Twitter is a cold medium that allows users to deliver content-rich but small packets of information to other users, and provides an opportunity for active and collaborative communication. In an education setting, this social media tool has potential to increase active learning opportunities, and increase student engagement with course content. The effects of Twitter on learning dynamics was tested in a field biology course offered by a large Canadian University: 29 students agreed to take part in the Twitter project and quantitative and qualitative data were collected, including survey data from 18 students. Students published 200% more public Tweets than what was required, and interacted frequently with the instructor and teaching assistant, their peers, and users external to the course. Almost 80% of students stated that Twitter increased opportunities for among-group communication, and 94% of students felt this kind of collaborative communication was beneficial to their learning. Although students did not think they would use Twitter after the course was over, 77% of the students still felt it was a good learning tool, and 67% of students felt Twitter had a positive impact on how they engaged with course content. These results suggest social media tools such as Twitter can help achieve active and collaborative learning in higher education. Using the hashtag, students have on-demand access to relevant course material .Twitter, a prevalent social media tool, has become a major player in education. While some skepticism remains by some teaching faculties, there are many who recognize the perceived benefits of using Twitter in the classroom, including the following:These insights provide a foundation to understand the possible positive effects of Twitter in an educational setting. However, there must be a clear recognition that the tool itself will influence the messages sent by students, and received by others. This relates toHot media deliver large packets of content in high definition that fill the recipient with information . Since hCold media, in contrast, send small amounts of information thereby requiring recipients to “fill in the blanks” in order to make meaning of the content , p. 22. This change between hot and cold media is dependent on the context in which the recipient engages with the material, suggesting the personal motivations of a student, for example, can impact their participation level, thereby influencing the medium. We posit that Twitter is a cold medium, since Twitter restricts content to a small amount of content and it follows that the recipients  are required to engage actively with the content through high levels of participation to make meaning of it. The medium, Twitter, would be directly responsible for shaping the message. We sought to directly quantify the effects of Twitter on learning dynamics in an undergraduate field biology classroom, and to place our findings in the context of how the medium shapes the message, and on the medium type. Our first objective was to test the effects of the medium, Twitter, on student engagement, and our second objective was to demonstrate how Twitter affected communication patterns and interactions within the course parameters.(iv) Communicate about Natural History & Ecology to general and specialized audiences (including the instructor), through a range of media ,” thus ensuring that Twitter integration into the course design was both purposeful and meaningful. To achieve this, students were provided an outreach strategy, using Twitter, worth 15%, in the course syllabus. The outreach strategy stated:A field biology course at a large Canadian post-secondary institution was the selected research site as part of the course was designed around student-led, group-based research projects about natural history (Outreach (15%) – during November, groups will be publishing blog posts about their selected study species and their research project. The post is to include an overview of the biology and natural history of the study species, outline of research project, personal anecdotes related to the projects, links to other resources and scientific literature, and photos or other media. This is worth 5% as each group member shall receive the same grade on the blog post. Throughout the term, groups will also use Twitter as a means to disseminate information about their study species, and as a means to network and collaborate about natural history within and beyond the course boundaries. Early in the term, the groups will make an Twitter account for their project, and will be required to tweet at least 4 times within a 24 hour time period of each field laboratory . Tweets will be ‘relevant’ to [course]. At least 10 tweets are to be released within 48 hours of each group’s blog post being published. Grading will be based on quality of tweets, evidence of communication within the course, and with people beyond the boundaries of the course. This is worth 10% and each group member shall receive the same grade.To maximize the potential for student success, students were given a “How-to” Twitter guide, the assignment details and a document containing reasons for using Twitter in the course.1. Quantity and format: groups met the minimum requirements for tweets, hashtags used appropriately.2. Etiquette and Professionalism: tweets and interactions on Twitter were mature, professional and written with appropriate tone.3. Engagement: evidence of collaboration, networking and engagement, both within the course and beyond; use of replies, direct tweets, and interactions.4. Quality: content of tweets of high quality, relevant to study species, research projects and the overall course content.5. Links and Photos: appropriate use of linked content in tweets, including photos .Within the Twitter assignment details, it was emphasized that Twitter was engaging, collaborative, and provided networking capabilities. It was explained that Twitter enables tracking and archiving of content, has academic and teaching value, and provides validation (i.e. it gives students an audience other than the professor and teaching assistant). There were explicit instructions and expectations, and student were given the following grading rubric as related to Twitter:The purpose of our project was to specifically assess how the use of Twitter in the field biology course affected learning dynamics, and as such, the tweeting process, and tweets themselves, became our quantitative data. Data about the students’ tweets, collected using various methods including, observations, tweet analysis, and individual student interviews, were conducted by the first author. First, pending consent, students were asked to provide a copy of their Twitter profile statistics, which were obtained through Twitter. Each groups’ tweets were analyzed in two distinct ways: content  and the tweet interactivity levels  in order to provide understanding of how students shaped content to fit within the Twitter restrictions and interaction levels. All participants were asked to complete a survey to gain insight into their thoughts around Twitter use; 18 students  participated in the survey. The complete survey can be found inBased purely on the number of tweets sent by our four study groups, students clearly used Twitter, sending over 200% more tweets than minimally required . Of thesActive learning pedagogy states that when students are actively involved in the learning process, it can result in enhanced memory recall and learning . Two impThe general results  illustraData from the survey are shown. The survey data provide quantitative information from all selected study participants.Click here for additional data file.Copyright: © 2015 Soluk L and Buddle CM2015Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).post hoc, and were able to separate the 521 tweets into the following distinct functions, in descending order of frequency: outreach , informal communication , promotion  and the 140-character restriction. Together, the limited restrictions gave students control of how to use Twitter (e.g. network building) and control over the content of tweets. Given that the students could control content, we categorized tweetsYeah, and many times I felt like well, come on I want this word in but how can I, you know, cause I didn’t want to shorten words cause it was still for the class. So I was trying to find a way to rephrase, and you’d rephrase that and because you rephrased it the second sentence would make sense. So you’d have to, it was much harder grammatically than like, that I expected. (Andrew)The 140-character restriction provided a limitation on tweet content. The students did not all view the 140-character favourably and found it frustrating. For example, Andrew (All names were given pseudonyms to protect student identity) explained,It’s hard to say because like since it’s limited it also gets people to write like meaningful things, like straight to the point instead of just dragging on sentences and stuff. (Emily)Despite this, the students indicated the restriction had an impact on their thinking: the limitation helped students clarify thoughts, as they had to consciously think about how to structure the text and content of the tweet. This idea is supported by Emily, as she stated,This restructuring of tweet content is a characteristic of active learning as the students actively manipulated the content to fit within the character restriction of a tweet.So it’s interesting because despite you saying that you don’t use Twitter you still have a very positive view of it? (Lauren)Yeah I know really because I kind of like, I don’t know how to say... because I kind of think about Facebook and I know how it’s like a great, because I just don’t know Twitter enough. But maybe if I knew Twitter how I know Facebook and if I used Twitter how I, how I used Facebook I know it would really like I would really like it. And I would really like students to go on there and to share things, and to, but it’s just the social media [the professor] chose I’m not really comfortable with. But I know it’s a great, a great idea to use social media to learn because, well Facebook I would have been really more comfortable but it’s okay. (Kristen) Given that 83% of the students had never used Twitter, the newness of the tool shaped the impact of the medium as the students had to learn how to use it. Despite this, the students evaluated Twitter and its associated benefits and drawbacks. There were few negative judgments of Twitter. For example, even a low-level Twitter user was able to see the potential benefits of using Twitter:Even though many students found that Twitter was a useful tool in the classroom, a large number of participants indicated that they would not continue to use it after the class finished: 67% of respondents said they would no longer continue to use Twitter outside of the classroom, where 28% said they would (one student said “maybe”) . These rSince Twitter was used in a group setting, it was anticipated that the medium would have an effect on group dynamics, intra-class dynamics, and communication with outsiders. Given that the Twitter responsibilities were associated with a particular group, not individuals, the integration of Twitter had strong implications for relations within groups, as the students had to identify individual Twitter roles. This design was purposeful as it was a strong belief of the second