Presence of non-Newtonian fluid in invasive pulmonary mucinous adenocarcinomas impacts fluorescence during intraoperative molecular imaging of lung cancer.

BACKGROUND: Intraoperative molecular imaging (IMI) with folate-targeted NIR tracers has been shown to improve lesion localization in more than 80% of lung adenocarcinomas. However, mucinous adenocarcinomas (MAs) and invasive mucinous adenocarcinomas (IMAs) of the lung, which are variants of adenocarcinoma, appear to have decreased fluorescence despite appropriate folate receptor expression on the tumor surface. We hypothesized that the etiology may be related to light excitation and emission through non-Newtonian fluid (mucin) produced by goblet and columnar cancer cells. METHODS: Intraoperative data for 311 subjects were retrospectively reviewed from a prospectively collected 6-year database. For standardization, all patients underwent infusion of the same targeted molecular optical contrast agent (pafolacianine, folate receptor-targeted NIR fluorochrome) for lung cancer resections. Then, the ratio of the mean fluorescence intensity of the tumors and background tissues (TBR) was calculated. Tumors were examined for mucin, FRa, FRb, and immunofluorescent tracer uptake by a board-certified pathologist. The optical properties of mucin analyzed by imaging software were used to create in vitro gel models to explore the effects on NIR tracer fluorescence intensity. RESULTS: A large proportion (192, 62%) of the patients were female, with an average of 62.8 years and a 34-year mean pack smoking history. There were no severe (Clavien-Dindo > III) complications related to pafolacianine infusion. A total of 195 lesions in the study were adenocarcinomas, of which 19 (6.1%) were of the mucinous subtype. A total of 14/19 of the patients had a smoking history, and more than 74% of the IMA lesions were in the lower lobes. IMA lesions had a lower in situ TBR than nonmucinous adenocarcinomas (2.64 SD 0.23) vs (3.45 SD 0.11), respectively (p < 0.05). Only 9/19 (47%) were localized in situ. Tumor bisection and removal of mucin from IMAs significantly increased pafolacianine fluorescence, with resultant TBR not being significantly different from the control group (4.67 vs 4.89) (p = 0.19). Of the 16 lesions that underwent FR expression analysis, 15/16 had FR presence on cancer cells or tumor-associated macrophages in the tumor microenvironment. There was no statistically significant difference in fluorescence intensity during immunofluorescence analysis (4.99 vs 5.08) (p = 0.16). Physical removal of mucin from IMAs improved the TBR from 3.11 to 4.67 (p < 0.05). In vitro analysis of the impact of synthetic non-Newtonian fluid (agarose 0.5%) on NIR tracer fluorescence showed a decrease in MFI by a factor of 0.25 regardless of the concentration for each 5 mm thickness of mucin. CONCLUSION: The mucinous subtype of lung adenocarcinomas presents a unique challenge in pafolacianine-targeted IMI-guided resections. The presence of non-Newtonian fluids presents a physical barrier that dampens the excitation of the tracer and fluorescence emission detected by the camera. Knowledge of this phenomenon can allow the surgeon to critically analyze lesion fluorescence parameters during IMI-guided lung cancer resections.

Targeted detection of cancer cells during biopsy allows real-time diagnosis of pulmonary nodules.

