Evaluation Of Gene Expression Research Articles

Prevalence of Hypothyroidism and Hyperthyroidism in Women of Reproductive Ages in Kabul

Introduction: The association of sex steroids with cancer cell proliferation has been reported in recent studies; however, the findings are still controversial. The present study aimed to determine the cytotoxic effects of progesterone and testosterone on colon cancer (SW480) cells and to evaluate the expression levels of Bcl-2 and Bax genes in SW480 cells. Materials and Methods: The SW480 cell line was divided into a control group (untreated) and groups treated with 125, 250, 500, and 1000 μg/mL of testosterone and progesterone. Cell viability was quantified by the MTT assay. qRT-PCR was performed to evaluate gene expression levels. Flow cytometry was used to assess the apoptosis in cancer cells. Data were analyzed using the student's t-test and ANOVA. Results: The expression level of the Bax gene significantly decreased in SW480 cells exposed to a cytotoxic dose of progesterone. Moreover, the expression level of the Bax gene significantly increased in the SW480 cells exposed to a cytotoxic dose of testosterone. Conclusion: The results of the present study showed that testosterone might affect the apoptosis of colon cancer cells at low concentrations. Studies have shown that progesterone can induce tumor cell death in cancer cells. Accordingly, the clinical use of testosterone and progesterone therapy for cancer treatment is highly controversial.

Utilizing Machine Learning Techniques for Categorizing Cancer Based on Gene Expression Data: A Review

Cancer is a group of diseases which share one common feature: the growth of abnormal cells, thus ranking as the second leading cause of death globally, after cardiovascular diseases in WHO's report. Evaluating gene expression is based on the fact that it is the genesis of early cancer detection, the concurrence of the molecular and genetic processes. By using DNA microarray techniques and RNA-sequencing approaches, researchers in computational genomics can give quantitative measures of gene expression levels providing very accurate input data for computational evaluation. The current paper is about machine learning technology, which identifies cancer subtypes according to patterns of gene expression. It embraces the two distinct methodologies which are traditional plus deep learning with high proficiency focused on the cancer-related gene. The outline includes the most popular deep neural network designs such as MLPs, CNN, RNN, GNN, and the recently emerged Transformer networks. The review describes common data collection methods used in this field and some of the essential datasets for supervised machine learning. In addition, the specific techniques developed to cover the complicated horizontal spread of gene expression data are also presented. The article explores theoretical possibilities for the promotion of machine learning-based gene expression analysis in cancer classification towards the end.

Skin transcriptome analysis in Brangus cattle under heat stress

Heat stress is a major factor that negatively affects animal welfare and production systems. Livestock should adapt to tropical and subtropical areas and to meet this, composite breeds have been developed. This work aimed to evaluate gene expression profiles in the skin of Brangus cattle under heat stress using a case-control design, and to correlate this with skin histological characteristics. Two groups of bulls were set using rectal temperature as a criterion to define stress conditions: stressed (N = 5) and non-stressed (N = 5) groups. Skin transcriptomics was performed and correlations between breed composition, phenotypic and skin histological traits were evaluated. Results showed 4309 differentially expressed genes (P < 0.01), 2113 downregulated and 2196 upregulated. Enrichment and ontology analyses revealed 132 GO terms and 67 pathways (P < 0.01), including thermogenesis, glycolysis, gluconeogenesis, mitochondrial activity, antioxidant and immune response, and apoptosis. The identity of the terms and pathways indicated the diversity of mechanisms directed to relieve the animals' suffering, acting from simple passive mechanisms (conduction, convection and radiation) to more complex active ones (behavioural changes, evaporation, vasodilation and wheezing). Furthermore, significant differences between phenotypic and skin histological traits and correlations between pairs of traits suggested a direction towards heat dissipation processes. In this sense, number of vessels was positively correlated with number of sweat glands (P < 0.001) and both were positively correlated with zebuine genetic content (P < 0.05 and P < 0.01, respectively), gland size was positively correlated with epidermal thickness and negatively with hair length (P < 0.05), and epidermal thickness was negatively correlated with gland-epidermis distance (P < 0.0005). These results support the notion that response to heat stress is physiologically complex, producing significant changes in the expression of genes involved in several biological pathways, while the animal's ability to face it depends greatly on their skin features.

