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Academy soccer players frequently train in the evening (i.e. 1700-2000 h), hence limited time to nutritionally prepare and recover due to schooling, travel and sleep schedules. Accordingly, we assessed timing and quantity of energy intake in the pre-training and post-training period. Over a 3-day in-season training period, male players (n=48; n=8 from under (U) 12, 13, 14, 15/16, 18 and 23 players) from an English Premier League academy self-reported dietary intake and physical activity levels (via the remote food photography method and activity diary, respectively) in the four hours pre- and post-training. Timing of pre-training energy intake ranged from 40 ± 28 mins (U15/U16 players) to 114 ± 71 mins (U18) before training and mean carbohydrate (CHO) intake ranged from 0.8±0.4 g.kg-1 (U23) to 1.5±0.9 g.kg-1 (U12). Timing of post-training energy intake ranged from 39 ± 27 mins (U14) to 70 ± 84 mins (U23) and mean CHO intake ranged from 1.6±0.8 g.kg-1 (U12) to 0.9±0.5 g.kg-1 (U14). In contrast to CHO, all age groups consumed sufficient protein intake in the post-training period (i.e. > 0.3 g.kg-1). We conclude academy soccer players habitually practice sub-optimal fuelling and recovery strategies, the consequence of which could impair growth, maturation and physical performance.

In response to the ongoing sex data gap, the present study provides a qualitative exploration of females’ nutritional experiences in elite sporting environments. Semistructured interviews were conducted with multiple participant groups (n = 18), including athletes (n = 7), practitioners (n = 6), and researchers (n = 5) across differing disciplines within professional sporting organiza-tions and/or national governing bodies. Combined content and thematic analysis provided an insight into the specific factors influencing current sport nutrition practices. A common theme highlighted among all participant groups was the paradoxical struggle between adequate fueling for training and competition demands, and the fear this may impact body mass and body composition goals. This tension was identified as being rooted within athletes’ perceptions of body image and driven by other participant groups and wider societal ideals. Each participant group also highlighted influences on cravings and approaches to food and dietary supplementation, centered around individual perceptions and challenges driven by symptomology associated with the female menstrual cycle and contraceptive use. To address these challenges, all participant groups called for more research to inform future change and continuing education pathways. In summary, this study contributes to providing a more complete understanding of elite female athlete sport nutrition experiences than currently exists. Multiple perspectives highlight the complexity of providing sport nutrition support to elite female athlete populations and directs future research, and practice, to reconsider one-size-fits-all approaches and acknowledge unique individual contexts which may influence these areas.

Purpose: This qualitative study explores player and stakeholder perceptions of the role of nutrition in supporting player development and performance in elite female soccer. Methods: Semi-structured interviews (36 ± 18 mins in length) were conducted with 47 participants, including players (n = 12), parents (n = 9), coaches (n = 9), sport scientists (n = 7), nutritionists (n = 5) and medical staff (n = 5). Via thematic analysis, data provided an insight into the nutrition culture within elite women’s soccer. Results and conclusions: Data demonstrate that considerable confusion and misconceptions exist amongst players and stakeholders regarding the theoretical underpinning and practical application of meeting energy requirements. As such, it is perceived that players ‘under-fuel’, which is likely caused by misunderstandings about the impact of carbohydrate intake on body composition, a fear of weight gain and the associated impacts upon body image. The ‘carbohydrate fear’ that is experienced by players is exacerbated by external pressures arising from social media, key stakeholders (e.g., coaches) and the skinfold culture surrounding measurement of body composition. Such cultural issues are amplified by the lack of full-time professionally accredited nutritionists overseeing the provision of nutrition support. Indeed, the infrastructure supporting the women’s game (e.g. staffing resource, on-site food provision, player education programmes, etc.) was considered incomparable to the men’s game. When taken together, our data provide a platform for which to develop organisational, stakeholder and player centred education and behaviour change interventions that strive to promote a positive performance nutrition culture within the women’s game.

Purpose: This qualitative study explores player and stakeholder perceptions of menstrual health support in elite female soccer. Methods: Semi-structured interviews were conducted with 47 participants including players (n = 12), parents (n = 9), coaches (n = 9), sport scientists (n = 7), nutritionists (n = 5) and medical staff (n = 5). Results: Via thematic analysis, data demonstrate that elite female soccer players experience a range of physical and psychological symptoms primarily at the onset of and during menses (as also perceived by stakeholders), with most participants perceiving these symptoms to impact performance. Nonetheless, menstrual health support is perceived as minimal and although players have their menstrual status tracked, they report little understanding as to why or how this information is used. This confusion was also present among stakeholders, often as a result of uncertainty about the evidence supporting the need for menstrual health support. The perceived lack of support may also be reflective of a culture where conversations about the menstrual cycle are not normalised. Overall, this may result in failure to identify and treat menstrual irregularities despite non-coaching staff members perceiving them to be common amongst players. Conclusion: These data support the need for individualised support based on the lived experiences of individual players and support staff. Furthermore, our research identifies the need for organisational, stakeholder, and player centred education programmes (led by experts in female athlete health) that create an environment where players receive personalised menstrual health support.

