Comprehensive Gene Expression Research Articles

B-cell hub genes play a cardiovascular pathogenic role of in childhood obesity and Kawasaki disease as revealed by transcriptomics-based analyses.

The study aims to explore the central genes that Kawasaki disease (KD) and Obesity (OB) may jointly contribute to coronary artery disease. Investigating single-cell datasets (GSE168732 and GSE163830) from a comprehensive gene expression database, we identified characteristic immune cell subpopulations in KD and OB. B cells emerged as the common immune cell characteristic subgroup in both conditions. Subsequently, we analyzed RNA sequencing datasets (GSE18606 and GSE87493) to identify genes associated with B-cell subpopulations in KD and OB. Lastly, a genome-wide association study and Mendelian randomization were conducted to substantiate the causal impact of these core genes on myocardial infarction. Quantitative real-time PCR (qRT-PCR) to validate the expression levels of hub genes in KD and OB. The overlapping characteristic genes of B cell clusters in both KD and OB yielded 70 shared characteristic genes. PPI analysis led to the discovery of eleven key genes that significantly contribute to the crosstalk. Employing receiver operating characteristic analysis, we evaluated the specificity and sensitivity of these core genes and scored them using Cytoscape software. The inverse variance weighting analysis suggested an association between TNFRSF17 and myocardial infarction risk, with an odds ratio of 0.9995 (95% CI = 0.9990-1.0000, p = 0.049). By employing a single-cell combined transcriptome data analysis, we successfully pinpointed central genes associated with both KD and OB. The implications of these findings extend to shedding light on the increased risk of coronary artery disease resulting from the co-occurrence of OB and KD.

Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

SPI1-mediated macrophage polarization aggravates age-related macular degeneration.

This study revealed a core regulator and common upstream mechanisms for the multifaceted pathological processes of age-related macular degeneration (AMD) and provided proof-of-concept for this new therapeutic target. Comprehensive gene expression analysis was performed using RNA sequencing of eye cup from old mice as well as laser-induced choroidal neovascularization (CNV) mouse model. Through integrative analysis and protein-protein interaction (PPI) analysis, common pathways and key transcription factor was identified simultaneously engaged in age-related retinal degeneration and CNV, the two typical pathological process of AMD. Subsequently, the expression changes of Spi1, the key regulator, as well as the alternation of the downstream mechanisms were validated in both models through qRT-PCR, Elisa, flow cytometry and immunofluorescence. Further, we assessed the impact of Spi1 knockdown in vitro and in vivo using gene intervention vectors carried by adeno-associated virus or lentivirus to test its potential as a therapeutic target. Compared to corresponding controls, we found 1,939 and 1,319 genes differentially expressed in eye cups of old and CNV mice respectively. The integrative analysis identified a total of 275 overlapping DEGs, of which 150 genes were co-upregulated. PPI analysis verified a central transcription factor, SPI1. The significant upregulation of Spi1 expression was then validated in both models, accompanied by macrophage polarization towards the M1 phenotype. Finally, SPI1 suppression significantly inhibited M1 polarization of BMDMs and attenuated neovascularization in CNV mice. This study demonstrates that SPI1 exerts a pivotal role in AMD by regulation of macrophage polarization and innate immune response, offering promise as an innovative target for treating AMD.

Comparative transcriptome analysis reveals genes involved in trichome development and metabolism in tobacco