Evaluation Gene Expression of Long Non-coding RNA and 5-Hydroxytriptamine 3A Receptor in Bronchial Asthma

Evaluation Gene Expression of Long Non-coding RNA and 5-Hydroxytriptamine 3A Receptor in Bronchial Asthma

Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data.

Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n=12) under different (patho)physiological conditions as well as in human-derived biofluids (n=3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications.

Licensing effects of inflammatory factors and TLR ligands on the regenerative capacity of adipose-derived mesenchymal stem cells.

Introduction: Adipose tissue-derived mesenchymal stem cells are promising contributors to regenerative medicine, exhibiting the ability to regenerate tissues and modulate the immune system, which is particularly beneficial for addressing chronic inflammatory ulcers and wounds. Despite their inherent capabilities, research suggests that pretreatment amplifies therapeutic effectiveness. Methods: Our experimental design exposed adipose-derived mesenchymal stem cells to six inflammatory factors for 24h. We subsequently evaluated gene expression and proteome profile alterations and observed the wound closure rate post-treatment. Results: Specific pretreatments, such as IL-1β, notably demonstrated an accelerated wound-healing process. Analysis of gene and protein expression profiles revealed alterations in pathways associated with tissue regeneration. Discussion: This suggests that licensed cells exhibit potentially higher therapeutic efficiency than untreated cells, shedding light on optimizing regenerative strategies using adipose tissue-derived stem cells.

Abstract 2710: Combination CXCR4 and PD-1 blockade increases T cell infiltration and effector function in fibrolamellar carcinoma (FLC)

Abstract Introduction: Fibrolamellar carcinoma (FLC), a rare liver cancer affecting young patients without cirrhosis, has a poor prognosis and few systemic therapy options. Current evidence implicates an immunosuppressive tumor microenvironment (TME), including our preliminary data suggesting preferential localization of T cells in stromal bands. We hypothesized that dual blockade of C-X-C chemokine receptor type 4 (CXCR4) and programmed cell death protein (PD-1) would enhance T cell infiltration and activation, respectively, and thereby increase tumor killing. Methods: Flow cytometry and single-nuclear RNA sequencing (snRNA-seq) were used to analyze immune cells and evaluate gene expression, respectively, in patient-derived FLC and non-tumor liver (NTL). Immunohistochemistry (IHC) was used to assess CD3+ T cell localization in relation to CXCL12, the chemokine ligand of CXCR4. Tumor slice cultures (TSCs) from fresh FLC resection specimens (n=11) were treated with control (IgG1), a small molecule CXCR4 inhibitor (AMD3100), anti-PD-1 blocking antibody (αPD-1), or combination CXCR4 and PD-1 blockade (AMD3100/αPD-1). IHC, RNA expression (Nanostring), and live microscopy were used to evaluate T cell mobilization and function. Results: Flow cytometry analysis demonstrated fewer CD45+ cells (16% vs 54% of live cells, p=0.01) and heterogenous PD-1 expression on T cells in FLC compared with NTL. SnRNA-seq analysis showed that CXCR4 expression is upregulated in FLC lymphocyte and macrophage populations relative to NTL (15% vs 10% and 20% vs 5% expressed), and CXCL12 is expressed primarily in stellate cells. CD3+ T cells colocalized with CXCL12 in the FLC stroma on IHC. Multiplex IHC of treated TSCs revealed more T cells in the carcinoma (CK7+) than the stromal (SMA+) compartment after AMD3100 compared with IgG1 (63% vs 34% T cells in CK7+ compartment, p=0.05). Nanostring indicated upregulation of effector pathways (apoptosis, cytotoxicity, lymphocyte activation) after AMD3100/αPD-1 compared with IgG1. Time-lapse live microscopy revealed a greater percentage of apoptotic carcinoma cells (SR-FLICA+CK7+) colocalized with CD3+ T cells (37% vs 18%, p=0.005) and longer CD3+ T cell track length (27 vs 22 μm, p=0.03) after AMD3100/αPD-1 than before treatment. Cleaved caspase-3 (CC3) IHC demonstrated significantly increased tumor apoptosis after AMD3100/αPD-1 compared with IgG1 and monotherapy (53% vs 32% (IgG1), 39% (AMD3100), and 42% (αPD-1) CC3+ cells, p=0.002, p=0.03, p=0.04). Conclusion: The CXCR4/CXCL12 axis mediates T cell exclusion in the FLC TME. Combination CXCR4 and PD-1 blockade overcomes immunosuppression in FLC by mobilizing T cells into the carcinoma compartment and activating their effector function, with resultant tumor apoptosis. These findings support consideration of clinical trials testing drugs targeting CXCR4 and PD-1 in FLC. Citation Format: Lindsay K. Dickerson, Renske van den Bijgaart, Xiuyun Jiang, Sara K. Daniel, Jason A. Carter, Alaa Farghli, Kevin M. Sullivan, Yongjun Liu, Heidi L. Kenerson, Raymond S. Yeung, Teresa S. Kim, Ian N. Crispe, Praveen Sethupathy, Kevin C. Barry, Venu G. Pillarisetty. Combination CXCR4 and PD-1 blockade increases T cell infiltration and effector function in fibrolamellar carcinoma (FLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2710.

