RNAscope In Situ Hybridization Research Articles

Longitudinal tissue-based evaluation of TIGIT expression in patients with pancreatic cancer: Effect of expression with advancing clinical stages and across racial groups.

e16314 Background: While ground has been gained, pancreatic ductal adenocarcinoma (PDAC) continues to have a low 5-year survival of 13%. This is owed partially to a lack of early detection biomarkers and resistance to standard therapeutic options. TIGIT, an immune checkpoint receptor, is a marker of T-cell exhaustion and plays a key role in the inhibition of anti-tumor immune responses. TIGIT inhibitors are being explored in clinical trials in PDAC. Here we evaluate TIGIT expression in a cohort of PDAC patients and correlate level and intensity of expression with clinical parameters. We also examine changes in expression as the disease progresses from primary to metastatic disease. Methods: We performed RNAscope in situ hybridization (ISH) with a probe specific for human TIGIT mRNA on 82 formalin-fixed paraffin-embedded (FFPE) tissue samples. The cohort of tissue samples included 9 biopsies, 67 primary resections and 6 metastatic lesions. We evaluated the total TIGIT expression (%) as well as the intensity of TIGIT expression (% of cells with 3+ punctae, signifying putative immune cells), and compared these values between samples. Utilizing linear regression for continuous outcomes and logistic regression for binary outcomes, we tested for associations between TIGIT expression and clinical covariates. Results: Staining analysis showed that TIGIT expression did not differ significantly between racial groups. The mean percentage of TIGIT positive cells was 64.0%. High expression of TIGIT was associated with more advanced clinical stage (p < 0.05). Evaluation of three longitudinal samples from the same patient revealed decreased TIGIT expression from the initial biopsy (41.6%) to resection (33.4%) and metastasis (2.8%). In these specimens, 3+ TIGIT expression also declined (2.1%, 1.2% and 0.02%, respectively). Conclusions: Anti-TIGIT therapy has potential to reverse immune suppression and, with other therapeutic modalities, may provide survival benefit. Here we demonstrate that increased TIGIT expression correlates with more advanced stage at diagnosis and also present data demonstrating that overall TIGIT expression, as detected by RNAscope ISH, may decrease as PDAC progresses to metastasis. As such, anti-TIGIT therapy may have important implications for evading an important mechanism of cancer progression.

TRPA1 Covalent Ligand JT010 Modifies T Lymphocyte Activation.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non-selective cation channel involved in sensitivity to a plethora of irritating agents and endogenous mediators of oxidative stress. TRPA1 influences neuroinflammation and macrophage and lymphocyte functions, but its role is controversial in immune cells. We reported earlier a detectable, but orders-of-magnitude-lower level of Trpa1 mRNA in monocytes and lymphocytes than in sensory neurons by qRT-PCR analyses of cells from lymphoid organs of mice. Our present goals were to (a) further elucidate the expression of Trpa1 mRNA in immune cells by RNAscope in situ hybridization (ISH) and (b) test the role of TRPA1 in lymphocyte activation. RNAscope ISH confirmed that Trpa1 transcripts were detectable in CD14+ and CD4+ cells from the peritoneal cavity of mice. A selective TRPA1 agonist JT010 elevated Ca2+ levels in these cells only at high concentrations. However, a concentration-dependent inhibitory effect of JT010 was observed on T-cell receptor (TcR)-induced Ca2+ signals in CD4+ T lymphocytes, while JT010 neither modified B cell activation nor ionomycin-stimulated Ca2+ level. Based on our present and past findings, TRPA1 activation negatively modulates T lymphocyte activation, but it does not appear to be a key regulator of TcR-stimulated calcium signaling.

