumour were assessed. The primary tumour and the xenotransplant counterpart were sectioned into 5 µm thick sections.Histological sections were deparaffinised, rehydrated and stained with haematoxylin and eosin following standard procedures.Primary tumour and the xenotransplant specimens were fixed in 2% glutaraldehyde and embedded in Epon 812. For contrast uranyl acetate and lead citrate were added to the ultra thin sections. Samples were analysed, special focus was placed on differentiation criteria [Diagnosis of the primary human liposarcomas was confirmed by independent reference histology. Tumour diagnosis and classification were determined according to WHO guidelines .The liposarcomas showed a pleomorphic growth pattern with atypical mitoses and a marked increase in nuclear and cell changes. Bizarre pleomorphic lipoblasts were seen next to spindle shaped tumour cells, even some giant cells were found. Myxoid changes were rare and tumour necrosis was seen in 15 % of areas. Pancytokeratines (MNF116), EMA, smooth muscle actin, desmin, S100 and CD34 were negative .Electron microscopic analysis showed malignant cells with mostly decreased rough endoplasmic reticulum and heterogeneity of cells and nuclei size and shape were present. Nuclear irregularities with multiple nucleoli were frequent. Nucleoli were increased in size and number. The sarcomatous cells had highly irregular nuclear profiles. Mitochondria were electron dark and elongated. Golgi complexes were present. No accumulation of glycogen was seen. Mitotic accumulation of lipid globules was a frequent finding. Few giant cells were present. Focally necrotic areas were seen .In all cases (n = 6) the liposarcomas in the chamber model demonstrated a moderate, partial severe inflammation with giant cells was detectable. All cases showed circumjacent to the tumour, slight to moderate proliferations of fibroblasts . Ultrastin vivo tumour model. Xenotransplantation of tumour cells into immune deficient mice has been well recognised and is a useful experimental model in cancer research [Animal models are indispensable tools for the study of liposarcomas as a paucity of clinical samples makes large-scale analysis of human samples challenging. Furthermore, the diversity of sarcoma subtypes complicates analyses of human sarcomas. Until recently, studies of sarcoma biology were limited to human cell lines and xenografted tumours. An in depth understanding of the tumour biology of these rare tumour entities is warranted to develop and improve sarcoma treatment thus demanding for a well characterized and reproducible research -19, 24. research -28. A 50research -28. In aresearch . The intresearch -28. Hencresearch . It can research -19. To oThe vascularised chamber was initially developed for cell transplantation in a tissue engineering setting at the Bernard O’Brien Institute of microsurgery . It has In the present, all of the xenotransplants into the groin chamber resulted in a survival of the transplanted liposarcomas. As expected from the tissue engineering experiments, an increased angiogenesis was also observed after xenotransplantation of tumour tissue within the groin chamber. All groin chambers displayed a dense functional newly formed vascular network. Tumour cells migrated within the matrix of the groin chamber and seemed to be evenly distributed throughout the chamber. A reduction of connective tissue within the tumour with an increased cell number after xenotransplantation has been described previously.in vivo. In addition, primary cell lines are known to acquire genetic alterations after tissue culture expansion. Therefore, in the present study we used tumour fragments transplanted directly after tumour resection to best be able to compare the growth pattern to the original human sarcoma. A potential disadvantage to the use of these tumour fragments includes the heterogeneity of the tumour fragment composition and therefore lack of standardisation.Immortalised tumour cell lines offer an attractive opportunity for cancer research. Their availability and standardised growth pattern make them suitable for routine testing, but their dedifferentiated nature and acquired additional genetic alterations during the immortalisation process may not reflect all aspects of the primary tumour -28. DiffNo metastases were observed in any experiment. The short three week observation period may explain this. The tissue engineering chamber produced viable and good tumour growth and seems to be a favourable model to analyze early tumour implantation and tumour stromal interactions. It also offers the chance to better assess the tumour angiogenesis early after transplantation. On the other hand the space limitation with in the silicon chamber might hinder extensive tumour growth.In conclusion, the vascularised tissue engineering chamber offers a suitable experimental sarcoma model. This model is valuable for sarcoma research because clinical samples are relatively rare and treatment regimens have not changed in decades for many sarcoma subtypes. The further development of models, including the vascularised tissue engineering chamber will continue to increase our understanding of soft tissue sarcoma biology. The susceptibility to hypoxia differs between the sarcoma subgroups and the type of soft tissue sarcoma, the vascularised tissue engineering chamber appears to have benefits in successful outcome of xenograft growth, in creating a more vital tumour that may be a more faithful model and on study of tumour stroma interaction and tumour angiogenesis


