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It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEV). The tumor-supportive CAFs possess a distinct phenotypic profile, compared with normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5β1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Furthermore, we show that NetG1 expression in CAFs is required for the prosurvival properties of sEVs. In addition, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5β1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5β1 were detected in sEVs collected from plasma of patients with PDAC, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor–stroma interactions and pathogenic stroma detection. Significance: Results from this study identified two unique types of tumor-supporting CAF EVs, with evidence of these being detected in patients. Thus, this study facilitates a novel avenue to further dissect the subtleties of the tumor–stroma interactions responsible for PDAC homeostasis and progression, as well as the possibility of establishing future means to detect and monitor dynamic stroma staging.

Spread sheet including a single tab per Figure panel conveying each graph's statistical analyses. Note that tabs are named accordingly. The tabs include the comprehensive statistical analyses that were performed for each experiment in which statistical significance was given. Each tab contains the full statistical output for its labeled experiment. The statistical test performed for each experiment is identified within the corresponding tab. Statistical tests were carried out using the software, Prism, version 7.05.

Spreadsheet including the metabolomic analyses corresponding to the experiments indicated by the naming of the assorted tabs. Specific analysis parameters for each experiment are provided within the respective main figure legends.

Supplementary Figure 1. Confirmation that CAFs produce ECMs distinct from NLFs. Supplementary Figure 2. Direct co-culture of CAFs support PDAC cells during nutrient-deprivation. Supplementary Figure 3. Additional CAF cell lines generate sEVs that rescue PDAC cells from nutrient deprivation-induced apoptosis. Supplementary Figure 4. NetG1+ CAF-sEVs support PDAC cell survival in a NGL-1 dependent manner. Supplementary Figure 5. NetG1 expression in CAFs is necessary for sEV-mediated survival of nutrient-deprived PDAC cells. Supplementary Figure 6. NetG1 ablation does not affect the uptake of sEV cargo in PDAC cells. Supplementary Figure 7. sEV supernatant contains DNPs enriched with sub-exosome sized EVs. Supplementary Figure 8. Enriched gene ontology clusters from proteomic and metabolomic analysis comparing sEV and DNP fractions. Supplementary Figure 9. EV cargo requires transfer in intact vesicles to provide tumor-supportive effect.

Spreadsheet including the proteomic analyses corresponding to the experiments indicated by the naming of the assorted tabs. Specific analysis parameters for each experiment are provided within the respective main figure legends.

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEV). The tumor-supportive CAFs possess a distinct phenotypic profile, compared with normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5β1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Furthermore, we show that NetG1 expression in CAFs is required for the prosurvival properties of sEVs. In addition, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5β1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5β1 were detected in sEVs collected from plasma of patients with PDAC, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor–stroma interactions and pathogenic stroma detection.Significance:Results from this study identified two unique types of tumor-supporting CAF EVs, with evidence of these being detected in patients. Thus, this study facilitates a novel avenue to further dissect the subtleties of the tumor–stroma interactions responsible for PDAC homeostasis and progression, as well as the possibility of establishing future means to detect and monitor dynamic stroma staging.

TMA Clinical Data

RNAseq

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC.Significance:This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211

Microarray

Amino Acid- Mass Spec

TMA IHC Scores

18 supplementary figures and corresponding legends

Lists all key resources and reagents

Sup Tables 5-7- Amino Acid Biochrom

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEVs). The tumor-supportive CAFs possess a distinct phenotypic profile, compared to normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α(5)β(1) localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1(+) CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Further, we show that NetG1 expression in CAFs is required for the pro-survival properties of sEVs. Additionally, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α(5)β(1) being enriched in exosomes. Finally, we found that NetG1 and Integrin α(5)β(1) were detected in sEVs collected from plasma of PDAC patients, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor-stroma interactions and pathogenic stroma detection.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. Significance: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function. See related commentary by Sherman, p. 230. This article is highlighted in the In This Issue feature, p. 211

This study investigates the impact of an immersive virtual reality (VR) simulation of herd immunity on vaccination intentions and its potential underlying mechanisms. In this preregistered field study, N = 654 participants were randomly assigned to one of the three VR conditions: (1) Gamified Herd Immunity; (2) Gamified Herd Immunity + Empathy (with additional narrative elements); (3) Control (gamified with no vaccination-related content). In the Gamified Herd Immunity simulation, participants embodied a vulnerable person and navigated a wedding venue trying to avoid getting infected. A total of 455 participants with below maximum intentions to take a novel vaccine and without severe cybersickness were analyzed. The Gamified Herd Immunity + Empathy and the Gamified Herd Immunity conditions increased vaccination intentions by 6.68 and 7.06 points on a 0–100 scale, respectively, compared to 1.91 for the Control condition. The Gamified Herd Immunity + Empathy condition enhanced empathy significantly more than the Gamified Herd Immunity condition but did not result in higher vaccination intentions. Experienced presence was related to the change in vaccination intentions. The results suggest that VR vaccination communication can effectively increase vaccination intentions; the effect is not solely due to the technological novelty and does not depend on empathy., Computers in Human Behavior, 139, ISSN:0747-5632, ISSN:1873-7692