author (based on previous experience) that not all students would gravitate towards the medium; the survey results and field observations supported this belief.Twitter was not my, I didn’t fully use Twitter. I posted one picture one time and some, some little comments, but most of the time I was like, I found some, some articles on the Internet. And I was like I tell them on Facebook I sent them, and [Marie] was like okay I’m gonna post this. And name was more like the, the poster and I, and I kind of did a little research. (Kristen)Most of the tweets I participate in is when we were doing the data collection in the-, so it was like on the moment I would say, “Oh, you should tweet that, it’s fun,” or something like at. (Carla)The interviews and field notes suggested that there was one group member who was often responsible for the majority of tweets and other group members’ involvement ranged from non-participation to active involvement with Twitter, suggesting the emergence of a Twitter hierarchy, yet despite the varying levels of Twitter use, 78% of participants still felt that they contributed to creating tweets. Some interviewees indicated that they participated through idea sharing and research:Despite the different levels of Twitter use, the excerpts also suggest that the students still worked together to form their tweets. The data provided supports the claims by the second author as there were different levels of Twitter users. It is reasonable to suggest that the group format directly contributed to the successful integration of Twitter as it provided those who favoured Twitter the opportunity to use the medium and those who were less inclined to use the medium and alternative way of contributing to the work without penalization.In the laboratory setting, the research groups were dispersed across a large forested reserve. Without communication devices, groups would have had limited contact with each other and integrating Twitter into the course design provided students with a tool to connect with peer groups and outsiders. The survey data demonstrates that Twitter provided student groups with a unique opportunity to interact with each other and the instructor in an online setting , and stuBecause we really get to see other like in a simple classroom like wouldn’t really know the other team’s research. But on Twitter it’s public and everyone can see it, and like everybody that’s not in the class also can add their own little thing to do it. I don’t know it’s more engaging and interactive. (Kristen) Twitter provided the students with an opportunity to interact and communicate with anonymous outsiders. To use Twitter effectively, students had to build publicly accessible networks and to do so required active participation from students. The students had to actively search and follow like-minded users, as it was their responsibility to develop networks for communication. Quantitative data from the individual group Twitter interactivity profiles demonstrated that students had numerous unique connections with outside users and they often had repeat connections with these users . The stuWell yeah I can say [it’s helpful] because some, some people when they were asking us questions we, it gave us another way of thinking about stuff. And, and sometimes they gave us also papers also to prove their point, so it did help me, well us in a team just to have more papers also to look at to answer our questions, so yeah. (Marie)Combined, 56% of interactions were with the instructor or teaching assistant, 27% of interactions were with peers groups, and 17% of interactions were with external users . ThroughThe various findings, including tweet content and creation and interactivity and communication suggest that student levels of engagement were affected by using Twitter. Engagement indicators are based on a variety of criteria, including academic challenge , learning with peers , experiences with faculty (student-faculty interactions and effective teaching practices), and campus environment  , p. 1. UOur results have implications for many educators. Twitter is determined by user interactions with the platform and in our work, the results were positive because the majority of students felt that they contributed, directly and indirectly, to the Twitter project. This resulted in increased communication, collaboration, and critical thinking, all of which was mostly related to course content. This demonstrates that students were actively involved in the learning process. A contributing factor to the successful integration of Twitter was its group form given that it allowed for differing levels of individual contribution based on comfort levels without impacting academic performance. The results showed that students had different levels of interactions with the medium. This meant that students who enjoyed using the medium could do so, and the students who were not drawn to the medium could have lower participation. Essentially, the group regulation, a by-product of classification, resulted in agreed upon Twitter roles that were comfortable for group members. As a functioning and regulated group they could effectively use Twitter and achieve the intended learning goals. If Twitter were integrated into course design as an individual-based project, we speculate that the results would not have been as positive since not all students are drawn to Twitter. This is evident by the differing levels of student interaction with Twitter. Also, It is also important to consider the interactivity levels of users for an additional purpose: given that not all students prefer to use the medium, the students must be provided with additional activities to engage with course material. Educators must employ various strategies to engage students; Twitter is only one tool that can facilitate engagement and collaboration.A key concern for educators is about how to integrate Twitter successfully into pedagogical practice. Educators must establish boundaries to guide student use of Twitter. In this study, students were required to create tweets related to course content. This restriction forced the students to use the medium primarily for educational purposes, thereby facilitating increased engagement directly with course content. In addition, there was a clear grading schema to assess and evaluation of student’s use of Twitter: this provided an important expectation and framework for the use of social media. Given the various interactivity levels with the medium, we would speculate that without a grade attachment, Twitter uptake would not be as successful. Also, given some students’ unfamiliarity with Twitter, providing documents or lessons on Twitter provides students with the tools to be effective Twitter users.To effectively build and establish networks, Twitter profiles must remain public. This allows for an easy, bi-directional relationship between followers and users. The benefit of network building is increased access to new and alternative learning materials through tweets. Also, with a larger Twitter network, students will have the opportunity to communicate and collaborate with various users. However, this also means that the instructors of teaching assistants should also be familiar with the tools, and leverage their own networks as required.required students to learn about Twitter and evaluate its usefulness in a classroom setting,facilitated engagement with course content,facilitated and enhanced intra-group dialogue,promoted and enhanced communication and collaboration with group outsiders, and,facilitated active learning with students being responsible for actively manipulating and restricting content to form, and response to, tweets.The purpose of the study was to investigate the effects of Twitter on undergraduate learning dynamics. To do so, we explored the functionality and purpose of tweets, Twitter’s effect on communication, and the impact of Twitter on engagement. The data analysis revealed that using Twitter:Twitter can be effectively used in an undergraduate classroom and can be effectively integrated into a course design. As a result of Twitter use, students actively participated in their learning, which resulted in collaboration, increased communication, and facilitated student engagement. The message of the medium is that active and collaborative learning can be achieved, it matters, and it should be recognized and supported.The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2015 Soluk L and Buddle CMData associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Figshare: Raw data from student survey on the use of Twitter to increase engagement in biology courses. Doi:http://dx.doi.org/10.6084/m9.figshare.1360179  its contributions to the scholarship of practice related to using Twitter in the classroom and 2) its methodology to measure the tool's impact. Innovative, is the pedagogical design of a group-level use of the tool. The strengths of the research begin with the strong link and alignment between the course-level learning outcomes and the use of Twitter. This finding, and the implications of the importance of choosing e-Learning tools aligned with student learning, are important contributions beyond the field of teaching and learning with social media. Broadly, I would encourage the authors to make explicit links to the teaching and learning literature in the opening of the paper to strengthen the underpinning pedagogical arguments. For example, there are also more recent (and compelling) references than McLuhan in the literature to support the claim that large-class lectures are more passive. They write that active student engagement is "…considered to be effective pedagogical practice"—again, a link to research that substantiates this claim would improve the argument being laid out.on smartphones was integral to the tool's positive impact on engagement across the forested reserve. In this case, it is notjust Twitter that had an impact, rather it was the combination of the platform (Twitter) and the internet-connected mobile device. This is a broad consideration for future research: how does the mobile, ubiquitous nature of smartphones, used in conjunction with a tool such as Twitter, impact student engagement and outcomes?While it may be implicit, I assume that students' use of TwitterI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. exactly one would use the medium. Moreover, cases that have demonstrated impacts are precious few. This piece addresses both of these shortcomings with a convincing and even handed approach to how one could use technology to augment an undergraduate field based class. They show that it works well for a given period of time, but that like all tools, there are appropriate and inappropriate uses for twitter. Having used the tool in several of my cases, I can wholeheartedly agree with the lack of engagement post-class - the medium simply isn't for everyone. However I think the authors here clearly show how using Twitter can help a class expand beyond the traditional boundaries and help students learn to engage in informal science discourse.While many people on twitter sing its praises for use in class, I think there is a real lack of worked examples of how I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The objective of the present study was to confirm the expression and localisation pattern of the urokinase-type plasminogen activator receptor (uPAR) focusing