Purpose: To audit hormonal contraceptive use and associated symptomology in elite women’s soccer in England. Methods: Seventy-five elite women’s soccer players from the Women’s Super League (WSL) completed a questionnaire to assess: hormonal contraceptive (HC) use or non-use, reasons for initiation and discontinuation and the symptoms experienced by HC and non-HC users. Results: Twenty-eight per cent reported current HC use, with 43% having used HCs previously. Combined HCs accounted for 62% of total usage, with progestin-only HCs making up the remainder. Eighty-six per cent pre-empted negative symptoms before commencing HCs, with 38% experiencing adverse symptoms. Negative symptoms were most common in progestin-only HC users (63%). Eighty-six per cent reported benefits associated with HC usage include pain management and the ability to predict or control their cycles. Six non-HC users reported amenorrhea, with one medically diagnosed. Negative MC-related symptoms were reported by 74%, with 4% unable to train due to these symptoms. Unfavorable symptoms typically occurred during the first days of menstruation (59%). Conclusion: Most WSL players do not currently use HCs (72%). Most HC users reported benefits of HC usage, whilst most non-HC users reported negative symptoms especially around menstruation. Practitioners should track players' MCs to help minimise discomfort and maximise performance.

Purpose: The purpose of this study is to quantify total daily energy expenditure (TEE) of international adult female soccer players. Methods: Twenty-four professional players were studied during a 12-d period where they participated in an international training camp (also inclusive of two competitive games) representing the English national team. The TEE was assessed via the doubly labeled water method during the full 12 d as well as the initial 4-d period before game one. Energy intake was also assessed (via weighed food analysis) during the initial 4-d period to permit estimation of energy availability (EA). Results: Mean TEE did not differ (P = 0.31) between the 12-d (2693 ± 432 kcal·d-1; range, 2105-3507 kcal·d-1; 54 ± 6 kcal·kg-1 fat-free mass [FFM]) versus the 4-d assessment period (2753 ± 359 kcal·d-1; range, 1942-3280 kcal·d-1; 56 ± 8 kcal·kg-1 FFM). Mean 4-d energy intake was 1923 ± 357 kcal·d-1 (range, 1639-2172 kcal·d-1) and mean activity energy expenditure was 1069 ± 278 kcal·d-1 (range, 155-1549 kcal·d-1). When assessed for estimated EA, 88% of players were categorized with low EA status according to the threshold of <30 kcal·kg-1 FFM. Mean daily carbohydrate intake equated to 3.3 ± 0.7 g·kg-1 body mass. Conclusions: When compared with previously published data from adult male players, we demonstrate that the relative daily energetic requirements of engaging in professional soccer training and match play are comparable between sexes. From a practical perspective, data suggest that practitioners should likely focus education and behavior change strategies on "fuelling"for match play and training to optimize both player health and performance.

BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow–induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

Football is a global game which is constantly evolving, showing substantial increases in physical and technical demands. Nutrition plays a valuable integrated role in optimising performance of elite players during training and match-play, and maintaining their overall health throughout the season. An evidence-based approach to nutrition emphasising, a 'food first' philosophy (ie, food over supplements), is fundamental to ensure effective player support. This requires relevant scientific evidence to be applied according to the constraints of what is practical and feasible in the football setting. The science underpinning sports nutrition is evolving fast, and practitioners must be alert to new developments. In response to these developments, the Union of European Football Associations (UEFA) has gathered experts in applied sports nutrition research as well as practitioners working with elite football clubs and national associations/federations to issue an expert statement on a range of topics relevant to elite football nutrition: (1) match day nutrition, (2) training day nutrition, (3) body composition, (4) stressful environments and travel, (5) cultural diversity and dietary considerations, (6) dietary supplements, (7) rehabilitation, (8) referees and (9) junior high-level players. The expert group provide a narrative synthesis of the scientific background relating to these topics based on their knowledge and experience of the scientific research literature, as well as practical experience of applying knowledge within an elite sports setting. Our intention is to provide readers with content to help drive their own practical recommendations. In addition, to provide guidance to applied researchers where to focus future efforts.

