Probes for INS

The release of urine, or micturition, serves a fundamental physiological function and, in many species, is critical for social communication. In mice, the pattern of urine release is modulated by external and internal factors and transmitted to the spinal cord via the pontine micturition center (PMC). Here, we exploited a behavioral paradigm in which mice, depending on strain, social experience, and sensory context, either vigorously cover an arena with small urine spots or deposit urine in a few isolated large spots. We refer to these micturition modes as, respectively, high and low territory-covering micturition (TCM) and find that the presence of a urine stimulus robustly induces high TCM in socially isolated mice. Comparison of the brain networks activated by social isolation and by urine stimuli to those upstream of the PMC identified the lateral hypothalamic area as a potential modulator of micturition modes. Indeed, chemogenetic manipulations of the lateral hypothalamus can switch micturition behavior between high and low TCM, overriding the influence of social experience and sensory context. Our results suggest that both inhibitory and excitatory signals arising from a network upstream of the PMC are integrated to determine context- and social-experience-dependent micturition patterns.

Chronic activation and dysregulation of the neuroendocrine stress response have severe physiological and psychological consequences, including the development of metabolic and stress-related psychiatric disorders. We provide the first unbiased, cell type–specific, molecular characterization of all three components of the hypothalamic-pituitary-adrenal axis, under baseline and chronic stress conditions. Among others, we identified a previously unreported subpopulation of Abcb1b+ cells involved in stress adaptation in the adrenal gland. We validated our findings in a mouse stress model, adrenal tissues from patients with Cushing’s syndrome, adrenocortical cell lines, and peripheral cortisol and genotyping data from depressed patients. This extensive dataset provides a valuable resource for researchers and clinicians interested in the organism’s nervous and endocrine responses to stress and the interplay between these tissues. Our findings raise the possibility that modulating ABCB1 function may be important in the development of treatment strategies for patients suffering from metabolic and stress-related psychiatric disorders.

Neuropathic pain is a chronic disorder resulting from damage to the afferent nerve fibers or central pain pathways and is often a complication in pathological conditions such as diabetes, shingles, multiple sclerosis, and stroke. The opioid epidemic has elucidated the need for more efficacious treatments for neuropathic pain. In 2019 alone, nearly 1.6 million people were diagnosed with an opioid use disorder and 48,000 people died from a synthetic opioid overdose. Despite the addictive properties, opioids are still the most frequently prescribed pain medication, even for chronic neuropathic pain. Heterotrimeric G-proteins consisting of the α, β, and γ subunits convey extracellular signals sensed by G-protein coupled receptors (GPCRs) to intracellular effectors. The Gβ5 subunit is a divergent member of the G-protein β subunit family as it does not bind to traditional γ subunits. Instead, Gβ5 complexes with the R7 subfamily of the regulators of G-protein signaling (R7-RGS) containing 4 members: RGS6, 7, 9 and 11. The Gβ5/R7-RGS complex acts as a GTPase accelerating protein (GAP) for G-protein αi/o (Gαi/o) subunits. Previous studies have established the integral role of R7-RGS proteins in pain transmission via their interactions with Gαi/o-coupled receptors including opioid and metabotropic gamma-aminobutyric acid (GABA-B) receptors. Our lab has shown the lack of Gβ5 in sensory ganglia diminishes mechanical, thermal, and chemical nociception. However, the conditional knockout of Gβ5 in Rgs7 expressing neurons reduces only mechanical nociception. This Gβ5/RGS7-dependent mechanical nociception relies on GABA-B receptor signaling as indicated by the rescue of mechanical nociception in Rgs7-Cre; Gnb5 fl/fl mice after treatment with 2-hydroxysaclofen, a GABA-B antagonist. We also established that Rgs9 expressing neurons regulate thermal nociception via a Gβ5-dependent pathway as assayed by the hotplate test in Rgs9-Cre; Gnb5 fl/fl mice. The purpose of this project has been to understand the molecular role of each R7-RGS member in the regulation of pain transmission. First, we confirmed co-localization between the Gnb5 transcript and all four R7-RGS mRNA transcripts in murine dorsal root ganglia (DRG) using the RNAscope HiPlex assay, a novel in situ hybridization technique. We then established the co-localization patterns between each R7-RGS member and various pain related receptors including Mrgprd, Trpa1, and Trpv1. Our RNAscope results support the behavioral tests since Rgs7 transcripts highly co-express with Mrgprd, a mechanical nociceptor, while Rgs9 transcripts most frequently co-express with Trpv1, a thermosensitive receptor. These results suggest that each R7-RGS member might regulate unique types of nociception. We have also shown that Rgs11 transcripts co-localize with Trpv1 and Trpa1 receptor transcripts which indicates Rgs11 might regulate the chemical nociception as tested by capsaicin and mustard oil administration in the eye-wipe test. Next, we aim to study the possible roles of Rgs6 and Rgs11 in regulating chemical nociception using conditional Gβ5 knockout mice mediated by Rgs6-cre and Rgs11-cre, respectively.