Less than 11% of pancreatic cancer patients survive 5-years post-diagnosis. The unique biology of pancreatic cancer includes a significant expansion of its desmoplastic tumor microenvironment, wherein cancer-associated fibroblasts (CAFs) and their self-produced extracellular matrix are key components. CAF functions are both tumor-supportive and tumor-suppressive, while normal fibroblastic cells are solely tumor-suppressive. Knowing that CAF-eliminating drugs are ineffective and can accelerate cancer progression, therapies that “normalize” CAF function are highly pursued. Eribulin is a well-tolerated anti-microtubule drug used to treat a plethora of neoplasias, including advanced/metastatic cancers. Importantly, eribulin can inhibit epithelial to mesenchymal transition via a mechanism akin to blocking pathways induced by transforming growth factor-beta (TGFβ). Notably, canonical TGFβ signaling also plays a pivotal role in CAF activation, which is necessary for the development and maintenance of desmoplasia. Hence, we hypothesized that eribulin could modulate, and perhaps “normalize” CAF function. To test this premise, we used a well-established in vivo-mimetic fibroblastic cell-derived extracellular matrix (CDM) system and gauged the effects of eribulin on human pancreatic CAFs and cancer cells. The pathophysiologic cell-culturing system was also used to query eribulin effects on CDM-regulated cancer cell survival and invasive spread. Results demonstrated that intact CDMs modestly restricted eribulin from obstructing cancer cell growth. Nonetheless, eribulin-treated CAFs generated CDMs that limited cancer cell survival under nutritional stress, similar to reported tumor-suppressive CDMs generated by TGFβ-deficient CAFs. Data from this study support the central proposed premise and suggest that eribulin could be used as a CAF-normalizing drug.

It is projected that, in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). This fibrous stroma, known as desmoplasia, causes the collapse of local blood vessels rendering a nutrient-deprived milieu. Hence, PDAC cells are nurtured by local CAF-secreted products, which include, among others, CAF-generated small extracellular vesicles (sEVs). It is well-accepted that upon culturing functionally tumor-promoting CAFs under pathophysiological-relevant conditions (e.g., within self-produced ECM), these cells express NetrinG1 (NetG1) and sustain endosomal pools rich in active α5β1-integrin; traits indicative of poor patient survival. We herein report that NetG1+ CAFs generate sEVs that rescue PDAC cells from nutrient-deprived induced apoptosis. Two unique sEVs, NetG1+ and α5β1-integrin+, were uncovered. The former constitutes cargo of CAF-generated exomeres, and the latter is detected in classic exosomes. Proteomic and metabolomic analyses showed that the sEV-dependent PDAC survival is, at least in part, dictated by the cargo packaged within sEVs in a NetG1-dependent manner. Indeed, despite producing a similar number of vesicles, selected key proteins and metabolites (e.g., glutamine) were incorporated within the unique sEVs. Finally, we found that NetG1 and α5β1-integrin were detected in sEVs collected from plasma of PDAC patients, while their concomitant levels were significantly lower in plasma of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF sEVs opens a new investigative avenue in tumor-stroma interactions and stroma staging detection.

Pancreatic ductal adenocarcinoma (PDAC) is soon to be the second deadliest types of cancer, with a 5-year survival of only 10%. The unique features of PDAC are the expansion of cancer-associated fibroblasts (CAFs), presence of a dense fibrous stroma with high bundled collagen I and immunosuppression, representing a challenge for therapies. Ways to overcome these unique pro-tumor features will be key for the development of better therapeutic strategies for PDAC. Herein we report that, in addition to the identified ectopic expression of NGL-1 in PDAC cells, it is also expressed in the stroma (CAFs and immune cells) of PDAC patients, inversely correlating with overall survival. Stromal NGL-1 was important for tumorigenesis in vivo, as NGL-1 knockout mice (KO) orthotopically allografted with pancreatic cancer cells presented less tumor burden. Further analysis showed that the tumors from KO mice presented more CD8+ T cells and less immunosuppressive cytokines, such as TGFβ. Single cell RNAseq analysis of these tumors showed decreased expression of pro-tumor factors, such as immune checkpoint molecules in T cells and TGFβ related genes across different cellular compartments (epithelial, immune and fibroblasts). In accordance with these results, these tumors presented a limited amount of desmoplastic bundled collagen, suggestive of a TGFβ-deficient environment. In order to further dissect between the NGL-1-dependent contributions of immune vs. local stroma (e.g., CAFs) cells, we generated bone marrow chimeras and performed orthotopic injections to generate tumors. The loss of NGL-1 in each cellular compartment alone failed to phenocopy the full body loss of NGL-1, suggesting that NGL-1 in both immune cells and local stromal are important for tumorigenesis. Functionally, NGL-1 deficient CAFs failed to support the survival of starved PDAC cells in vitro, downregulated important myofibroblastic molecules such as p-smad, and produced less immunosuppressive cytokines, suggesting a role for NGL-1 in key pro-tumor features of CAFs. In fact, the fibroblastic NGL-1 dependent immunomodulatory effects were confirmed with human CD8+ T cells from healthy donors, which lost their cytotoxic profile in the presence of conditioned media (CM) from NGL-1+ CAFs, but were able to keep this profile when exposed to CM from NGL-1 deficient CAFs. Bone marrow derived macrophages from KO mice produced less pro-tumor cytokines and CD8+ T cells lacking NGL-1 proliferated more than those from wild type animals, when stimulated in vitro. Mechanistically, while immune cells and CAFs deficient in NGL-1 are both tumor suppressive, the latter can regain pro-tumor functions in response to TGFβ, explaining the need for a global modulation of NGL-1 expression for an anti-tumor effect. Finally, NGL-1 KO mice orthotopically allografted with PDAC cells responded better (smaller tumors) to chemotherapeutical regimen (FOLFIRINOX) compared to WT animals. All these results point to NGL-1 as a potential new target to modulate immunosuppression and tumorigenesis in pancreatic cancer. Citation Format: Debora Barbosa Vendramini Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Esteban Martinez, Stephen Sykes, Nina Steele, Marina Pasca di Magliano, Dmitry Zhigarev, Charline Ogier, Huamin Wang, Igor Astsaturov, Kerry Campbell, Edna Cukierman. Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR017.