We assessed the effects of post-exercise cold-water immersion (CWI) in modulating PGC-1α expression in response to exercise commenced with low muscle glycogen availability. In a randomized repeated-measures design, nine recreationally active males completed an acute two-legged high-intensity cycling protocol (8 x 5 min at 82.5% peak power output) followed by 10 minutes of two-legged post-exercise CWI (8°C) or control conditions (CON). During each trial, one limb commenced exercise with low (LOW: <300 mmol.kg-1 dw) or very low (VLOW: <150 mmol.kg-1 dw) pre-exercise glycogen concentration, achieved via completion of a one-legged glycogen depletion protocol undertaken the evening prior. Exercise increased (P<0.05) PGC-1α mRNA at 3 h post-exercise. Very low muscle glycogen attenuated the increase in PGC-1α mRNA expression compared with the LOW limbs in both the control (CON VLOW ~3.6-fold vs. CON LOW ~5.6-fold: P = 0.023, ES 1.22 Large) and CWI conditions (CWI VLOW ~2.4-fold vs. CWI LOW ~8.0 fold: P = 0.019, ES 1.43 Large). Furthermore, PGC-1α mRNA expression in the CWI-LOW trial was not significantly different to the CON LOW limb (P = 0.281, ES 0.67 Moderate). Data demonstrate that the previously reported effects of post-exercise CWI on PGC-1α expression (as regulated systemically via β-adrenergic mediated cell signalling) are offset in those conditions in which local stressors (i.e. high-intensity exercise and low muscle glycogen availability) have already sufficiently activated the AMPK- PGC-1α signaling axis. Additionally, data suggest that commencing exercise with very low muscle glycogen availability attenuates PGC-1α signaling.

PURPOSE: To evaluate the effects of probiotic supplementation on gastrointestinal (GI) symptoms, circulatory markers of GI permeability, damage, and markers of immune response during a marathon race. METHODS: Twenty-four recreational runners were randomly assigned to either supplement with a probiotic (PRO) capsule [25 billion CFU Lactobacillus acidophilus (CUL60 and CUL21), Bifidobacterium bifidum (CUL20), and Bifidobacterium animalis subs p. Lactis (CUL34)] or placebo (PLC) for 28 days prior to a marathon race. GI symptoms were recorded during the supplement period and during the race. Serum lactulose:rhamnose ratio, and plasma intestinal-fatty acid binding protein, sCD14, and cytokines were measured pre- and post-races. RESULTS: Prevalence of moderate GI symptoms reported were lower during the third and fourth weeks of the supplement period compared to the first and second weeks in PRO (p < 0.05) but not PLC (p > 0.05). During the marathon, GI symptom severity during the final third was significantly lower in PRO compared to PLC (p = 0.010). The lower symptom severity was associated with a significant difference in reduction of average speed from the first to the last third of the race between PLC (- 14.2 ± 5.8%) and PRO (- 7.9 ± 7.5%) (p = 0.04), although there was no difference in finish times between groups (p > 0.05). Circulatory measures increased to a similar extent between PRO and PLC (p > 0.05). CONCLUSION: Probiotics supplementation was associated with a lower incidence and severity of GI symptoms in marathon runners, although the exact mechanisms are yet to be elucidated. Reducing GI symptoms during marathon running may help maintain running pace during the latter stages of racing.

Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302. Intravital imaging facilitates the real-time tracking and targeting of moving hypoxic regions within pancreatic ductal adenocarcinoma. Using this approach, Conway et al. alleviate hypoxia-induced resistance to a dual mTORC1/2 inhibitor AZD2014, improving PI3K pathway inhibition and demonstrating a powerful dual imaging modality applicable to targeting other pathways and cancers.

DNA methylation is an important epigenetic modification that can regulate gene expression following environmental encounters without changes to the genetic code. Using Infinium MethylationEPIC BeadChip Arrays (850,000 CpG sites) we analysed for the first time, DNA isolated from untrained human skeletal muscle biopsies (vastus lateralis) at baseline (rest) and immediately following an acute (single) bout of resistance exercise. In the same participants, we also analysed the methylome following a period of muscle growth (hypertrophy) evoked via chronic (repeated bouts-3 sessions/wk) resistance exercise (RE) (training) over 7-weeks, followed by complete exercise cessation for 7-weeks returning muscle back to baseline levels (detraining), and finally followed by a subsequent 7-week period of RE-induced hypertrophy (retraining). These valuable methylome data sets described in the present manuscript and deposited in an open-access repository can now be shared and re-used to enable the identification of epigenetically regulated genes/networks that are modified after acute anabolic stimuli and hypertrophy, and further investigate the phenomenon of epigenetic memory in skeletal muscle.