Tumour heterogeneity refers to the fact that different tumour cells can show distinct morphological and phenotypic profiles, including cellular morphology, gene expression, metabolism, motility, proliferation and metastatic potential. This phenomenon occurs both between tumours (inter-tumour heterogeneity) and within tumours (intra-tumour heterogeneity), and it is caused by genetic and non-genetic factors. The heterogeneity of cancer cells introduces significant challenges in using molecular prognostic markers as well as for classifying patients that might benefit from specific therapies. Thus, research efforts for characterizing heterogeneity would be useful for a better understanding of the causes and progression of disease. It has been suggested that the study of heterogeneity within Circulating Tumour Cells (CTCs) could also reflect the full spectrum of mutations of the disease more accurately than a single biopsy of a primary or metastatic tumour. In previous years, many high throughput methodologies have raised for the study of heterogeneity at different levels . The aim of the current review is to stress clinical implications of tumour heterogeneity, as well as current available methodologies for their study, paying specific attention to those able to assess heterogeneity at the single cell level. In oncology, molecular, cellular and architectural variability are frequently referred to with the term “heterogeneity”, a concept that increases the complexity of the pathogenesis of malignant tumours. In terms of cell phenotype, cell density or cell location, cell heterogeneity can be observed between tumours that occur in the same organ and/or between patients. Inter-tumour heterogeneity leads to the classification of tumour subtypes, which can be distinguished by whether or not their molecular profile correlate with their morphologies and expression of specific markers. In addition, tissue variation also occurs within individual tumours (intra-tumour heterogeneity), meaning that tumour cells can have various functional properties and can express several markers ,2. Such Ideally, tumour heterogeneity should be monitored over time, and more specifically in relation to therapeutic strategies. However, the invasiveness of biopsies makes it impractical to perform them frequently. The risk of cell dissemination, the procedures cost, as well as sometimes the existence of multiple metastases and the time needed must also be taken into consideration. Circulating tumour cells (CTCs) are a potential surrogate for tissue-based cancer diagnostic and may thus provide the opportunity for monitoring serial changes in tumour biology. Recent progress has made possible accurate and reliable quantification and molecular characterization of CTCs ,5. The pIntra-tumour heterogeneity was demonstrated by Fidler and Hart more than 30 years ago in murine models . It refeIntra-tumour heterogeneity was initially explained by means of the cancer stem-like cell (CSC) or clonal-evolution models  shown inInter-tumour heterogeneity defines differences between tumours of the same origin in different patients. These tumour subtypes have specific individual molecular signatures, different biological behaviours and, as a result, have a differential impact on clinical outcomes . Two maiEarly findings suggested that genetic heterogeneity was a result of treatment pressure . HoweverNowell proposed that most neoplasms have their origin in a single cell, and tumour progression results from acquired genetic variability within the original clone making possible sequential selection of more aggressive subpopulations . NowadayChromosomal instability is one of processes that generate variability. Cells capable of surviving with this genetic instability generate a mutational load and genetic diversity. Increased genetic instability is considered to be one of the cancer hallmarks ,27. The Nongenetic factors are associated with development of so-called deterministic and stochastic heterogeneities. Stochastic heterogeneity corresponds to differences between genetically identical cells caused by spontaneous modifications to biochemical processes in the cell when deterministic diversity develops . These pThe tumour microenvironment, including inflammatory cells, hypoxia conditions or the extracellular matrix, is the main