on its possible clinical relevance in patients with urothelial neoplasia of the bladder. uPAR is a central molecule in tissue remodelling during cancer invasion and metastasis and is an established prognostic marker in various cancer diseases other than bladder cancer. Formalin-fixed and paraffin-embedded tumour-tissue blocks from 186 patients treated with radical cystectomy were analysed. uPAR expression was scored as either negative or positive as well as by the actual score. Separate scores were obtained for cancer cells, macrophages and myofibroblasts at the invasive front and in tumour core. We were able to confirm, in an independent patient cohort, the tissue expression and localisation pattern of uPAR as investigated by Immunohistochemistry as well as a significant association between uPAR positivity and increasing tumour stage and tumour grade. This demonstrates the robustness of our previous and current findings. In addition the association between uPAR positive myofibroblasts and poor survival was reproduced. The highest hazard ratios for survival were seen for uPAR positive myofibroblasts both at the invasive front and in tumour core. Evaluating uPAR expression by the actual score showed a significant association between uPAR positive myofibroblasts in tumour core and an increased risk of cancer specific mortality. Our investigations have generated new and valuable biological information about the cell types being involved in tumour invasion and progression through the plasminogen activation system. Extracellular proteolysis is crucial during tumour growth, invasion and metastasis, because of its ability to degrade the extracellular matrix allowing the tumour cells to invade the surrounding tissue including the vascular bed. The matrix degradation is catalysed by a pericellular network of interacting proteolytic systems, of which the plasminogen activation system is a central player. The plasminogen activation system converts plasminogen to plasmin, which both directly and indirectly, degrades components of the basement membrane and extracellular matrix . PlasminGiven the potential clinical relevance of uPAR expression in bladder cancer tissue, the purpose of the present study was to confirm our previous findings in an independent patient cohort.in situ (CIS) (n = 1) in the cystectomy specimen. In these cases, the prior transurethral resection of the bladder specimen was used for immunohistochemistry and evaluation.Retrospectively collected, routine formalin-fixed and paraffin-embedded (FFPE) tumour tissue blocks from 186 consecutive patients treated with RC and bilateral pelvic lymphadenectomy during the period 2000 to 2005 at a single academic centre  were analysed. Eligible for inclusion were patients (age 18+ years) with histopathological proven urothelial neoplasia of the bladder. Indications for RC were high-risk non-muscle invasive or muscle invasive disease without signs of lymph node or distant metastases. None of the patients received neoadjuvant or adjuvant chemotherapy. Patient characteristics are presented in The study was approved by The Committees of Health Research Ethics in the Capital Region of Denmark (H-1-2012-003) and the Danish Data Protection Agency (2007-58-0015). The investigation was carried out according to the Helsinki Declaration II and in accordance with the REMARK guidelines .All cases were histopathologically reclassified by one pathologist (BRI) with expertise in genitourinary pathology. For each patient, the pathologist reviewed all available FFPE tumour samples from the cystectomy and the block representing the deepest invasive site was selected for further evaluation. The criteria by the Union for International Cancer Control were used for pathological staging and the World Health Organisation classification for pathological grading ,16. All The polyclonal antibody (pAb) against uPAR has previously been described and validated ,18. Mono2O2) for 15 minutes and thereafter washed in Tris-buffered saline  and then manually mounted on Shandon racks with immunostaining cover plates . The primary antibodies were diluted in Antibody Diluent with Background-Reducing Components  and incubated at the following concentrations overnight at 4°C: uPAR 2.8 μg/ml, CK-mix (CK-pan 0.4 μg/ml + CK7 0.5 μg/ml), CD68 0.3 μg/ml and α-SMA 0.4 μg/ml. The primary antibodies were detected with EnVision Rabbit or Mouse reagents for 45 minutes. The sections were then developed with NovaRed  for 9 minutes. Each incubation step was followed by washes in TBS-T. At last, the sections were counterstained using Mayer’s haematoxylin for 30 seconds, and thereafter dehydrated in ethanol and mounted with pertex using a CoverSlipper from Dako.All stainings were performed in the same laboratory by one person. Whole-slide, 3 μm paraffin sections from each of the blocks were mounted on glass slides and deparaffinised with xylene and hydrated through ethanol/water dilutions. Antigen retrieval for uPAR, cytokeratins (CK-pan and CK7 mixed) and CD68 was performed by Protease K (5 μg/μl) in a Proteinase K-buffer  at 37°C for 15 minutes. Antigen retrieval for α-SMA was performed at 98°C in TEG-buffer  for 10 min in a T/T Micromed microwave processor . Sections were blocked for endogenous peroxidase activity by incubating in 1% hydrogen peroxide . The primary endpoint was overall survival (OS). In addition we explored recurrence free survival (RFS) and cancer specific survival (CSS) [14. The p2-test for independence in the relevant contingency tables. Spearman rank correlation was used as a measure of association between the uPAR scores and tests comparing levels between categories were done using the Wilcoxon rank sum test. Univariate as well as multivariate analysis of time to event data  were done using the Cox proportional hazards model. Results are presented by hazard ratios (HR) with 95% confidence intervals (CI). The Cox models have been assessed based on martingale residuals. The uPAR scores have been evaluated by the actual score and dichotomized based on no expression versus a score > 0. In addition, the estimated survival curves based on the Cox regression model for predefined levels of the covariates are presented. The level of significance was set to 5%. All statistical calculations have been done using SAS .Descriptive statistics for continuous covariates are presented by the median as well as the minimum and maximum and categorical variables by the frequencies. The associations of dichotomised uPAR scores to clinicopathological covariates were done using the χin situ, were uPAR negative. The invasive front has been analysed in 180 specimens and tumour core in all 186 specimens. As no invasive front is present in non-invasive neoplasias (Ta = 6) these samples were excluded. uPAR immunoreactivity was detected in 173/180 (96%) and 162/186 (87%) of the neoplasias at the invasive front and tumour core, respectively. The cellular source of uPAR was primarily confined to myofibroblasts and macrophages in the surrounding stroma as well as some cancer cells . The adjThe uPAR score (0–6) of the three cell types was assessed and found higher at the invasive front than in tumour core. In both locations the scores for myofibroblasts were the highest . CorrelaA significant association between uPAR positivity and advanced tumour stage was found for myofibroblasts, and macrophages both at the invasive front and in tumour core (p≤0.015), but not for cancer cells . An inteThe 5-year OS, RFS and CSS for the entire cohort were 47.3%, 95% CI: 40.0–54.3; 64.2%, 95% CI: 56.1–71.1; 66.9%, 58.9–73.8, respectively. 65 (35%) patients experienced disease recurrence. 127 patients had died (68%), 57 (31%) of UCB. 3 patients died perioperative. The median follow-up was 11.6 years (range 8.9–14.9) for those patients alive at time of analyses.In the univariate analyses no significant association was seen between uPAR positivity and OS, RFS or CSS. The highest HRs for survival was seen for uPAR positive myofibroblasts both at the invasive front and in tumour core . To explIncreasing tumour stage (cystectomy specimen), lymph node metastases, lymph vascular invasion and positive resection margin were associated with significantly worse OS . No signThe significant association between uPAR score and cancer specific survival found in the univariate analysis was not significant in the multivariable analyses (p = 0.41). In the multivariate analyses for the primary endpoint, OS, the statistical significance were retained for the clinical baseline values organ confined disease , non-organ confined disease , vascular invasion  and positive resection margin .In the present study we were able to confirm, in an independent cohort of patients with urothelial neoplasia of the bladder, that uPAR as evaluated by Immunohistochemistry is primarily expressed by myofibroblasts and macrophages in the tumour associated stroma. In addition, we were able to confirm the significant association between uPAR positivity in the tumour tissue and increasing tumour stage and tumour grade, which were recently published by our group . This dein situ hybridisations have shown that uPA-mRNA is expressed by the malignant urothelial cells themselves [It is increasingly well accepted that the tumour stroma plays an important role in carcinogenesis. We have in two independent studies shown that in urothelial neoplasia of the bladder uPAR is primarily expressed by myofibroblasts and macrophages and to a lesser extent by cancer cells. In contrast, investigations of its ligand uPA by emselves . Generalemselves . Data reemselves . In addiemselves . We do ne.g. as to whether a patient should receive adjuvant chemotherapy or to avoid such after cystectomy [Urothelial carcinoma of the bladder is an extremely heterogeneous group of malignancies. For patients with high-risk non-muscle invasive and muscle-invasive disease without signs of metastasis, RC with lymphadenectomy is considered the standard treatment. Neoadjuvant chemotherapy is recommended in patients with muscle-invasive node negative disease . Adjuvanstectomy ,28. Despstectomy . The devstectomy ,31.uPAR consists of three domains denoted domain I, II and III. uPA bound to uPAR is capable of cleaving neighbouring cell-bound uPAR. Therefore the cleaved uPAR forms reflect the activity of the plasminogen activation system. The cleaved uPAR forms measured in blood have shown to be stronger prognostic biomarkers than the levels of the collective amount of uPAR in several types of cancers ,13,32–34Our investigations have generated new and valuable biological information about the cell types being involved in tumour invasive and progression through the plasminogen activation system. We were able to confirm our previous published findings of tissue expression and localisation pattern of uPAR, as well as a significant association between uPAR positivity and increasing tumour stage in tumour tissue from patients with urothelial neoplasia of the bladder. In addition we are able to reproduce the association between uPAR positive myofibroblasts in the tumour core and poor survival.At present, uPAR expression as evaluated by immunohistochemistry cannot be recommended for routine use for prediction of clinical outcome in urothelial bladder cancer. The found biological importance of uPAR in urothelial carcinoma of the bladder may have clinical implications when uPAR forms are measured in body fluids such as blood.


