Protocol for real-time monitoring of CD8+ T and myeloid cell behavior in human high-grade serous ovarian cancer slices.

Studying cell behavior in live human tumors is crucial to understand and improve response to immunotherapies. Here, we present a protocol to slice human ovarian tumors ex vivo, maintain their viability for 24 h, and monitor the behavior of CD8+ T and myeloid cells in real time. Furthermore, we detail procedures to semi-automatically analyze cell movements and aggregate and process behavior data. This protocol can potentially be applied for multiple tumor types and mouse cancer models. For complete details on the use and execution of this protocol, please refer to Laforets et al.1.

Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFß-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

The relations between growth mindset, motivational beliefs, and career interest in math intensive fields in informal STEM youth programs.

Past research has shown that growth mindset and motivational beliefs have an important role in math and science career interest in adolescence. Drawing on situated expectancy-value theory (SEVT), this study extends these findings by investigating the role of parental motivational beliefs (e.g., expectancy beliefs, utility values) and parent growth mindset in math on adolescent career interest in math-intensive fields (e.g., mathematics, computer science, statistics, and engineering; MCSE) through adolescent motivational beliefs in math. Structural equation modeling was used to test the hypothesized model using data from 290 adolescents (201 girls, 69.3%; Mage = 15.20), who participate in informal STEM (science, technology, engineering, mathematics) youth programs, and their parents (162 parents, 87.7% female) in the United Kingdom and the United States. As hypothesized, adolescent expectancy beliefs, utility values, and growth mindset in math had a significant direct effect on MCSE career interest. Further, there was a significant indirect effect of parental expectancy beliefs in math on MCSE career interest through adolescents' expectancy beliefs. Similarly, there was a significant indirect effect from parental utility values in math to MCSE career interest through adolescents' utility values. The findings suggest that parents' math motivational beliefs play a critical role in adolescent math motivational beliefs and their career interest in math-intensive fields.

Children's and adolescents' evaluations of wealth-related STEM inequality.

The fields of science, technology, engineering, and mathematics (STEM) are rife with inequalities and under-representation that have their roots in childhood. While researchers have focused on gender and race/ethnicity as two key dimensions of inequality, less attention has been paid to wealth. To this end, and drawing from the Social Reasoning Development approach, we examined children's and adolescents' perceptions of STEM ability and access to opportunities as a function of wealth, as well as their desire to rectify such inequalities. Participants (n = 234: early childhood, n = 70, mean age = 6.33, SD = .79; middle childhood, n = 92, mean age = 8.90, SD = .83 and early adolescence, n = 62, mean age = 12.00; SD = 1.16) in the U.K. (64% White British) and U.S. (40% White/European American) read about two characters, one high-wealth and one low-wealth. In early childhood, participants reported that the high-wealth character would have greater STEM ability and were just as likely to invite either character to take part in a STEM opportunity. By middle childhood, participants were more likely to report equal STEM abilities for both characters and to seek to rectify inequalities by inviting the low-wealth character to take part in a STEM opportunity. However, older participants reported that peers would still prefer to invite the high-wealth character. These findings also varied by ethnic group status, with minority status participants rectifying inequalities at a younger age than majority status participants. Together these findings document that children are aware of STEM inequalities based on wealth and, with age, will increasingly seek to rectify these inequalities.

Developmental Trajectories of Adolescents' Math Motivation: The Role of Mindset and Perceptions of Informal STEM Learning Site Inclusivity.

Motivation is a key factor in engagement, achievement, and career choices in science, technology, engineering, and mathematics (STEM). While existing research has focused on student motivation toward math in formal school programs, new work is needed that focuses on motivation for those involved in informal STEM programs. Specifically, the role of math mindset and perceived inclusivity of informal STEM sites (to those of varying gender and ethnic backgrounds) on longitudinal trajectories of adolescents' math motivation has not been explored. This study investigates longitudinal changes in math expectancy, interest, and utility values and the effects of math fixed mindset, math growth mindset, and perceptions of the inclusivity of informal STEM learning sites on these changes for adolescents participating in STEM programs at these informal sites in the United Kingdom and the United States (n = 249, MT1age = 15.2, SD = 1.59). Three latent growth curve models were tested. The data suggest that math expectancy, interest, and utility values declined over three years. Growth mindset positively predicted changes in utility, while fixed mindset negatively predicted changes in utility. Inclusivity positively influenced the initial levels of utility. Girls reported lower initial expectancy than boys. Age influenced both the initial levels and rate of change for expectancy. Older adolescents had lower levels of expectancy compared to their younger counterparts; however, they had a less steep decline in expectancy over three years. These findings suggest that designing inclusive learning environments and promoting growth mindset may encourage math motivation.