external factor that promotes cell diversity by selecting cells adapted to various conditions in the microenvironment . The mosDifferences in DNA mutational status and RNA or protein expression profiles between the primary tumour and metastases, or within a single tumour, has been widely reported ,40,41,42® system (Veridex), which is the only assay for enriching and enumerating CTCs in clinics that has been cleared by the US Food and Drug Administration, or the DEParray™ technology , the only automated instrument that can identify, quantify and recover individual cells after initial pre-enrichment step [Different studies have shown that CTCs exhibit considerable cell-to-cell diversity ,47,48. Ient step ,52. We pRecent findings have identified the importance of CTC clusters in tumour dissemination. Contrary to common belief, Au et al. demonstrated that grouped CTCs are able to pass through the narrow vessels to reach distant organs . This maThe main reason why cancer treatments fail is that treatment programmes are not designed to address tumour heterogeneity. Most of the methods used to characterise tumours lead to a global overview of the characteristics of the cancer tissue, corresponding to an average picture of all the tumour clones and their microenvironment. The intra- and inter-tumour variations of biomarkers may affect the success of biomarker-driven clinical trials if the biomarker used to both predict the therapeutic response and stratify the patients into subgroups shows spatial variability. In addition, divergent evolution of metastatic tumour cells and different microenvironments at the metastatic sites may contribute to the change in the expression of biomarkers initially identified in the primary tumour ,54. TreaIt is possible that new targeted therapies introduced into clinical practice will induce temporary benefits until the tumour becomes resistant. Monitoring accumulated genetic or plastic changes in cancer during patient’s treatment might guide selection of the next therapy, or even the addition of a subsequent therapy. Ideally, researchers would obtain multiple regions from each tumour or metastasis in order to capture spatial heterogeneity. Epigenetic and other analyses also need to be performed. Clinical annotation of the samples and phenotypic correlation are essential at each step. At present, medical protocols envisage obtaining biopsies to establish diagnosis and determine whether the predictive biomarkers are in agreement or instead present differences between the metastases and primary tumours. However, most metastases are difficult to access, and biopsies are invasive, inconvenient and costly. Obtaining subsequent biopsies is thus almost impossible from a logistic point of view. To overcome these problems, detecting and characterising CTCs might be an excellent alternative.Studies on CTCs have focused on their prognostic significance, their utility in real-time therapies monitoring, the identification of therapeutic targets, resistance mechanisms and understanding the metastatic process ,56. ReceIn the following section, we will review available methodologies for studying tumour heterogeneity in tissues and at the single cell level. Assessing heterogeneity in tissue samples has been limited to methods based on microscopy, such as immunohistochemistry (IHC), immunofluorescence (IF) and fluorescence in situ hybridisation (FISH). In situ hybridisation techniques make it possible to detect RNA, DNA and protein molecules with high sensitivity in both frozen and formalin-fixed paraffin-embedded (FFPE) tissues ,70. ThesImmuno-FISH identifies genomic imbalances and specific DNA translocations, in particular cell subpopulations by combining the hybridisation of probes with the immunodetection of antigenic markers . ComparaHeterogeneity at the protein level has mainly been investigated by targeted assays, using antibodies for IHC, or after extraction by reverse phase protein array or liquid chromatography. Matrix-assisted laser desorption/ionisation (MALDI) and multiplexed ion beam imaging are performed in situ while preserving the morphological characteristics . EvaluatAs mentioned in the previous sections, single-cell analysis is the most reliable approach for studying tumour heterogeneity. This section briefly summarises the technologies most used today to identify molecular differences at the single-cell level. Advances in Whole Genome Analysis (WGA), Next Generation Sequence (NGS), fluorescence-activated cell sorting (FACS) and other techniques have made it possible to analyse multiple markers in single tumour cells isolated from fresh or fixed primary tumours and metastases ,79,95,96Single-cell technologies have evolved considerably in the area of genome and transcriptome sequencing in the last ten years. The first challenge is obtaining a suspension of viable single cells from complex solid tissues by means of mechanical or enzymatic dissociation, or