BACKGROUND: The diagnostic yield of biopsies of solitary pulmonary nodules (SPNs) is low, particularly in sub-solid lesions. We developed a method (NIR-nCLE) to achieve cellular level cancer detection during biopsy by integrating (i) near-infrared (NIR) imaging using a cancer-targeted tracer (pafolacianine), and (ii) a flexible NIR confocal laser endomicroscopy (CLE) system that can fit within a biopsy needle. Our goal was to assess the diagnostic accuracy of NIR-nCLE ex vivo in SPNs. METHODS: Twenty patients with SPNs were preoperatively infused with pafolacianine. Following resection, specimens were inspected to identify the lesion of interest. NIR-nCLE imaging followed by tissue biopsy was performed within the lesion and in normal lung tissue. All imaging sequences (n = 115) were scored by 5 blinded raters on the presence of fluorescent cancer cells and compared to diagnoses by a thoracic pathologist. RESULTS: Most lesions (n = 15, 71%) were adenocarcinoma-spectrum malignancies, including 7 ground glass opacities (33%). Mean fluorescence intensity (MFI) by NIR-nCLE for tumor biopsy was 20.6 arbitrary units (A.U.) and mean MFI for normal lung was 6.4 A.U. (p < 0.001). Receiver operating characteristic analysis yielded a high area under the curve for MFI (AUC = 0.951). Blinded raters scored the NIR-nCLE sequences on the presence of fluorescent cancer cells with sensitivity and specificity of 98% and 97%, respectively. Overall diagnostic accuracy was 97%. The inter-observer agreement of the five raters was excellent (κ = 0.95). CONCLUSIONS: NIR-nCLE allows sensitive and specific detection of cancer cells in SPNs. This technology has far-reaching implications for diagnostic needle biopsies and intraprocedural decision-making.

Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency.

Defining the variables that impact the specificity of CRISPR/Cas9 has been a major research focus. Whereas sequence complementarity between guide RNA and target DNA substantially dictates cleavage efficiency, DNA accessibility of the targeted loci has also been hypothesized to be an important factor. In this study, functional data from two genome-wide assays, genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) and circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), have been computationally analyzed in conjunction with DNA accessibility determined via DNase I-hypersensitive sequencing from the Encyclopedia of DNA Elements (ENCODE) Database and transcriptome from the Sequence Read Archive to determine whether cellular factors influence CRISPR-induced cleavage efficiency. CIRCLE-seq and GUIDE-seq datasets were selected to represent the absence and presence of cellular factors, respectively. Data analysis revealed that correlations between sequence similarity and CRISPR-induced cleavage frequency were altered by the presence of cellular factors that modulated the level of DNA accessibility. The above-mentioned correlation was abolished when cleavage sites were located in less accessible regions. Furthermore, CRISPR-mediated edits were permissive even at regions that were insufficient for most endogenous genes to be expressed. These results provide a strong basis to dissect the contribution of local chromatin modulation markers on CRISPR-induced cleavage efficiency.

Bose glass and Mott glass of quasiparticles in a doped quantum magnet.

The low-temperature states of bosonic fluids exhibit fundamental quantum effects at the macroscopic scale: the best-known examples are Bose-Einstein condensation and superfluidity, which have been tested experimentally in a variety of different systems. When bosons interact, disorder can destroy condensation, leading to a 'Bose glass'. This phase has been very elusive in experiments owing to the absence of any broken symmetry and to the simultaneous absence of a finite energy gap in the spectrum. Here we report the observation of a Bose glass of field-induced magnetic quasiparticles in a doped quantum magnet (bromine-doped dichloro-tetrakis-thiourea-nickel, DTN). The physics of DTN in a magnetic field is equivalent to that of a lattice gas of bosons in the grand canonical ensemble; bromine doping introduces disorder into the hopping and interaction strength of the bosons, leading to their localization into a Bose glass down to zero field, where it becomes an incompressible Mott glass. The transition from the Bose glass (corresponding to a gapless spin liquid) to the Bose-Einstein condensate (corresponding to a magnetically ordered phase) is marked by a universal exponent that governs the scaling of the critical temperature with the applied field, in excellent agreement with theoretical predictions. Our study represents a quantitative experimental account of the universal features of disordered bosons in the grand canonical ensemble.

Unusual Mott transition in multiferroic PbCrO3.

The Mott insulator in correlated electron systems arises from classical Coulomb repulsion between carriers to provide a powerful force for electron localization. Turning such an insulator into a metal, the so-called Mott transition, is commonly achieved by "bandwidth" control or "band filling." However, both mechanisms deviate from the original concept of Mott, which attributes such a transition to the screening of Coulomb potential and associated lattice contraction. Here, we report a pressure-induced isostructural Mott transition in cubic perovskite PbCrO3. At the transition pressure of ∼3 GPa, PbCrO3 exhibits significant collapse in both lattice volume and Coulomb potential. Concurrent with the collapse, it transforms from a hybrid multiferroic insulator to a metal. For the first time to our knowledge, these findings validate the scenario conceived by Mott. Close to the Mott criticality at ∼300 K, fluctuations of the lattice and charge give rise to elastic anomalies and Laudau critical behaviors resembling the classic liquid-gas transition. The anomalously large lattice volume and Coulomb potential in the low-pressure insulating phase are largely associated with the ferroelectric distortion, which is substantially suppressed at high pressures, leading to the first-order phase transition without symmetry breaking.