Generation and characterization of collecting duct specific GPR56 knockout mice

GPR56 is a multifunctional adhesin G protein-coupled receptor involved in diverse biological processes, including neurodevelopment and disease, immune regulation, reproduction, hemostasis, and tumorigenesis. The role of GPR56 in the kidneys has been understudied. A recent study demonstrated that GPR56 in the glomerular endothelial cells promoted diabetic kidney disease progression via regulation of eNOS. Using RNAscope in situ hybridization (ISH) for GPR56 and aquaporin 2 (AQP2) followed by immunofluorescent labeling with NKCC2 (thick ascending limb, TAL marker), we detected GPR56 mRNA highly expressed in the collecting duct (both AQP2 positive principal cells and AQP2 negative intercalated cells) and TAL of the loop of Henle. In contrast, GPR56 mRNA is not detected in the proximal tubule or glomeruli. This expression pattern is further confirmed by quantitative RT-qPCR for GPR56 in microdissected inner medulla/inner stripe outer medulla and renal cortex. Of note, inner medulla/ inner stripe outer medulla is primarily composed of the collecting duct and the loop of Henle. GPR56 mRNA exhibited over a 6-fold higher expression levels in the inner medulla/inner stripe outer medulla compared to renal cortex, which is consistent with limited expression in the proximal tubule. To determine the physiological role of GPR56 in the collecting duct, we generated a collecting duct-specific GPR56 knockout (GPR56CD-KO) mouse model by crossing GPR56flox (Control, Con) with cadherin 16 Cre (Ksp-Cre) mice. The deletion of GPR56 in the collecting duct was confirmed by RNAscope ISH for GPR56 and AQP2 followed by fluorescent labeling with NKCC2. GPR56CD-KO mice were born at predicted Mendelian frequencies, appeared grossly indistinguishable from Con mice, and developed normally. For baseline phenotypic characterization, we found no differences in blood pH (7.21±0.01 vs 7.19±0.01, NS), blood HCO3− (17.4±0.5 vs 16.8±1.1 mmol/L, NS), blood Na+ (143±0 vs 144±0 mmol/L, NS), or blood K+ (4.3±0.1 vs 4.3±0.1 mmol/L, NS) between GPR56CD-KO (n=5, males) and Con mice (n=6, males). We also conducted metabolic cage experiments. No differences were observed for fluid intake (4.7±0.5 vs 4.5±0.2 mL/(day*30 g bodyweight), NS), urine volume (629±87 vs 662±108 mL/day, NS), urinary pH (5.75±0.03 vs 5.69±0.03, NS) or urine osmolality (2456±151 vs 2776±253 mOsm/kg, NS) between genotypes in baseline. After 24hr water deprivation, urine osmolality in both mice groups had significantly increased compared to that in baseline, however, no difference (Δ change: 558±82 vs 583±60 mOsm/kg, NS) was found between GPR56CD-KO and Con mice. In conclusion, we successfully generated collecting duct-specific GPR56 knockout mouse and found no defective urine concentrating ability in GPR56CD-KO mice. This mouse model will be useful to delineate the collecting duct-specific role of GPR56 for renal function, including acid-base regulation. We will also generate whole kidney GPR56 knockout mice to determine the isolated role of GPR56 in the loop of Henle compared to that in the collecting duct. This study is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (4R00DK127215). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Viral DNA in submandibular gland tissue with an inflammatory disorder

ABSTRACT Background The etiology behind different types of chronic sialadenitis (CS), some of which exhibit IgG4 overexpression, is unknown. Further, IgG4-related disease (IgG4-RD) commonly affects the submandibular gland, but its relationship to IgG4-overexpressing CS, and the antigen triggering IgG4 overexpression, remain unknown. Materials and Methods By qPCR, we assessed the presence of 21 DNA-viruses causing IgG4 overexpression in submandibular gland tissue from patients with IgG4-positive and IgG4-negative CS. Healthy submandibular glands and glands with sialolithiasis without CS were used as controls. We examined the distribution of HHV-7, HHV-6B and B19V DNA, within virus PCR-positive tissues with RNAscope in-situ hybridization (RISH). Results We detected DNA from seven viruses in 48/61 samples. EBV DNA was more prevalent within the IgG4-positive samples (6/29; 21%) than the IgG4-negative ones (1/19; 5.3%). B19V DNA was more prevalent within the IgG4-negative samples (5/19; 26%) than the IgG4-positive ones (4/29; 14%). The differences in virus prevalence were not statistically significant. Of the IgG4-RD samples (n = 3) one contained HHV-6B DNA. RISH only showed signals of HHV-7. Conclusions None of the studied viruses are implicated as triggering IgG4-overexpression in CS. Although our results do not confirm viral etiology in the examined conditions, they provide valuable information on the prevalence of viruses in both diseased and healthy submandibular gland tissue.

Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope in situ hybridization signal amplification.

RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.

Expression of genes with biomarker potential identified in skin from DSLD-affected horses increases with age.

Degenerative Suspensory Ligament Desmitis (DSLD) negatively impacts connective tissues in horses, which often leads to progressive chronic pain and lameness. DSLD has been shown to be a systemic disorder that affects multiple body systems, including tendons, sclerae, and the aorta. Currently, the diagnosis is confirmed by post mortem histological examination of a tendon or suspensory ligament. Histology reveals inappropriate accumulations of proteoglycans in the tendons and other tissues in DSLD-affected horses. Unfortunately, there is no reliable method to diagnose DSLD in living horses. Recently, bone morphogenetic protein 2 (BMP2) was identified in active DSLD lesions. In addition, recent data from RNA sequencing (RNA-seq) showed overexpression of numerous genes, among them BMP2, FOS and genes for keratins in DSLD skin biopsies-derived RNA. We hypothesized that some of these genes can be used as biomarkers for diagnosis of DSLD in a panel. Overexpression of some of them was verified in quantitative real time PCR. Immunohistochemistry and RNAscope in-situ hybridization (ISH) assays were used to determine the level of overexpression of specific genes in skin biopsies from control and DSLD-affected horses. The RNAscope ISH assay has shown to be more reliable and more specific that immunohistochemistry. ISH confirmed a significant increase in KRT83 and BMP-2 in hair follicles in DSLD cases, as well as abnormally high expression of FOS in the epidermis, especially in aging horses. Because statistically relevant specificity and sensitivity was documented only for FOS and BMP2, but not KRT83 we recommend the use of FOS and BMP2 panel to diagnose DSLD. We conclude that a panel of two markers from the studied group (BMP2 and FOS) can serve as an additional diagnostic tool for DSLD in living horses, especially in older animals. Further studies are necessary to confirm if this biomarker panel could be used as a prospective tool to identify DSLD in horses as they age.