Effective interventions for increasing people's intention to get vaccinated are crucial for global health, especially considering COVID-19. We devised a novel intervention using virtual reality (VR) consisting of a consultation with a general practitioner for communicating the benefits of COVID-19 vaccination and, in turn, increasing the intention to get vaccinated against COVID-19. We conducted a preregistered online experiment with a 2×2 between-participant design. People with eligible VR headsets were invited to install our experimental application and complete the ten minute virtual consultation study at their own discretion. Participants were randomly assigned across two age conditions (young or old self-body) and two communication conditions (with provision of personal benefit of vaccination only, or collective and personal benefit). The primary outcome was vaccination intention (score range 1–100) measured three times: immediately before and after the study, as well as one week later. Five-hundred-and-seven adults not vaccinated against COVID-19 were recruited. Among the 282 participants with imperfect vaccination intentions (, Vaccine, 39 (46), ISSN:0264-410X, ISSN:1358-8745

Here, we report the genome sequence of PA291, a nonmucoid, multidrug-resistant strain of Pseudomonas aeruginosa isolated from cystic fibrosis sputum. Short reads were de novo assembled into 190 contigs and scaffold assembled to a length of 6.26 Mbp. PhiSpy predicts that PA291 is free of prophages.

International audience; In Virtual Reality (VR), users can be immersed in emotionally intense and cognitively engaging experiences. Yet, despite strong interest from scholars and a large amount of work associating VR and Affective and Cognitive States (ACS), there is a clear lack of structured and systematic form in which this research can be classified. We define "Affective and Cognitive VR" to relate to works which (1) induce ACS, (2) recognize ACS, or (3) exploit ACS by adapting virtual environments based on ACS measures. This survey clarifies the different models of ACS, presents the methods for measuring them with their respective advantages and drawbacks in VR, and showcases Affective and Cognitive VR studies done in an immersive virtual environment (IVE) in a non-clinical context. Our article covers the main research lines in Affective and Cognitive VR. We provide a comprehensive list of references with the analysis of 63 research articles and summarize future directions.

Vaccine hesitancy poses one of the largest threats to global health. Informing people about the collective benefit of vaccination has great potential in increasing vaccination intentions. This research investigates the potential for engaging experiences in immersive virtual reality (VR) to strengthen participants’ understanding of community immunity, and therefore, their intention to get vaccinated. In a pre-registered lab-in-the-field intervention study, participants were recruited in a public park (tested: n = 232, analyzed: n = 222). They were randomly assigned to experience the collective benefit of community immunity in a gamified immersive virtual reality environment (2/3 of sample), or to receive the same information via text and images (1/3 of sample). Before and after the intervention, participants indicated their intention to take up a hypothetical vaccine for a new COVID-19 strain (0–100 scale) and belief in vaccination as a collective responsibility (1–7 scale). The study employs a crossover design (participants later received a second treatment), but the primary outcome is the effect of the first treatment on vaccination intention. After the VR treatment, for participants with less-than-maximal vaccination intention, intention increases by 9.3 points (95% CI: 7.0 to 11.5, p < 0.001). The text-and-image treatment raises vaccination intention by 3.3 points (difference in effects: 5.8, 95% CI: 2.0 to 9.5, p = 0.003). The VR treatment also increases collective responsibility by 0.82 points (95% CI: 0.37 to 1.27, p < 0.001). The results suggest that VR interventions are an effective tool for boosting vaccination intention, and that they can be applied “in the wild” – providing a complementary method for vaccine advocacy., Scientific Reports, 12 (1), ISSN:2045-2322