Effects of number of training generations on genomic prediction for various traits in a layer chicken population.

BACKGROUND: Genomic estimated breeding values (GEBV) based on single nucleotide polymorphism (SNP) genotypes are widely used in animal improvement programs. It is typically assumed that the larger the number of animals is in the training set, the higher is the prediction accuracy of GEBV. The aim of this study was to quantify genomic prediction accuracy depending on the number of ancestral generations included in the training set, and to determine the optimal number of training generations for different traits in an elite layer breeding line. METHODS: Phenotypic records for 16 traits on 17,793 birds were used. All parents and some selection candidates from nine non-overlapping generations were genotyped for 23,098 segregating SNPs. An animal model with pedigree relationships (PBLUP) and the BayesB genomic prediction model were applied to predict EBV or GEBV at each validation generation (progeny of the most recent training generation) based on varying numbers of immediately preceding ancestral generations. Prediction accuracy of EBV or GEBV was assessed as the correlation between EBV and phenotypes adjusted for fixed effects, divided by the square root of trait heritability. The optimal number of training generations that resulted in the greatest prediction accuracy of GEBV was determined for each trait. The relationship between optimal number of training generations and heritability was investigated. RESULTS: On average, accuracies were higher with the BayesB model than with PBLUP. Prediction accuracies of GEBV increased as the number of closely-related ancestral generations included in the training set increased, but reached an asymptote or slightly decreased when distant ancestral generations were used in the training set. The optimal number of training generations was 4 or more for high heritability traits but less than that for low heritability traits. For less heritable traits, limiting the training datasets to individuals closely related to the validation population resulted in the best predictions. CONCLUSIONS: The effect of adding distant ancestral generations in the training set on prediction accuracy differed between traits and the optimal number of necessary training generations is associated with the heritability of traits.

Response and inbreeding from a genomic selection experiment in layer chickens.

BACKGROUND: Genomic selection (GS) using estimated breeding values (GS-EBV) based on dense marker data is a promising approach for genetic improvement. A simulation study was undertaken to illustrate the opportunities offered by GS for designing breeding programs. It consisted of a selection program for a sex-limited trait in layer chickens, which was developed by deterministic predictions under different scenarios. Later, one of the possible schemes was implemented in a real population of layer chicken. METHODS: In the simulation, the aim was to double the response to selection per year by reducing the generation interval by 50 %, while maintaining the same rate of inbreeding per year. We found that GS with retraining could achieve the set objectives while requiring 75 % fewer reared birds and 82 % fewer phenotyped birds per year. A multi-trait GS scenario was subsequently implemented in a real population of brown egg laying hens. The population was split into two sub-lines, one was submitted to conventional phenotypic selection, and one was selected based on genomic prediction. At the end of the 3-year experiment, the two sub-lines were compared for multiple performance traits that are relevant for commercial egg production. RESULTS: Birds that were selected based on genomic prediction outperformed those that were submitted to conventional selection for most of the 16 traits that were included in the index used for selection. However, although the two programs were designed to achieve the same rate of inbreeding per year, the realized inbreeding per year assessed from pedigree was higher in the genomic selected line than in the conventionally selected line. CONCLUSIONS: The results demonstrate that GS is a promising alternative to conventional breeding for genetic improvement of layer chickens.

Emerging methods for genome-scale metabolic modeling of microbial communities.