Abstract 2543: Evaluating biomarkers of immunotherapy response in a real-world metastatic NSCLC cohort

Abstract Background: Non-small cell lung cancer (NSCLC) is the leading cause of global cancer deaths, since a large proportion of cases are initially diagnosed at advanced stages. Therapeutic developments have shifted first-line (1L) therapy in the metastatic setting to immunotherapy (IO)-based regimens, albeit with heterogeneity in response. Patients harboring KEAP1 and STK11 mutations have been reported to exhibit both poorer outcomes and IO treatment resistance. Recent work suggests gene expression signatures which characterize mutant phenotypes may better identify those with compromised IO response. Leveraging a molecular real-world dataset of metastatic NSCLC patients treated with 1L IO, we aimed to evaluate gene expression signatures representing KEAP1 and STK11-mutated phenotypes and their impact on real-world outcomes. Methods: Metastatic NSCLC patients who received 1L anti-PD(L)1 as a monotherapy or in combination were identified from the multimodal real-world database of Tempus Labs, Inc. All patients in the analytical cohort had tissue biopsies sequenced on Tempus targeted DNA and whole transcriptome RNA assays within 90 days prior to the start of the 1L IO regimen. Patients were negative for actionable mutations with record duration of at least 30 days from 1L initiation. KEAP1 and STK11 mutants were identified with likely pathogenic and/or pathogenic mutations as annotated by Tempus bioinformatic pipelines. Gene expression signatures related to these mutations were obtained from recently published literature. Evaluation of mutations or signatures as genomic markers for IO non-response were conducted using real-world progression-free survival (rwPFS) and overall survival (rwOS). Results: A total of 332 patients were included in the study (45% IO monotherapy, 55% IO combination therapy). Previously validated biomarkers, including tumor mutation burden, T-cell inflamed GEP signature, and PD(L)1 expression, were associated with prolonged rwPFS. KEAP1 mutations were observed in 9% of analytical cohort with a higher proportion in non-squamous (11%) compared with squamous (4%) samples. For STK11, 10% were identified as mutants with a similar histology distribution (12% vs. 3%). Neither KEAP1 mutations nor related gene signatures evaluated in this study were significantly associated with real-world survival outcomes. Patients harboring STK11 mutations trended towards poor outcomes; furthermore, those with higher STK11 inactivation signature score were associated with shorter rwPFS and rwOS. Conclusions: STK11 inactivation phenotype described by a gene expression signature was able to distinguish patients with significantly poorer survival outcomes. This finding would need validation in independent cohorts. This real-world cohort study adds supporting evidence for the utility of gene expression signatures encompassing mutant phenotypes for patient stratification. Citation Format: Katherina C. Chua, Assieh Saadatpour, Ariel Chen, Aisha Hasan, Bevan Emma Huang, Stewart Bates. Evaluating biomarkers of immunotherapy response in a real-world metastatic NSCLC cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2543.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Mythimna loreyi (Lepidoptera: Noctuidae).

Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA-box, GAPDH, AK, Actin, EF, α-tubulin, SOD, 18S rRNA, and FTZ-F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ-F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.

Influence of epicardial adipose tissue inflammation and adipocyte size on postoperative atrial fibrillation in patients after cardiovascular surgery.

Epicardial adipose tissue (EAT) is an active endocrine organ that is closely associated with occurrence of atrial fibrillation (AF). However, the role of EAT in the development of postoperative AF (POAF) remains unclear. We aimed to investigate the association between EAT profile and POAF occurrence in patients who underwent cardiovascular surgery. We obtained EAT samples from 53 patients to evaluate gene expression, histological changes, mitochondrial oxidative phosphorylation (OXPHOS) capacity in the EAT, and protein secretion in EAT-conditioned medium. EAT volume was measured using computed tomography scan. Eighteen patients (34%) experienced POAF within 7 days after surgery. Although no significant difference was observed in EAT profile between patients with and without POAF, logistic regression analysis identified that the mRNA expression levels of tumor necrosis factor-alpha (TNF-α) were positively correlated and adipocyte size in the EAT was inversely correlated with onset of POAF, respectively. Mitochondrial OXPHOS capacity in the EAT was not associated with POAF occurrence; however, it showed an inverse correlation with adipocyte size and a positive correlation with adiponectin secretion. In conclusion, changes in the secretory profile and adipocyte morphology of the EAT, which represent qualitative aspects of the adipose tissue, were present before the onset of AF.

Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance.

The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors involved in the molecular response to hypoxia, to overcome GBM radioresistance. We used the U87 cell line subjected to chemical hypoxia generated by CoCl2 and exposed to 2 Gy of X-rays, as single or combined treatments, and evaluated gene expression changes of biomarkers involved in the Warburg effect, cell cycle control, and survival to identify the best molecular targets to be knocked-down, among those directly activated by the HIF-1α transcription factor. By this approach, glut-3 and pdk-1 genes were chosen, and the effects of their morpholino-induced gene silencing were evaluated by exploring the proliferative rates and the molecular modifications of the above-mentioned biomarkers. We found that, after combined treatments, glut-3 gene knockdown induced a greater decrease in cell proliferation, compared to pdk-1 gene knockdown and strong upregulation of glut-1 and ldha, as a sign of cell response to restore the anaerobic glycolysis pathway. Overall, glut-3 gene knockdown offered a better chance of controlling the anaerobic use of pyruvate and a better proliferation rate reduction, suggesting it is a suitable silencing target to overcome radioresistance.

Hyperinsulinemia impairs decidualization via AKT-NR4A1 signaling: new insight into polycystic ovary syndrome (PCOS)-related infertility.

Investigating the underlying molecular mechanisms responsible for endometrial dysfunction in women with PCOS is essential, particularly focusing on the role of hyperinsulinemia. We explored the role of insulin in the decidualization process using a synthetic decidualization assay. To dissect the effects of PI3K/AKT-NR4A signaling, we employed small interfering RNAs (siRNAs) targeting the NR4A genes and inhibitors of the PI3K/AKT pathway. We also investigated the disruption of AKT-NR4A1 signaling in the endometrium of PCOS female rats induced with dehydroepiandrosterone (DHEA). Quantitative real-time PCR (qRT-PCR) and Western blot (WB) analyses were utilized to evaluate gene expression regulation. Insulin was found to suppress the expression of decidualization markers in human endometrial stromal cells (hESC) in a dose-dependent manner, concurrently triggering an inappropriate activation of the PI3K/AKT pathway. Members of the NR4A family, as downstream effectors in the PI3K/AKT pathway, were implicated in the insulin-induced disruptions during the decidualization process. Moreover, the endometrium of PCOS models showed significantly elevated levels of phosphorylated (Ser473) AKT, with a corresponding reduction in Nr4a1 protein. Our research demonstrates that insulin negatively regulates decidualization in hESC via the PI3K/AKT-NR4A pathway. In vivo analysis revealed a significant dysregulation of the AKT-NR4A1 pathway in the endometrium of PCOS rats. These findings offer novel insights into the pathogenesis of infertility and endometrial disorders associated with hyperinsulinemia in PCOS.