BackgroundThe glandular trichomes of tobacco (Nicotiana tabacum) can efficiently produce secondary metabolites. They act as natural bioreactors, and their natural products function to protect plants against insect-pests and pathogens and are also components of industrial chemicals. To clarify the molecular mechanisms of tobacco glandular trichome development and secondary metabolic regulation, glandular trichomes and glandless trichomes, as well as other different developmental tissues, were used for RNA sequencing and analysis.ResultsBy comparing glandless and glandular trichomes with other tissues, we obtained differentially expressed genes. They were obviously enriched in KEGG pathways, such as cutin, suberine, and wax biosynthesis, flavonoid and isoflavonoid biosynthesis, terpenoid biosynthesis, and plant–pathogen interaction. In particular, the expression levels of genes related to the terpenoid, flavonoid, and wax biosynthesis pathway mainly showed down-regulation in glandless trichomes, implying that they lack the capability to synthesize certain exudate compounds. Among the differentially expressed genes, 234 transcription factors were found, including AP2-ERFs, MYBs, bHLHs, WRKYs, Homeoboxes (HD-ZIP), and C2H2-ZFs. These transcription factor and genes that highly expressed in trichomes or specially expressed in GT or GLT. Following the overexpression of R2R3-MYB transcription factor Nitab4.5_0011760g0030.1 in tobacco, an increase in the number of branched glandular trichomes was observed.ConclusionsOur data provide comprehensive gene expression information at the transcriptional level and an understanding of the regulatory pathways involved in glandular trichome development and secondary metabolism.

Comprehensive gene expression analysis using RNA sequencing between male and female patients with idiopathic carpal tunnel syndrome

AbstractIdiopathic carpal tunnel syndrome is the most common entrapment neuropathy in hand surgery, and it is characterized by Noninflammatory fibrosis of subsynovial connective tissues. The prevalence and incidence differ between male and female individuals, and the mechanism underlying this difference remains largely unclear. In the present study, we collected subsynovial connective tissues from six male and six female patients diagnosed with idiopathic carpal tunnel syndrome during surgery. We performed a comprehensive gene expression analysis using RNA sequencing to compare the gene expression profiles between male and female patients with idiopathic carpal tunnel syndrome. We identified 26 genes with significantly different expressions between male and female patients, in which POSTN, COL1A1, and COL3A1, which are involved in extracellular matrix organization, and IGF1, an important fibrotic factor, were significantly upregulated in male patients. Immunohistochemistry confirmed the expression of proteins encoded by these genes in tissues, and male patients tended to show increased POSTN expression. Our results indicate that fibrosis of subsynovial connective tissues is induced by different mechanisms in male and female patients, and genes involved in extracellular matrix organization, especially POSTN, might be important factors in male patients. This study provides insight into the pathogenesis of idiopathic carpal syndrome and might contribute to the development of new treatment strategies.

Analysis of human neuronal cells carrying ASTN2 deletion associated with psychiatric disorders

Recent genetic studies have found common genomic risk variants among psychiatric disorders, strongly suggesting the overlaps in their molecular and cellular mechanism. Our research group identified the variant in ASTN2 as one of the candidate risk factors across these psychiatric disorders by whole-genome copy number variation analysis. However, the alterations in the human neuronal cells resulting from ASTN2 variants identified in patients remain unknown. To address this, we used patient-derived and genome-edited iPS cells with ASTN2 deletion; cells were further differentiated into neuronal cells. A comprehensive gene expression analysis using genome-edited iPS cells with variants on both alleles revealed that the expression level of ZNF558, a gene specifically expressed in human forebrain neural progenitor cells, was greatly reduced in ASTN2-deleted neuronal cells. Furthermore, the expression of the mitophagy-related gene SPATA18, which is repressed by ZNF558, and mitophagy activity were increased in ASTN2-deleted neuronal cells. These phenotypes were also detected in neuronal cells differentiated from patient-derived iPS cells with heterozygous ASTN2 deletion. Our results suggest that ASTN2 deletion is related to the common pathogenic mechanism of psychiatric disorders by regulating mitophagy via ZNF558.