Genome-scale metabolic models (GEMs) are consolidating as platforms for studying mixed microbial populations, by combining biological data and knowledge with mathematical rigor. However, deploying these models to answer research questions can be challenging due to the increasing number of available computational tools, the lack of universal standards, and their inherent limitations. Here, we present a comprehensive overview of foundational concepts for building and evaluating genome-scale models of microbial communities. We then compare tools in terms of requirements, capabilities, and applications. Next, we highlight the current pitfalls and open challenges to consider when adopting existing tools and developing new ones. Our compendium can be relevant for the expanding community of modelers, both at the entry and experienced levels.

Human Tumor-Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis.

The clinical benefits of tumor-targeting antibodies (tAb) are modest in solid human tumors. The efficacy of many tAbs is dependent on Fc receptor (FcR)-expressing leukocytes that bind Fc fragments of tAb. Tumor-associated macrophages (TAM) and neutrophils (TAN) represent the majority of FcR+ effectors in solid tumors. A better understanding of the mechanisms by which TAMs and TANs regulate tAb response could help improve the efficacy of cancer treatments. Here, we found that myeloid effectors interacting with tAb-opsonized lung cancer cells used antibody-dependent trogocytosis (ADT) but not antibody-dependent phagocytosis. During this process, myeloid cells "nibbled off" tumor cell fragments containing tAb/targeted antigen (tAg) complexes. ADT was only tumoricidal when the tumor cells expressed high levels of tAg and the effectors were present at high effector-to-tumor ratios. If either of these conditions were not met, which is typical for solid tumors, ADT was sublethal. Sublethal ADT, mainly mediated by CD32hiCD64hi TAM, led to two outcomes: (i) removal of surface tAg/tAb complexes from the tumor that facilitated tumor cell escape from the tumoricidal effects of tAb; and (ii) acquisition of bystander tAgs by TAM with subsequent cross-presentation and stimulation of tumor-specific T-cell responses. CD89hiCD32loCD64lo peripheral blood neutrophils (PBN) and TAN stimulated tumor cell growth in the presence of the IgG1 anti-EGFR Ab cetuximab; however, IgA anti-EGFR Abs triggered the tumoricidal activity of PBN and negated the stimulatory effect of TAN. Overall, this study provides insights into the mechanisms by which myeloid effectors mediate tumor cell killing or resistance during tAb therapy. SIGNIFICANCE: The elucidation of the conditions and mechanisms by which human FcR+ myeloid effectors mediate cancer cell resistance and killing during antibody treatment could help develop improved strategies for treating solid tumors.

Genome-wide association study for Marek's disease mortality in layer chickens.

A genome-wide association study (GWAS) using Bayesian variable selection was performed to determine genomic regions associated with mortality due to Marek's disease virus (MDV) infection in layers. Mortality (%) under experimental disease challenge (500 plaque-forming units of a very virulent plus MDV strain) was recorded for progeny groups (average 15.5 birds; range 3 to 30) of 253 genotyped sires from four generations of a brown-egg layer line. An additional generation of 43 sires with progeny data was used to validate results. Sires were genotyped with a 42K Illumina single-nucleotide polymorphism (SNP) chip. Methods BayesB (pi = 0.995) and BayesCpi, with or without weighting residuals by the size of progeny groups were applied. The proportion of genetic variance contributed by SNPs within each 1-megabase (Mb) genomic region was quantified. Average mortality was 33% but differed significantly between generations. Genetic markers explained about 11% of phenotypic variation in mortality. Correlations between genomic estimated breeding values and percentage of progeny mortality for the validation generation (sons of individuals in training) were 0.12, 0.17, 0.02, and 0.16 for BayesB, weighted BayesB, BayesCpi, and weighted BayesCpi, respectively, when using the whole genome, and 0.03, 0.20, -0.06, and 0.14, when using only SNP from the 10, 1-Mb regions, explaining the largest proportion of genetic variance according to each method. Results suggest that regions on chromosomes 2, 3, 4, 9, 15, 18, and 21 are associated with Marek's disease resistance and can be used for selection and that accounting for the size of progeny groups has a large impact on correct localization of such genomic regions.

Pedigree and genomic analyses of feed consumption and residual feed intake in laying hens.

