A 3D in vitro assay to study combined immune cell infiltration and cytotoxicity.

Immune cell-mediated killing of cancer cells in a solid tumor is prefaced by a multi-step infiltration cascade of invasion, directed migration, and cytotoxic activities. In particular, immune cells must invade and migrate through a series of different extracellular matrix (ECM) boundaries and domains before reaching and killing their target tumor cells. These infiltration events are a central challenge to the clinical success of CAR T cells against solid tumors. The current standard in vitro cell killing assays measure cell cytotoxicity in an obstacle-free, two-dimensional (2D) microenvironment, which precludes the study of 3D immune cell-ECM interactions. Here, we present a 3D combined infiltration/cytotoxicity assay based on an oil-in-water microtechnology. This assay measures stromal invasion following extravasation, migration through the stromal matrix, and invasion of the solid tumor in addition to cell killing. We compare this 3D cytotoxicity assay to the benchmark 2D assay through tumor assembloid cocultures with immune cells and engineered immune cells. This assay is amenable to an array of imaging techniques, which allows direct observation and quantification of each stage of infiltration in different immune and oncological contexts. We establish the 3D infiltration/cytotoxicity assay as an important tool for the mechanistic study of immune cell interactions with the tumor microenvironment.

Engineering self-propelled tumor-infiltrating CAR T cells using synthetic velocity receptors.

Chimeric antigen receptor (CAR) T cells express antigen-specific synthetic receptors, which upon binding to cancer cells, elicit T cell anti-tumor responses. CAR T cell therapy has enjoyed success in the clinic for hematological cancer indications, giving rise to decade-long remissions in some cases. However, CAR T therapy for patients with solid tumors has not seen similar success. Solid tumors constitute 90% of adult human cancers, representing an enormous unmet clinical need. Current approaches do not solve the central problem of limited ability of therapeutic cells to migrate through the stromal matrix. We discover that T cells at low and high density display low- and high-migration phenotypes, respectively. The highly migratory phenotype is mediated by a paracrine pathway from a group of self-produced cytokines that include IL5, TNFα, IFNγ, and IL8. We exploit this finding to "lock-in" a highly migratory phenotype by developing and expressing receptors, which we call velocity receptors (VRs). VRs target these cytokines and signal through these cytokines' cognate receptors to increase T cell motility and infiltrate lung, ovarian, and pancreatic tumors in large numbers and at doses for which control CAR T cells remain confined to the tumor periphery. In contrast to CAR therapy alone, VR-CAR T cells significantly attenuate tumor growth and extend overall survival. This work suggests that approaches to the design of immune cell receptors that focus on migration signaling will help current and future CAR cellular therapies to infiltrate deep into solid tumors.

An artificial intelligence model correlated with morphological and genetic features of blastocyst quality improves ranking of viable embryos.

RESEARCH QUESTION: Can better methods be developed to evaluate the performance and characteristics of an artificial intelligence model for evaluating the likelihood of clinical pregnancy based on analysis of day-5 blastocyst-stage embryos, such that performance evaluation more closely reflects clinical use in IVF procedures, and correlations with known features of embryo quality are identified? DESIGN: De-identified images were provided retrospectively or collected prospectively by IVF clinics using the artificial intelligence model in clinical practice. A total of 9359 images were provided by 18 IVF clinics across six countries, from 4709 women who underwent IVF between 2011 and 2021. Main outcome measures included clinical pregnancy outcome (fetal heartbeat at first ultrasound scan), embryo morphology score, and/or pre-implantation genetic testing for aneuploidy (PGT-A) results. RESULTS: A positive linear correlation of artificial intelligence scores with pregnancy outcomes was found, and up to a 12.2% reduction in time to pregnancy (TTP) was observed when comparing the artificial intelligence model with standard morphological grading methods using a novel simulated cohort ranking method. Artificial intelligence scores were significantly correlated with known morphological features of embryo quality based on the Gardner score, and with previously unknown morphological features associated with embryo ploidy status, including chromosomal abnormalities indicative of severity when considering embryos for transfer during IVF. CONCLUSION: Improved methods for evaluating artificial intelligence for embryo selection were developed, and advantages of the artificial intelligence model over current grading approaches were highlighted, strongly supporting the use of the artificial intelligence model in a clinical setting.