Background. Vaccine hesitancy poses one of the biggest threats to global health. Informing people about the collective benefit of vaccination due to community immunity has great potential in increasing vaccination intentions. Novel communication formats are needed to increase people’s interest in and engagement with such information, boosting the intervention’s effectiveness. This research investigates the potential for virtual reality (VR) to strengthen participants’ understanding of community immunity, and therefore, their intention to get vaccinated.Methods and Findings. In this pre-registered lab-in-the-field intervention study, participants (n= 222) were recruited in a public park. They either experienced the collective benefit of community immunity in a gamified immersive virtual reality environment (2/3 of sample), or received the same information via text and images (1/3 of sample). Before and after the intervention, participants indicated their intention to take up a hypothetical vaccine for a new COVID-19 strain (0–100 scale) and belief in vaccination as a collective responsibility (1–7 scale). After the VR treatment, for participants with imperfect vaccination intention, intention increases by 9.3 points (95% CI:7.0 to 11.5, p

This is the RAST annotation dataset for the P. aeruginosa Strain PA291 isolated from cystic fibrosis sputum.

It is predicted that pancreatic ductal adenocarcinoma (PDAC) will become the second most lethal cancer in the US by 2030. PDAC includes a fibrous-like stroma, desmoplasia, encompassing most of the tumor mass, which is produced by cancer-associated fibroblasts (CAFs) and includes their cell-derived extracellular matrices (CDMs). Since elimination of desmoplasia has proven detrimental to patients, CDM reprogramming, as opposed to stromal ablation, is therapeutically desirable. Hence, efforts are being made to harness desmoplasia's anti-tumor functions. We conducted biomechanical manipulations, using variations of pathological and physiological substrates in vitro, to culture patient-harvested CAFs and generate CDMs that restrict PDAC growth and spread. We posited that extrinsic modulation of the environment, via substrate rigidity, influences CAF's cell-intrinsic forces affecting CDM production. Substrates used were polyacrylamide gels of physiological (~1.5 kPa) or pathological (~7 kPa) stiffnesses. Results showed that physiological substrates influenced CAFs to generate CDMs similar to normal/control fibroblasts. We found CDMs to be softer than the corresponding underlying substrates, and CDM fiber anisotropy (i.e., alignment) to be biphasic and informed via substrate-imparted morphological CAF aspect ratios. The biphasic nature of CDM fiber anisotropy was mathematically modeled and proposed a correlation between CAF aspect ratios and CDM alignment; regulated by extrinsic and intrinsic forces to conserve minimal free energy. Biomechanical manipulation of CDMs, generated on physiologically soft substrates, leads to reduction in nuclear translocation of pERK1/2 in KRAS mutated pancreatic cells. ERK2 was found essential for CDM-regulated tumor cell spread. In vitro findings correlated with in vivo observations; nuclear pERK1/2 is significantly high in human PDAC samples. The study suggests that altering underlying substrates enable CAFs to remodel CDMs and restrict pancreatic cancer cell spread in an ERK2 dependent manner.

International audience; This paper describes an "all-in-one" solution for the real-time recognition of users' mental workloads in virtual reality through the cus-tomization of a commercial HMD with physiological sensors. First, we describe the hardware and software solution employed to build the system. Second, we detail the machine learning methods used for the automatic recognition of the users' mental workload, which are based on the well-known Random Forest algorithm. In order to gather data to train the system, we conducted an extensive user study with 75 participants using a VR flight simulator to induce different levels of mental workload. In contrast to previous works which label the data based on a standardized task (e.g. n-back task) or on a pre-defined task-difficulty, participants were asked about their perceived mental workload level along the experiment. With the data collected, we were able to train the system in order to classify four different levels of mental workload with an accuracy up to 65%. In addition, we discuss the role of the signal normalization procedures, the contribution of the different physiological signals on the recognition accuracy and compare the results obtained with the sensors embedded in the HMD with commercial grade systems. Preliminary results show our pipeline is able to recognize mental workload in real-time. Taken together, our results suggest that such all-in-one approach, with physiological sensors directly embedded in the HMD, is a promising path for VR applications in which the real-time or off-line estimation of Mental Workload assessment is beneficial.

Purpose Widespread antibiotic-resistant bacteria are threatening the arsenal of existing antibiotics. Not only are antibiotics less likely to be effective today, but their extensive use continues to drive the emergence of multidrug-resistant pathogens. A new-old antibacterial strategy with bacteriophages (phages) is under development, namely, phage therapy. Phages are targeted bacterial viruses with multiple antibacterial effector functions, which can reduce multidrug-resistant infections within the human body. This review summarizes recent phage therapy clinical trials and patient cases and outlines the fundamentals behind phage treatment strategies under development, mainly through bench-to-bedside approaches. We discuss the challenges that remain in phage therapy and the role of phages when combined with antibiotic therapy. Methods This narrative review presents the current knowledge and latest findings regarding phage therapy. Relevant case reports and research articles available through the Scopus and PubMed databases are discussed. Findings Although recent clinical data suggest the tolerability and, in some cases, efficacy of phage therapy, the clinical functionality still requires careful definition. The lack of well-controlled clinical trial data and complex regulatory frameworks have driven the most recent human data generation on a single-patient compassionate use basis. These cases often include the concomitant use of antibiotics, which makes it difficult to draw conclusions regarding the effectiveness of phages alone. However, human data support using antibiotics as phage potentiators and resistance breakers; thus, phage adjuvants are a promising avenue for near-term clinical development. Current knowledge gaps exist on the appropriate routes of administration, phage selection, frequency of administration, dosage, phage resistance, and pharmacokinetic and pharmacodynamic properties of the phages. In addition, we highlight that some phage therapies have mild adverse effects in patients. Implications Although more translational research is needed before the clinical implementation is feasible, phage therapy may well be pivotal in safeguarding humans against antibiotic-resistant infections.