© 2018 The Author(s). It is unknown if adult human skeletal muscle has an epigenetic memory of earlier encounters with growth. We report, for the first time in humans, genome-wide DNA methylation (850,000 CpGs) and gene expression analysis after muscle hypertrophy (loading), return of muscle mass to baseline (unloading), followed by later hypertrophy (reloading). We discovered increased frequency of hypomethylation across the genome after reloading (18,816 CpGs) versus earlier loading (9,153 CpG sites). We also identified AXIN1, GRIK2, CAMK4, TRAF1 as hypomethylated genes with enhanced expression after loading that maintained their hypomethylated status even during unloading where muscle mass returned to control levels, indicating a memory of these genes methylation signatures following earlier hypertrophy. Further, UBR5, RPL35a, HEG1, PLA2G16, SETD3 displayed hypomethylation and enhanced gene expression following loading, and demonstrated the largest increases in hypomethylation, gene expression and muscle mass after later reloading, indicating an epigenetic memory in these genes. Finally, genes; GRIK2, TRAF1, BICC1, STAG1 were epigenetically sensitive to acute exercise demonstrating hypomethylation after a single bout of resistance exercise that was maintained 22 weeks later with the largest increase in gene expression and muscle mass after reloading. Overall, we identify an important epigenetic role for a number of largely unstudied genes in muscle hypertrophy/memory.

The purpose of the present study was to investigate the prevalence of gastrointestinal symptoms (GIS) amongst recreational runners during a marathon race, and potential nutritional factors that may contribute. Recreational runners of the 2017 Liverpool (n = 66) and Dublin (n = 30) marathons were recruited. GIS were reported post-marathon and we considered GIS in the 7 days prior to the marathon and during the marathon using the Gastrointestinal Symptom Rating Scale (GSRS). Nutritional intake was recorded using food diaries for the day before the race, morning of the race, and during the race; 43% of participants reported moderate (≥4) GIS in the 7 days prior to the marathon and 27% reported moderate symptoms during the marathon with most common symptoms being flatulence (16%) during training, and nausea (8%) during the marathon race. Correlations between all nutritional intake and GIS were not statistically significant (p > 0.05). There were significant correlations between total GIS score (r = 0.510, p < 0.001), upper GIS score (r = 0.346, p = 0.001) and lower GIS score (r = 0.483, p < 0.001) in training and during the marathon. There appears to be a modest prevalence of GIS in recreational runners, in the week prior to a marathon and during marathon running, although there was no association with nutritional intake before or during the race.

OBJECTIVE: Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4. DESIGN: Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens). RESULTS: Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach. CONCLUSION: This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.

Mechanisms mediating post-exercise cold-induced increases in PGC-1α gene expression in human skeletal muscle are yet to be fully elucidated, but may involve local cooling effects on AMPK and p38 MAPK related signalling and/or increased systemic β-adrenergic stimulation. We aimed to therefore examine whether post-exercise cold-water immersion enhancement of PGC-1α mRNA is mediated through local or systemic mechanisms. Ten subjects completed acute cycling (8x5 min at ~80% peak power output) followed by seated-rest (CON) or single leg cold-water immersion (CWI; 10 min, 8°C). Muscle biopsies were obtained pre-, post- and 3 h post-exercise from a single limb in the CON condition but from both limbs in CWI (thereby providing tissue from a CWI and non-immersed limb, NOT). Muscle temperature decreased up to 2 h post-exercise following CWI (-5°C) in the immersed limb, with lesser changes observed in CON and NOT (-3°C; P<0.05). No differences between limbs were observed in p38MAPK phosphorylation at any time point (P<0.05), whilst a significant interaction effect was present for AMPK phosphorylation (P=0.031). Exercise (CON) increased gene expression of PGC-1α 3 h post-exercise (~5-fold; P<0.001). CWI augmented PGC-1α expression above CON in both the immersed (CWI; ~9-fold; P=0.003) and NOT limbs (~12-fold; P=0.001). Plasma Normetanephrine concentration was higher in CWI vs. CON immediately post-immersion (860 vs. 665 pmol/L; P=0.034). We report for the first time that local cooling of the immersed limb evokes transcriptional control of PGC1-α in the non-immersed limb, suggesting increased systemic β-adrenergic activation of AMPK may mediate, in part, post-exercise cold-induction of PGC-1α mRNA.