What are the cellular composition and single-cell transcriptomic differences between myometrium and leiomyomas as defined by single-cell RNA sequencing?We discovered cellular heterogeneity in smooth muscle cells (SMCs), fibroblast and endothelial cell populations in both myometrium and leiomyoma tissues.Previous studies have shown the presence of SMCs, fibroblasts, endothelial cells and immune cells in myometrium and leiomyomas. However, there is no information on the cellular heterogeneity in these tissues and the transcriptomic differences at the single-cell level between these tissues.We collected five leiomyoma and five myometrium samples from a total of eight patients undergoing hysterectomy. We then performed single-cell RNA sequencing to generate a cell atlas for both tissues. We utilized our single-cell sequencing data to define cell types, compare cell types by tissue type (leiomyoma versus myometrium) and determine the transcriptional changes at a single-cell resolution between leiomyomas and myometrium. Additionally, we performed MED12-variant analysis at the single-cell level to determine the genotype heterogeneity within leiomyomas.We collected five MED12-variant positive leiomyomas and five myometrium samples from a total of eight patients. We then performed single-cell RNA sequencing on freshly isolated single-cell preparations. Histopathological assessment confirmed the identity of the samples. Sanger sequencing was performed to confirm the presence of the MED12 variant in leiomyomas.Our data revealed previously unknown heterogeneity in the SMC, fibroblast cell and endothelial cell populations of myometrium and leiomyomas. We discovered the presence of two different lymphatic endothelial cell populations specific to uterine leiomyomas. We showed that both myometrium and MED12-variant leiomyomas are relatively similar in cellular composition but differ in cellular transcriptomic profiles. We found that fibroblasts influence the leiomyoma microenvironment through their interactions with endothelial cells, immune cells and SMCs. Variant analysis at the single-cell level revealed the presence of both MED12 variants as well as the wild-type MED12 allele in SMCs of leiomyomatous tissue. These results indicate genotype heterogeneity of cellular composition within leiomyomas.The datasets are available in the NCBI Gene Expression Omnibus (GEO) using GSE162122.Our study focused on MED12-variant positive leiomyomas for single-cell RNA sequencing analyses. Leiomyomas carrying other genetic rearrangements may differ in their cellular composition and transcriptomic profiles.Our study provides a cellular atlas for myometrium and MED12-variant positive leiomyomas as defined by single-cell RNA sequencing. Our analysis provides significant insight into the differences between myometrium and leiomyomas at the single-cell level and reveals hitherto unknown genetic heterogeneity in multiple cell types within human leiomyomas. Our results will be important for future studies into the origin and growth of human leiomyomas.This work was supported by funding from the National Institute of Child Health and Human Development (HD098580 and HD088629). The authors declare no competing interests.