Pancreatic Ductal Adenocarcinoma (PDAC) has a five-year survival under 10%. Treatment is compromised due to a fibrotic-like stromal remodeling process, known as desmoplasia, which limits therapeutic perfusion, supports tumor progression, and establishes an immunosuppressive microenvironment. These processes are driven by cancer-associated fibroblasts (CAFs), functionally activated through transforming growth factor beta1 (TGFβ1). CAFs produce a topographically aligned extracellular matrix (ECM) that correlates with reduced overall survival. Paradoxically, ablation of CAF populations results in a more aggressive disease, suggesting CAFs can also restrain PDAC progression. Thus, unraveling the mechanism(s) underlying CAF functions could lead to therapies that reinstate the tumor-suppressive features of the pancreatic stroma. CAF activation involves the f-actin organizing protein palladin. CAFs express two palladin isoforms (iso3 and iso4) which are up-regulated in response to TGFβ1. However, the roles of iso3 and iso4 in CAF functions remain elusive. Using a CAF-derived ECM model, we uncovered that iso3/iso4 are required to sustain TGFβ1-dependent CAF activation, secrete immunosuppressive cytokines, and produce a pro-tumoral ECM. Findings demonstrate a novel role for CAF palladin and suggest that iso3/iso4 regulate both redundant and specific tumor-supportive desmoplastic functions. This study highlights the therapeutic potential of targeting CAFs to restore fibroblastic anti-tumor activity in the pancreatic microenvironment.

Three-dimensional (3D) culturing models, replicating in vivo tissue microenvironments that incorporate native extracellular matrix (ECM), have revolutionized the cell biology field. Fibroblastic cells generate lattices of interstitial ECM proteins. Cell interactions with ECMs and with molecules sequestered/stored within these are crucial for tissue development and homeostasis maintenance. Hence, ECMs provide cells with biochemical and biomechanical cues to support and locally control cell function. Further, dynamic changes in ECMs, and in cell-ECM interactions, partake in growth, development, and temporary occurrences such as acute wound healing. Notably, dysregulation in ECMs and fibroblasts could be important triggers and modulators of pathological events such as developmental defects, and diseases associated with fibrosis and chronic inflammation such as cancer. Studying the type of fibroblastic cells producing these matrices and how alterations to these cells enable changes in ECMs are of paramount importance. This chapter provides a step-by-step method for producing multilayered (e.g., 3D) fibroblastic cell-derived matrices (fCDM). Methods also include means to assess ECM topography and other cellular traits, indicative of fibroblastic functional statuses, like naive/normal vs. inflammatory and/or myofibroblastic. For these, protocols include indications for isolating normal and diseased fibroblasts (i.e., cancer-associated fibroblasts known as CAFs). Protocols also include means for conducting microscopy assessments, querying whether fibroblasts present with fCDM-dependent normal or CAF phenotypes. These are supported by discrete semi-quantitative digital imaging analyses, providing some imaging processing advice. Additionally, protocols include descriptions for effective fCDM decellularization, which renders cellular debris-free patho/physiological in vivo-like scaffolds, suitable as 3D substrates for subsequent cell culturing.