Validation of an RNAscope assay for the detection of avian influenza A virus.

Highly pathogenic avian influenza (HPAI) is an acute viral disease associated with high mortality and great economic losses. Immunohistochemistry (IHC) is a common diagnostic and research tool for the demonstration of avian influenza A virus (AIAV) antigens within affected tissues, supporting etiologic diagnosis and assessing viral distribution in both naturally and experimentally infected birds. RNAscope in situ hybridization (ISH) has been used successfully for the identification of a variety of viral nucleic acids within histologic samples. We validated RNAscope ISH for the detection of AIAV in formalin-fixed, paraffin-embedded (FFPE) tissues. RNAscope ISH targeting the AIAV matrix gene and anti-IAV nucleoprotein IHC were performed on 61 FFPE tissue sections obtained from 3 AIAV-negative, 16 H5 HPAIAV, and 1 low pathogenicity AIAV naturally infected birds, including 7 species sampled between 2009 and 2022. All AIAV-negative birds were confirmed negative by both techniques. All AIAVs were detected successfully by both techniques in all selected tissues and species. Subsequently, H-score comparison was assessed through computer-assisted quantitative analysis on a tissue microarray comprised of 132 tissue cores from 9 HPAIAV-infected domestic ducks. Pearson correlation of r = 0.95 (0.94-0.97), Lin concordance coefficient of ρc = 0.91 (0.88-0.93), and Bland-Altman analysis indicated high correlation and moderate concordance between the 2 techniques. H-score values were significantly higher with RNAscope ISH compared to IHC for brain, lung, and pancreatic tissues (p ≤ 0.05). Overall, our results indicate that RNAscope ISH is a suitable and sensitive tool for in situ detection of AIAV in FFPE tissues.

Abstract P2-22-01: Nuclear long non-coding RNA LINC00355 expression and treatment using antisense oligonucleotides in Estrogen Receptor positive late-stage relapse breast cancer

Abstract Late-stage relapse also known as late recurrence breast cancer is a metastatic disease that occurs more than five years after initial adjuvant endocrine therapy treatment. The 5-year relative survival rate is less than 30% and there are limited treatment options for patients with late-stage relapse because of poor performance status. We hypothesize that long non-coding RNA (lncRNA) protein interactions regulate genes to promote late-stage relapse and these interactions can be targeted using RNA therapeutics. To address this, we used RNA-sequencing to identify LINC00355 to be up-regulated in 24 late-stage relapse samples compared to a unique cohort of 72 primary breast cancer samples. Analysis of 480 primary breast cancer samples from The Cancer Genome Atlas (TCGA) also shows decrease expression of LINC00355 compared to late-stage relapse. To better assess LINC00355 cell type expression, we analyzed 26 publicly available primary breast cancer single cell sequencing data samples and detected 0.90% or less of cells expressing LINC00355, which was restricted to cancer epithelial cells. Due to cellular localization playing an important role in lncRNA function, we isolated nuclear and cytoplasmic lysates and orthogonally validated with RNAscope in situ hybridization (RNA-ISH) to show that LINC00355 is located in the nucleus of MCF7 long-term estrogen deprived (MCF7 LTED) cells. LTED cells are deprived of estrogen for longer than three years meant to recapitulate the acquired resistance to aromatase inhibitors. Next, we determined that LINC00355 binds to MENIN protein to regulate p27KIP expression to promote proliferation with UV crosslinking immunoprecipitation and chromatin immunoprecipitation qPCR of LINC00355 overexpression and mutant constructs. Furthermore, RNA-ISH assays validated high nuclear expression of LINC00355 and RNA-ISH protein co-detection assays showed co-localization of LINC00355-MENIN interaction in a panel of late-stage relapse breast cancer patient tissues compared to primary tissues. Lastly to determine clinical significance, we treated MCF7 LTED cells with two locked nucleic acid antisense oligos (ASOs) targeting LINC00355 to observe a significant decrease in proliferation, cell viability, and invasion. Overall, this is the first study to show the importance of lncRNA-protein interactions and therapeutically target lncRNAs with ASOs in late-stage relapse breast cancer. Moving forward, we intend to further understand how LINC00355 contributes to the progression of late-stage relapse with the intent of creating novel cancer diagnostics and therapies. Citation Format: DeAnna Wells, Kyla Gelev, Prasanth Thunuguntla, Reyka Jayasinghe, Li Ding, Jieya Shao, Cynthia Ma, Jessica Silva-Fisher. Nuclear long non-coding RNA LINC00355 expression and treatment using antisense oligonucleotides in Estrogen Receptor positive late-stage relapse breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-22-01.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1A IN ARCHIVAL TISSUE USING RNASCOPE® IN SITU HYBRIDIZATION.