However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment.Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for  Intravital microscopy has been used in laboratory animals to visualise the blood vessels in tumours. Here, the authors use this technique in melanoma patients undergoing surgery and show that vessels in situ have a larger diameter than excised tissue Common clinical imaging procedures such as retinography (≤ × 15 magnification) have proved useful for examination of vessels at the macrolevel. However, preclinical intravital microscopy (IVM) at typical magnifications of ≥ × 100 magnification is fundamentally more powerful for the real-time microscopic evaluation of the dynamic nature of functional blood vessels within living organisms at a resolution capable of distinguishing cellular and subcellular features within the surrounding microenvironmentRecent advances in the preclinical imaging of tumours have generated an unprecedented understanding of the nature of tumour vascular structure, blood flow and tumour-immune cell interactions. As the gateway to the tumour microenvironment, tumour vessels are critically important sites that serve to enhance or limit host interactions. Immunohistochemistry (IHC) and pathologic examination remain the clinical standard for defining tumour characteristics including vascularity, but provide only a static ‘snapshot' that may not reflect the active processes occurring As a research tool, preclinical IVM in murine systems has contributed to the elucidation of inherent tumour functions including angiogenesis123579101112131415912post hoc movie analyses of recorded observations. Preclinical animal models often utilize window chambers that facilitate visual access to underlying tissue during IVM  and subsequent vibration-limiting stability when interfaced with the tumour. To create a working space between the microscope objective and the patient positioned on the OR table, a simple cantilever construction was chosen . The canMelanoma patients enrolled in our Roswell Park Cancer Institute review board-approved protocol  met strict eligibility criteria that included having a melanoma that was either palpably or visibly ≥0.5 cm that required excision in the OR as their standard of care . EligiblDuring surgery, microscopic observations were performed first, before the definitive oncologic surgical procedure to eliminate any potential artifact. In some superficial melanomas that attenuated the overlying skin, direct IVM observations were performed without any manipulation of the tumour. For the observation of deeper lesions, the incision for the microscopic observation was delineated in a manner consistent with standard oncologic resection, yet also allowed for a ‘portal' to observe the tumour surface directly without disrupting tumour-feeding blood vessels . The plaPost hoc application of image stabilization software  did not appreciably change the degree of motion artifact.Measurements of vessel density and diameter were possible in some patients even before the injection of fluorescein due to the absorptive capacity of haemoglobin in red blood cells. However, due to the dense melanin pigmentation of the melanoma tumours visualization of the vessels was greatly improved following injection of fluorescein. Typical images of patient melanoma tumours included evidence of surrounding adipose tissue and vessels of varying depths within the tumour tissue . Tumour n=7) of vessels were deemed non-functional at the time of observation despite most patients having a mean arterial pressure exceeding 65, which is considered adequate for tissue perfusionn=7) of tumour vessels that were non-functional, further suggesting that this is an inherent characteristic of the undisturbed tumour.During the course of observation, a variable percentage of vessels within the tumour appeared patent, but did not manifest any evidence of blood flow or intravascular fluorescence following fluorescein injection . For theIn three patients  intravascular fluorescein could not be detected in the exposed tumour, and thus, blood flow velocity and per cent of non-functional vessels could not be calculated. Patient #4 had a profound desmoplastic reaction around his tumour, which likely was a physical barrier preventing visualization of underlying tumour vessels. Subsequent histology of this tumour confirmed a vigorous fibrotic reaction . In Pati−1  and wall shear rate (77±5.6 s−1) in human tumours than would have been predicted based on studies in B16 melanoma murine tumours shown here  3132ctively)  and in octively)  suggestictively) .Another potential explanation for the differences in human tumour vessel diameters measured by IVM versus IHC could be related to tissue shrinkage following formalin fixation. To address this possibility, we examined how vessel diameters were altered in the B16 murine model by taking the IVM observed murine tumours and examining the vessel diameters by IHC. The murine tumours were treated in an identical fashion to the patient tumours including the same fixation time, paraffin embedding process, and tissue sectioning and IHC staining. The average diameter of murine tumour vessels measured by IHC was 9±1 μm and demonstrated a significant reduction in diameter as compared with the diameters detected by IVM in mice (15±2 μm), which suggested process-related tissue shrinkage . HoweverA potential explanation for the disparity between tumour vessel diameters measured by IVM and IHC could have been that the interstitial pressure in tumours is variable between the core and the peripheryCurrent innovations have made human IVM possible and allowed the transition of this technology from the laboratory to the clinical study of cancer. In the current feasibility trial, we have demonstrated the ability of IVM to successfully measure dynamic tumour vascular features for the first time in humans with a spatial resolution of 1 μm. The method of IVM observation of tumours was seamlessly incorporated during surgical resection and did not interfere with the standard operative procedures. Our platform was capable of accessing any anatomic location on a patient and digital movie recordings demonstrated high resolution and manageable motion artifact that allowed for precise measurements of tumour-associated vessels. The results demonstrate that live-imaging of human tumour dynamics at a microscopic level (≥ × 100 magnification) is feasible, consistent and reproducible.Intravital imaging in melanoma patients confirmed expectations for several key vascular parameters including the presence of tortuous, disorganized architecture and non-functional tumour vessels14394143The major finding from this clinical trial was the consistent and significantly larger tumour vessel diameter measurements obtained by IVM versus IHC in patient tumours. As confirmation that the differing measurements were not due to sampling bias, IHC measurements were performed in the same fields observed during transcutaneous IVM. Tumour vessel diameter measurements by IHC remained significantly smaller despite matching the fields observed by IVM. Also, the ‘shrinkage' of tissue that typically occurs with formalin-fixation45−1, could be considered as a surrogate for common chemotherapeutic agents with a similar size such as 5-FU (130 g mol−1), cyclophosphamide (261 g mol−1) and oxaliplatin (397 g mol−1). Moreover, certain chemotherapy agents, including doxorubicin, are auto-fluorescentThe finding of consistently larger tumour vessels by human IVM could be profound when considering how the geometry of blood vessels influences tumour tissue perfusion and drug delivery as well as trafficking of immune cells into the tumour site. For example, a recent review on tumour vessel normalization cited a standard tumour vessel diameter of 15 μm ref. , which i3132313233in vitro and in vivo studies have further documented that mechanical forces associated with vessel diameter and wall shear stress have a profound impact on lymphocyte trafficking. In this regard, a narrow window of wall shear stress ranging from ∼0.5 to <2 dyn cm−2 is necessary for optimal lymphocyte adhesion to vessel walls during trafficking, whereas adhesion within vessels declines precipitously when the shear stress exceeds 3 dyn cm−2  and 50% saline solution (negative control) were placed appropriately. The skin underlying each solution was individually pricked with separate and sterile Duotip lancets . After 30 min, the appearance of a wheal >3 mm in diameter was considered a positive test result in the context of the appropriate positive and negative control. If a positive test was noted to fluorescein, the patient was no longer eligible for this study.Patients with a visible  or palpable melanoma lesion that required excision in the OR were evaluated for entry into clinical trial I 231512 ‘A Pilot Study of Feasibility of Performing Intravital Microscopy in Melanoma Patients' at Roswell Park Cancer Institute, Buffalo, NY  approved by the Institute Review Board of Roswell Park Cancer Institute, Buffalo, NY. Inclusion and exclusion criteria are listed in Patient tumours were observed at × 100 magnification using a highly modified Olympus microscopy system. The microscope was attached to a cantilevered arm to extend over the patient. Given the high magnification required to observe individual vessels and flow, vibrations in the microscope were dampened using a weighted marble base of over 360 kg. Fluorescein was illuminated using a 467–498-nm excitation, 513–556 emission dichronic filter set  with an X-Cite 120 Led light source . Images were captured using a Luca EMCCD camera  controlled through the Solis acquisition and analysis programme. Images were acquired with a minimum of a 0.05-s exposure at 20 frames a second. Offline quantification of vessel diameter and blood velocity was performed using the ImageJ software suite.Before definitive surgical excision, melanoma tumours were exposed in a consistent manner. The skin overlying the tumour was prepped with ChloraPrep  and sterile towels were applied for exposure of the area. A curvilinear incision within the field of planned resection was created with a scalpel in line with the longitudinal axis of the patient. With care to keep the vasculature intact, the skin was elevated bluntly and retracted laterally with the use of zero silk sutures. Electrocautery was avoided as much as possible to prevent underlying tissue damage. The sutures were anchored to a retractor affixed to the OR table on the opposite side of the planned microscopic observation. For deeper melanoma tumours, a self-retaining weitlaner retractor was used following incision of the skin and underlying subcutaneous tissue. In all instances, a saline-soaked gauze was placed over top of the tissue to prevent desiccation before microscopic interface. After exposure of the underlying tumour mass, the microscope was positioned for observation. At the completion of the observation period, the microscope was withdrawn and the skin flap was closed with a running 3-0 silk suture. The area was then re-prepped for the definitive oncologic surgery including a new skin prep, sterile towels and drapes, and new gown and gloves.x and y axis were performed by using the built-in motor controls. Once the microscope was directly overlying the exposed tumour tissue, the fluorescent light source and image acquisition software were activated. Vertical control of the microscope was performed manually by the surgeon until tumour vessels came into view on the digital monitor. Tumour vessels were clearly recognized by their bizarre architecture, coiling, and branching patterns and observations focused on these areas. To facilitate stabilization of the images, respirations were temporarily held by anaesthesia for a maximum of 30 s. When a stable view of the vessels was achieved, 0.5 ml of 25% (250 mg ml−1) fluorescein  was injected via a peripheral intravenous catheter. An observation was completed when fluorescein was noted to have extravasated into the tumour tissue and the vessels no longer manifested a detectable fluorescent signal. The microscope was then re-positioned with fine motor controls to observe a different tumour area. The microscopic observations were finalized when the 2 ml of fluorescein was exhausted.The microscope was grossly moved into position by the surgeon over the field of interest and locked into place. Fine adjustments in the 6 B16/F10 melanoma cellsD) of vessels was measured in offline observations of five mice. 4T-1 mammary carcinoma cells and B16/F10 melanoma cells were implanted in C57BL/6 mice  and EMT6 mammary carcinoma cells and CT26 colon carcinomas in BALB/c mice  for correlative window chamber studies as reportedFor intraoperative IVM experiments 10D) and radius (r) at the vessel's largest width. Blood flow velocity (v) was evaluated by determining the time ΔT distinct features in the fluorescent dye would take to travel a known distance ΔS and then averaging ΔS/ΔT for at least 10 points per vessel. Per cent functionality was determined as mean±s.e.m. on a per field basis. Wall shear stress was computed as . Wall shear rate was determined using the formula (w=v*8/D).Vessels were defined morphologically and a single vessel was described as beginning at a branch point continuing to the next branch point. To be measured, vessels had to be 100 μm in length with no branch points. Vessel density was established by enumerating vessels within a field of observation. ImageJ software was used to measure vessel diameter . Stained sections were scanned with an Aperio Scanscope XT  and evaluated using Aperio Spectrum software. Vessel density was assessed using standard ‘hot spot' methodologyt-tests were utilized to examine differences between vessel diameters measured by human IVM, IHC and murine IVM, IHC, comparing each set separately using Graph Pad Prism following testing for data normality with the D'Agostino & Pearson omnibus normality test. No statistical method was used to predetermine sample size. Statistical significance for all comparisons was accepted at P<0.05. The numbers of samples per group (n), or the numbers of experiments, are specified in the figure legends.Unpaired Student's How to cite this article: Fisher, D. T. et al. Intraoperative intravital microscopy permits the study of human tumour vessels. Nat. Commun. 7:10684 doi: 10.1038/ncomms10684 (2016).Supplementary Figures 1-5 and Supplementary Tables 1-3Representative video depicting IVM observation of Patient #1 tumour.Representative video depicting IVM observation of Patient #2 tumour.Representative video depicting IVM observation of Patient #10 tumour.Representative video depicting methods for analysis of IVM observation of Patient #3 tumour.