Study question Which genes are differentially expressed between patients with and without testicular fibrosis? Summary answer This study revealed three X-related genes MXRA5, DCX and VC3BX, which may be involved in Klinefelter-related testicular fibrosis. What is known already Klinefelter syndrome (KS; 47,XXY) affects 1-2 in 1000 males. Most KS men suffer from azoospermia due to a loss of spermatogonial stem cells. Additionally, testicular fibrosis is detected from puberty onwards. However, mechanisms responsible for fibrosis and germ cell loss remain unknown. Previous genomics studies on KS tissue focused on germ cell loss, however, differential gene expression analyses focused on testicular fibrosis have not been performed before. This study aimed to identify factors involved in the fibrotic remodeling of KS testes by analyzing the transcriptome of (non-)fibrotic testicular tissue. Study design, size, duration Transcriptome analysis on extracted RNA from testicular biopsies was performed. RNA scope analysis and immunohistochemistry were performed as validation for the findings of the transcriptomics study. Participants/materials, setting, methods RNA sequencing was performed to compare the genetic profile of testicular biopsies from patients with (KS and testis atrophy) and without (Sertoli cell-only syndrome and fertile controls) testicular fibrosis (n = 5, each). Next, differentially expressed genes (DEGs) between KS and testis atrophy samples were compared. To gain insight in potential functions of DEGs (significant when p < 0.01 and log2FC > 2), gene-ontology and KEGG analyses were performed. To validate the gene expression results, immunohistochemistry and RNA scope were performed. Main results and the role of chance A first transcriptomic analysis of fibrotic versus non-fibrotic testis tissue resulted in 734 significant DEGs (167 up- and 567 downregulated), of which 26 were X-linked. In the top upregulated biological functions, DEGs involved in the extracellular structure organization were found, including vascular cell adhesion molecule 1 (VCAM1). KEGG analysis showed an upregulation of genes involved in the TGF-β pathway. The second analysis of KS versus testis atrophy samples resulted in 539 significant DEGs (59 up- and 480 downregulated). One of the biological functions found though gene ontology analysis was the chronic inflammatory response. When looking at the overlap of DEGs on the X-chromosome from the first and second analysis, three genes were found: matrix-remodeling associated 5 (MXRA5), doublecortin (DCX) and variable charge X-Linked 3B (VCX3B). Through validation by immunohistochemistry and RNA scope, an overexpression of VCAM1, MXRA5 and DCX was found within the fibrotic group compared to the non-fibrotic group. Limitations, reasons for caution The study included fresh testis tissue from adult KS patients, however these are quite scarce, resulting in a low number of included patients per group (n = 5). Wider implications of the findings This study revealed genes which may play a role in testicular fibrosis, including VCAM1. In addition, fibrotic genes on the X-chromosome were revealed: MXRA5, DCX and VCX3B. Up- or downregulation of these genes may prevent testicular fibrosis and thus enhance the chances at retrieving spermatozoa from KS patients. Trial registration number NA

Background: Chronic bacterial infections pose a significant problem for individuals living with cystic fibrosis (CF) because of their correlation with lung function decline. Although there are several antibiotic therapies to treat and manage infections caused by Pseudomonas aeruginosa and other species, there are limited interventions for nontuberculous mycobacterium (NTM) infections, which affect approximately 10% of CF patients. Although clearance of NTM infection slows the rate of lung function decline, culture conversion and cure rates remain low despite intensive treatment regimens. Improved therapeutics are needed to address NTM lung infections. Exogenous nitric oxide (NO) delivery by gas has been proposed as a therapy to treat a range of conditions, including NTM lung infections. Herein, we describe the use of a small carbon-bound diazeniumdiolate molecule, MD3, that releases NO under physiological conditions to treat NTM infections. Methods: NO release was measured using a Sievers 280i chemiluminescence NO analyzer (NOA). The antimicrobial activity of MD3 was compared with that of several NTM species using minimum inhibitory concentration and minimum bactericidal concentration assays. For biofilm studies, bacteria were grown on pegs of a Calgary device and treated for 18 to 24 hours with varying concentrations of MD3 before being disrupted by sonication and serially diluted to determine CFU/mL. The maximal tolerated dose (MTD) was determined in SCID mice dosed once daily for 3 consecutive days. MD3 efficacy was evaluated in vivo using the acute NTM infection model. SCID mice were infected with Mycobacterium abscessus intratracheally, followed by dosing with an MD3 solution once daily for 8 consecutive days. Animals were sacrificed 24 hours after the last dose, followed by enumeration of surviving bacteria in the lung, liver, and spleen. Results: MD3 proved effective against multiple NTM species grown in vitro, including drug-resistant clinical isolates. MD3 activity is NO-dependent, as shown by the poor activity of NO-liberated MD3. MD3 was also effective against NTM grown in biofilms. The MTD for MD3 given intratracheally to SCID mice was more than 32 mg/kg. Given the encouraging results in vitro, we evaluated MD3 in a mouse model of acute NTM infection and found that MD3 was capable of reducing the bacterial load of M. abscessus in the lungs of infected mice by 2.8 logs after 8 days of daily dosing. Conclusion: Antibiotics are a mainstay of CF therapy to prevent and treat chronic lung infections, but new therapeutic approaches are desperately needed, particularly for difficult-to-treat infections such as NTM. This study provides support for an innovative therapeutic alternative to antibiotics for the treatment of NTM infections in CF. We demonstrate that MD3 is effective against multiple species and strains of NTM grown planktonically and in biofilms, with the ability to reduce bacterial burden significantly in a mouse model of acute NTM infection. Future work will evaluate MD3 in a model of chronic NTM lung infection.

Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Private grant from Kowa Pharmaceuticals to Brigham and Woman's Hospital Calcific aortic valve stenosis (CAVS) is the most prevent valvular heart disease in the western world increasing exponentially with age, with an 112% increase in CAVS deaths in the last three decades; however no therapeutic treatment is currently available. Recently, lipoprotein(a) [Lp(a)] has been demonstrated to be an independent and causal risk factor for CAVS, yet the understanding of its cellular uptake and catabolism is limited thus underscoring the need for further investigation. This study aimed to determine a target receptor, unique for Lp(a) on the surface of valvular interstitial cells (VICs) and ascertain the role of the receptor on the development of VIC calcification. Unbiased ligand-receptor capture mass spectrometry (TriCEPS) was used to identify target receptor, with western blotting, ELISA, qPCR, alizarin red calcium staining and immunofluorescence used to validate the targets in vitro via siRNA inhibition and overexpression. Transmission electron microscopy (TEM) was used to determine uptake of Lp(a) within excised human valves. Identification of small molecule inhibitors was assessed computationally via the L1000 dataset, with the top hit candidate validated in vitro. Genotype-phenotype studies were examined using the United Kingdom Biobank (UKBB) and the Millions Veterans Program. Linear regression was used to evaluate association between aortic stenosis and plasma Lp(a) levels, and a phenotype-wide association analysis was then performed against this generated ‘genotype’. Ligand-receptor capture mass spectrometry was used to detect novel membrane proteins with specific binding to Lp(a); MFSD5, MRC2, LDLR were identified as possible candidates. MFSD5 RNAscope demonstrated its presence in human aortic valves. Lp(a) uptake in VICs was confirmed via western blot and TEM. MFSD5 siRNA significantly reduced dil-labelled Lp(a) uptake in human VICs (p=0.003) and HEPG2 cells (p=0.0003), conversely MFSD5 overexpression increased uptake (p=0.0345, p=0.0318), whilst specificity of MFSD5 to Lp(a) alone was shown via no change in LDL uptake following MFSD5 inhibition (p=0.616, p=0.991). MFSD5 inhibition reduced RUNX2 (p=0.0124) and Osteocalcin (p<0.001) RNA expression and reduced alizarin red staining following culture in Lp(a) osteogenic media for 21 days (p<0.0033). Druggability of MFSD5 was confirmed by the L1000 database, which identified aminopurvalanol as a binding partner for MFSD5 and significantly reduced Lp(a) uptake within VICs (p=0.0091). MFSD5-loss of function within the UKBB showed no significant cardiovascular association, however 50kb +/- of the MFSD5 gene showed nominal association with hyperlipidaemia and atrial fibrillation. The current study demonstrates the novel Lp(a) receptor MFSD5 may be responsible for uptake of Lp(a) within VICs, resulting in the development of aortic valve calcification, highlighting the need for further exploration into the role of MFSD5 in aortic valve disease.