Hemorrhagic disease due to elephant endotheliotropic herpesvirus infection (EEHV-HD) is an important cause of calf mortality in managed and free-ranging Asian (Elephas maximus) and African elephant (Loxodonta spp.) populations. Consequently, infection has profound implications for elephant population growth and sustainability. The mechanisms of disease caused by EEHV (i.e., infection, dissemination, shedding, latency) are relatively undefined, in part because of a lack of robust validated assays for detecting viral gene products in relevant samples. To address this issue, we used RNAscope® in situ hybridization (ISH) based on EEHV1A DNA polymerase and terminase genes to detect EEHV1A RNA in archival formalin-fixed, paraffin-embedded Asian elephant heart and tongue from PCR-confirmed cases (n = 4) of EEHV-HD and Asian elephants (n = 2) that died from other causes. EEHV1A-positive cases had positive hybridization signal in endothelial cell nuclei of both tissues for both DNA polymerase and terminase. EEHV-negative cases lacked signal. In positive cases, the number of positive nuclei was manually assessed to provide an estimate of the viral load and compare sensitivity of the two probes. In all cases, heart had greater signal than tongue for both probes (Wilcoxon rank test; P ≤ 0.01). Overall, terminase hybridization signal was greater than DNA polymerase signal (Wilcoxon rank test; P ≤ 0.01). Results indicate RNAscope ISH is a valuable tool for detection of EEHV in archival samples and for confirming infection. Additionally, the terminase gene is the optimal target and heart is preferable to tongue for detection in cases of EEHV-HD. Results will inform future investigations of viral tropism in EEHV-HD cases due to EEHV1A.

Analysis of gene expression in poultry red mite, Dermanyssus gallinae, by RNAscope in situ hybridization.

The poultry red mite (PRM; Dermanyssus gallinae) is a hematophagous ectoparasite that mainly infests chickens, and its infestation causes significant economic losses to the poultry industry. In this study, we examined the use of RNAscope-based in situ hybridization (ISH) to characterize gene expression in PRM. We analyzed the mRNA expression of Dermanyssus gallinae cathepsin D-1 (Dg-CatD-1) and Dermanyssus gallinae cystatin (Dg-Cys). RNAscope ISH analysis revealed that mRNA expression of Dg-CatD-1 was observed in the digestive tract, and Dg-Cystatin mRNA was expressed in the ovaries in addition to the digestive tract. RNAscope ISH could be applicable for the analysis of gene expression in each tissue of PRM and is an effective method to investigate the characteristics of target genes.

Abstract 060: Angiotensin Type-2 Receptors In The Median Preoptic Nucleus Demarcate A Novel Depressor Neural Circuit In Mice

Blood pressure (BP) is under constant control and regulation by an extensive neural network that spans a number of brain regions. This includes the median preoptic nucleus (MnPO) which directly projects to the pre-autonomic neurons of the hypothalamus to increase BP. While angiotensin type-1 receptors (AT 1 R) are expressed in the MnPO and project to the hypothalamus to increase BP, the role of angiotensin type-2 receptors (AT 2 R) is not clear. Here, we combine the use of AT 2 R-Cre mice, neuroanatomical techniques, in vivo optogenetics, pharmacology, and cardiovascular physiology to test the hypothesis that AT 2 R neurons of MnPO (MnPO AT2R ) elicit cardioprotective effects. We found an abundance of AT 2 R-containing neurons in the MnPO that expressed the mRNA(s) for AT 2 R with high fidelity (~77%; 241 of 313 neurons; n=7). Using RNAscope in situ hybridization, we found that ~ 42% (153 of 364 neurons; n=8) of MnPO AT2R expressed genetic markers for GABA, an inhibitory neurotransmitter. Next, we delivered a Cre-inducible AAV to direct expression of channelrhodopsin-2 (ChR2) and/or enhanced yellow fluorescent protein (eYFP) within MnPO AT2R of AT 2 R-Cre mice. Using in vitro electrophysiology, we confirmed functional expression of ChR2 within MnPO AT2R , with pulses of light reliably producing action potentials (n= 12 neurons/3 animals). Subsequently, we found that activation of MnPO AT2R elicited inhibitory post-synaptic currents in neighboring AT 2 R-negative neurons. The in vivo optogenetic excitation of MnPO AT2R elicited depressor responses in both adult male (ΔSBP: +2.8 ± 1.3 vs. -4.1 ± 1.9 mmHg, p<0.05 eYFP vs. ChR2, n=15) and female mice (ΔSBP: +0.8 ± 2.2 vs.-5.3 ± 1.9 mmHg, p<0.05 eYFP vs. ChR2, n=5). These responses were abolished in the presence of the autonomic ganglion blocker, hexamethonium. Finally, to assess whether the depressor effects were AT 2 R-mediated, we administered the selective AT 2 R-agonist, compound 21, into the MnPO and observed depressor responses (ΔSBP: +0.5 ± 0.3 vs.-4.9 ± 1.0 mmHg, p<0.05 Saline vs. C21, n=7). In conclusion, our results demonstrate that MnPO AT2R are depressor inhibitory neurons that can be targeted with compound 21 to lower blood pressure.