Cell Trafficking at the Intersection of the Tumor-Immune Compartments.

Migration is an essential cellular process that regulates human organ development and homeostasis as well as disease initiation and progression. In cancer, immune and tumor cell migration is strongly associated with immune cell infiltration, immune escape, and tumor cell metastasis, which ultimately account for more than 90% of cancer deaths. The biophysics and molecular regulation of the migration of cancer and immune cells have been extensively studied separately. However, accumulating evidence indicates that, in the tumor microenvironment, the motilities of immune and cancer cells are highly interdependent via secreted factors such as cytokines and chemokines. Tumor and immune cells constantly express these soluble factors, which produce a tightly intertwined regulatory network for these cells' respective migration. A mechanistic understanding of the reciprocal regulation of soluble factor-mediated cell migration can provide critical information for the development of new biomarkers of tumor progression and of tumor response to immuno-oncological treatments. We review the biophysical andbiomolecular basis for the migration of immune and tumor cells and their associated reciprocal regulatory network. We also describe ongoing attempts to translate this knowledge into the clinic.

Robotic arm material characterisation using LIBS and Raman in a nuclear hot cell decommissioning environment.

Material characterisation in nuclear environments is an essential part of decommissioning processes. This paper explores the feasibility of deploying commercial off the shelf (COTS) laser induced breakdown spectroscopy (LIBS) and Raman spectroscopy, for use in a decommissioning hot cell environment, to inform waste operation decision making. To operate these techniques, adapters and probes were designed and constructed, for each instrument, to form tools that a robotic arm could pick up and operate remotely from an isolated control room. The developed instrumentation successfully returned live measurement data to a control room for saving and further analysis (e.g. material classification/identification). Successful testing of the solutions was performed for contact LIBS, contact Raman and stand-off Raman on a PaR M3000 robotic arm, in a simulated hot cell environment and the limitations identified. Data obtained by the techniques are analysed, classified and presented in a 3D virtual environment. The spectral data collected by a basic COTS LIBS showed potential for use in contamination identification (beryllium is used as example). Potential for COTS, LIBS and Raman in decommissioning is established and improvements to the hardware, the measurement processes and how the data is stored and used, are identified.

Estimating nutrient uptake requirements for soybean using QUEFTS model in China.

Estimating balanced nutrient requirements for soybean (Glycine max [L.] Merr) in China is essential for identifying optimal fertilizer application regimes to increase soybean yield and nutrient use efficiency. We collected datasets from field experiments in major soybean planting regions of China between 2001 and 2015 to assess the relationship between soybean seed yield and nutrient uptake, and to estimate nitrogen (N), phosphorus (P), and potassium (K) requirements for a target yield of soybean using the quantitative evaluation of the fertility of tropical soils (QUEFTS) model. The QUEFTS model predicted a linear-parabolic-plateau curve for the balanced nutrient uptake with a target yield increased from 3.0 to 6.0 t ha-1 and the linear part was continuing until the yield reached about 60-70% of the potential yield. To produce 1000 kg seed of soybean in China, 55.4 kg N, 7.9 kg P, and 20.1 kg K (N:P:K = 7:1:2.5) were required in the above-ground parts, and the corresponding internal efficiencies (IE, kg seed yield per kg nutrient uptake) were 18.1, 126.6, and 49.8 kg seed per kg N, P, and K, respectively. The QUEFTS model also simulated that a balanced N, P, and K removal by seed which were 48.3, 5.9, and 12.2 kg per 1000 kg seed, respectively, accounting for 87.1%, 74.1%, and 60.8% of the total above-ground parts, respectively. These results were conducive to make fertilizer recommendations that improve the seed yield of soybean and avoid excessive or deficient nutrient supplies. Field validation indicated that the QUEFTS model could be used to estimate nutrient requirements which help develop fertilizer recommendations for soybean.

