Wholemount _in situ_ hybridization has been widely used to explore gene expression distribution in both tissues and sections (Hauptmann and Gerster, 1994; Nieto _et al._, 1996). In the field of developmental biology, information on the spatial and temporal distribution of gene expression revealed by _in situ_ hybridization has facilitated the identification of master regulators of embryogenesis. In our recent study, we reported that _Pou3f1_ is an important regulator of mouse neuroectoderm development by combining wholemount _in situ_ hybridization and multiple functional analyses (Zhu _et al._, 2014). We optimized a wholemount RNA _in situ_ hybridization protocol that uses digoxigenin labeled RNA probes and an anti-digoxigenin antibody conjugated with alkaline phosphatase to detect the enrichment of _Pou3f1_ in the anterior embryonic region of the mouse gastrula, which indicated potential biological functions of _Pou3f1_ in embryonic ectoderm development. Thereafter, more lineage regulators of the mouse gastrulation have been revealed and validated using this optimized protocol (Yang _et al._, 2018 and 2019; Peng _et al._, 2016 and 2019). The current protocol exhibits strong experimental robustness and displays application potential in a wide range of biological studies. Thus, we summarize the protocol here, in the hope its application can facilitate the study of gene expression.

African swine fever (ASF) has evolved from an exotic animal disease to a threat to global pig production. An important avenue for wide-spread transmission of animal diseases is the dissemination of viruses through boar semen used for artificial insemination (AI). In this context, we investigated the role of male reproductive organs in ASF. Mature domestic boars and adolescent wild boar inoculated with different ASF virus strains were investigated by means of virological and pathological methods. Additionally, electron microscopy was employed to investigate in vitro inoculated sperm. Viral genome, antigen and infectious virus could be found in all gonadal tissues and accessory sex glands. The viral antigen and viral mRNAs were mainly found in mononuclear cells of the respective tissues. However, some other cell types, including Leydig, endothelial and stromal cells were also found positive. Using RNAScope, p72 mRNA could be found in scattered halo cells of the epididymal duct epithelium which could point to disruption of the barrier. No direct infection of spermatozoa was observed by immunohistochemistry or electron microscopy. Taken together, our results strengthen the assumption that ASFV can be transmitted via boar semen. Future studies are needed to explore excretion dynamics and transmission efficiency.

The development and subsequent adaptation of mass cytometry for the histological analysis of tissue sections has allowed the simultaneous spatial characterization of multiple components. This is useful to find the correlation between the genotypic and phenotypic profile of tumor cells and their environment in clinical-translational studies. In this revision, we provide an overview of the most relevant hallmarks in the development, implementation and application of multiplexed imaging in the study of cancer and other conditions. A special focus is placed on studies based on imaging mass cytometry (IMC) and multiplexed ion beam imaging (MIBI). The purpose of this review is to help our readers become familiar with the verification techniques employed on this tool and outline the multiple applications reported in the literature. This review will also provide guidance on the use of IMC or MIBI in any field of biomedical research.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths with a 5-year survival rate of approximately 7%. PDAC may originate from acinar cell trans-differentiation into ductal-like cells, termed acinar-to-ductal metaplasia (ADM), triggered by chronic pancreatitis and/or mutations in K-Ras. The progression to PDAC is associated with a dense fibrotic stroma, including cancer-associated fibroblasts (CAFs). YAP is a tension-stimulated CAF activator that promotes ECM stiffening, creating a permissive microenvironment for cancer progression. We hypothesize that the Hippo pathway may coordinate fibroinflammatory signals emanating from the stromal compartment during regenerative responses to acinar cell injury and progression towards PDAC. Methods: To resolve the transcriptional changes occurring during the transition to ADM and PDAC, we mapped the in situ expression of over 1800 RNA targets in patient-derived tissues using NanoString Technologies’ Digital Spatial Profiling (DSP) technology. We also performed immune-profiling and evaluated Yap expression in human ADM by immunohistochemistry. To study the in vivo role of Hippo signaling in stromal cells, we conditionally deleted Yap/Taz in Collagen1a2-producing cells in a murine model of caerulein-induced pancreatitis, which recapitulates many of the features associated with human ADM. I will analyze the resulting phenotype by immunostaining for metaplastic, proliferative, immune and stromal markers. Results: DSP analysis revealed genes implicated in fibroblast activation, epithelial-to-mesenchymal transition (EMT), neutrophil activation and IFNγ signaling as potential key drivers of ADM. I will further evaluate the expression of candidate genes and survey Yap expression at the single cell level in human ADM tissue by multiplexed RNAscope in situ hybridization. We found up-regulation of CD4+ and CD8+ T cells in ADM, and an increasing trend of neutrophil and macrophage accumulation in the progression from normal parenchyma to ADM to PDAC. Conclusions: This work will provide an in-depth understanding of epithelial-stroma crosstalk in ADM and a foundation for the development of new therapeutic strategies for treating non-invasive precursor lesions like ADM, thereby preventing pancreatic cancer progression. Source of Funding: This research is supported by the Fonds de Recherce du Quebec - Santé (FRQS), Canadian Institutes of Health Research (CIHR) and the Research Institute of the McGill University Health Centre (RI-MUHC).