Clinicopathological features of mixed cervical carcinoma with adenoid cystic pattern

Objective: To investigate the clinicopathological characteristics, immunophenotype, molecular characteristics, differential diagnosis, clinical treatment and prognosis of mixed carcinoma of cervix with adenoid cystic pattern. Methods: Three cases of mixed cervical carcinoma with adenoid cystic pattern were collected at the Affiliated Hospital of Xuzhou University Medical School from 2018 to 2021.The clinicopathological characteristics were analyzed, immunohistochemistry (IHC) and in situ hybridization (ISH) were performed. The related literature was reviewed. Results: The three patients were postmenopausal women with a median age of 74.7 years. The clinical symptom was vaginal bleeding without obvious causes. One case was an endophytic tumor, and the others were exophytic. The median diameter of the three cases was 3.3 cm. Two patients underwent hysterectomy, the tumors infiltrated the external 1/3 and middle 1/3 of the cervix respectively. All the lymph nodes were negative. One patient had a previous biopsy. Microscopically, all three tumors were characterized by a cribriform structure, which were filled with basophilic myxoid substance and surrounded by tubules lined by two layers of cells. The tumor cells had scanty cytoplasm and showed the characteristics of cervical basal-like cells. All three cases were accompanied by high-grade squamous intraepithelial lesions and squamous cell carcinoma, and one also showed a non-specific spindle cell sarcomatoid component. Within the double-layered epithelial structure, the outer epithelium was positive for p63, CD117, p16INK4a (clone E6H4) and MYB protein and negative for S-100 by IHC. The combined positive score of PD-L1 (clone 22C3) was less than 1 in all three cases. Human papillomavirus (HPV) types 16 and 18 were detected in one patient preoperatively, while high-risk HPV were positive in the other two patients by RNAscope ISH postoperatively. None of the three cases showed MYB gene rearrangement by FISH. The mean follow-up time was 23.3 months (36, 28 and 6 months, respectively). Two patients underwent hysterectomy and radiotherapy survived without disease. One patient survived with tumor just by radiotherapy and drug therapy. Conclusions: Mixed cervical carcinoma with adenoid cystic pattern is extremely rare. It is a high-grade malignancy with poor prognosis. The tumor is associated with high-risk HPV infection, without MYB gene rearrangement, and with low PD-L1 immunoreactivity. Radical surgery combined with radiotherapy and chemotherapy is the mainstay of treatment at present.

Dysregulation of CXCL14 promotes malignant phenotypes of esophageal squamous carcinoma cells via regulating SRC and EGFR signaling

The present study was to identify abnormal methylation genes implicated in esophageal squamous cell carcinoma (ESCC). Genomic methylation alterations in ESCC tissues were analyzed using laser-microdissection and whole-genome bisulfite sequencing. CXCL14 promoter was frequently hypermethylated in ESCC tissues. The correlation of CXCL14 hypermethylation status and the mRNA and protein expression levels were validated using nested methylation-specific PCR (nMS-PCR), RNAscope in situ hybridization (RISH) and Western blot. RISH results showed completely negative CXCL14 expression in 34.3% (34/99) ESCC, compared with those in the basal layer cells of normal epithelia. Low expression of CXCL14 was more present in patients with lower differentiation. The anticancer role of CXCL14 has been commonly associated with immune regulation in the literature. Here, we observed by functional analysis that CXCL14 can also act as a tumor suppressor in ESCC cells. 5-Aza-dC treatment suppressed CXCL14 methylation and up-regulated the expression of CXCL14. Ectopic expression of CXCL14 suppressed the proliferation, invasion, tumor growth, and lung metastasis of ESCC cells. Both ectopic expression and induction of CXCL14 with 5-Aza-dC inhibited the activity of SRC, MEK1/2 and STAT3 in ESCC cells, while activated EGFR. Importantly, a combination of CXCL14 expression and SRC or EGFR inhibitor dramatically repressed the proliferation of ESCC cells and the growth of xenografts. Our findings revealed a direct tumor suppressor role of CXCL14, but not through the immune system. The data suggest that for ESCC patients with low level CXCL14, increasing CXCL14 expression combined with inhibition of SRC or EGFR might be a promising therapeutic strategy.

Abstract P4-07-22: Evaluation of human epididymis protein 4 serum and tissue expression in ductal carcinoma in situ of the breast