Tumor origin identification through machine learning and gene expression profiling.

e13597 Background: Accurate identification of tumor origin is crucial for effective diagnosis and treatment, particularly in cases of metastatic tumors. Interpretable machine learning models have displayed significant potential in addressing this problem when imaging and immunohistochemistry (IHC) examinations are ineffective. In this study, we developed a panel using gene expression profiles from various tumors and constructed a robust machine learning model for precise identification of tumor origin. Methods: RNA sequencing (RNA-seq) data of 9462 tumor samples originating from 21 different organs were collected from The Cancer Genome Atlas (TCGA). We conducted feature engineering through unsupervised clustering and differential gene expression analysis, selecting a refined panel of 164 genes from a pool of over 60,000 identifiers. Subsequently, a machine learning classifier grounded in Logistic regression (LR) was trained on 9462 samples with the constructed 164-gene panel. To enhance the adaptability of our model to this task, 10-fold cross-validation was employed in the multi-class mode. Two independent test sets, the Primary tumor set (PT, n=3420, including 19 tumor types) and the Metastatic tumor set (MTP, n=100, all originating from the prostate and spread to bone, liver, etc.), were established with samples from Gene Expression Omnibus (GEO) and other published studies. Notably, all samples underwent FPKM normalization and there was no overlap between training and testing samples. Model performance was assessed using accuracy, specificity, and sensitivity metrics. Results: The 164-gene expression panel achieved a cross-validation accuracy of 96.73%. In the assessment of the PT test set, our model achieved an overall 99.62% specificity, 92.31% accuracy and 89.79% sensitivity, exhibiting performance comparable to similar models in other studies. Additionally, our model accurately traced 91% of metastatic tumors in the MTP test set to the prostate, surpassing previous lines of work by a large margin. Conclusions: Gene expression patterns reveal organ-specific characteristics that could be used to identify tumor origin. The combination of a condensed yet comprehensive gene expression panel with a robust machine learning model serves as a promising tool for tumor diagnosis. Ongoing correlative studies aim to extend predictions from tissue organs to cancer subtypes.

Cryptic Resilience: Decoding Molecular Networks in Pearl Millet for Enhanced Heat Stress

Pearl millet (<em>Pennisetum glaucum</em>), a vital cereal crop renowned for its drought tolerance, is a cornerstone for smallholder farmers in arid and semi-arid regions, ranking as the fifth most significant cereal globally. Despite its resilience, the molecular mechanisms underlying its tolerance to heat stress remained elusive. To address this knowledge gap, we subjected ten-day-old pearl millet seedlings to an unprecedented temperature of 50°C for 60 seconds. Subsequent next-generation RNA sequencing aimed to unravel differential gene expression in heat-stressed seedlings compared to control conditions. Our analysis revealed a remarkable 29.8% differential expression in the genome sequence in response to heat stress. Heat-stressed pearl millet leaves exhibited differential expression in 11,483 genes, with fold changes ranging from 2 to 18.6 compared to the control group. Of these, 3,612 genes displayed upregulation, while 7,871 genes exhibited downregulation. These genes play roles in diverse biological processes involving crucial enzymes such as aminoacyl-tRNA synthetases, ligases, methyltransferases, oxidoreductases, and DNA-directed RNA polymerases. The Photosystem II Type I Chlorophyll-a/b-binding protein and heat shock proteins displayed the most significant fold changes in heat-stressed leaves. Moreover, various transcription factor families, including bHLH, ERF, NAC, WRKY, MYB-related, C2H2, bZIP, MYB, FAR1, and B3, vital in controlling pearl millet's response to heat stress, were linked to over 100 differentially expressed genes. The dataset generated through this research, shedding light on the molecular processes enabling pearl millet to withstand heat, holds immense value given the crop's role in food security and resilience to extreme weather. In the context of climate change and global warming, this knowledge lays the foundation for further studies on metabolic engineering and selecting crops resilient to high temperatures. Our transcriptomics approach provides comprehensive gene expression profiles of heat-stressed plants. It elucidates pearl millet's response to heat stress, offering a crucial resource for future investigations into crop adaptation strategies.