A,B, Expression correlation analysis in 78 PDAC patient tumors12 [/articles/s43018-021-00258-w#ref-CR12] (E-MTAB-6134 [http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6134/]) with high tumor cellularity between cJUN and VIM (A) as well as GATA6 and VIM (B). RMA-normalized probe intensity values were plotted. A linear regression with 95% CI is shown in orange. Pearson’s correlation (_R_) and corresponding two-tailed _P_ value are indicated. C, Representative bright-field images of GCDX62 cells transduced with empty vector (EV) or cJUN overexpression (cJUN-OE) constructs. Morphology was monitored over several passages. D-F, RNA-seq analysis was performed on GCDX62 cells transduced with EV or cJUN-OE. n = 3 independent cultures. D, PCA plot. E,F, Enrichment plots for gene set enrichment analysis between cJUN-OE and EV samples for ‘classical’ and ‘quasi-mesenchymal’ PDAC13 [/articles/s43018-021-00258-w#ref-CR13] (E), as well as the top genes up- and downregulated following TNFα treatment in CLA (CAPAN1) cells (F). G, WB for indicated targets in CAPAN1 cells transduced with EV or cJUN-OE. Representative of n = 3 independent experiments. H, Representative bright-field images of CAPAN1 cells transduced with EV or cJUN-OE. Morphology was monitored over several passages. C,H, Scale bar: 200 µm. I,J, Trans-well invasion assay of CAPAN1 cells transduced with EV or cJUN-OE, showing representative DAPI staining of invaded cells (I) as well as quantification thereof (J). I, Scale bar: 100 µm. J, Data given as average counts per F.o.V., with means ± s.d. Unpaired, two-tailed Student’s t-test. n = 3 independent experiments. K-M, Mean cell viability ± s.d. at different concentrations of gemcitabine (K), oxaliplatin (L) and SN38 (M) in CAPAN1 cells transduced with EV or cJUN-OE. IC50 values for each drug are indicated. n = 3 independent experiments.

Guidelines regulating the development of advanced therapy medicinal products (ATMPs) request nonclinical data for toxicity, biodistribution and tumorigenicity before mesenchymal stromal cell (MSC) products can be administered in large clinical trials. We assessed the biodistribution/persistence, safety and tumorigenicity of MC0518, a human allogeneic MSC product from pooled bone marrow mononuclear cells of eight healthy, adult, unrelated donors, which is currently investigated for the treatment of steroid-refractory acute Graft-versus-Host Disease (aGvHD) after hematopoietic stem cell transplantation. In our GLP studies, immuno-deficient mice were administered repeat doses of MC0518 (once weekly for 6 weeks, i.v.) at doses exceeding the proposed human clinical dose 20-60-fold. No signs of toxicity were observed in the combined biodistribution/toxicity study. Human MSCs in mouse tissues were detected by quantitative PCR (qPCR) and in situ hybridization (ISH). MC0518 showed initial trapping in the lung, occasional distribution into other organs and low tissue persistence beyond 24 h after application. No MSC-induced tumors of human origin were identified after a follow-up of six months. Additionally, we found that the combination of different detection methods (qPCR and ISH) is crucial for a reliable interpretation of biodistribution results. Our data suggest that MC0518 is safe for use in human.

Background A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. Methods Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. Findings In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. Conclusions LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics.

Vamorolone is a first-in-class steroidal anti-inflammatory drug with novel structure/activity relationships with glucocorticoid and mineralocorticoid receptor targets compared to deflazacort or prednisone. Published open-label dose-finding studies (0.25-6.0 mg/kg/day) in DMD showed significant motor function improvement over 24 weeks for 2.0 and 6.0 mg/kg/day dose groups (n=48; age 4 to 6-month delay and maintained a higher dose through the remaining study period. Analyses of disease trajectories showed a strong effect of age at initiation of treatment. Subjects initiating treatment at 4-5 years showed highest clinical outcome performance levels as well as a delayed decline of motor function compared to subjects initially treated with low doses (0.25 or 0.75 mg/kg/day). Stratification of participants by treatment period at high doses (2.5 years; or delayed start 2.0 years), and/or by age showed data consistent with a disease modifying effect for many outcome measures. Vamorolone treatment was not associated with typical safety concerns of corticosteroid treatment (slowing of linear growth, insulin resistance, decreases in osteocalcin).

The test was aimed to study the expression and regulation of nuclear paraspeckle assembly transcript 1 (NEAT1) in uterine tissue during early pregnancy of mice, and provided scientific evidence for revealing the mechanism of NEAT1 in mouse embryo implantation. The models of early pregnancy, pseudopregnancy, artificial decidualization and steroid hormone treatments were constructed respectively, and the expression of NEAT1 in the uterine tissue was detected by RNAscope and Real-time quantitative PCR techniques. The results showed that the expression of NEAT1 was dynamically changing in uterine tissue in early pregnancy. NEAT1 was detected in endometrium and muscular layer on days 1-5 of pregnancy and mainly observed in endometrial luminal and glandular epithelial cells. NEAT1 was highly expressed in decidual cells on days 6-8 of pregnancy. There was no detectable NEAT1 staining at conceptus. Similar with those of early pregnancy, the expression of NEAT1 in pseudopregnant uteri was observed in endometrium and muscular layer. In the artificial decidulization model, NEAT1 was mainly localized in the decidualised cells and the expression level was extremely significant higher than that on the control group (_P_

The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.

Fingerprints are of long-standing practical and cultural interest, but little is known about the mechanisms that underlie their variation. Using genome-wide scans in Han Chinese cohorts, we identified 18 loci associated with fingerprint type across the digits, including a genetic basis for the long-recognized "pattern-block" correlations among the middle three digits. In particular, we identified a variant near EVI1 that alters regulatory activity and established a role for EVI1 in dermatoglyph patterning in mice. Dynamic EVI1 expression during human development supports its role in shaping the limbs and digits, rather than influencing skin patterning directly. Trans-ethnic meta-analysis identified 43 fingerprint-associated loci, with nearby genes being strongly enriched for general limb development pathways. We also found that fingerprint patterns were genetically correlated with hand proportions. Taken together, these findings support the key role of limb development genes in influencing the outcome of fingerprint patterning.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.