Abstract Background: Human epididymis protein 4 (HE4), also known as WFDC2, is a member of whey acidic protein (WAP) family with a presumptive role in natural immunity. HE4 expression has been increased in many cancer types, particularly in gynecologic and pulmonary cancers. HE4 has been identified as an important serum biomarker in the diagnosis and monitoring of epithelial ovarian cancer. Recent researches have demonstrated that HE4 levels in serum and tissues are elevated in patients with breast cancer and HE4 is thought be involved in breast cancer progression. In contrast, expression of HE4 in ductal carcinoma in situ (DCIS) has not been well characterized yet. The present work was undertaken to evaluate serum and tissue levels of HE4 in DCIS and their correlations with clinicopathological features of DCIS. Materials and methods: Preoperative serum HE4 levels in 59 DCIS patients was analyzed using the ARCHITECT assay. Tissue microarray containing DCIS and adjacent normal tissues from the same cohort were tested for HE4 mRNA and protein by RNAscope in situ hybridization (ISH) and immunohistochemistry analysis, respectively. Tissue microarray containing only DCS tissues from 41 patients were also tested for HE4 mRNA and protein. To validate prognostic potential of HE4 in breast cancer patients, the BreastMark database was used. Results: HE4 levels in 59 DCIS patients ranged from 23.5 to 86.3 pmol/L (mean ± SD: 39.4 ± 11.9). Based on the cutoff level, there were no abnormal cases for HE4. Serum HE4 levels did not differ according to clinicopathological parameters except for menopause status. RNAscope ISH and immunohistochemistry confirmed an increase in HE4 in DCIS tissues compared with their corresponding normal tissues. Spearman’s correlation analyses revealed no significant correlation between serum and tissue levels of HE4. Among 100 DCIS tissues, there was a positive correlation between HE4 mRNA ISH scores and HE4 immunohistochemical staining scores (r = 0.771, P < 0.001). High mRNA and protein expression of HE4 were observed in 25 of 99 (25.3%) and 34 of 99 (34.3%) cases, respectively. High mRNA HE4 expression was significantly associated with low stromal tumor infiltrating lymphocyte (TIL) density scores, ER positivity, HER2 negativity, and HR+/HER2- subtype. High protein HE4 expression was also significantly associated with absence of comedo-type necrosis, low stromal TIL density scores, ER positivity, HER2 negativity, and HR+/HER2- subtype. HE4 mRNA and protein expression did not correlate with recurrence of DCIS. In breast cancer patients, high HE4 expression was significantly associated with good survival in the overall group (HR = 0.838, P = 0.003, n = 2,652) and lymph node negative group (HR = 0.791, P = 0.029, n = 1,183). Conclusions: Our study revealed that serum HE4 is not elevated in patients with DCIS. High expression of HE4 mRNA and protein in DCIS tissues are associated with good clinicopathological characteristics, which warrant. further investigations. Citation Format: Ji Shin Leee, Nah Ihm Kim, Min Ho Park. Evaluation of human epididymis protein 4 serum and tissue expression in ductal carcinoma in situ of the breast [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-22.

TRPM4 Contributes to Subthreshold Membrane Potential Oscillations in Multiple Mouse Pacemaker Neurons.

Select neuronal populations display steady rhythmic neuronal firing that provides tonic excitation to drive downstream networks and behaviors. In noradrenergic neurons of the locus coeruleus (LC), circadian neurons of the suprachiasmatic nucleus (SCN), and CO2/H+-activated neurons of the brainstem retrotrapezoid nucleus (RTN), large subthreshold membrane potential oscillations contribute to the pacemaker-like action potential discharge. The oscillations and firing in LC and SCN involve contributions from leak sodium (NALCN) and L-type calcium channels while recent work from RTN suggested an additional pivotal role for a secondary calcium-activated and voltage-gated cationic current sensitive to TRPM4 channel blockers. Here, we tested whether TRPM4 contributes to subthreshold oscillations in mouse LC and SCN. By RNAscope in situ hybridization, Trpm4 transcripts were detected in both cell groups. In whole-cell recordings from acute slice preparations, prominent voltage-dependent membrane potential oscillations were revealed in LC and SCN after blocking action potentials. These oscillations were inhibited by two chemically-distinct blockers of TRPM4, 9-phenanthrol (9-pt) and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). Under whole-cell voltage clamp, inward currents evoked by oscillation voltage waveforms were inhibited in LC by blocking L-type calcium channels and TRPM4. These data implicate TRPM4 in the large subthreshold membrane potential oscillations that underlie tonic action potential discharge in LC and SCN, providing a voltage-dependent and calcium-dependent cationic current to augment the depolarizing inward Na+ and Ca2+ currents previously associated with this distinctive electroresponsive property.

Abstract 58: Angiotensin-(1-7) Mas Receptors In The Arcuate Nucleus Of The Hypothalamus Protect Against High Fat Diet-induced Insulin Resistance In Female Mice

Angiotensin (Ang)-(1-7) is a protective hormone of the renin-angiotensin system that improves insulin sensitivity, glucose tolerance, and energy balance in obese rodents. Our recent findings suggest that Ang-(1-7) activates mas receptors (MasR) in the arcuate nucleus of the hypothalamus (ARC), a brain region critical to control of energy balance and glucose homeostasis, to induce these positive metabolic effects. The distribution of MasR in the ARC and their role in metabolic regulation, however, is unknown. We hypothesized: (1) MasR are expressed in the ARC; and (2) deletion of ARC MasR leads to worsened metabolic outcomes following high fat diet (HFD). To test this, male and female C57Bl/6J mice were fed a 60% HFD or matched control diet ad libitum for 12 weeks. RNAscope in situ hybridization was performed on coronal ARC sections in rostral-middle-caudal regions to determine percentage of MasR positive neurons (n=5/group). In a second experiment, we assessed body composition and insulin and glucose tolerance in transgenic mice with deletion of MasR in ARC neurons (MasR-flox with AAV5-hsyn-GFP-Cre). RNAscope revealed a wide distribution on MasR-positive cells throughout the rostral to caudal extent of the ARC. The average percentage of MasR positive neurons was increased in females versus males, with HFD tending to increase MasR expression in both sexes (control diet male: 11±2; control diet female: 17±3; HFD male: 15±5; HFD female: 24±2; p sex : 0.030; p diet : 0.066; p int : 0.615; two-way ANOVA). Deletion of MasR in ARC neurons worsened insulin sensitivity in HFD but not control diet females (area under the curve for change in glucose from baseline: -1989±1359 HFD control virus vs. 2530±1762 HFD Cre virus; p=0.016), while fasting glucose, glucose tolerance, and body composition did not change. There was no effect of ARC MasR deletion on metabolic outcomes in control diet or HFD male mice. These findings suggest females have more MasR positive neurons in the ARC compared to males, which may be a sex-specific protective mechanism for glucose homeostasis. While further studies are needed to explore the role of ARC MasR in metabolic regulation, these findings support targeting Ang-(1-7) as an innovative strategy in obesity.