International audience; In the context of the IEEE VR 2020 3DUI Contest entitled “Embodiment for the Difference”, this paper presents a VR application to highlight the challenges that people with physical disabilities face on their daily life. Two-armed users are placed in a world where people normally have three arms, making them effectively physically disabled. The scenario takes the user through the process of struggling with everyday interactions (designed for humans with three arms), then receiving a third arm prosthesis and thus recovering some level of autonomy. The experience is intended to generate a sense ofdifference and empathy for physically disabled persons.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer, with a 5-year survival of 10%. A major feature of PDAC is the presence of a dense fibrous stroma, due to the expansion of cancer associated fibroblasts (CAFs) and their extracellular matrix. This unique environment represents a challenge for therapies as it promotes immunosuppression, limits access to nutrients, and excludes or inactivates antitumor immune cells. Recently, we identified the ectopic expression of the neuronal protein Netrin G1 Ligand (NGL-1) in PDAC tissue, including its novel expression in immune cells and CAFs. However, the roles of NGL-1 in the tumor microenvironment (TME) of PDAC and in immune cell function are unknown and warranted further investigation. The contribution of NGL-1 to PDAC tumorigenesis was assessed by measuring the expression of NGL-1 in different models of PDAC and by orthotopically injecting PDAC cells in wild type (WT) or NGL-1 full body knockout mice (KO). Using our in vitro 3D system we evaluated if NGL-1+ CAFs, compared to NGL-1 knockdown (KD) CAFs, produced less immunosuppressive factors and were able to rescue PDAC cell survival under nutrient deprivation. For NGL-1 dependent immune cell functions we isolated naïve immune cells from WT and KO mice and performed ex-vivo functional assays. NGL-1 expression in fibroblasts correlated with disease development in different models of PDAC, and myeloid, T and NK cells from tumor bearing mice tended to overexpress NGL-1 when compared with cells from naïve mice. Accordingly, NGL-1 KO mice orthotopically injected with PDAC cells developed smaller tumors with decreased secretion of immunosuppressive factors, increased presence of CD8+ T cells and CD4+ T cells expressing less pro-tumor markers. Single cell RNA sequencing data from tumors from KO mice showed downregulation of pro-tumor genes in different cell populations, with the fibroblastic populations differing between WT and KO mice. In order to evaluate the contribution of the immune system for tumorigenesis in WT and KO mice, we performed bone marrow chimeras and depletion of specific immune cells. Functionally, CD8+ and CD4+ T cells from KO mice proliferated more when stimulated in vitro, suggesting that NGL-1 could represent a functional brake for T cells, inhibiting their anti-tumor capacity. The lack of NGL-1 in stimulated bone marrow-derived macrophages decreased pro-inflammatory cytokine secretion, further suggesting a functional role for NGL-1 in myeloid cells. Of note, NGL-1 KD CAFs did not support PDAC cell survival in vitro and produced less immunosuppressive cytokines, which was phenocopied by the treatment with a peptide targeting NGL-1. Translationally, we assessed the overall survival of 140 PDAC patients according to NGL-1 expression in the TME, where low expression of NGL-1 in CAFs and immune cells correlated with better survival of PDAC patients. Overall, this suggests NGL-1 as potential new target in PDAC, that could be manipulated in different compartments in pancreatic cancer. Citation Format: Debora Barbosa Vendramini Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Nina Steele, Benjamin Allen, Marina Pasca di Magliano, Charline Ogier, Igor Astsaturov, Kathy Q. Cai, Andres J. Klein-Szanto, Huamin Wang, Kerry Campbell, Edna Cukierman. The synaptic protein Netrin G1 ligand (NGL-1) modulates tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-096.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly types of cancer, with a 5-year survival of only 10%. One of the major features of PDAC is the dense fibrous stroma, due to the expansion of cancer associated fibroblasts (CAFs) and their extracellular matrix (ECM). This unique environment promotes immunosuppression, lack of nutrients and exclusion or inactivation of antitumor immune cells, representing a challenge for therapies. Recently, we identified the ectopic expression of the neuronal protein Netrin G1 Ligand (NGL-1) in PDAC tissue, including its novel expression in immune cells and CAFs. However, the roles of NGL-1 in the microenvironment of PDAC and in immune cell function are unknown and deserved further investigation. The effects in tumorigenesis were evaluated by orthotopically injecting pancreatic tumor cells in wild-type and NGL-1 full body knockout mice. The roles of NGL-1 in the pro-tumor functions of CAFs were evaluated using our in vitro 3D system, and questioning if NGL-1+ CAFs, compared to NGL-1 knockdown CAFs (CRISPRi), are able to rescue PDAC cell survival under nutrient deprivation, and discern their immunosuppressive profile. For the translational approach, we assessed the overall survival of 140 PDAC patients according to NGL-1 expression in the TME. Myeloid, T and NK cells from tumor-bearing mice tended to overexpress NGL-1 when compared with cells from naïve mice. Moreover, CD8+ and CD4+ T cells from NGL-1 KO mice proliferated more when stimulated in vitro, suggesting that NGL-1 could represent a functional brake for T cells. Moreover, the lack of NGL-1 in stimulated bone marrow-derived macrophages decreased pro-inflammatory cytokine secretion, further suggesting a functional role for NGL-1 in myeloid cells. Of note, NGL-1 KO mice orthotopically injected with PDAC cells developed smaller tumors with decreased secretion of immunosuppressive factors. In accordance, NGL-1 knockdown CAFs did not support PDAC cell survival in vitro and produced less immunosuppressive cytokines, which was phenocopied by the treatment with a peptide targeting NGL-1. Finally, the low expression of NGL-1 in CAFs and immune cells correlated with better survival of PDAC patients, therefore underscoring NGL-1 as a potential new target in PDAC, that could be manipulated in different compartments in pancreatic cancer (cancer cells, CAFs, immune cells). Citation Format: Debora B. Vendramini-Costa, Ralph Francescone, Tiffany Luong, Janusz Franco-Barraza, Igor Astsaturov, Kathy Q. Cai, Andres J. Klein-Szanto, Huamin Wang, Kerry S. Campbell, Edna Cukierman. Stromal expression of the synaptic protein netrin G1 ligand (NGL-1) promotes tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 116.