Sex-specific developmental gene expression atlas unveils dimorphic gene networks in C. elegans

Sex-specific traits and behaviors emerge during development by the acquisition of unique properties in the nervous system of each sex. However, the genetic events responsible for introducing these sex-specific features remain poorly understood. In this study, we create a comprehensive gene expression atlas of pure populations of hermaphrodites and males of the nematode Caenorhabditis elegans across development. We discover numerous differentially expressed genes, including neuronal gene families like transcription factors, neuropeptides, and G protein-coupled receptors. We identify INS-39, an insulin-like peptide, as a prominent male-biased gene expressed specifically in ciliated sensory neurons. We show that INS-39 serves as an early-stage male marker, facilitating the effective isolation of males in high-throughput experiments. Through complex and sex-specific regulation, ins-39 plays pleiotropic sexually dimorphic roles in various behaviors, while also playing a shared, dimorphic role in early life stress. This study offers a comparative sexual and developmental gene expression database for C. elegans. Furthermore, it highlights conserved genes that may underlie the sexually dimorphic manifestation of different human diseases.

IMPAQT reveals that adequate mitohormesis from TFAM overexpression leads to life extension in mice.

Mitochondrial transcription factor A, TFAM, is essential for mitochondrial function. We examined the effects of overexpressing the TFAM gene in mice. Two types of transgenic mice were created: TFAM heterozygous (TFAM Tg) and homozygous (TFAM Tg/Tg) mice. TFAM Tg/Tg mice were smaller and leaner notably with longer lifespans. In skeletal muscle, TFAM overexpression changed gene and protein expression in mitochondrial respiratory chain complexes, with down-regulation in complexes 1, 3, and 4 and up-regulation in complexes 2 and 5. The iMPAQT analysis combined with metabolomics was able to clearly separate the metabolomic features of the three types of mice, with increased degradation of fatty acids and branched-chain amino acids and decreased glycolysis in homozygotes. Consistent with these observations, comprehensive gene expression analysis revealed signs of mitochondrial stress, with elevation of genes associated with the integrated and mitochondrial stress responses, including Atf4, Fgf21, and Gdf15. These found that mitohormesis develops and metabolic shifts in skeletal muscle occur as an adaptive strategy.

Transgenerational acclimation to acidified seawater and gene expression patterns in a sea urchin

Transgenerational responses of susceptible calcifying organisms to progressive ocean acidification are an important issue in reducing uncertainty of future predictions. In this study, a two-generation rearing experiment was conducted using mature Mesocentrotus nudus, a major edible sea urchin that occurs along the coasts of northern Japan. Morphological observations and comprehensive gene expression analysis (RNA-seq) of resulting larvae were performed to examine transgenerational acclimation to acidified seawater. Two generations of rearing experiments showed that larvae derived from parents acclimated to acidified seawater tended to have higher survival and show less reduction in body size when exposed to acidified seawater of the same pH, suggesting that a positive carry-over effect occurred. RNA-seq analysis showed that gene expression patterns of larvae originated from both acclimated and non-acclimated parents to acidified seawater tended to be different than control condition, and the gene expression pattern of larvae originated from acclimated parents was substantially different than that of larvae of non-acclimated and control parents.

Ferulic acid in synergy with retinol alleviates oxidative injury of HaCaT cells during UVB-induced photoaging.

Application of retinol (Vitamin A, VA) in skincare is limited for instability, poor water solubility, and skin intolerance that combats skin aging. We employed computer-aided virtual screening and cell experiments with transcriptomics, thereby unveiling the comprehensive gene expression and regulation pathway of photoaging HaCaT cell treated with ferulic acid (FA) in synergizing with VA. Through network pharmacology analysis, the combined use of VA and FA exhibited highly correlated cross-targets with skin aging acting on EGFR, PTPN1, ESR2, GSK3B, BACE1, PYGL, PTGS2 and APP. The indicators of oxidative stress, such as SOD, GSH, MDA, CAT and ROS in HaCaT cells after co-administration, were significantly improved from those in photoaging group (p<0.0001). 155 differential expressed genes (DEGs) were specific between groups, while reducing the expression of PTGS2 was identified as an important regulatory factor in photoaging HaCaT cells by VA and FA. Those DEGs of co-administration group focused on oxidative-reduction enzyme activity, skin growth, keratinization, and steroid biosynthesis. Apparently, the co-administration of VA and FA effectively mitigated the process of UVB-induced photoaging by reducing oxidative stress injury, inflammation responses, and regulating cell growth. This synergistic approach significantly slowed down the photoaging progression and improved the applied performance of VA in HaCaT cells.