Background. Literature data suggest that cells such as mast cells (MCs), are involved in angiogenesis. MCs can stimulate angiogenesis by releasing of several proangiogenic cytokines stored in their cytoplasm. In particular MCs can release tryptase, a potent in vivo and in vitro proangiogenic factor. Nevertheless few data are available concerning the role of MCs positive to tryptase in primary pancreatic cancer angiogenesis. This study analyzed MCs and angiogenesis in primary tumour tissue from patients affected by pancreatic ductal adenocarcinoma (PDAC). Method. A series of 31 PDAC patients with stage T2-3N0-1M0 (by AJCC for Pancreas Cancer Staging 7th Edition) was selected and then underwent surgery. Tumour tissue samples were evaluated by means of immunohistochemistry and image analysis methods in terms of number of MCs positive to tryptase (MCDPT), area occupied by MCs positive to tryptase (MCAPT), microvascular density (MVD), and endothelial area (EA). The above parameters were related to each other and to the main clinicopathological features. Results. A significant correlation between MCDPT, MCAPT, MVD, and EA group was found by Pearson's t-test analysis . No other significant correlation was found. Conclusion. Our pilot data suggest that MCs positive to tryptase may play a role in PDAC angiogenesis and they could be further evaluated as a novel tumour biomarker and as a target of antiangiogenic therapy. Tumour tissue samples were evaluated by means of immunohistochemistry and image analysis methods, obtaining a significant correlation between MCDPT, MCAPT, MVD, and EA group. Our pilot data suggest that MCs positive to tryptase may play a role in PDAC angiogenesis and they could be further evaluated as a novel tumour biomarker and as a target of antiangiogenic therapy.Inflammatory cells, such as macrophages, lymphocytes, and mast cells (MCs), play a major role in tumour angiogenesis by means of angiogenic cytokines stored in their cytoplasm. MCs are involved in neovascularization in experimentally induced tumour, accumulate near to tumour cells before the angiogenesis onset, and participate in the metastatic spreading of primary tumours. MCs intervene in angiogenic process releasing classical proangiogenic factors, such as vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP), fibroblast growth factor-2 (FGF-2), and the nonclassical proangiogenic factor, namely, tryptase stored in their secretory granules –9. The rThe clinicopathological features of selected patients are summarized in μm thick serial sections of formalin-fixed and paraffin-embedded surgically removed tumour samples were deparaffinised. Then, for antigen retrieval, sections were microwaved at 500 W for 10 min, after which endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution. Next, adjacent slides were incubated with the monoclonal antibodies anti-CD31  diluted 1 : 40 for 30 min at room temperature and anti-tryptase  diluted 1 : 100 for 1 h at room temperature. The bound antibody was visualised using biotinylated secondary antibody, avidin-biotin peroxidase complex, and fast red. Nuclear counterstaining was performed with Gill's haematoxylin number 2 . Primary antibody was omitted in negative controls.For the evaluation of MCDPT, MCAPT, MVD, and EA, a three-layer biotin-avidin-peroxidase system was utilized . BrieflyAn image analysis system (Semiquantimet 400 Nikon) was employed.2 area; Figures 2 area) [The five most vascularized areas (“hot spots”) were selected at low magnification and both MCDPT  and indi 2 area) . Finallyr). Correlation between MCDPT, MCAPT, MVD, and EA groups and the main clinicopathological features were analysed by chi-square test. In all analyses a P < 0.05 was considered significant. All statistical analyses were performed with the SPSS statistical software package .Linear correlations between MCDPT, MCAPT, MVD, and EA groups were quantified by means of Pearson's correlation coefficient , between MCAPT and MVD , between MCDPT and EA , between MCAPT and EA , between MVD and EA , and between MCDPT and MCAPT   . No corrMCs' involvement in tumour angiogenesis has been demonstrated in several animals models and human malignancies , 18–20.MCs are recruited and activated via several factors secreted by tumour cells, such as the C-Kit receptor or stem cells factor, VEGF, FGF-2, and TP. In tumour microenvironment, MCs secrete both gelatinases A and B which, in turn, degrade extracellular matrix, releasing stored angiogenic factors –33. in vitro and in vivo experimental models and it is also an agonist of the PAR-2 in vascular endothelial cells that, in turn, induces angiogenesis. Interestingly in several human malignancies but not in pancreatic cancer, MCDPT and MCAPT have been associated with tumour angiogenesis. In this regard experimental results suggested that MCDPT may stimulate pancreatic cancer cells contributing to pancreatic tumour progression [On the other hand, MCs may induce angiogenesis by several proangiogenic factors stored in their secretory granules, such as VEGF, FGF-2, tumour necrosis factor alpha, and interleukin 8, transforming growth factor beta, heparin, and tryptase. With special reference to the last, it is involved in tumour angiogenesis stimulating the formation of vascular tubes ingression –40.Published data from Esposito et al.  showed t2 as previously published in other tumours types [In this pilot study, we have evaluated the correlations between MCDPT, MCAPT, MVD, and EA in a series of 31 PDACP having undergone surgery and our results suggest an association between tryptase and microvascular bed. We found this correlation in double way: first in terms of number of positive tryptase cells and immunostained microvessels and second in terms of extension of positive tryptase area and immunostained microvessels area. To avoid methodological bias the evaluation of MCDPT, MCAPT, MVD, and EA has been performed by means of an image analysis system at ×400 magnifications in a well-defined microscopic area of 0.19 mmrs types –45. Furt


TAMs) represent pivotal components of tumour microenvironment promoting angiogenesis, tumour progression and invasion. In colorectal cancer (CRC), there are no conclusive data about the role of TAMs in angiogenesis‐mediated tumour progression. In this study, we aimed to evaluate a correlation between TAMs, TAM immunostained area (TAMIA) microvascular density (MVD), endothelial area (EA) and cancer cells positive to VEGF‐A (CCP‐VEGF‐A) in primary tumour tissue of locally advanced CRC patients undergone to radical surgery. A series of 76 patients with CRC were selected and evaluated by immunohistochemistry and image analysis. An anti‐CD68 antibody was employed to assess TAMs and TAMIA expression, an anti‐CD34 antibody was utilized to detect MVD and EA expression, whereas an anti‐VEGF‐A antibody was used to detect CCP‐VEGF‐A; then, tumour sections were evaluated by image analysis methods. The mean ± S.D. of TAMs, MVD and CCP‐VEGF‐A was 65.58 ± 21.14, 28.53 ± 7.75 and 63% ± 37%, respectively; the mean ± S.D. of TAMIA and EA was 438.37 ± 124.14μ2 and 186.73 ± 67.22μ2, respectively. A significant correlation was found between TAMs, TAMIA, MVD and EA each other . The high level of expression of TAMs and TAMIA in tumour tissue and the significant correlation with both MVD and EA illustrate that TAMs could represent a marker that plays an important role in promoting angiogenesis‐mediated CRC. In this context, novel agents killing TAMs might be evaluated in clinical trials as a new anti‐angiogenic approach.Tumour‐associated macrophages ( Colorectal cancer (CRC) represents the second cause of cancer death in Europe after breast cancer in women and lung cancer in men Macrophages are myeloid cells and derive from CD34‐positive bone marrow progenitors In fact, it has been demonstrated that TAMs are particularly abundant and involved in tumour angiogenesis, development and progression in several animal and human malignancies Concerning the role of TAMs in CRC, published results have been not conclusive. In CRC murine and human models, it has been demonstrated a relationship between angiogenesis and TAMs in tumour progression A series of 76 CRC patients observed at the Clinical Surgery Unit of the ‘Magna Graecia’ University of Catanzaro were selected For the evaluation of TAMs, TAMIA, MVD, EA and CCP‐VEGF‐A, a three‐layer biotin–avidin–peroxidase system was utilized 2 area). In the same sections, TAM and MVD were also evaluated in terms of immunostained area, so called TAMIA and EA.An image analysis system  was utilized r) analysis. The correlations between the above indexes and the clinicopathological features listed in Table Tumour‐associated macrophages, TAMIA, MVD, EA and CCP‐VEGF‐A mean values ± 1 S.D. were evaluated by two independent observers (G.R. and V.Z.) for each tumour sample and in all series of sections. Correlations between TAMs, TAMIA, MVD and EA were calculated using Pearson's , TAMIA and MVD , TAMs and EA , TAMIA and EA , TAMs and TAMIA (r = 0.81 P = 0.001) and MVD and EA  was found. No other significant correlations between the above indexes and the main clinicopathological features were found  ligands 1 and 2 and PD‐1 receptor expressed on T‐cells In this context, due to the sensitivity of CRC to anti‐angiogenic therapy, novel drugs killing TAMs, such as trabectedin, anti‐PD‐L1 antibodies, a pro‐apoptotic peptide M2 and PLX3397 (CSF‐1R tyrosine kinase inhibitor), might be evaluated in clinical trials as a new anti‐angiogenic strategy The authors confirm that there are no conflicts of interest.