Efficiency of production is increasingly important with the current escalation of feed costs and demands to minimize the environmental footprint. The objectives of this study were 1) to estimate heritabilities for daily feed consumption and residual feed intake and their genetic correlations with production and egg-quality traits; 2) to evaluate accuracies of estimated breeding values from pedigree- and marker-based prediction models; and 3) to localize genomic regions associated with feed efficiency in a brown egg layer line. Individual feed intake data collected over 2-wk trial periods were available for approximately 6,000 birds from 8 generations. Genetic parameters were estimated with a multitrait animal model; methods BayesB and BayesCπ were used to estimate marker effects and find genomic regions associated with feed efficiency. Using pedigree information, feed efficiency was found to be moderately heritable (h(2) = 0.46 for daily feed consumption and 0.47 for residual feed intake). Hens that consumed more feed and had greater residual feed intake (lower efficiency) had a genetic tendency to lay slightly more eggs with greater yolk weights and albumen heights. Regions on chromosomes 1, 2, 4, 7, 13, and Z were found to be associated with feed intake and efficiency. The accuracy from genomic prediction was higher and more persistent (better maintained across generations) than that from pedigree-based prediction. These results indicate that genomic selection can be used to improve feed efficiency in layers.

Glycoprotein Receptor CEACAM5-Targeted Intraoperative Molecular Imaging Tracer in Non-Small Cell Lung Cancer.

BACKGROUND: Intraoperative molecular imaging has emerged as a potential tool in addressing challenges faced during lung cancer surgery by localizing small lesions, ensuring negative margins, and identifying synchronous cancers. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) glycoprotein has emerged as a potential target in fluorescent labeling of non-small cell lung cancer given the high antigen density in tumor cells and absence of expression in normal parenchyma. The goal of our study was to determine whether anti-CEACAM5 targeted near-infrared fluorochrome could be a suitable target in non-small cell lung cancer. METHODS: The CEACAM5 expression was evaluated in AB-12 (known negative control), HT29 (known positive control), and H460 (non-small cell lung cancer) cell lines by polymerase chain reaction. SGM-101, a CEACAM5 antibody, coupled with a BM-104 near-infrared fluorescent tracer was evaluated with dose escalation, in vitro cellular localization, and immunofluorescence microscopy. Subsequently, in vivo validation was performed in 52 athymic nude xenografts. RESULTS: Polymerase chain reaction analysis demonstrated 3000x relative expression of CEACAM5 in HT-29 cells compared with AB-12. The H460 cells showed 1000x relative expression compared with AB12 (P < .05). Both HT29 and H460 cells showed tracer internalization with signal to background ratio of 4.5 (SD 0.34) whereas there was minimal uptake by AB12 cells with signal to background ratio 1.1 (SD 0.1; P < .05). There was linear fluorescence increase with increasing tracer dosing in receptor expressing cell lines. In preclinical models, HT-29 and H460 cells lines produced near-infrared fluorescence with average tumor to background ratio of 3.89 (SD 0.25) irrespective of tumor size compared with no fluorescence by AB12 tumors (P < .05). The CEACAM5 expressing tumors had excellent dye uptake compared with AB12 tumors. CONCLUSIONS: CEACAM5 serves as a possible receptor for targeted intraoperative molecular imaging resections in lung cancer. This study sets a path for evaluation of CEACAM5 targets in future clinical trials.

Sodium Multivitamin Transporter-Targeted Fluorochrome Facilitates Enhanced Metabolic Evaluation of Tumors Through Coenzyme-R Dependent Intracellular Signaling Pathways.