Donor regulatory T cells rapidly adapt to recipient tissues to control murine acute graft-versus-host disease

The adoptive transfer of regulatory T cells is a promising strategy to prevent graft-versus-host disease after allogeneic bone marrow transplantation. Here, we use a major histocompatibility complex-mismatched mouse model to follow the fate of in vitro expanded donor regulatory T cells upon migration to target organs. Employing comprehensive gene expression and repertoire profiling, we show that they retain their suppressive function and plasticity after transfer. Upon entering non-lymphoid tissues, donor regulatory T cells acquire organ-specific gene expression profiles resembling tissue-resident cells and activate hallmark suppressive and cytotoxic pathways, most evidently in the colon, when co-transplanted with graft-versus-host disease-inducing conventional T cells. Dominant T cell receptor clonotypes overlap between organs and across recipients and their relative abundance correlates with protection efficacy. Thus, this study reveals donor regulatory T cell selection and adaptation mechanisms in target organs and highlights protective features of Treg to guide the development of improved graft-versus-host disease prevention strategies.

Decreased histone expression in chronic rhinosinusitis with nasal polyps.

Histones have been associated with human diseases. However, the implication of extranuclear histone proteins and their potential mechanism in the pathophysiology of chronic rhinosinusitis (CRS) have not been thoroughly investigated. This study was designed to evaluate the role of histones in patients with CRS by comparing histone expression between patients and controls. Nasal polyp (NP) tissues were obtained, and their comprehensive gene expression profiles were investigated by microarray analysis. Differences in expression were verified by reverse transcriptase polymerase chain reaction and immunohistochemical staining. Cell culture and flow cytometry were used to evaluate the role of histones in the pathogenesis of polyps. Significant differences in the microarray analysis were observed between the patient and control groups (P < 0.01). It was found by flow cytometry that the histone (H2BK) can promote cell apoptosis in NPs. Our results indicate that reduced expression of H2BK may contribute to the imbalance process of cell proliferation and apoptosis in CRS with NP.

Hippocampal Lactate-Infusion Enhances Spatial Memory Correlated with Monocarboxylate Transporter 2 and Lactylation.

Lactate has emerged as a key player in regulating neural functions and cognitive processes. Beyond its function as an energy substrate and signal molecule, recent research has revealed lactate to serve as an epigenetic regulator in the brain. However, the molecular mechanisms by which lactate regulates spatial memory and its role in the prevention of cognitive disorders remain unclear. Herein, we injected L-lactate (10 μmol/kg/d for 6 d) into the mouse's hippocampus, followed by the Morris water maze (MWM) test and molecular analyses. Improved spatial memory performances were observed in mice injected with lactate. Besides, lactate upregulated the expression of synaptic proteins post-synaptic density 95 (PSD95), synaptophysin (SYP), and growth associated protein 43 (GAP43) in hippocampal tissues and HT22 cells, suggesting a potential role in synaptic transmission and memory formation. The facilitative role of monocarboxylate transporter 2 (MCT2), a neuron-specific lactate transporter, in this process was confirmed, as MCT2 antagonists attenuated the lactate-induced upregulation of synaptic proteins. Moreover, lactate induced protein lactylation, a post-translational modification, which could be suppressed by MCT2 inhibition. RNA sequencing of lactated-injected hippocampal tissues revealed a comprehensive gene expression profile influenced by lactate, with significant changes in genes associated with transcriptional progress. These data demonstrate that hippocampal lactate injection enhances spatial memory in mice, potentially through the upregulation of synaptic proteins and induction of protein lactylation, with MCT2 playing a crucial role in these processes. Our findings shed light on the multi-faceted role of lactate in neural function and memory regulation, opening new avenues for therapeutic interventions targeting cognitive disorders.