Reciprocal Associations Between Science Efficacy, STEM Identity and Scientist Career Interest Among Adolescent Girls within the Context of Informal Science Learning.

Limited research has explored the longitudinal pathway to youth career interests via identity and efficacy together. This study examined the longitudinal associations between science efficacy, STEM (science, technology, engineering and math) identity, and scientist career interest among girls who are historically considered as an underrepresented group among scientists. The sample included 308 girls (M age = 15.22, SD age = 1.66; 42.8% White) from six STEM youth programs, each at a different informal science learning site within the U.K. and the U.S. Longitudinal structural equation modelling demonstrated that science efficacy consistently predicted STEM identity and scientist career interest, and similarly, STEM identity consistently predicted science efficacy over a two-year period. Scientist career interest at 12 months predicted science efficacy at 24 months. The coefficients of efficacy predicting STEM identity and scientist career interest were significantly larger compared to STEM identity and scientist career interest in predicting science efficacy from 12 months to 24 months. Further mediation analysis supported a significant pathway from STEM identity at 3 months to scientist career interest at 24 months via 12-month science efficacy. The findings highlight that science efficacy and STEM identity for girls relate to their scientist career interest and these longitudinal associations are reciprocal. This study suggests that science efficacy and STEM identity mutually influence each other, and enhancing science efficacy and STEM identity is key to promoting adolescents' interest in being a scientist.

Investigating the impact of London's ultra low emission zone on children's health: children's health in London and Luton (CHILL) protocol for a prospective parallel cohort study.

BACKGROUND: Air pollution harms health across the life course. Children are at particular risk of adverse effects during development, which may impact on health in later life. Interventions that improve air quality are urgently needed both to improve public health now, and prevent longer-term increased vulnerability to chronic disease. Low Emission Zones are a public health policy intervention aimed at reducing traffic-derived contributions to urban air pollution, but evidence that they deliver health benefits is lacking. We describe a natural experiment study (CHILL: Children's Health in London and Luton) to evaluate the impacts of the introduction of London's Ultra Low Emission Zone (ULEZ) on children's health. METHODS: CHILL is a prospective two-arm parallel longitudinal cohort study recruiting children at age 6-9 years from primary schools in Central London (the focus of the first phase of the ULEZ) and Luton (a comparator site), with the primary outcome being the impact of changes in annual air pollutant exposures (nitrogen oxides [NOx], nitrogen dioxide [NO2], particulate matter with a diameter of less than 2.5micrograms [PM2.5], and less than 10 micrograms [PM10]) across the two sites on lung function growth, measured as post-bronchodilator forced expiratory volume in one second (FEV1) over five years. Secondary outcomes include physical activity, cognitive development, mental health, quality of life, health inequalities, and a range of respiratory and health economic data. DISCUSSION: CHILL's prospective parallel cohort design will enable robust conclusions to be drawn on the effectiveness of the ULEZ at improving air quality and delivering improvements in children's respiratory health. With increasing proportions of the world's population now living in large urban areas exceeding World Health Organisation air pollution limit guidelines, our study findings will have important implications for the design and implementation of Low Emission and Clean Air Zones in the UK, and worldwide. CLINICALTRIALS: GOV: NCT04695093 (05/01/2021).

Interferon epsilon and ovarian cancer.

Most biologists know of the interferons IFNα, IFNß, and IFNγ and their roles in immunity and infection, but they may not have heard of IFNε. A recent study in Nature suggests that IFNε can act as a tumor suppressor in serous ovarian cancers.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

Preparing the Next Generation for STEM: Adolescent Profiles Encompassing Math and Science Motivation and Interpersonal Skills and Their Associations With Identity and Belonging.

Science, technology, engineering, and math (STEM) workers need both motivation and interpersonal skills in STEM disciplines. The aims of the study were to identify clusters of adolescents who vary in math and science motivation and interpersonal skills and to explore what factors are related to membership in a high math and science motivation and interpersonal skills cluster. Participants included 467 adolescents (312 female; Mage = 15.12 to SD = 1.71 year) recruited from out-of-school STEM programs in the US and UK. Findings from latent class analyses revealed four clusters, including a "High Math and Science Motivation and Interpersonal Skills" group, as well as groups that exhibited lower levels of either motivation or interpersonal skills. STEM program belonging, and STEM identity are related to membership in the high motivation and skills cluster. Findings provide insight into factors that may encourage motivation and interpersonal skills in adolescents, preparing them for STEM workforce entry.

The impact of pericytes on the stability of microvascular networks in response to nanoparticles.