BACKGROUND: Intraoperative molecular imaging (IMI)-guided resections have been shown to improve oncologic outcomes for patients undergoing surgery for solid malignancies. The technology utilizes fluorescent tracers targeting cancer cells without the use of any ionizing radiation. However, currently available targeted IMI tracers are effective only for tumors with a highly specific receptor expression profile, and there is an unmet need for IMI tracers to label a broader range of tumor types. Here, we describe the development and testing of a novel tracer (CR)-S0456) targeted to the sodium multivitamin transporter (SMVT). METHODS: Preclinical models of fibrosarcoma (HT-1080), lung (A549), breast (4T1), and renal cancers (HEK-293 T) in vitro and in vivo were used for assessment of (CR)-S0456 specific tumor labeling via sodium-mediated SMVT uptake in dipotassium phosphate or choline chloride-containing media buffer. Additionally, pharmacologic inhibition of multiple intracellular coenzyme-R obligate signaling pathways, including holocarboxylase synthetase (sulconazole nitrate), PI3K/AKT/mTOR (omipalisib), and calmodulin-dependent phosphatase (calmidazolium), were investigated to assess (CR)-S0456 uptake kinetics. Human fibrosarcoma-bearing xenografts in athymic nude mice were used for tumor and metabolic-specific labeling. Novel NIR needle confocal laser endomicroscopic (nCLE) intratumoral sampling was performed to demonstrate single-cell specific labeling by CR-S0456. RESULTS: CR-S0456 localization in vitro correlated with highly proliferative cell lines (MTT) and doubling time (p < 0.05) with the highest microscopic fluorescence detected in aggressive human fibrosarcomas (HT-1080). Coenzyme-R-specific localization was demonstrated to be SMVT-specific after competitive inhibition of internal localization with excess administration of pantothenic acid. Inhibiting the activity of SMVT by affecting sodium ion hemostasis prevented the complete uptake of CR-S0456. In vivo validation demonstrated (CR)-S0456 localization to xenograft models with accurate identification of primary tumors as well as margin assessment down to 1 mm3 tumor volume. Systemic treatment of xenograft-bearing mice with a dual PI3K/mTOR inhibitor suppressed intratumoral cell signaling and (CR)-S0456 uptake via a reduction in SMVT expression. Novel analysis of in vivo intratumoral cytologic fluorescence using near-infrared confocal laser endomicroscopy demonstrated the absence of coenzyme-R-mediated NIR fluorescence but not fibroblast activation protein (FAP)-conjugated fluorochrome, indicating specific intracellular inhibition of coenzyme-R obligate pathways. CONCLUSION: These findings suggest that a SMVT-targeted NIR contrast agent can be a suitable tracer for imaging a wide range of malignancies as well as evaluating metabolic response to systemic therapies, similar to PET imaging with immune checkpoint inhibitors.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule Type 5 Receptor-Targeted Fluorescent Intraoperative Molecular Imaging Tracer for Lung Cancer: A Nonrandomized Controlled Trial.

Importance: Localization of subcentimeter ground glass opacities during minimally invasive thoracoscopic lung cancer resections is a significant challenge in thoracic oncology. Intraoperative molecular imaging has emerged as a potential solution, but the availability of suitable fluorescence agents is a limiting factor. Objective: To evaluate the suitability of SGM-101, a carcinoembryonic antigen-related cell adhesion molecule type 5 (CEACAM5) receptor-targeted near-infrared fluorochrome, for molecular imaging-guided lung cancer resections, because glycoprotein is expressed in more than 80% of adenocarcinomas. Design, Setting, and Participants: For this nonrandomized, proof-of-principal, phase 1 controlled trial, patients were divided into 2 groups between August 1, 2020, and January 31, 2022. Patients with known CEACAM5-positive gastrointestinal tumors suggestive of lung metastasis were selected as proof-of-principle positive controls. The investigative group included patients with lung nodules suggestive of primary lung malignant neoplasms. Patients 18 years or older without significant comorbidities that precluded surgical exploration with suspicious pulmonary nodules requiring surgical biopsy were included in the study. Interventions: SGM-101 (10 mg) was infused up to 5 days before index operation, and pulmonary nodules were imaged using a near-infrared camera system with a dedicated thoracoscope. Main Outcomes and Measures: SGM-101 localization to pulmonary nodules and its correlation with CEACAM5 glycoprotein expression by the tumor as quantified by tumor and normal pulmonary parenchymal fluorescence. Results: Ten patients (5 per group; 5 male and 5 female; median [IQR] age, 66 [58-69] years) with 14 total lesions (median [range] lesion size, 0.91 [0.90-2.00] cm) were enrolled in the study. In the control group of 4 patients (1 patient did not undergo surgical resection because of abnormal preoperative cardiac clearance findings that were not deemed related to SGM-101 infusion), the mean (SD) lesion size was 1.33 (0.48) cm, 2 patients had elevated serum CEA markers, and 2 patients had normal serum CEA levels. Of the 4 patients who underwent surgical intervention, those with 2+ and 3+ tissue CEACAM5 expression had excellent tumor fluorescence, with a mean (SD) tumor to background ratio of 3.11 (0.45). In the patient cohort, the mean (SD) lesion size was 0.68 (0.22) cm, and no elevations in serum CEA levels were found. Lack of SGM-101 fluorescence was associated with benign lesions and with lack of CEACAM5 staining. Conclusions and Relevance: This in-human proof-of-principle nonrandomized controlled trial demonstrated SGM-101 localization to CEACAM5-positive tumors with the detection of real-time near-infrared fluorescence in situ, ex vivo, and by immunofluorescence microscopy. These findings suggest that SGM-101 is a safe, receptor-specific, and feasible intraoperative molecular imaging fluorochrome that should be further evaluated in randomized clinical trials. Trial Registration: ClinicalTrials.gov identifier: NCT04315467.