Objectives: The increasing global burden of oral cancer has driven much of the focus of research to the determination of reliable prognostic markers which may have significant effects on survival and the control of post-treatment morbidity. This study was undertaken to evaluate tumour associated tissue eosinophilia (TATE) quantitatively in oral cancer specimens and observe for its possible association with tumour stage, patterns of locoregional recurrence and overall prognosis.Study Design: 14 patients undergoing surgical resection for primary oral squamous cell carcinoma (OSCC) were subjected to grey scale ultrasonography (USG) to assess tumour dimensions. The findings were compared with the cTNM stage initially documented. TATE was evaluated along the invasive tumour front (ITF) using H & E stained sections of histopathological specimens for 10 continuous high power fields (HPF) and graded as mild, moderate or intense. Patients were followed up over 5 years and observed for patterns of recurrence.Results: Loco regional recurrence was significantly associated with intense degree of TATE. (p<0.001) cTNM stage as well as USG stage did not correlate with the degree of TATE with p=0.419 and 0.772 respectively. None of the patients with mild/ moderate dysplasia developed locoregional recurrence within the period of follow up.Conclusions: Analysis of TATE in OSCC patients may provide an early indication of future locoregional recurrence. Identification of an appropriate biopsy site representing the ITF where TATE analysis can be performed may be a simple, inexpensive method of obtaining valuable prognostic information at the time of diagnosis. Key words:Tumour associated tissue eosinophilia, oral cancer, prognosis. With an annual estimated incidence of around 275,000 and two-thirds of the cases occurring in developing countries alone, oral cancer is a matter of serious concern in these parts of the world. Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cavity cancers . Poorer Over treatment may also be a result of improper staging of oral cancer patients. Clinical methods used for staging are not sufficient to form the basis for treatment planning and adjunctive imaging modalities must be considered wherever possible to avoid under or over estimation of dimensions. Reliable indicators of locoregional recurrence would also greatly improve survival rates in OSCC patients . DespiteTumour associated tissue eosinophilia (TATE) has been reported in diverse sites -14 incluThe aim of our study was to analyse TATE quantitatively along the invasive front of the tumour in histopathological specimens of OSCC patients and correlate it with clinical staging, recurrence patterns and overall prognosis. It has already been established that ultrasonography (USG) gives an accurate measure of tumour dimensions comparable with histological measurements . To avoi-Patients:14 patients undergoing surgical resection for primary OSCC at M.S. Ramaiah Medical Hospital between January 2009 to January 2010 were selected for the study out of a total 45 patients initially screened, upon fulfilment of inclusion and exclusion criteria. A detailed case history was recorded, clinical examination and necessary investigations were performed and cTNM staging for the tumours was documented.Patients with primary OSCC confirmed by biopsy who had not undergone radiotherapy, chemotherapy, or other any treatment prior to surgery were included in the study. Meanwhile patients with primary tumours at other anatomic locations, unresectable tumours and difficulty in mouth opening were excluded from the study. A major requirement was the availability of tumour tissue for microscopic analysis obtained from the invasive tumour front (ITF). Thus, incisional biopsies were excluded from the study. Clinical follow-up was maintained including data on locoregional recurrence and death. Duration of follow up was from January 2009 to December 2013.-Ultrasonographic examination:A grey scale USG evaluation (GE Volusun 350 at 7.5MHz) of the primary tumour was performed with an intra-oral probe to assess its transverse dimension and identify the appropriate biopsy site.An incisional biopsy was performed to confirm the diagnosis following which the patients underwent surgical treatment.-TATE analysis:Paraffin embedded blocks of the tumour tissue were obtained from the archives of the Department of Pathology, M.S. Ramaiah Medical Hospital, Bangalore, India and the block representing the invasive front of the tumour was identified for each patient. 3µ sections were obtained and stained routinely with Haematoxylin and Eosin stain for TATE analysis. Eosinophils at the invasive front were counted under a high power objective (x 400) for 10 continuous high power fields (HPF) using a light microscope (OLYMPUS C-20 i). The mean values obtained were then graded according to the criteria given by Goldsmith et al.25 In this criteria, score ‘0’ corresponded with absence of eosinophils, ‘1+’ with presence of 5 to 10 eosinophils/HPF, ‘2+’with 10 to 20 eosinophils/HPF, ‘3+’ with 20 to 30 eosinophils/HPF and ‘4+’ with more than 30 eosinophils/HPF. We considered scores ‘0’ and ‘1+’ as absent/mild eosinophilia, scores ‘2+’ and ‘3+’ as moderate eosinophilia and ‘4+’ as intense eosinophilia,  demonstrated mild TATE, 3 cases (21.4%) demonstrated moderate TATE and the remaining 4 (28.6%) cases showed intense TATE. None of the patients with mild/moderate TATE showed locoregional recurrence. Loco regional recurrence was significantly associated with intense degree of TATE with p<0.001  when compared with USG findings, there were no statistically significant differences between the cTNM and USG stages with  p<0.001 . These pely Fig. , Table 3The complexities of the interactions between carcinomatous tissue and the underlying stroma has been an area of prime focus in cancer research with various features such as increased angiogenesis, presence of varying degrees of inflammatory infiltrates, desmoplastic stromal reaction and microinvasion recognized as important indicators of the general biology and behaviour of the tumour cell population .The invasive tumour front (ITF) provides a ‘fertile ground’ for the assessment of such interactions as it houses the most aggressive cells of the tumour population, readily demonstrates molecular events of importance in tumour spread and is also less differentiated than the residual parts of the tumour . The ITFEosinophils are bone marrow-derived, tissue-dwelling granulocytes found transiently in the blood circulation en-route to tissue inflammatory sites. They are mainly associated with parasitic infections and allergic diseases but are also known to participate in tissue remodelling and modulation of the host immune response . Their pMany have theorized eosinophils to have a tumour protective role due to secretion of cytotoxic mediators such as major basic protein, eosinophil cationic protein and eosinophil peroxidase and their ability to increase permeability of tumour killing cytokines into tumour cells . On the In this study we attempted to analyze TATE along the ITF of patients with OSCC. The patients were preoperatively subjected to a grey scale USG examination to determine the tumour dimensions and were compared with clinical measurements. A number of studies have validated the accuracy of tumour size assessment using USG . The traet al. (et al. (et al. (Previous reports have found significant association between the presence of high degrees of TATE and stromal invasion in OSCC ,31. An iet al. . A recenet al. . On the  (et al.  and Lore (et al.  did not A plausible explanation for this disparity in results of various studies could be due to the lack of a standard criteria for grading TATE that is universally followed,  use of bet al. however reported the H & E technique to be as reliable as the immunostaining technique for identification of eosinophils. 32 In our study, eosinophils were easy to identify on H & E sections of 3 µ thickness but were often obscured in 4-5 µ sections due to overlapping of structures. Thus we studied only 3 µ sections.Although intact eosinophils are identified with relative ease in routine H & E stained sections of tissue, it may get difficult to identify these cells in inflamed or fibrous tissue which is where special techniques such as autofluorescence and immunohisto-chemistry are recommended. Lorena Locoregional recurrence develops much earlier than metachronous disease and carries the worst prognosis. Patients who survive a first encounter with this disease have up to a 20 fold increased risk of developing a second cancer. In developing countries like India, OSCC is more prevalent among low socioeconomic status groups. Such patients are often lost to follow up and do not comply with lifestyle modifications. Thus, early identification of recurrence at the time of diagnosis will be useful in identifying patients at risk and employing customized treatment strategies and follow up policies for them ,12.The use of adjunctive imaging modalities such as USG to confirm the most appropriate biopsy site that should include the advancing front of the tumour or ITF may be helpful as TATE analysis can be done even in biopsied tumour tissue in such cases and based on the results, individualized treatment plans can be formulated. This method is simple, cost effective and can be performed routinely. Further studies recruiting larger samples are required to validate the findings.