Recapitulating the normal physiology of the microvasculature is pivotal in the development of more complex in-vitro models and organ-on-chip designs. Pericytes are an important component of the vasculature, promoting vessel stability, inhibiting vascular permeability and maintaining the vascular hierarchical architecture. The use of such co-culture for the testing of therapeutics and nanoparticle safety is increasingly considered for the validation of therapeutic strategies. This report presents the use of a microfluidic model for such applications. Interactions between endothelial cells and pericytes are first explored. We identify basal conditions required to form stable and reproducible endothelial networks. We then investigate interactions between endothelial cells and pericytes via direct co-culture. In our system, pericytes prevented vessel hyperplasia and maintained vessel length in prolonged culture (> 10 days). In addition, these vessels displayed barrier function and expression of junction markers associated with vessel maturation, including VE-cadherin, ß-catenin and ZO-1. Furthermore, pericytes maintained vessel integrity following stress (nutrient starvation) and prevented vessel regression, in contrast to the striking dissociation of networks in endothelial monocultures. This response was also observed when endothelial/pericyte co-cultures were exposed to high concentrations of moderately toxic cationic nanoparticles used for gene delivery. This study highlights the importance of pericytes in protecting vascular networks from stress and external agents and their importance to the design of advanced in-vitro models, including for the testing of nanotoxicity, to better recapitulate physiological response and avoid false positives.

Semi-supervised analysis of myeloid and T cell behavior in ex vivo ovarian tumor slices reveals changes in cell motility after treatments.

Studies of the high-grade serous ovarian cancer (HGSOC) tumor microenvironment, the most lethal gynecological cancer, aim to enhance the efficiency of established therapies. Cell motility is an important process of anti-tumor response. Using ex vivo human and mouse HGSOC tumor slices combined with time-lapse imaging, we assessed the motility of CD8+ T and myeloid cells. We developed a semi-supervised analysis of cell movements, identifying four cell behaviors: migrating, long migrating, static, and wobbling. Tumor slices were maintained 24h ex vivo, retaining viability and cell movements. Ex vivo treatments with lipopolysaccharide altered CD8+ T and myeloid cell behavior. In vivo chemotherapy reduced ex vivo cell movements in human and mouse tumors and differentially affected CD8+ T and myeloid cells in chemo-sensitive and chemo-resistant mouse models. Ex vivo tumor slices can extend in vivo mouse studies to human, providing a stepping stone to translate mouse cancer studies to clinical trials.

A role for macrophages under cytokine control in mediating resistance to ADI-PEG20 (pegargiminase) in ASS1-deficient mesothelioma.

BACKGROUND: Pegylated arginine deiminase (ADI-PEG20; pegargiminase) depletes arginine and improves survival outcomes for patients with argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM). Optimisation of ADI-PEG20-based therapy will require a deeper understanding of resistance mechanisms, including those mediated by the tumor microenvironment. Here, we sought to reverse translate increased tumoral macrophage infiltration in patients with ASS1-deficient MPM relapsing on pegargiminase therapy. METHODS: Macrophage-MPM tumor cell line (2591, MSTO, JU77) co-cultures treated with ADI-PEG20 were analyzed by flow cytometry. Microarray experiments of gene expression profiling were performed in ADI-PEG20-treated MPM tumor cells, and macrophage-relevant genetic "hits" were validated by qPCR, ELISA, and LC/MS. Cytokine and argininosuccinate analyses were performed using plasma from pegargiminase-treated patients with MPM. RESULTS: We identified that ASS1-expressing macrophages promoted viability of ADI-PEG20-treated ASS1-negative MPM cell lines. Microarray gene expression data revealed a dominant CXCR2-dependent chemotactic signature and co-expression of VEGF-A and IL-1α in ADI-PEG20-treated MPM cell lines. We confirmed that ASS1 in macrophages was IL-1α-inducible and that the argininosuccinate concentration doubled in the cell supernatant sufficient to restore MPM cell viability under co-culture conditions with ADI-PEG20. For further validation, we detected elevated plasma VEGF-A and CXCR2-dependent cytokines, and increased argininosuccinate in patients with MPM progressing on ADI-PEG20. Finally, liposomal clodronate depleted ADI-PEG20-driven macrophage infiltration and suppressed growth significantly in the MSTO xenograft murine model. CONCLUSIONS: Collectively, our data indicate that ADI-PEG20-inducible cytokines orchestrate argininosuccinate fuelling of ASS1-deficient mesothelioma by macrophages. This novel stromal-mediated resistance pathway may be leveraged to optimize arginine deprivation therapy for mesothelioma and related arginine-dependent cancers.