APOLLO: A genome-scale metabolic reconstruction resource of 247,092 diverse human microbes spanning multiple continents, age groups, and body sites.

Computational modelling of microbiome metabolism has proved instrumental to catalyse our understanding of diet-host-microbiome-disease interactions through the interrogation of mechanistic, strain- and molecule-resolved metabolic models. We present APOLLO, a resource of 247,092 human microbial genome-scale metabolic reconstructions spanning 19 phyla and accounting for microbial genomes from 34 countries, all age groups, and five body sites. We explored the metabolic potential of the reconstructed strains and developed a machine learning classifier able to predict with high accuracy the taxonomic strain assignments. We also built 14,451 sample-specific microbial community models, which could be stratified by body site, age, and disease states. Finally, we predicted faecal metabolites enriched or depleted in gut microbiomes of people with Crohn's disease, Parkinson disease, and undernourished children. APOLLO is compatible with the human whole-body models, and thus, provide unprecedented opportunities for systems-level modelling of personalised host-microbiome co-metabolism. APOLLO will be freely available under https://www.vmh.life/.

Protocol for fluorescence-activated cell sorting of human EpCAM+ lung cancer cells for gene expression analysis of Rac guanine-nucleotide exchange factors.

Here, we describe a protocol for fluorescence-activated cell sorting (FACS) of human EpCAM+ cells from fresh surgically resected specimens. We then use Q-PCR to identify specific molecular targets associated with the metastatic phenotype. This combined approach enables a qualitative and quantitative gene expression analysis of lung cancer samples. We describe how to use the protocol for Rac GEFs, but it can be applied broadly to other molecular targets. For complete details on the use and execution of this protocol, please refer to Cooke et al. (2021) and Quatromoni et al. (2015).

PDLIM2 is highly expressed in Breast Cancer tumour-associated macrophages and is required for M2 macrophage polarization.

The PDZ-LIM domain-containing protein 2 (PDLIM2) regulates cell polarity and the protein stability of key transcription factors in epithelial and hemopoietic cells. We previously reported that PDLIM2 is more highly expressed in Triple Negative Breast Cancer (TNBC) than in other breast cancer types or normal breast tissue. In the course of the TNBC study, it was noted that PDLIM2 was highly expressed in the stroma of PDLIM2-expressing tumours. Here, we investigated the phenotype of these stromal cells and whether any infiltrating immune population was linked to PDLIM2 expression. We found that high PDLIM2 expression in breast tumours was associated with higher levels of infiltrating M2 macrophages, but was not associated with infiltrating T cell sub-populations. We then tested whether PDLIM2 contributes to macrophage differentiation or function by using cultures of bone marrow-derived macrophages from wildtype and Pdlim2 knockout mice. This demonstrated that PDLIM2 is required for naïve macrophage migration and for the full adoption of IL-4-induced M2 polarization, including expression of M2 phenotypic markers, cell adhesion and cell migration. TLR4-, TLR3- or IFNγ-induced M1 macrophage activity was less dependent on PDLIM2. Finally, analysis of publicly available breast cancer datasets showed that high PDLIM2 expression is associated with increased M2 macrophage infiltration. We conclude that PDLIM2 expression influences the tumour associated stroma and, in particular, M2 macrophage infiltration that may contribute to the progression of TNBC or other subsets of breast cancer.