Gene expression associated with vaginal bleeding in women using the 52-mg levonorgestrel hormonal intrauterine device: A prospective study.

To evaluate gene expression associated with vaginal bleeding in the 52-mg hormonal intrauterine device (IUD) users. We conducted a prospective study involving 100 women seeking to use the 52-mg hormonal IUD for contraception. We excluded women with a history or current condition of abnormal uterine bleeding and who were unable to attend a 1-year follow up. Women who expelled the device, removed it for reasons unrelated to vaginal bleeding, or were lost to follow up were discontinued. We collected endometrial biopsies immediately before IUD placement and assessed 20 selected genes using reverse transcription quantitative polymerase chain reaction. Users maintained a uterine bleeding diary for 12 months following IUD insertion. For statistical analysis, participants were categorized into groups with or without vaginal bleeding at 3 and 12 months. Women with elevated CXCL9 expression had an 8.15-fold higher likelihood of experiencing vaginal bleeding at 3 months (odds ratio [OR] 8.15, 95% confidence interval [CI] 2.24-29.61, P = 0.001). At 12 months of follow up, women with increased TIMP1 expression had a 2.74-fold higher chance of experiencing vaginal bleeding (OR 2.74, 95% CI 1.08-6.95, P = 0.033). CXCL9 ≥ 1.5 and IL17A ≥ 0.68 were associated with a higher probability of vaginal bleeding at 3 months, while TIMP1 levels ≥0.943 were linked to an increased risk of bleeding at 12 months. Users of the 52-mg hormonal IUD with elevated relative CXCL9 expression face an increased risk of vaginal bleeding at 3-month follow up, whereas those with heightened TIMP1 expression are more likely to experience vaginal bleeding at 12 months.

The role of androgen response pathway in association with tumor biology and response to neoadjuvant immune-checkpoint inhibitors (ICI) in muscle-invasive urothelial bladder carcinoma (MIBC).

662 Background: Emerging findings from ICI trials are suggestive of differential outcomes in females compared with males. Sex-specific differences in tumor biology, microbiome, and outcomes in MIBC are well-established. Conversely, limited data are available regarding the role of the androgen response pathway in explaining subtype differences between males and females and, most importantly, response to ICI. Methods: We retrospectively evaluated the biomarker findings of the transurethral resection of the bladder tumors (TURBT) and baseline clinical characteristics of patients (pts) with cT2-4N0M0 MIBC who received neoadjuvant pembrolizumab and radical cystectomy (RC) in the PURE-01 study. Transcriptome-wide expression profiling was performed on 102 TURBT samples. We focused on androgen receptor (AR) gene expression and the hallmark androgen response pathway expression quantifying 117 different genes related to the androgen response pathway as previously reported (PMID: 26771021). Pt and tumor characteristics were compared between subgroups using χ2 tests and two-sided Wilcoxon rank-sum tests. Kaplan-Meier curves and log rank tests evaluated gene expression and signature scores in association with the event-free survival (EFS), calculated from start of pembrolizumab treatment. Signature scores were analyzed as continuous values and were split by median and quartile values. Results: Pts received pembrolizumab and RC from 2017 to 2022. There were 87 (85.3%) males and 15 (14.7%) females. AR gene expression was not significantly different between males and females (p = 0.5), whereas androgen response signature was significantly higher in male (p = 0.03). The AR gene expression and androgen response signature scores were more highly expressed in luminal tumors vs rest of molecular subtypes (p = 0.005 and p &lt; 0.001). Despite AR gene expression was not associated with ypT0N0 response (p = 0.425), hallmark androgen response signature scores were significantly lower in ypT0N0 responders (p = 0.033). Higher hallmark androgen response signature scores were statistically significantly associated with shorter EFS (p = 0.028), whereas AR gene expression was not (p = 0.152). Conclusions: For the first time to our knowledge, we reported the androgen response pathway expression to be a potential biomarker of ICI benefit in MIBC. This signature was also associated with a luminal subtype, linked to inferior responsiveness to ICI. If confirmed at a larger scale these analyses will strengthen the need for sex-specific approaches and potentially new combination therapies in MIBC.