The prevalence of parasitic nematode infections are a major human and animal health concern. There are still no effective vaccines available, hence anthelmintic drugs have remained the cornerstone for prophylaxis and treatment. The repertoire of available anthelmintics is limited, with treatment relying heavily on three major chemical classes of anthelmintics. These are the imidazothiazoles/tetrahydropyrimidines (levamisole, pyrantel, morantel, oxantel); benzimidazoles (mebendazole, flubendazole, thiabendazole, albendazole); and the macrocyclic lactones (ivermectin, moxidectin, abamectin), all of which act on parasitic nematode ion channels, except for the benzimidazoles. Ion-channels are crucial components of excitable tissues and valuable targets for anthelmintics. Prolong treatment and incorrect use of these anthelmintics however, have led to the development of resistance worldwide. Additionally, the development of new anthelmintics is slow-paced, with only three drug classes being developed and approved for animal use in the since the year 2000. This includes the amino-acetonitrile (monepantel), cyclooctadepsipeptide (emodepside) and the spiroindole (derquantel). Hence, there is an urgent need for the development of new, more effective anthelmintic drugs that can alleviate the morbidity and mortality caused by existing parasite infections. Additionally, significant gaps in our understanding of anthelmintic resistance need to be improved so that we can provide practical solutions on improving drug efficacy and delaying the onset of resistance. We have confirmed the expression of four nicotinic acetylcholine receptor (nAChR) subunits: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8 that constitute the putative levamisole receptor in adult female Ascaris suum intestine. We then validated these findings by using RNAscope in situ hybridization to localize the subcellular distribution of the subunits in the intestine. Quantitative real-time PCR (qPCR) was also used to confirm the mRNA expression levels of each subunit in both muscle and intestine cells. To determine whether these subunits formed functional receptors that were responsive to cholinergic agonists, we employed calcium imaging. Our calcium imaging results demonstrated that both acetylcholine and levamisole elicited intracellular calcium responses in the intestinal tissue. These findings suggest that the presence of functional nAChRs in the intestine may not be limited to neuromuscular transmission, but an acetylcholine paracrine function. Hence, A. suum intestine can be a suitable target for therapeutic exploitation. Secondly, we expressed two receptor subtypes, namely Ode-levamisole and Ode pyrantel/tribendimidine from the pig parasite Oesophagostomum dentatum in Xenopus laevis oocytes. We demonstrated that compounds from the two macrocyclic lactone sub-family, the avermectins (abamectin and ivermectin) and the milbemycin, moxidectin are positive allosteric modulators (PAMs) on the Ode levamisole receptor. In contrast, abamectin and ivermectin acted as negative allosteric modulators (NAMs) on the Ode pyrantel/tribendimidine receptor subtype, while moxidectin maintained its PAM action. These findings suggest that the macrocyclic lactones are allosteric modulators of nAChRs and structural differences between each drug or the presence or absence of a subunit, namely ACR-8 may influence the allosteric modulatory effects. Hence combination therapy that includes macrocyclic lactones and cholinergic anthelmintics, might improve drug efficacy and delay anthelmintic resistance. Finally, we investigated the adaptation of Brugia malayi to levamisole exposure. We showed that B. malayi recovered motility with loss of sensitivity to levamisole within 4 hours of exposure. Molecular analysis also revealed up-regulation of mRNA levels for one AChR subunit, unc-38 and down-regulation of a gene that encodes for an ER retention protein, nra-2. Patch-clamp experiments on 4 hour recovered worms also showed that muscle responses to levamisole had desensitized. Knock down of nra-2 by RNAi resulted in faster recovery in motility, significant reduction in levamisole currents and no change in acetylcholine currents. This suggest that loss of NRA-2 facilitates the insertion of pentameric AChR subtypes in the muscle that are insensitive to levamisole, thus leading to faster recovery in motility in the presence of levamisole. Additionally, simultaneous knockdown of AChR subunits, namely, unc-38, acr-26 and acr-16, inhibited recovery of motility in the worms. These findings are notable and highlights the dynamic mechanisms used to by the parasite to vary AChR subunit composition that generates various receptor subtypes, thus facilitating recovery of motility and insensitivity to anthelmintic exposure (levamisole). This process of habituation can be interpreted as a mechanism of resistance that can be used by parasitic nematodes.

Biological processes are largely governed by the RNA molecules and resulting peptides that are encoded by an organism’s DNA. For decades, our understanding of biology has been vastly enhanced through study of the distribution and abundance of RNA molecules. Studies of the inner ear are no exception, and approaches like qPCR, RNA-seq, and in situ hybridization (ISH) have contributed greatly to our understanding of inner ear development and function. While qPCR and RNA-seq provide sensitive and broad measures of RNA quantity, they can be limited in their ability to resolve RNA localization. Thus, ISH remains a vital technique for inner ear studies. However, traditional ISH approaches can be technically challenging, time-consuming, suffer from high background, and are generally limited to the investigation of only a single RNA of interest. Recent advances in ISH approaches have overcome many of these limitations allowing for speed, high signal-to-noise, and the ability to perform multiplexed ISH where several transcripts of interest can be visualized in the same tissue or section. One such approach is RNAscope which is a commercially available option that allows for ease of use and, for many transcripts, the ability to achieve absolute quantification of RNA molecules per cell. Here we outline RNAscope methods that have been optimized for inner ear (and related) tissues and allow for relatively rapid labeling of RNA transcripts of interest in fixed tissues. Furthermore, these methods elucidate how RNAscope labeling can be imaged with brightfield or fluorescence microscopy, how it allows for quantification as well as localization, how it can be multiplexed to visualize multiple transcripts simultaneously, and how it can be combined with immunocytochemistry so that RNA and proteins may be visualized in the same sample.