A Prostate-Specific Membrane Antigen-Targeted Near-Infrared Conjugate for Identifying Pulmonary Squamous Cell Carcinoma during Resection.

Pulmonary squamous cell carcinoma is the second most common lung cancer subtype and has a low 5-year survival rate at 17.6%. Complete resection with negative margins can be curative, but a high number of patients suffer early postoperative recurrence due to inadequate disease clearance at the index operation. Intraoperative molecular imaging (IMI) with tumor-targeted optical contrast agents is effective in improving resection completeness for other tumor types, but there are no IMI tracers targeted to pulmonary squamous cell carcinoma. In this report, we describe the use of a novel prostate-specific membrane antigen (PSMA)-targeted near-infrared conjugate (OTL78) to identify pulmonary squamous cell carcinoma. We identified PSMA as a viable target by examining its expression in human lung tumor specimens from a surgical cohort. Ninety-four percent of tumors expressed PSMA in either the pulmonary squamous cells or the tumor neovasculature. Using in vitro and in vivo models, we found that OTL78 reliably localized pulmonary squamous cell carcinoma in a PSMA-dependent manner. Finally, we found that IMI with OTL78 markedly improved surgeons' ability to identify residual disease after surgery in a preclinical model. Ultimately, this novel optical tracer may aid surgical resection of pulmonary squamous cell carcinoma and potentially improve long-term outcomes.

Targeted detection of cancer at the cellular level during biopsy by near-infrared confocal laser endomicroscopy.

Suspicious nodules detected by radiography are often investigated by biopsy, but the diagnostic yield of biopsies of small nodules is poor. Here we report a method-NIR-nCLE-to detect cancer at the cellular level in real-time during biopsy. This technology integrates a cancer-targeted near-infrared (NIR) tracer with a needle-based confocal laser endomicroscopy (nCLE) system modified to detect NIR signal. We develop and test NIR-nCLE in preclinical models of pulmonary nodule biopsy including human specimens. We find that the technology has the resolution to identify a single cancer cell among normal fibroblast cells when co-cultured at a ratio of 1:1000, and can detect cancer cells in human tumors less than 2 cm in diameter. The NIR-nCLE technology rapidly delivers images that permit accurate discrimination between tumor and normal tissue by non-experts. This proof-of-concept study analyzes pulmonary nodules as a test case, but the results may be generalizable to other malignancies.

Tumor factors stimulate lysosomal degradation of tumor antigens and undermine their cross-presentation in lung cancer.

Activities of dendritic cells (DCs) that present tumor antigens are often suppressed in tumors. Here we report that this suppression is induced by tumor microenvironment-derived factors, which activate the activating transcription factor-3 (ATF3) transcription factor and downregulate cholesterol 25-hydroxylase (CH25H). Loss of CH25H in antigen presenting cells isolated from human lung tumors is associated with tumor growth and lung cancer progression. Accordingly, mice lacking CH25H in DCs exhibit an accelerated tumor growth, decreased infiltration and impaired activation of intratumoral CD8+ T cells. These mice do not establish measurable long-term immunity against malignant cells that undergo chemotherapy-induced immunogenic cell death. Mechanistically, downregulation of CH25H stimulates membrane fusion between endo-phagosomes and lysosomes, accelerates lysosomal degradation and restricts cross-presentation of tumor antigens in the intratumoral DCs. Administration of STING agonist MSA-2 reduces the lysosomal activity in DCs, restores antigen cross presentation, and increases therapeutic efficacy of PD-1 blockade against tumour challenge in a CH25H-dependent manner. These studies highlight the importance of downregulation of CH25H in DCs for tumor immune evasion and resistance to therapy.