Molecular effects of photodynamic therapy with curcumin on Leishmania major promastigotes.

Leishmaniasis is a neglected disease mainly affecting low-income populations. Conventional treatment involves several side effects, is expensive, and, in addition, protozoa can develop resistance. Photodynamic therapy (PDT) is a promising alternative in treating the disease. PDT involves applying light at a specific wavelength to activate a photosensitive compound (photosensitizer, PS), to produce reactive oxygen species (ROS). Curcumin and its photochemical characteristics make it a good candidate for photodynamic therapy. Studies evaluating gene expression can help to understand the molecular events involved in the cell death caused by PDT. In the present study, RNA was extracted from promastigotes from the control and treated groups after applying PDT. RT-qPCR was performed to verify the expression of the putative ATPase beta subunit (ATPS), ATP synthase subunit A (F0F1), argininosuccinate synthase 1 (ASS), ATP-binding cassette subfamily G member 2 (ABCG2), glycoprotein 63 (GP63), superoxide dismutase (FeSODA), and glucose-6-phosphate dehydrogenase (G6PDH) genes (QR). The results suggest that PDT altered the expression of genes that participate in oxidative stress and cell death pathways, such as ATPS, FeSODA, and G6PD. The ATP-F0F1, ASS, and GP63 genes did not have their expression altered. However, it is essential to highlight that other genes may be involved in the molecular mechanisms of oxidative stress and, consequently, in the death of parasites.

Abstract A085: Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment

Abstract Breast cancer is the most common cancer diagnosis worldwide, and an unfortunately high number of women die every year due to a lack of effective therapeutic strategies for metastatic breast cancer. Patients with brain metastases in particular have a significantly poorer prognosis than women with metastasis to other sites (e.g., lung, liver, bone). Previous studies by our group identified a ligand-receptor complex in the tumor microenvironment (TME) that promotes metastasis of breast cancer to the brain. This interaction between platelet-derived growth factor-BB (PDGFB), produced by cancerous breast epithelial cells, and its receptor, platelet-derived growth factor receptor beta (PDGFRβ), expressed by mesenchymal cells, is also prognostic of breast cancer metastasis to the brain in patients. The mechanism of how the PDGFB-PDGFRβ pathway mediates brain metastasis is still unclear and is the focus of our current work. To further analyze the biological effects of PDGFB, our group evaluated gene expression changes between primary human breast tumors expressing high PDGFB versus those expressing low PDGFB and found significantly more immune signaling in the low-expressing tumors. These results led us to hypothesize that the PDGFB-PDGFRβ pathway creates an immunosuppressive microenvironment, allowing for increased metastasis of cancerous cells. To test this, flow cytometry was completed on the brains, spleens, lungs, and livers of control mice and mutant mice expressing stromal-specific hyperactive PDGFRβ following intracardiac injection of PDGFB-expressing mammary tumor cells. No significant changes in immune cell populations were observed in non-tumor-bearing mice. However, in the brains of tumor-bearing mutant mice, there are significant changes to the myeloid lineage. Specifically, analysis at day 10 post-injection (prior to detectable metastatic lesions) revealed diminished CD45+CD11b+ populations. Segregating by CD45hi/CD45lo confirmed a profound decrease in CD45loCD11b+F4/80+ microglia, likely a tumor-suppressive population. Looking at day 16 post-injection (detectable metastatic lesions), there is a shift towards an increase in CD45+ CD11b+GR1+ infiltrating myeloid-derived suppressor cells (MDSCs) in experimental mice, likely a tumor-promoting population. Minimal changes were observed in the lungs and livers at any time point. Confirmatory immunofluorescence staining showed a decrease in tumor-infiltrating microglia (Iba1+) and a significant increase in tumor-infiltrating peripheral macrophages (F4/80+), mirroring the flow data. This experiment was then repeated to test the contribution of the ligand (PDGFB), where a significant increase of infiltrating microglia (Iba1+) was observed upon tumor cell knockdown of PDGFB. Together, these results suggest that the PDGFB-PDGFRβ pathway creates an immunosuppressive environment in the brain TME at least in part through reorchestration of the myeloid populations. Citation Format: Alexis A Mossing, Johnathon G Schiebel, Rebecca L Packard, Amrendra Kumar, Sarah A Steck, Nathaniel S Grabinski, Katie A Thies, Bri Wasik, Tasneem Ariswala, Gary Tozbikian, Johnathan Godbout, Steven T Sizemore, Paul R Lockman, Anna E Vilgelm, Gina M Sizemore. Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A085.