Is there an effect of Virtual Reality (VR) Head-Mounted Display (HMD) on the user's mental effort? In this paper, we compare the mental effort in VR versus in real environments. An experiment $(\mathrm{N}=27)$ was conducted to assess the effect of being immersed in a virtual environment (VE) using an HMD on the user's mental effort while performing a standardized cognitive task (the well-known N-back task, with three levels of difficulty, $N\in\{1,2,3\})$ . In addition to test the effect of the environment (i.e., virtual versus real), we also explored the impact of performing a dual task (i.e., sitting versus walking) in both environments on mental effort. The mental effort was assessed through self-report, task performance, behavioural and physiological measures. In a nutshell, the analysis of all measurements revealed no significant effect of being immersed in the VE on the users' mental effort. In contrast, natural walking significantly increased the users' mental effort. Taken together, our results support the fact there is no specific additional mental effort related to the immersion in a VE using a VR HMD.

International audience; In this paper, we present the design of ten different 3D user interactions using passive haptics and embedded in an escape game scenario in which users have to escape from a pyramid in a limited time. Our solution is innovative by its modularity, allowing interactions with virtual objects using tangible props manipulated either directly using the hands and feet or indirectly through a single prop held in the hand, in order to perform several interactions with the virtual environment (VE). We also propose a navigation technique based on the “impossible spaces” design, allowing users to naturally walk through several overlapping rooms of the VE. All together, our different interaction techniques allow the users to solve several enigmas built into a challenging scenario inside a pyramid.

Radiation, combined with radio-sensitizing chemotherapy, is used preoperatively as neoadjuvant therapy (NT) for borderline resectable pancreatic ductal adenocarcinoma (PDAC) with the intent of facilitating a curative surgical intervention. PDAC is uniquely characterized by an extensive fibrous microenvironment, desmoplasia, which can unintendedly be aggravated by NT to foster its pro-tumoral function. Radiation, as part of NT, is aimed at providing a margin adjacent to un-resectable vessels and sterilizing regional lymph nodes. Yet, to avoid the unintended desmoplastic aggravation, and because of a risk of toxicities associated with high doses due to the radio-sensitivity of adjacent small bowel and stomach, the total dose of NT radiation delivered to PDAC patients is classically modest. Therefore, NT radiation in PDAC patients is often lower than optimal for effective tumor cell elimination. Pulsed low-dose-rate (PLDR) radiation improves the safety of radiation treatment as it allows time for DNA damage repair in non-tumorous cells/tissues while simultaneously remaining as effective as continuous dose rate (CDR) radiation in cancer cells. In theory, PLDR could be amendable for increased radiation dosage. Of interest, the use of PLDR in pre-clinical animal studies revealed a systemic lowering of transforming growth factor beta (TGFβ), a known immunosuppressive factor. Hence, we posit that since cancer-associated fibroblasts (CAFs) produce TGFβ and constitute one of the most abundant cells in PDAC desmoplasia, variations in TGFβ levels in response to NT will inform on desmoplastic dynamic changes. Our team has developed means to assess CAF functions and activation statuses. These efforts were guided by generating a desmoplastic biomarker signature obtained from patient-harvested CAFs during the production of extracellular matrix (ECM), using our well-established in vitro 3D system. We employed this in vivo-mimetic system to test the hypothesis that PLDR limits pro-tumoral desmoplastic CAF aggravation. As part of the CAF functional signature, we tested levels of immunosuppressive TGFβ secretion, expression of netrin-G1, palladin, and others; together with the biogenesis of unique extracellular vesicles, and the production of ECMs capable of nurturing PDAC cells under starvation. During a 5-day assay, human PDAC 3D ECM producing CAFs were treated with gemcitabine (Gem; 5nm) plus 4Gy or 8Gy PLDR vs. CDR. Results indicated that Gem alone or with CDR indeed aggravated CAF’s pro-PDAC phenotype and function, while Gem combined with PDLR limited and sometimes reverted this functional pro-tumor CAF signature. Based on these results, we are poised to test this functional CAF/desmoplasia signature, as laboratory correlatives, in an ongoing clinical trial at Fox Chase Cancer Center. The phase I trial is a dose-escalation study of PLDR radiation and chemotherapy at standard and intensified doses in preoperative pancreatic cancer patients with toxicity, histopathologic, and CAF-informing translational endpoints. Citation Format: Janusz Franco-Barraza, Tiffany Luong, Jessica K. Wong, Debora B. Vendramini-Costa, Ralph Francescone, Jaye C. Gardiner, Kristopher S. Raghavan, Joshua E. Meyer, Edna Cukierman. Pulsed low-dose-rate radiation (PLDR) limits pancreatic pro-tumor stroma aggravation: Pre-clinical basis for an ongoing PLDR trail [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-006.

Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest cancers, features a highly immunosuppressive environment, with expansion of cancer-associated fibroblasts (CAFs), and absence/inactivation of antitumor immune cells. Therefore, ways to manipulate this environment, favoring the antitumor cells, will be critical for effective therapies for PDAC. Recently, we identified the neuronal protein Netrin G1 Ligand (NGL-1) to be overexpressed in PDAC tissue, including its novel expression in immune cells and CAFs. However, the role that NGL-1 plays in the tumor microenivronment (TME) is unknown and warrented further investigation. Using wild-type and NGL-1 full body knockout mice orthotopically injected or not with pancreatic tumor cells, we assessed tumorigenesis and secretion of immunosuppresive factors. Moreover, using our in vitro 3D system we explored the ability of NGL-1+ CAFs to rescue PDAC cell survival under nutrient deprivation, their immunosuppressive profile and the ability of a peptide targeting NGL-1 to reverse these features. Finally, we assessed the overall survival of 140 PDAC patients according to NGL-1 expression in the TME. Comparing immune cells from naïve and tumor-bearing mice we observed that myeloid, T and NK cells from tumor-bearing mice tend to overexpress NGL-1. Moreover, CD8+ and CD4+ T cells from NGL-1 KO mice proliferated more when stimulated in vitro, suggesting that NGL-1 could represent a functional brake for T cell activation and proliferation. Interestingly, the absence of NGL-1 in bone marrow-derived macrophages stimulated in vitro led to decreased production of pro-inflammatory cytokines, further suggesting a functional role for NGL-1 in myeloid cells. Importantly, NGL-1 KO mice orthotopically injected with PDAC cells developed smaller tumors and these produced less immunosuppressive factors. In accordance, CAFs lacking NGL-1 were not supportive of PDAC cell survival in vitro and produced less immunosuppressive cytokines, which was phenocopied by the treatment with the peptide targeting NGL-1. Finally, data from PDAC patients showed that low expression of NGL-1 in CAFs and immune cells correlated with better survival of these patients, therefore highlighting NGL-1 as a potential new target that could be manipulated in different compartments in pancreatic cancer (cancer cells, CAFs, immune cells). This represents an innovative perspective for such a complex disease. Citation Format: Debora Barbosa Vendramini-Costa, Ralph Francescone, Tiffany Luong, Janusz Franco-Barraza, Igor Astsaturov, Kathy Q. Cai, Andres J. Klein-Szanto, Huamin Wang, Kerry Campbell, Edna Cukierman. The synaptic protein netrin G1 ligand (NGL-1) modulates the immunosuppressive environment in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT019.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. NetG1+ cancer-associated fibroblasts (CAFs) supported PDAC survival, through a NetG1 mediated effect on glutamate/glutamine metabolism. NetG1+ CAFs were intrinsically immunosuppressive and inhibited NK cell mediated killing of tumor cells. These pro-tumor functions were controlled by a signaling circuit downstream to NetG1, which was comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally blocking NetG1 with a neutralizing antibody stunted in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC.SignificancePDAC is a devastating disease lacking effective therapies. A major hallmark of PDAC is desmoplasia, characterized by the expansion of CAFs and their extracellular matrix, creating a unique microenvironment that limits blood-supplied nutrition and is highly immunosuppressive. A better understanding of the role of CAFs in PDAC may lead to the identification of new targets for therapeutic intervention. Here, we uncovered roles for NetG1 in CAFs to promote tumorigenesis. NetG1 was important for two major CAF functions: the metabolic support of PDAC cells and the intrinsic immunosuppressive capacity of CAFs. Our results helped clarify the role that CAFs play in PDAC, by defining CAF phenotypes through NetG1 expression. Moreover, we established a link between CAF driven metabolism and their intrinsic immunosuppressive capacity, and identified a signaling circuit that governs NetG1 functions. Finally, we demonstrated the therapeutic potential of inhibiting NetG1 in vivo by limiting tumorigenesis in mice with a neutralizing antibody, illustrating that targeting stromal NetG1 could be an attractive therapeutic approach.

International audience; In this paper, we propose 3D user interfaces (3DUI) that are adapted to specific Virtual Reality (VR) tasks: climbing a ladder using a puppet metaphor, piloting a drone thanks to a 3D virtual compass and stacking 3D objects with physics-based manipulation and time control. These metaphors have been designed to provide the user with an intuitive, playful and efficient way to perform each task.

Enteropathogenic Escherichia coli (EPEC) is a major etiology for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse has led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy. Due to the drawbacks of EPEC in vivo models, a surrogate is the mouse-restricted gut pathgoen Citrobacter rodentium. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus and CrRp10 is a new strain within the genus Tequatrovirus. Neither phage carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 and CrRp10 appear to have independently evolved from E. coli phages. CrRp3 appears to be the more ‘potent’ being 24x more likely to find a host cell and has a shorter lytic cycle, while CrRp10 at MOI 0.001 was able to maintain bacterial density below the limit of detection after 18 h. We found that hypoxia (5% O2 and 5% CO2) inhibited CrRp3 ability to reverse exponential bacterial growth. It is unclear whether the subtle characteristic differences between CrRp3 and CrRp10 will influence treatment efficacy in future phage therapy in vivo investigations.