Repeated cocaine administration upregulates CB2 receptor expression in striatal medium-spiny neurons that express dopamine D1 receptors in mice.

Cannabinoid CB2 receptors (CB2R) are importantly involved in drug reward and addiction. However, the cellular mechanisms underlying CB2R action remain unclear. We have previously reported that cocaine self-administration upregulates CB2R expression in midbrain dopamine (DA) neurons. In the present study, we investigated whether cocaine or heroin also alters CB2R expression in striatal medium-spiny neuronsthat express dopamineD1 or D2 receptors (D1-MSNs, D2-MSNs) and microglia. Due to the concern of CB2R antibody specificity, we developed three mouse CB2-specific probes to detect CB2R mRNA using quantitative RT-PCR and RNAscope in situ hybridization (ISH) assays. We found that a single injection of cocaine failed to alter, while repeated cocaine injections or self-administration dose-dependently upregulated CB2R gene expression in both brain (cortex and striatum) and periphery (spleen). In contrast, repeated administration ofheroin produced a dose-dependent reduction in striatal CB2 mRNA expression. RNAscope ISH assays detected CB2R mRNA in striatal D1- and D2-MSNs, not in microglia. We then used transgenic CX3CR1eGFP/+ microglia reporter mice and D1- or D2-Cre-RiboTag mice to purify striatal microglia or ribosome-associated mRNAs from CX3CR1eGFP/+,D1-MSNs, or D2-MSNs, respectively. We found that CB2R upregulation occurred mainly in D1-MSNs, not in D2-MSNs or microglia, in the nucleus accumbens rather than the dorsal striatum. These findings indicate that repeated cocaine exposure may upregulate CB2R expression in both brain and spleen, with regional and cell type-specific profiles. In the striatum, CB2R upregulation occurs mainly in D1-MSNs in the nucleus accumbens. Given the important role of D1-MSNs in brain reward function, the present findings provide new insight into mechanisms by which brain CB2Rs modulate cocaine action.

Abstract 2681: Co-detection of a tumor-infiltrating lymphocyte immunofluorescence (IF) panel and cytokine RNA in-situ hybridization (ISH) markers in non-small cell lung cancer (NSCLC) tumor microenvironment using combined MultiOmyx and RNAscope platforms

Abstract Co-detection of RNA and protein can greatly expand the data output from a single specimen, providing critical information such as the source of secreted proteins (e.g. cytokines) or cell type specific transcript levels. MultiOmyx is a proprietary immunofluorescence (IF) platform for the visualization and characterization of up to 60 protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) section. RNAscope Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. Combination of MultiOmyx IF with RNAscope Multiplex ISH would therefore provide a novel and powerful platform to co-detect multiple RNA and protein markers in a single slide/sample. However, the RNAscope Multiplex assay includes a protease pretreatment step, which may compromise downstream antibody-antigen interaction and thereby the IF signal. This study is a validation of the sensitivity, specificity, reproducibility, and repeatability of an integrated MultiOmyx IF and RNAscope ISH assay. Depending on the context, cytokines interleukin-10 (IL10) and interferon gamma (IFNγ) have both been shown to either induce immunosuppression and favor tumor growth or promote an anti-tumor response. The mechanisms and cues determining the pro- or anti-tumor activity of IL10 and IFNγ are still poorly understood. Spatiotemporal characterization of IFNG and IL10 is therefore critical to help define the dynamic relationship of cytokines and the immune system within the tumor microenvironment. Therefore, for validation of the integrated MultiOmyx-RNAscope platform, RNA ISH markers for IL10 and IFNγ were combined with the MultiOmyx 12 marker tumor infiltrating lymphocyte (TIL) panel (CD3, CD4, CD8, CD20, CD68, CD56, CD45RO, PD-1, PD-L1, CTLA4, FOXP3, and tumor marker PanCK) on FFPE human NSCLC samples. This combined MultiOmyx-RNAscope workflow was performed for three individual runs using triplicate NSCLC samples for each run. Intensity and cell classification for each ISH and TIL marker was quantified using the proprietary MultiOmyx Analytics pipeline. The results demonstrate that the integrated assay maintains sensitivity and specificity of the TIL IF markers in the integrated workflow when benchmarked to an IF alone workflow. Furthermore, these results can be used to characterize expression of IL10 and IFNγ within immune cell subsets represented by the TIL IF panel (e.g. T cells, NK cells, macrophages). This integrated approach can be used to spatially correlate the distribution of cytokine and immune cell expression within the tumor microenvironment. Therefore, the RNAscope-MultiOmyx IF assay provides a robust and powerful platform to simultaneously co-detect RNA with protein IF in a single specimen. Citation Format: Courtney Todorov, Maricel Gozo, Harry Nunns, Erinn Parnell, Judy Kuo, Eric Leones, Mate Nagy, Qingyan Au. Co-detection of a tumor-infiltrating lymphocyte immunofluorescence (IF) panel and cytokine RNA in-situ hybridization (ISH) markers in non-small cell lung cancer (NSCLC) tumor microenvironment using combined MultiOmyx and RNAscope platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2681.