The intrafollicular concentrations of biologically active cortisol in women rise abruptly shortly before ovulation and follicular rupture.

What is the temporal activity and the concentration in follicular fluid (FF) of the anti-inflammatory steroid cortisol during the ovulatory process in humans? Intrafollicular concentrations of cortisol become massively upregulated close to ovulation concomitant with an exceptionally high biological activity securing a timely and efficient termination of inflammatory processes. Ovulation has been described as a local, controlled inflammatory process resulting in the degeneration of the follicle wall which facilitate oocyte extrusion. Ovulation also affects the glucocorticoid metabolism of granulosa cells (GCs) and although de novo synthesis of cortisol only occurs in the adrenal cortex, the mid-cycle surge has been shown to induce a change from high expression of HSD11B2, inactivating cortisol to cortisone, to high expression of HSD11B1 which reversibly catalyses cortisol production from cortisone. Furthermore, high concentrations of progesterone and 17OH-progesterone within follicles may cause dislodging of cortisol from cortisol binding protein (CBP) thereby activating the biological activity of cortisol. This prospective cohort study included 50 women undergoing fertility treatment according to a standard antagonist protocol at a university hospital-affiliated fertility clinic in Denmark. Women donated FF and GCs from one follicle for research purpose aspirated at one of four time points during the process of final maturation of follicles: T = 0 h, T = 12 h, T = 17 h, T = 32 h. A second sample was collected at oocyte pick up at T = 36 h. The concentration of cortisol and cortisone together with a range of sex steroids was measured by LC-MS/MS in FF collected at the five time points mentioned above. Whole genome microarray data, validated by q-PCR analysis, was used to evaluate gene expression of CYP11B1, CYP21A2, HSD11B1, HSD11B2, and NR3C1 in GCs at the same time points. The concentration of cortisol was significantly increased from a few nM at 0 h to around 100-140 nM (P ≤ 0.0001) at 32-36 h, whilst cortisone was almost constant from 0 to 17 h at a concentration of between 90 and 100 nM being significantly reduced to 25-40 nM (P ≤ 0.0001) at 32-36 h. This was paralleled by a 690-fold upregulation of HSD11B1 from 0 to 12 h increasing to a more than 20.000-fold change at 36 h. HSD11B2 was quickly downregulated 15- to 20-fold after ovulation induction. Concentrations of progesterone and 17OH-progesterone increased during the ovulatory process to high levels which in essence displaces cortisol from its binding protein CBP due to similar binding affinities. Furthermore, a significant decrease in 11-deoxycortisol expression was seen, but CYP11B1 expression was below detection limit in GCs. The study included women undergoing ovarian stimulation and results may differ from the natural cycle. More observations at each specific time point may have strengthened the conclusions. Furthermore, we have not been able to measure the actual active biological concentration of cortisol. For the first time, this study collectively evaluated the temporal pattern of cortisol and cortisone concentrations during human ovulation, rendering a physiological framework for understanding potential dysregulations in the inflammatory reaction of ovulation. This research was supported by the University Hospital of Copenhagen, Rigshospitalet, and Novo Nordisk Foundation grant number NNF21OC00700556. Interreg V ÔKS through ReproUnion (www.reprounion.eu); Region Zealand Research Foundation. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. N/A.