Interest, Mindsets and Engagement: Longitudinal Relations in Science Orientations for Adolescents in Informal Science Programs.

Little is known about the factors that influence engagement for adolescents participating in informal youth science programs. This study examined longitudinal reciprocal associations between adolescents' science engagement, interest, and growth mindset. Participants were adolescents (Mage = 15.06, SD = 1.82 years, 66.8% female) from the UK (n = 168) and the US (n = 299). A cross lagged path analysis indicated that participants' science growth mindset at baseline was positively related to interest, and engagement at year 1, and science interest at year 1 was positively related to growth mindset at year 2. Additionally, girls had lower science growth mindsets than boys. This evidence suggests that informal programs may encourage positive STEM trajectories by fostering engagement, growth mindset and interest.

Promoting Diverse Youth's Career Development through Informal Science Learning: The Role of Inclusivity and Belonging.

Little research has examined the associations between perceived inclusivity within informal science learning sites, youth program belonging and perceptions of program career preparation. This study explored relations between these factors at three timepoints (T1 = start of program, T2 = 3 months and T3 = 12 months after start). Participants were a diverse sample of 209 adolescents participating in STEM youth programs within informal science learning sites situated in the United States and United Kingdom (70% females: M age = 15.27, SD age = 1.60), with 53.1% British and 64.1% non-White. Path analysis revealed that only perceptions of inclusivity for own social identity group (i.e., gender, ethnicity) at T1 were associated with T2 STEM youth program belonging. There was a significant indirect effect of T1 perceptions of inclusivity for one's own social identity groups on T3 perceptions of program career preparation via T2 program belonging. This study highlights that, over time, perceptions of inclusivity around youth's own social identity groups (i.e., gender and ethnicity/culture) are related to a sense of youth program belonging, which in turn is later associated with perceptions of program career preparation.

Gender Stereotypes and Peer Selection in STEM Domains Among Children and Adolescents.

Gender stereotypes are harmful for girls' enrollment and performance in science and mathematics. So far, less is known about children's and adolescents' stereotypes regarding technology and engineering. In the current study, participants' (N = 1,206, girls n = 623; 5-17-years-old, M = 8.63, SD = 2.81) gender stereotypes for each of the STEM (science, technology, engineering, and mathematics) domains were assessed along with the relation between these stereotypes and a peer selection task in a STEM context. Participants reported beliefs that boys are usually more skilled than are girls in the domains of engineering and technology; however, participants did not report gender differences in ability/performance in science and mathematics. Responses to the stereotype measures in favor of one's in-group were greater for younger participants than older participants for both boys and girls. Perceptions that boys are usually better than girls at science were related to a greater likelihood of selecting a boy for help with a science question. These findings document the importance of domain specificity, even within STEM, in attempts to measure and challenge gender stereotypes in childhood and adolescence.

The Tumor Microenvironment of Clear-Cell Ovarian Cancer.

Some patients with advanced clear-cell ovarian cancer (CCOC) respond to immunotherapy; however, little is known about the tumor microenvironment (TME) of this relatively rare disease. Here, we describe a comprehensive quantitative and topographical analysis of biopsies from 45 patients, 9 with Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stage I/II (early CCOC) and 36 with FIGO stage III/IV (advanced CCOC). We investigated 14 immune cell phenotype markers, PD-1 and ligands, and collagen structure and texture. We interrogated a microarray data set from a second cohort of 29 patients and compared the TMEs of ARID1A-wildtype (ARID1Awt) versus ARID1A-mutant (ARID1Amut) disease. We found significant variations in immune cell frequency and phenotype, checkpoint expression, and collagen matrix between the malignant cell area (MCA), leading edge (LE), and stroma. The MCA had the largest population of CD138+ plasma cells, the LE had more CD20+ B cells and T cells, whereas the stroma had more mast cells and αSMA+ fibroblasts. PD-L2 was expressed predominantly on malignant cells and was the dominant PD-1 ligand. Compared with early CCOC, advanced-stage disease had significantly more fibroblasts and a more complex collagen matrix, with microarray analysis indicating "TGFß remodeling of the extracellular matrix" as the most significantly enriched pathway. Data showed significant differences in immune cell populations, collagen matrix, and cytokine expression between ARID1Awt and ARID1Amut CCOC, which may reflect different paths of tumorigenesis and the relationship to endometriosis. Increased infiltration of CD8+ T cells within the MCA and CD4+ T cells at the LE and stroma significantly associated with decreased overall survival.

A Therapeutically Actionable Protumoral Axis of Cytokines Involving IL-8, TNFα, and IL-1ß.

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1ß upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1ß induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1ß inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated. SIGNIFICANCE: IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1ß are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007.