POS-080 RNASCOPE MULTIPLEX IN SITU HYBRIDIZATION: A POWERFUL COMPLEMENT FOR STUDIES OF PROTEIN-PROTEIN INTERACTIONS IN COMPLEX TISSUES.

Protein-protein interactions form the basis most physiological and pathophysiological mechanisms, but studying them at the tissue level is challenging, especially when they are transient, weak or involve low-abundance molecules in complex tissues. The problem is compounded when using antibody-based approaches, due to the paucity of rigorously validated antibodies for in situ studies. To circumvent these limitations, novel in situ hybridization (ISH) techniques present a number of advantages: 1) simple design of highly sensitive and specific probes for any species; 2) rapid probe synthesis; 3) uniform staining protocol across all probes; 4) efficient and reliable multiplexing, including in combination with antibodies or alternative staining approaches. We are using RNAscope (ACD/Bio-Techne) ISH to examine the role of interactions involving the epithelial adapter protein Dok-4 injury models in wild type (WT) and Dok-4 null (KO) mice obtained by CRISPR/CAS9-induced frame shift. Novel interactions were identified by yeast two-hybrid and BioID method. For ISH, paraffin-embedded tissue was stained according to standard protocol either with chromogenic (DAB) method or multiplex fluorescence method. Dok-4 RNAscope probes were obtained from ACD and designed to be either overlapping or not with the short Dok-4 mRNA mismatch present in the KO mice. This allowed us to validate the specificity of the assay on one hand and to pinpoint the expected sites of Dok-4 protein expression (in WT) and deficiency (in KO). For EdU staining, EdU (ClickChemistry) was administered 2 hours before euthanasia and detected with Azide-Fluor 488 (Thermo). In both kidney and intestine, multiplex ISH demonstrated overlapping Dok-4 and EGFR mRNA expression. Partner identification could be also multiplexed with ISH probes for nephron segment identity (UMOD) or cellular injury (KIM1). Additionally, ISH could be multiplexed with validated immunofluorescence antibodies, such as to the proximal tubule marker AQP1, and with a marker of proliferation (EdU staining). In a mouse model of irradiation enteritis, directionally opposed but overlapping epithelial mRNA gradients of Dok-4 (dominant in differentiated villi) and its novel partner EGF receptor (dominant in undifferentiated crypts) could be demonstrated. Notably, increased EdU staining found 3 days after irradiation was concentrated in this area of Dok-4/EGFR overlap. Multiplex ISH is a highly sensitive, specific and flexible approach to complement proteomics and biochemical interaction studies. Multiplexing for partner molecules can confirm the in vivo relevance of protein-protein interactions defined and validated in more rudimentary systems. Multiplexing can also be done with markers of nephron segment identity, tissue injury or repair, and more. Notably, in transgenic models where mRNA is minimally altered (such as with CRISPR/CAS9 methods), ISH can allow more focused phenotypic studies by delineating the predominant sites of targeted protein deficiency at the single cell level.

Orexin-A Intensifies Mouse Pupillary Light Response by Modulating Intrinsically Photosensitive Retinal Ganglion Cells.

We show for the first time that the neuropeptide orexin modulates pupillary light response, a non-image-forming visual function, in mice of either sex. Intravitreal injection of the orexin receptor (OXR) antagonist TCS1102 and orexin-A reduced and enhanced pupillary constriction in response to light, respectively. Orexin-A activated OX1Rs on M2-type intrinsically photosensitive retinal ganglion cells (M2 cells), and caused membrane depolarization of these cells by modulating inward rectifier potassium channels and nonselective cation channels, thus resulting in an increase in intrinsic excitability. The increased intrinsic excitability could account for the orexin-A-evoked increase in spontaneous discharges and light-induced spiking rates of M2 cells, leading to an intensification of pupillary constriction. Orexin-A did not alter the light response of M1 cells, which could be because of no or weak expression of OX1Rs on them, as revealed by RNAscope in situ hybridization. In sum, orexin-A is likely to decrease the pupil size of mice by influencing M2 cells, thereby improving visual performance in awake mice via enhancing the focal depth of the eye's refractive system.SIGNIFICANCE STATEMENT This study reveals the role of the neuropeptide orexin in mouse pupillary light response, a non-image-forming visual function. Intravitreal orexin-A administration intensifies light-induced pupillary constriction via increasing the excitability of M2 intrinsically photosensitive retinal ganglion cells by activating the orexin receptor subtype OX1R. Modulation of inward rectifier potassium channels and nonselective cation channels were both involved in the ionic mechanisms underlying such intensification. Orexin could improve visual performance in awake mice by reducing the pupil size and thereby enhancing the focal depth of the eye's refractive system.