Fibromyalgia (FM) is a chronic pain condition characterized by widespread musculoskeletal pain with significant movement-evoked pain. Transcutaneous electrical nerve stimulation (TENS), a non-pharmacological treatment, is efficacious for reduction of pain and fatigue in FM. While physical therapists are trained in the use of TENS, it is an underutilized tool in physical therapy (PT) clinics. To this end, we designed a pragmatic clinical trial to test if using TENS in a PT setting is feasible and if FM patients using TENS show reduced movement-evoked pain, improved adherence to PT, are more likely to reach their therapeutic goals. During a 1-year planning period the study team designed and set up procedures to implement the trial into 24 physical therapy clinics within 5 healthcare systems. Surveys and interviews with PT sites and interactions with the NIH Collaboratory group guided design of this pragmatic trial. The 24 PT clinics will be cluster-randomized and stratified by healthcare system into two groups: (1) No TENS (PT usual care) or (2) TENS+ (TENS plus PT usual care) with a goal of enrolling 600 participants the trial. Assessments will be collected at baseline then at 30-, 60-, 90- and 180-days from randomization through electronic data capture from participants directly and from the electronic health record. The primary outcome is a reduction in movement-evoked pain during a sit and stand test (baseline to 60 days) with secondary outcomes across multiple domains including pain, fatigue, sleep, disease impact, medication usage, psychosocial variables, patient-specific functional goals, and adherence to PT. Successful completion of this trial will provide generalizable effectiveness data on TENS for referring providers, physical therapists, patients, and insurers and will inform future pragmatic trials of non-pharmacological treatments conducted in PT practices. NIH; 4UH3AR076387-02 REVISED.

Ureteric bud induction and branching morphogenesis is fundamental to the establishment of the renal architecture and is a key determinant of nephron number. Defective ureteric bud morphogenesis could give rise to a spectrum of malformations associated with congenital anomalies of the kidney and urinary tract (CAKUT). Signaling involving glial cell line-derived neurotrophic factor and its receptor RET and coreceptor GFRA1 appears to be particularly important in ureteric bud development. Recent epigenome profiling studies have uncovered dynamic changes of histone H3 lysine K4 (H3K4) methylation during metanephros development, and dysregulated H3K4 methylation has been associated with a syndromic human CAKUT.To investigate whether and how inactivation of Ash2l, which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide H3K4 methylation, might contribute to CAKUT, we inactivated Ash2l specifically from the ureteric bud lineage in C57BL/6 mice and examined the effects on genome-wide H3K4 methylation and metanephros development. Genes and epigenome changes potentially involved in these effects were screened using RNA-seq combined with CUT&Tag-seq.Ureteric bud-specific inactivation of Ash2l caused CAKUT-like phenotypes mainly involving renal dysplasia at birth, which were associated with deficient H3K4 trimethylation. Ash2l inactivation slowed proliferation of cells at the ureteric bud tip, delaying budding and impairing ureteric bud branching morphogenesis. These effects were associated with downregulation of Ret, Gfra1, and Wnt11, which participate in RET/GFRA1 signaling.These experiments identify ASH2L-dependent H3K4 methylation in the ureteric bud lineage as an upstream epigenetic regulator of RET/GFRA1 signaling in ureteric bud morphogenesis, which, if deficient, may lead to CAKUT.

Canine apocrine gland anal sac adenocarcinoma (AGASACA) is an aggressive canine tumor originating from the anal sac glands. Surgical resection, with or without adjuvant chemotherapy, represents the standard of care for this tumor, but the outcome is generally poor, particularly for tumors diagnosed at an advanced stage. For this reason, novel treatment options are warranted, and a few recent reports have suggested the activation of the immune checkpoint axis in canine AGASACA. In our study, we developed canine-specific monoclonal antibodies targeting PD-1 and PD-L1. A total of 41 AGASACAs with complete clinical and follow-up information were then analyzed by immunohistochemistry for the expression of the two checkpoint molecules (PD-L1 and PD-1) and the presence of tumor-infiltrating lymphocytes (CD3 and CD20), which were evaluated within the tumor bulk (intratumor) and in the surrounding stroma (peritumor). Seventeen AGASACAs (42%) expressed PD-L1 in a range between 5% and 95%. The intratumor lymphocytes were predominantly CD3+ T-cells and were positively correlated with the number of PD-1+ intratumor lymphocytes (ρ = 0.36; p = 0.02). The peritumor lymphocytes were a mixture of CD3+ and CD20+ cells with variable PD-1 expression (range 0-50%). PD-L1 expression negatively affected survival only in the subgroup of dogs treated with surgery alone (n = 14; 576 vs. 235 days). The presence of a heterogeneous lymphocytic infiltrate and the expression of PD-1 and PD-L1 molecules support the relevance of the immune microenvironment in canine AGASACAs and the potential value of immune checkpoints as promising therapeutic targets.