Abstract 5494: Spatial organization between claudin and the tumor microenvironment in head and neck squamous cell carcinomas

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading form of cancer worldwide and one of the most difficult and challenging malignancies, with a high level of heterogeneity and a wide range of treatment responses. Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer and accounts for over 90% of cancers that develop within the mucosal epithelium of the oral cavity. Additional carcinogenic risk factors, including tobacco, alcohol and HPV infection are also associated with the pathogenesis of oral carcinogenesis. Unfortunately, today the failure of standard treatment modalities, such as surgery, radiotherapy, and chemotherapy underscore the need to identify better biomarkers of this disease. Advances, especially in spatial technology have created unprecedented opportunities to identify cell types and biomarkers of disease processes, revealing important relationships within the tumor microenvironment (TME) that allow a detailed characterization of specific cell phenotypes defined by co- or lack of expression of multiple markers that may help in predicting clinical responses and mechanisms of resistance to therapy. Recent findings indicate that abnormal expression of claudins (CLDNs) is closely related to the occurrence, progression and prognosis of many solid tumors. Specifically, CLDN6 is expressed in numerous tumors but rarely found in adult normal tissues making it a potential therapeutic target in head and neck tumor types. To investigate this further, expression of CLDN6 and the its relationship with changes within the TME of cohort OSCC tumors was determined using a proprietary comprehensive spatial workflow and gene expression analysis. Methods: A retrospective analysis was performed on biopsied Formalin-Fixed Paraffin-Embedded (FFPE) samples from OSCC patients with and without a history of smoking and treated +/- chemotherapy. Patient samples representing matched normal tissue to serve as controls were compared to samples derived from OSCC tumors. In order to perform a comprehensive expression profiling of the TME from OSCC tumors we used the spatial MultiOmyx™ immunofluorescence (IF) multiplexing assay with transcriptomic information from bulk mRNA gene expression acquired using the NanoString nCounter® PanCancer IO 360™ panel that includes the Tumor Inflammation Signature (TIS), as well as targets that characterize immune hot, desert, and cold regions in the stroma and tumor epithelium. Results: We characterized the expression of CLDN6 across the different cohorts of OSCC samples and further investigated the relationship within the tumor immune microenvironment coupled to genes related to the prognosis of OSCC. A better understanding of the TME and gene expression profiling of OSCC tumors and the effect of smoking will allow us to tailor the treatment of patients to continue to improve outcomes. Citation Format: Kirsteen Maclean, Lakshmi Chandramohan, Maryam Rohafza, Harry Nunns, Erinn Parnell, Qingyan Au, Anna Juncker-Jensen. Spatial organization between claudin and the tumor microenvironment in head and neck squamous cell carcinomas [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 5494.