Reason for Corrigendum:Due to an oversight by the authors, Figures This research was supported by a grant from the National University of Singapore, Singapore to LS (HSS R-581-000-178-646), to a Ministry of Education Tier 1 Academic Research Fund (FY2013-FRC2-009) grant to LS, and to a grant from the Singapore Children's Society to CF.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The correct funding information is as follows: Plan Estatal I+D+i and Instituto de Salud Carlos III-Subdirección General de Evaluación y Fomento de la Investigación (Grant no.: PI13/00794)


Inter-patient variations in tumour growth rate are usually interpreted as biological heterogeneity among patients due to, e.g., genetic variability. However, these variations might be a result of non-exponential, e.g. the Gompertzian, tumour growth kinetics. The aim was to study if the natural tumour growth deceleration, i.e. non-exponential growth, is a dominant factor in such variations.The correlation between specific growth rate (SGR) and the logarithm of tumour volume, Ln(V), was calculated for tumours in patients with meningioma, hepatocellular carcinoma, pancreatic carcinoma, primary lung cancer, post-chemotherapy regrowth of non-small cell lung cancer (NSCLC), and in nude mice transplanted with human midgut carcinoid GOT1, a tumour group which is biologically more homogeneous than patient groups.The correlation between SGR and Ln(V) was statistically significant for meningioma, post-chemotherapy regrowth of NSCLC, and the mouse model, but not for any other patient groups or subgroups, based on differentiation and clinical stage.This method can be used to evaluate the homogeneity of tumour growth kinetics among patients. Homogeneity of post-chemotherapy regrowth pattern of NSCLC suggests that, in contrast to untreated tumours, the remaining resistant cells or stem cells (if exist) might have similar biological characteristics among these patients. Mathematical modelling of tumour growth can provide not only key insights into tumour biology but also tools for, e.g., optimization of screening programs, cancer patient prognosis, schedul0 and t, the following equation can be used for calculation of SGR[However, one of the main limitations for using tumour growth rate in patient studies is that therapy is usually started soon after diagnosis and the natural growth of tumours can be followed only for a limited period of time, during which the growth of tumours is usually well described by the exponential model. Tumour volumes are usually estimated by delineating tumours in computed tomography (CT) or magnetic resonance imaging (MRI) slice, multiplying the measured area with slice thickness, and adding all volumes together. There are, therefore, uncertainties involved in estimated volumes in form of intra- and inter-investigator variations of estimated volumes. However, growth rate of exponentially growing tumours can be quantified with tumour volume doubling time (DT), given in days or months. However, the specific growth rate (SGR) of tumours, given in, e.g. percent per day, is mathematically more accurate and biologically more relevant than DT for quantification of tumour growth rate,8. If thn of SGR:0 and V are the volume of tumour at t = t0 and t, respectively.where VAccording to the exponential model, the growth rate, i.e. SGR, is constant and independent of tumour age or volume. However, studies have shown that tumour growth rate may decelerate as tumour grows-11. Grow0 is the initial growth rate of tumour at t = t0 and λ is the growth deceleration constant.where SGRFitting the Gompertzian model to the natural growth of tumours needs at least three tumour volume values measured on occasions spread over a relatively long period of time, which is rarely obtainable in clinical observations. We have previously developed a method for estimation of growth deceleration constant, λ, from the linear regression of SGR with the logarithm of tumour volume:The above equation enabled us estimating the growth model, including Gompertzian growth deceleration constant in Equation B (λ) and formation times of metastases in individual patients.Tumour response to a specific treatment varies largely among patients. Beside other biological factors, tumour growth kinetics is important for how a tumour responds to therapy,22. TumoAccording to Equation C, a significant correlation between SGR and ln(V) in a group of tumours indicates that tumours probably follow the same growth curve and the difference in their growth rate is a result of difference in their volume. In other words, such a correlation is a measure of similarity of growth pattern among tumours, e.g., in a group of patients. Then, a unique curve can describe the growth of all tumours in the group, e.g., the curve that we derived for growth of liver metastases from a primary ileum carcinoid in a previous article.The aim of this study was to assess the homogeneity of the growth kinetics of tumours of the same type in a population of patients. Data from different groups of patients, including meningioma, hepatocellular carcinoma, pancreatic carcinoma, and primary lung cancer, were analysed. The model was also applied to data from a mouse model bearing transplanted human midgut carcinoid GOT1 tumours, a tumour group which is biologically more homogeneous than patient groups.SGR was calculated for each pair of tumour volume measurements using Equation A. Correlation between SGR and ln(V) was calculated between all SGR values and their corresponding tumour volume, i.e., the geometric mean of the two volumes used for calculation of SGR.Data from clinical studies were retrieved from the literature based on the availability of tumour volume estimates and corresponding measurement time intervals. Tumour volumes were estimated by delineating tumours in computed tomography (CT) and/or magnetic resonance imaging (MRI) slices and multiplying the measured area with slice thickness. Correlation between SGR and the logarithm of the volume of tumours was calculated for the following types of tumours.Data on the growth of non-small cell lung cancer (NSCLC) tumours in 18 patients were used. Tumour Another set of data was found for the growth rate and characteristics of small peripheral lung tumours as they appear on CT images. The tumour types included were rapidly growing (DT < 150 days) small cell lung cancer, adenocarcinoma, and squamous cell carcinoma tumours.Data for untreated pancreatic carcinoma in nine patients who underwent serial examinations by helical computed tomography was used for calculations. The mea0.14. This study showed that smaller tumours had shorter DT values, i.e. grew faster, than larger tumours and, therefore, may require shorter follow-up time to observe progression[The growth of hepatocellular carcinoma (HCC) was studied in 11 untreated patients where serial CT or MRI images were available . Calculagression.In an analyses of data from 34 HCC patients, the growth rate of most tumours could be estimated properly only using histological parameters, e.g. Ki-67index, apoptotic index, and histologic grade, available at a single time point (DT range was 17–274 days)[Another data set on HCC included tumours less than 3 cm in diameter at first observation in 21 patients. The nat3 and DT ranged from 1.27 to 143 years . DT was shorter for younger patients, but was not correlated with tumour size. We determined SGR and tumour volumes based on data from that article[The natural history of incidental meningiomas was studied in asymptomatic patients. Average article. The relIn order to test the validity of our method, the correlation between SGR and ln(V) was studied on tumours in the GOT1 nude mouse model (human midgut carcinoid tumours), which should biologically be more homogeneous compared with patients. Data frTo test the significance of correlations, the t-values for Pearson correlation were calculated and then converted to p-values using one tailed T.DIST function in Microsoft Excel. P-value < 0.05 was assumed to be statistically significant. All patient data used in this study were retrieved retrospectively from already published articles. This type of information is exempt from ethical approval. The animal experiments were approved by the Ethical Committee for Animal Research at University of Gothenburg.The correlation between SGR and the logarithm of the volume of different types of tumours are shown in Figure The correlation between SGR of tumour and the logarithm of its volume was statistically significant for data from nude mice bearing GOT1 tumours (p < 1E-11)  showed that the presented method is a useful tool to assess if growth deceleration is an important factor influencing difference in tumour growth rates observed among patients.vice versa. This provides the possibility for further development of mathematical models for elaborating this correlation in more accurate efficacy assessment of new drugs or combination of treatments. However, lack of correlation between SGR and the logarithm of tumour volume indicates that biological factors other than growth deceleration are more important for explaining the differences in the tumour growth rate observed in a population of patients with the same tumour type. These tumours may grow exponentially with different growth rates, or according to the Gompertzian model, and the model constants, SGR0 and λ, are heterogeneously distributed among tumours[A significant correlation between SGR and the logarithm of tumour volume in a group of tumours also indicates that the smaller tumours represent the growth of larger tumours when they were of small size and  tumours.In this study, the correlation between SGR and the logarithm of tumour volume was statistically significant for the growth of tumours in meningioma patients. Further analysis by dividing the material into small and large tumours also supported this result. A similar growth model observed for a group of patients with one tumour type corroborates that the response rate in this group might be a suitable measure to assess the efficacy of novel treatments. The correlation between tumour growth rate and the logarithm of its volume was, however, not statistically significant for the studied patient cohorts with hepatocellular carcinoma, pancreatic carcinoma, and primary lung cancer. This result was expected for the primary lung cancer cohort, since that included different types of lung cancer. A lack of correlation indicates that the growth of these types of tumours varies between patients with similar tumour type and, therefore, growth rate should be taken into account as an independent variable in efficacy assessment of treatment of patients with these types of tumours both in clinical trials and for individualized therapy planning.However, in the present study we were in general not able to include factors such as histologic grade and differentiation in our analyses, due to the limited number of tumours/patients or lack of information for other types of tumours. The only exception was for hepatocellular carcinoma, where subgroups could be categorized according to their differentiation level and clinical stage. However the correlation between tumour growth rate and the logarithm of its volume was not statistically significant for any of the two patient cohorts studied. It would otherwise be possible that stratification of the tumours according to, e.g., histopathological information would have given better correlation within subgroups of each tumour type. The result of the animal study indicates such a situation.Nevertheless, the significant correlation between tumour growth rate and the logarithm of its volume for regrowth of primary lung cancer tumours after chemotherapy was interesting, because this can be interpreted as homogeneity of these tumours among patients. Post-chemotherapy tumours consisting of resistant cancer cells or cancer stem cell clones (if exist) of non-small cell lung cancer might be more homogeneous in terms of cellular and histological characteristics and, therefore, respond more similarly to therapy. Such information is valuable for further treatments in, e.g., neoadjuvant therapy.The main difference between meningioma and other types of tumours in the present study is that meningiomas are benign. It is possible that the reason for meningioma being the only tumour type that gave significant correlation between tumour size and tumour growth rate in the patient cohorts is that it is benign, with low proliferation rate and histologically more homogeneous than malignant tumours.In the original study on meningioma, no correlation between tumour DT and its volume was found. On the In conclusion, the presented method, i.e. estimation of the Gompertzian growth deceleration constant using limited clinical data, can be used to evaluate the homogeneity of tumour growth pattern among patients. Tumour growth kinetics was largely heterogeneous among patients with the same type of tumour, except for the meningioma group. This implies that the tumour growth kinetics in each patient should be considered in efficacy assessment of new drugs and for optimization of treatment in individual patients using, e.g., the tumour response model in ref,22. FurtThe authors declare that they have no competing interests.EM and EFA initiated the study. EM developed the model and analyzed the data. EM and EFA drafted the manuscript. Both authors read and approved the final manuscript.