by using more sophisticated technologies such as laser-assisted microdissection . Once inIt is thus necessary to define the type of genomic interrogation of the amplified genomes. Depending on the objective of the study, it is possible to query specific loci of interest  ; sequencIf the aim of the study is to explore transcriptome heterogeneity (Whole Transcriptome Sequencing (WTS)), single-cell RNA sequencing methods must be applied. Nowadays, there are many protocols available for RNA sequencing at the single-cell level, Quartz-seq, CEL-seq, STRT-seq, Smart-seq or Drop-Seq are the best known, each one having both advantages and disadvantages ,86. A veRegardless of which single cell analysis is performed, WGS or WTS, and given high number of technical errors that emerge during the process, the results must be validated, as these technical errors are often interpreted as real biological variations . DespiteAnother source of tumour cell heterogeneity is related to epigenetics, including histone modification, and DNA base modifications (such as methylation and hydroxymethylation), as mentioned previously. Robust technologies have been developed to provide genome-wide maps of most epigenetic marks. ChIP-seq, suitable for studying histone marks at the single-cell level, combines chromatin precipitation with next generation sequencing . To asseFluorescent Activated Cell Sorting (FACS) data allows disrupted expression patterns in proteins on cells in cancer to be identified, as well as to analyse signalling networks (phosphorylation) and their link to the outcome of the disease. The instruments grouped under the term “flow cytometer” now extend to microfluidics, and miniaturise the components into lab-on-a-chip devices ,93. ThesOne of the drawbacks of the flow cytometry analysis is that some pathways may be sensitive to disruption when cells are taken out of context, leading to changes in the intracellular signalling, and this may thus alter results of cell heterogeneity analysis. In whole blood–based assays , this tends to be minimised. However, despite proteomic analyses at the single cell level which have been already successful in haematological malignancies ,107,108,Studying proteomics at single-cell level is also possible with Cytometry time of flight (CyTOF), developed by Bandura et al. . CyTOF wCancer is a heterogeneous disease, with variations in terms of morphology, immunophenotype and genotype. Analysing conventional genetic profiling on bulk single biopsy tissues is often used today as the standard for evaluating tumours, thus limiting diagnostic and treatment decisions to the analysed region. Profiling of bulk samples may also unravel the phenotypes obtained from previous selective pressures which are not relevant to the status of the current metastatic disease. Furthermore, different regions of the same tumour can contain signatures for either good or bad prognoses; a single biopsy may thus not be sufficiently representative of the tumour’s composition.Circulating tumour cells (CTCs) can be isolated and interrogated as a potential window into the genetics of a tumour by means of non-invasive sampling, making it possible to evaluate the tumour’s temporal heterogeneity during the clinical course of the disease. To overcome the averaging approach determined by using bulk CTC analysis, single-cell isolation can only be achieved with sophisticated instrumentation, requiring expert operators such as the DEPArray™ system,  and time-consuming protocols which can only guarantee the single-cell level on rare occasions. Moreover, depending on the methodology used for their isolation, the number of recovered CTCs may be different and thus the analysis of heterogeneity will report different results ,113. CTCOne of the key questions is to determine to what extent CTCs represent heterogeneity within the primary tumour or metastases. It is possible that the cells detected with the currently available techniques are merely those that were shed and are only the “tip of the iceberg”. For example, it is clear that a high number of CTCs detected are associated with poor prognosis, but it is not possible to know if these cells actually have malignant behaviour themselves. Perhaps they are just terminally differentiated cells that merely reflect the presence of more malignant, but uncaptured cancer cells. These cells do not necessarily represent the biology of the underlying tumour . InteresIn the last ten years, many new technologies have been developed in order to characterise single-cell heterogeneity at the DNA, RNA or protein level. Some of these technologies are reviewed in It is important to note that the tumour microenvironment itself is also heterogeneous. This microenvironment, which defines an additional barrier or an asset for therapy, is responsible for differently directed selective pressure within the same tumour, changing the evolutional trajectories of the tumour cells . This op