Abstract 7501: Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system

Abstract Background: Targeting rare cell types such as circulating tumor cells (CTCs), considered to be metastatic precursors in cancer progression, may provide long-term clinical benefits by enhancing patient outcomes. The study of CTCs comes with technical challenges due to their heterogeneity and low cell numbers, leading to difficulties in genomic characterization. This study aims to overcome these technical challenges by developing a comprehensive gene expression profiling workflow using qPCR and RNA sequencing (RNA-Seq) on rare cells enriched with Genesis System with Celselect SlidesTM. Methods: To mimic CTCs in blood, we created a rare cell model sample by spiking A549 non-small cell lung cancer cells at concentrations of 50, 100, 500 and 1000 cells/ml in whole blood from healthy donors. The Genesis System, which can capture rare cells of 8µm-30µm, isolated 83.73% of spiked A549 cells in whole blood. Using the same blood samples without A549 spiked in, we enriched cells using the Genesis and it was used as a baseline for gene expression controls. High quality RNA was extracted from each of the enriched samples and used for both qPCR and RNA-Seq to detect the gene expression signature of the non-small cell lung cancer cells. Results: Our analyses revealed significant differential expression of cancer-related genes (p &amp;lt; 0.05) in all A549-spiked samples when compared to the blood-only controls. Notably, in the 50 cells/ml dilution, RNA-Seq analysis identified over 3,000 differentially expressed genes, encompassing protein-coding genes, long non-coding RNAs, and small non-coding RNAs. Among these, 759 genes were consistently present across all enriched samples, suggesting a common expression pattern. To investigate the potential utility of these genes as non-small cell lung cancer biomarkers, we constructed a custom qPCR array comprising 38 of these genes and pre-amplified their targets using 50pg of RNA from the enriched cell samples. Notably, six genes from this panel (MET, TOP2A, NF1, SPP1, GAPDH, and ANXA5) exhibited significant differential expression (+/- 4-fold, p&amp;lt;0.05) in the enriched samples. MET and TOP2A, in particular, are well-known biomarkers for non-small cell lung cancer. Conclusion: Our liquid biopsy workflow demonstrates the recovery of high-quality RNA from enriched rare cells, including CTCs. It enables comprehensive profiling of the tumor transcriptome, encompassing RNAs of various sizes, including miRNAs. Our approach has the potential to facilitate the identification of patient-specific biomarkers, enabling swift monitoring using preamplification and qPCR in advancement of targeted oncology therapy and research. Citation Format: Srikanth Perike, Angelica Olcott, Cissy Jiang, Nish Kumar, Nathan Knapp, Jennifer Placek, Candice Cox, Yoon-Tae Kang, Linda Lingelbach, Elizabeth Dreskin. Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system [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 7501.

Comprehensive pan-cancer analysis of 33 human cancers reveals immunotherapeutic value of focal adhesion tyrosine kinase.

The immune environment in tumors is the key factor affecting the survival and immunotherapeutic response of patients. This research aimed to explore the underlying association between focal adhesion tyrosine kinase (FAK/PTK2) and cancer immunotherapy in 33 human cancers. Gene expression data and clinical features of 33 cancers were retrieved from the Cancer Genome Atlas Database. The immunotherapy cohorts included GSE67501, GSE78220, and IMVIGOR210, which were derived from the comprehensive gene expression database or from previous studies. Clinical parameters including patient age, gender, survival rate, and tumor stage were analyzed to evaluate the prognostic value of FAK/PTK2. FAK/PTK2 activity was detected by single-sample gene set enrichment analysis and used to compare the difference between FAK/PTK2 transcriptome and protein expression levels. To better understand the role of FAK/PTK2 in cancer immunotherapy, we analyzed its correlations with tumor microenvironment and with immune processes/elements (e.g., immune cell infiltration, immunosuppressants, and stimulants) and major histocompatible complexes. Potential pathways associated with FAK/PTK2 signaling in cancers were also explored. Correlations between FAK/PTK2 and 2 immunotherapeutic biomarkers (tumor mutation load and microsatellite instability) were studied. Finally, the 3 independent immunotherapy cohorts were used to study the relationship between FAK/PTK2 and immunotherapeutic response. Although FAK/PTK2 is not closely associated with age (13/33), gender (5/33), or tumor stage (5/33) in any of the studied human cancers, it has potential prognostic value for predicting patient survival. Consistency between FAK/PTK2 activity and expression exists in some cancers (3/33). Generally, FAK/PTK2 is robustly correlated with immune cell infiltration, immune modulators, and immunotherapeutic markers. Moreover, high FAK/PTK2 expression is significantly related to immune-relevant pathways. However, FAK/PTK2 is not significantly correlated with the immunotherapeutic response. Research on the immunotherapeutic value of FAK/PTK2 in 33 human cancers provides evidence regarding the function of FAK/PTK2 and its role in clinical treatment. However, given the use of a bioinformatics approach, our results are preliminary and require further validation.