Evaluating Gene Expression and Signaling Activities in Lipopolysaccharide Induced Human Peripheral Blood Mononuclear Cells

Evaluating Gene Expression and Signaling Activities in Lipopolysaccharide Induced Human Peripheral Blood Mononuclear Cells

Molecular and cellular regulation on sexual fate and gonadal development in hermaphrodite yellowfin seabream Acanthopagrus latus

Yellowfin seabream (Acanthopagrus latus) is a hermaphroditic fish with male prematurity, which is also an economically important mariculture species in coastal areas of China. Although the production yield has been increasing in recent years, artificial breeding of this species remains imperfect due to the lack understanding of its sex differentiation mechanism. In this study, we conducted transcriptomic sequencing to screen for sex-related genes in different stage of the testis, ovotestis, ovary, hypothalamus, and pituitary tissues of yellowfin seabream. During gonad development (from intersex to mature stage), Gnrh3, Kiss2, and Dbh were expressed in the hypothalamus and Lhb and Fshb were expressed in the pituitary. Sex-related genes such as Hsd11b1, Foxl2l, Amh, and Gsdf were among the top 10 downregulated genes in the intersex testis compared with the mature testis. The expression of Hsd11b, Wnt, and Sox families genes differed significantly in the sex change process among functional mature gonads. In addition, treatment with estradiol resulted in feminizing of the ovotestis. Inhibition of androgens or estrogens promoted ovarian or testicular steps of ovotestis development. In summary, we systematically evaluated gene expression profiles during yellowfin seabream gonad development from intersex to functional male or female. Our results confirmed that regulation by the hypothalamus-pituitary-gonad (HPG) axis and the promotion of sex steroid hormones acted on this process, and they provide a reference for further studies of the involvement of the HPG axis in sex change and of sex induction methods in yellowfin seabream.

Neurotropin® ameliorates chronic pain associated with scar formation in a mouse model: A gene expression analysis of the inflammatory response.

Background: Scar formation after trauma and surgery involves an inflammatory response and can lead to the development of chronic pain. Neurotropin® (NTP) is a nonprotein extract of inflamed skin of rabbits inoculated with vaccinia virus. It has been widely used for the treatment of chronic pain. However, the in vivo effects of NTP on painful scar formation have not been determined. To investigate the molecular mechanisms underlying the effects of NTP on the inflammatory response, we evaluated gene expression in the scar tissues and dorsal root ganglions (DRGs) of mice administered NTP and control mice. Methods and results: Mice injected with saline or NTP were used as controls; other mice were subjected to surgery on the left hind paw to induce painful scar formation, and then injected with saline or NTP. Hind paw pain was evaluated by measuring the threshold for mechanical stimulation using the von Frey test. The paw withdrawal threshold gradually returned to pre-operative levels over 4weeks post-operation; NTP-treated mice showed a significantly shortened recovery time of approximately 3weeks, suggesting that NTP exerted an analgesic effect in this mouse model. Total RNA was extracted from the scarred hind paw tissues and DRGs were collected 1 week post-operation for a microarray analysis. Gene set enrichment analysis revealed that the expression of some gene sets related to inflammatory responses was activated or inhibited following surgery and NTP administration. Quantitative real-time reverse transcription-polymerase chain reaction analysis results for several genes were consistent with the microarray results. Conclusion: The administration of NTP to the hind paws of mice with painful scar formation following surgery diminished nociceptive pain and reduced the inflammatory response. NTP inhibited the expression of some genes involved in the response to surgery-induced inflammation. Therefore, NTP is a potential therapeutic option for painful scar associated with chronic pain.