Separation, Sizing, and Quantitation of Engineered Nanoparticles in an Organism Model Using Inductively Coupled Plasma Mass Spectrometry and Image Analysis.

For environmental studies assessing uptake of orally ingested engineered nanoparticles (ENPs), a key step in ensuring accurate quantification of ingested ENPs is efficient separation of the organism from ENPs that are either nonspecifically adsorbed to the organism and/or suspended in the dispersion following exposure. Here, we measure the uptake of 30 and 60 nm gold nanoparticles (AuNPs) by the nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove noningested AuNPs. Both conventional inductively coupled plasma mass spectrometry (ICP-MS) and single particle (sp)ICP-MS are utilized to measure the total mass and size distribution, respectively, of ingested AuNPs. Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) imaging confirmed that traditional nematode washing procedures were ineffective at removing excess suspended and/or adsorbed AuNPs after exposure. Water rinsing procedures had AuNP removal efficiencies ranging from 57 to 97% and 22 to 83%, while the sucrose density gradient procedure had removal efficiencies of 100 and 93 to 98%, respectively, for the 30 and 60 nm AuNP exposure conditions. Quantification of total Au uptake was performed following acidic digestion of nonexposed and Au-exposed nematodes, whereas an alkaline digestion procedure was optimized for the liberation of ingested AuNPs for spICP-MS characterization. Size distributions and particle number concentrations were determined for AuNPs ingested by nematodes with corresponding confirmation of nematode uptake via high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and in vivo quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment.

Yield Gap, Indigenous Nutrient Supply and Nutrient Use Efficiency for Maize in China.

Great achievements have been attained in agricultural production of China, while there are still many difficulties and challenges ahead that call for put more efforts to overcome to guarantee food security and protect environment simultaneously. Analyzing yield gap and nutrient use efficiency will help develop and inform agricultural policies and strategies to increase grain yield. On-farm datasets from 2001 to 2012 with 1,971 field experiments for maize (Zea mays L.) were collected in four maize agro-ecological regions of China, and the optimal management (OPT), farmers' practice (FP), a series of nutrient omission treatments were used to analyze yield gap, nutrient use efficiency and indigenous nutrient supply by adopting meta-analysis and ANOVA analysis. Across all sites, the average yield gap between OPT and FP was 0.7 t ha-1, the yield response to nitrogen (N), phosphorus (P), and potassium (K) were 1.8, 1.0, and 1.2 t ha-1, respectively. The soil indigenous nutrient supply of N, P, and K averaged 139.9, 33.7, and 127.5 kg ha-1, respectively. As compared to FP, the average recovery efficiency (RE) of N, P, and K with OPT increased by percentage point of 12.2, 5.5, and 6.5, respectively. This study indicated that there would be considerable potential to further improve yield and nutrient use efficiency in China, and will help develop and inform agricultural policies and strategies, while some management measures such as soil, plant and nutrient are necessary and integrate with advanced knowledge and technologies.

World fertilizer nutrient reserves: a view to the future.

The increasing need for food production in subtropical regions likely translates to a need for additional plant nutrients. As a consequence, knowledge of world fertilizer nutrient reserves is of particular relevance to sustainable agriculture in the subtropics. The stewardship responsibilities of agriculture include the wise use of the raw materials from which commercial fertilizers are produced. Development and implementation of fertilizer best management practices with focus on the 4Rs-right source, right rate, right time, right place-are timely not only for short-term economic and environmental reasons, but also for the wise stewardship of the non-renewable nutrient resources upon which food, feed, fiber, and fuel production depend.