© 2016 Cancer Research UK Beatson Institute. Published under the terms of the CC BY 4.0 license Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a KrasG12D/p53R172Hmouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three-dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK-induced genes that facilitate extracellular matrix remodeling, with greatest fold-changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13. MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three-dimensional contexts. Treatment of KrasG12D/p53R172HPDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor-associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time. Nobis et al. generated a RhoA-FRET biosensor mouse to characterize and quantify the spatiotemporal distribution of RhoA activity in native mammalian tissues in vivo during development and disease progression. They show that RhoA activity is tightly regulated during various normal biological processes and is co-opted in disease settings, such as invasive breast and pancreatic cancers.

Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.

Pancreatic cancer has a devastating prognosis, with an overall 5-year survival rate of ∼8%, restricted treatment options and characteristic molecular heterogeneity. SerpinB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma (PDAC) and our genomic analysis revealed that SERPINB2 is frequently deleted in PDAC. We show that SerpinB2 is required by stromal cells for normal collagen remodelling in vitro, regulating fibroblast interaction and engagement with collagen in the contracting matrix. In a pancreatic cancer allograft model, co-injection of PDAC cancer cells and SerpinB2 -/- mouse embryonic fibroblasts (MEFs) resulted in increased tumour growth, aberrant remodelling of the extracellular matrix (ECM) and increased local invasion from the primary tumour. These tumours also displayed elevated proteolytic activity of the primary biochemical target of SerpinB2 - urokinase plasminogen activator (uPA). In a large cohort of patients with resected PDAC, we show that increasing uPA mRNA expression was significantly associated with poorer survival following pancreatectomy. This study establishes a novel role for SerpinB2 in the stromal compartment in PDAC invasion through regulation of stromal remodelling and highlights the SerpinB2/uPA axis for further investigation as a potential therapeutic target in pancreatic cancer.

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments. Erami et al. generate an E-cadherin-GFP mouse to demonstrate real-time quantification of E-cadherin mobility using intravital photobleaching in a range of tissue types. They show that changes in E-cadherin mobility correlate with changes in cell junction integrity and invasiveness while demonstrating applications of the mouse for future drug discovery studies.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related mortality. Despite significant advances made in the treatment of other cancers, current chemotherapies offer little survival benefit in this disease. Pancreaticoduodenectomy offers patients the possibility of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found that LOX expression is upregulated in metastatic tumors from Pdx1-Cre Kras(G12D/+) Trp53(R172H/+) (KPC) mice and that inhibition of LOX in these mice suppressed metastasis. Mechanistically, LOX inhibition suppressed both migration and invasion of KPC cells. LOX inhibition also synergized with gemcitabine to kill tumors and significantly prolonged tumor-free survival in KPC mice with early-stage tumors. This was associated with stromal alterations, including increased vasculature and decreased fibrillar collagen, and increased infiltration of macrophages and neutrophils into tumors. Therefore, LOX inhibition is able to reverse many of the features that make PDAC inherently refractory to conventional therapies and targeting LOX could improve outcome in surgically resectable disease.

OBJECTIVE: Pancreatic cancer is a leading cause of cancer-related death in the Western world. Current chemotherapy regimens have modest survival benefit. Thus, novel, effective therapies are required for treatment of this disease. DESIGN: Activating KRAS mutation almost always drives pancreatic tumour initiation, however, deregulation of other potentially druggable pathways promotes tumour progression. PTEN loss leads to acceleration of Kras(G12D)-driven pancreatic ductal adenocarcinoma (PDAC) in mice and these tumours have high levels of mammalian target of rapamycin (mTOR) signalling. To test whether these KRAS PTEN pancreatic tumours show mTOR dependence, we compared response to mTOR inhibition in this model, to the response in another established model of pancreatic cancer, KRAS P53. We also assessed whether there was a subset of pancreatic cancer patients who may respond to mTOR inhibition. RESULTS: We found that tumours in KRAS PTEN mice exhibit a remarkable dependence on mTOR signalling. In these tumours, mTOR inhibition leads to proliferative arrest and even tumour regression. Further, we could measure response using clinically applicable positron emission tomography imaging. Importantly, pancreatic tumours driven by activated KRAS and mutant p53 did not respond to treatment. In human tumours, approximately 20% of cases demonstrated low PTEN expression and a gene expression signature that overlaps with murine KRAS PTEN tumours. CONCLUSIONS: KRAS PTEN tumours are uniquely responsive to mTOR inhibition. Targeted anti-mTOR therapies may offer clinical benefit in subsets of human PDAC selected based on genotype, that are dependent on mTOR signalling. Thus, the genetic signatures of human tumours could be used to direct pancreatic cancer treatment in the future.