We assessed the impact of fluid accumulation on the development of acute kidney injury (AKI) and need for continuous renal replacement therapy in cardiac surgical patients. Fluid accumulation has been associated with negative outcomes including development of AKI in critically ill patients -3. As can = 3,207). The fluid accumulation percentage  /weight (kg) × 100) was calculated for 18 hours post surgery as most patients were in the ICU for this period. Acute Kidney Injury Network (AKIN) creatinine criteria were used to classify AKI using creatinine adjusted for fluid balance.We performed a retrospective analysis of prospectively collected data on all patients admitted after cardiac surgery to St Vincent's Hospital ICU, Melbourne, Australia from 1 July 2004 to 30 June 2012 . The fluid accumulation percentage was associated with an 8% increase in odds for AKI (OR (Cl), 1.08 (1.04 to 1.12)), and a 13% increase in odds for requiring renal replacement therapy (1.13 (1.05 to 1.21)) for each percent increase in fluid accumulation (l/kg%) after cardiac surgery, after adjusting for variables including APACHE score, cardiac failure, type of surgery, and inotrope use in multivariate analysis.In this relatively homogeneous patient group undergoing cardiac surgery, postoperative percent fluid accumulation at 18 hours was associated with AKI and need for renal replacement therapy. Whether there is residual confounding due to indication for fluid use or unmeasured risk factors requires further investigation in controlled trials.


Bodily state plays a critical role in our perception. In the present study, we asked the question whether and how bodily experience of weights influences time perception. Participants judged durations of a picture (a backpack or a trolley bag) presented on the screen, while wearing different weight backpacks or without backpack. The results showed that the subjective duration of the backpack picture was dilated when participants wore a medium weighted backpack relative to an empty backpack or without backpack, regardless of identity  of the visual backpack. However, the duration dilation was not manifested for the picture of trolley bag. These findings suggest that weight experience modulates visual duration estimation through the linkage between the wore backpack and to-be-estimated visual target. The congruent action affordance between the wore backpack and visual inputs plays a critical role in the functional linkage between inner experience and time perception. We interpreted our findings within the framework of embodied time perception. Our cognition and perception are grounded in bodily state as well as its interaction with environment . For exaNot only spatial perception, time perception can also be better understood within the framework of embodiment . StudiesTo explain interactions between subjective time and interoceptive (bodily) states, However, the body-related events  are often accompanied with the changes of arousal, indicated by physiological body response  caused by target stimuli are critical for duration distortion of the target stimuli . In mostThe present study was designed to investigate whether and how weighted experience, wearing a 5.7 kg backpack, influences visual duration judgments. In particular, whether congruent action affordance between weight experience and visual target plays a key role in subjective time distortion. Participants were asked to judge the duration of computer-presented pictures, either a backpack or trolley bag, while wearing a real weighted or empty backpack. The function of backpack is ‘wearing’, whereas the function of trolley bag is ‘pulling’. Thus, a backpack picture with affordance of ‘wearing’, regardless of its feature , might activate its functional linkage to weight experience through the congruent affordance. Then the weighted experience induced by the wore weighted backpack, associated with more energy costs, might dilate subjective duration of the backpack picture via such functional linkage, whereas the ‘pulling’ trolley bag is incongruent in affordance with the ‘wearing’ backpacks, such that the weight experience may have little influence on duration judgments of the visual trolley bag. Alternatively, the general arousal account  would prSD = 2.7). The numbers of females were 11, 12, and 14 in Experiments 1, 2, and 3, respectively. All participants had normal or corrected-to-normal visual acuity and no somatosensory disorders. All participants were naive to the purpose of experiments. The experiments were approved by the ethics committee of Jiangnan University. Informed consent in accordance with the Declaration of Helsinki was obtained from each participant before the start of the experiment.Fifty-five students from Jiangnan University took part in the experiments . Participants were asked to keep standing and holding a light response box during blocks. The viewing distance was kept at 57 cm. Visual stimuli presentation was controlled by Matlab program using Psychophysics Toolbox , small gray business trolley bag , orange backpack , or small trolley bag  in pictures were the same as the wore blue backpack (weighted or empty).In each trial, the to-be-estimated visual duration was the exposure time of a picture, which could be a blue backpack (Experiment 1), an orange backpack (Experiment 2), or a gray trolley bag (Experiment 3). During all experiments, participants wore a blue backpack (44 cm × 32 cm × 35cm) depicted in A classic temporal bisection task was used in the experiments. Participants were first trained to discriminate two visual anchor durations: a short one (200 ms) and a long one (600 ms). The anchor stimulus was a white rectangle , same size as the pictures used in the experiments. The training session ended when participants reached 100% accuracy of discrimination for consecutive 20 trials.Figure 2, each trial started with a fixation cross for 500 ms, followed by a blank display randomly for 500∼800 ms. Then a target picture  was presented for a given probe duration, randomly selected from 200, 300, 400, 500, or 600 ms. After the picture presentation, a question mark was shown to prompt for a response. Participants had to judge whether the duration of the picture was closer to the short anchor (200 ms) or the long anchor (600 ms) as accurately as possible by pressing the left or right key on the response box, respectively. The inter-trial interval (ITI) varied randomly from 1000 to 1500 ms.In the subsequent test session, illustrated in The test session consisted of three conditions of wearing weights block-wisely: the weighted backpack (5.7 kg), empty backpack (0.7 kg), and no backpack (baseline) conditions. Each weight condition was repeated twice, and randomly intermixed with the other conditions. Within each block, five probe durations were repeated randomly for 10 times, yielding 50 trials per block. Thus, the test session consisted of 300 trials. To refresh participants about the short and long anchors, each of the two anchors was presented for five times at the beginning of each block. Participants took a rest about 2 min by taking off the backpack between blocks. The length of the test session was around 50 min.After the test session, participants were asked to rate the valence and arousal using the paper sheet of the affective-rating Self-Assessment-Manikin (SAM) in order to compare the arousal levels among three conditions of wearing weights. The SAM evaluation is 9-point scales rating, ranging from sad to pleasant for the ‘valence’ and from calm to activated for the ‘arousal’ . To makeThe proportions of ‘long’ responses for the five probe durations were calculated and fitted by a logistic function for each participant at each weight condition. The points of subjective equality (PSEs) of the temporal bisection were then estimated corresponding to the duration at the 50th percentile of the fitted curves. To measure the sensitivity of duration judgments, the just-noticeable differences (JNDs) were estimated by taking half the difference in durations between the 25th and 75th percentiles  were 373 ± 9, 391 ± 9, and 394 ± 13 ms for the weighted backpack, empty backpack, and baseline conditions (Table 1). Repeated measures ANOVA revealed a significant influence of wearing weights on the visual duration judgment, F = 3.66, p < 0.05, p < 0.05), and between the weighted backpack and baseline conditions , but not between the empty backpack and baseline conditions (p = 0.70). The JNDs (±SE) were 53 ± 6, 50 ± 4, and 55 ± 3 ms for the weighted backpack, empty backpack, and baseline conditions (Table 1). A repeated-measures ANOVA failed to show any significant difference on JNDs among these three conditions, F = 0.69, p = 0.51, Experiment 1 examined the influences of wearing a backpack on the duration judgment of the same backpack picture. Figure 4). The mean PSEs (±SE) were 388 ± 11, 412 ± 10, and 404 ± 13 ms for the weighted, empty backpacks and baseline conditions, respectively (Table 1). The ANOVA revealed that the influence of the feeling of weight on visual duration judgments was significant, F = 3.5, p < 0.05, p < 0.05), but no significant difference between the empty backpack and baseline conditions (p = 0.44). A further ANOVA on discrimination sensitivity (JNDs) showed a marginal significance among three conditions, F = 3.3, p = 0.05, p < 0.05), while no differences were shown in other comparison conditions .Experiment 2 changed the identity of the backpack picture, yielding similar results as those of Experiment 1 . The mean PSEs (±SE) were in similar magnitudes for the three conditions: 398 ± 12, 395 ± 13, and 401 ± 12 ms for the weighted, empty backpacks and baseline, respectively (Table 1), and failed to reveal any main effect of perceiving weight on the visual duration judgment, F = 0.28, p = 0.76, F = 1.23, p = 0.31, Experiment 3, on the other hand, revealed different outcomes  = 5.14, p < 0.05, p < 0.05), but there was no evidence of significant difference between the weighted and empty backpack conditions (p = 0.12).Given that subjective ratings of arousal were similar across three experiments, we collapsed arousal ratings across three experiments for the weighted backpack, empty backpack, and baseline conditions. The total results showed that the subjective ratings of arousal significantly differed among the three conditions  lengthened subjective duration of a backpack picture, regardless of the identity of the backpack. In contrast, weight experience failed to impact duration judgments of a trolley bag picture. The findings suggest that the effect of weight experience on visual duration judgments depends on a functional link between weight experience and the to-be-estimated picture.Our findings of differential impacts of weight experiences on visual duration judgments could hardly be explained by the arousal-based account  or the aAlternatively, our findings can be better explained by the awareness theory based on the embodiment framework , accordiOne might argue, however, the same category (‘backpack’), rather than congruent affordance, between weight experience and visual blue or orange backpacks, contributed to the linkage. Both the visual blue and orange backpacks can be categorized as ‘backpack’, but trolley bag cannot. Thus, the category linkage between visual backpacks and the wore backpack might be proposed to induce the impact of weight on visual timing. The present research extends the evidence of embodied timing by revealing that wearing a weighted backpack dilates subjective visual duration through a functional linkage. The congruent action affordance between wearing behavior of weight and visual target is critical for such functional role of weight experience on visual timing. Note that we only applied three types of stimuli in visual modality. Thus, future work should expand stimuli to more general categories, and focus on influences of various types of action linkage between weight experience and duration estimation by using different types of sensory inputs, not limited to visual modality.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.