Abstract 6775: 3D culture aggregation morphology depends on cell phenotype and gene expression profiles in lung cancer

Abstract Background: Three-dimensional (3D) cell culture allows for the reproduction of tissue structure and function in a state more akin to the in vivo environment compared to two-dimensional (2D) cell culture. It is established that 3D culture more faithfully replicates cell morphology, differentiation, proliferation, gene and protein expression, and responsiveness to stimulation, as well as drug metabolism. Focusing on the observation that lung cancer cell lines exhibit different clustering morphologies in 3D cell culture, we conducted a comparative study on the relationship between these clustering morphologies and the characteristics of the lung cancer cell lines. Methods: We cultured 30 lung cancer cell lines using 3D culture plate, and classified the types of cell clustering morphologies formed. We evaluated the molecular and cellular biology characteristics of all cell lines. Additionally, comprehensive gene expression analysis using RNA sequencing was performed on the 30 3D-cultured cell samples, and the characteristics of gene expression between different clustering types were compared. Results: The 3D culture aggregation morphology of 30 lung cancer cell lines were classified into three types (monolayer sheet, NOS, spheroid). Monolayer sheet-type cells exhibited higher proliferation, migration, and invasion capabilities compared to spheroid-type cells. In the transcriptomic bioinformatics analysis of the acquired gene expression data, GSEA analysis using Hallmark gene sets showed that the enrichment of epithelial-mesenchymal transition (EMT) and cancer metastasis-related genes in the monolayer type cells significantly compared to the spheroid type cells. Conversely, cell adhesion and intercellular binding-related gene sets were highly enriched in the spheroid type cells. Conclusion: 3D culture aggregation morphology represents phenotypic properties and transcriptomic profiles in non-small cell lung cancer cells. Citation Format: Naohiro Hayashi, Shimpei Tsudaka, Ken Suzawa, Tomoaki Higashihara, Yin Min Thu, Atsushi Matsuoka, Daisuke Mizuno, Ryo Yoshichika, Fumiaki Mukohara, Mao Yoshikawa, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. 3D culture aggregation morphology depends on cell phenotype and gene expression profiles in lung cancer [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 6775.

The RNA sequencing results revealed the expression of different genes and signaling pathways during chemotherapy resistance in peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) is a subtype of non-Hodgkin's lymphoma that occurs primarily at extranodal sites and is commonly treated using chemotherapy and radiotherapy. PTCL is more malignant than other lymphoid tumors, resulting in a poor prognosis.The 5-year recurrence rate remains high, and there is a lack of standard treatment for patients with relapse-resistant disease. However, the molecular mechanisms underlying the resistance of peripheral T-cell lymphoma cells to chemotherapeutic drugs, as well as identifying strategies to overcome drug resistance remains unclear. In this study, we aimed to identify pivotal genes and signaling pathways associated with chemotherapy resistance in PTCL.MethodsIn this study, a total of 5 healthy controls and 7 clinical patients were enrolled; 4 patients were classified as chemotherapy sensitive, and 3 patients were classified as chemotherapy resistant. Peripheral blood samples were collected from each participant, and total RNA was extracted from the white blood cells. RNA sequencing was conducted on the Illumina HiSeq platform to obtain comprehensive gene expression profiles. Subsequently, the expression patterns of the DEGs associated with the most enriched signaling pathways, with a special focus on cancer-related genes, were validated using quantitative real-time polymerase chain reaction (qRT–PCR) in peripheral TCL patients.ResultsRNA sequencing (RNA-seq) analysis revealed 4063 differentially expressed genes (DEGs) in peripheral T-cell lymphoma specimens from patients with chemotherapy resistance, of which 1128 were upregulated and 2935 were downregulated. Subsequent quantitative gene expression analysis confirmed a differential expression pattern in all the libraries, with 9 downregulated genes and 10 upregulated genes validated through quantitative real-time PCR in 6 clinical specimens from patients with chemotherapy resistance. KEGG pathway analysis revealed significant alterations in several pathways, with 6 downregulated pathways and 9 upregulated pathways enriched in the DEGs. Notably, the TNF signaling pathway, which is extensively regulated, was among the pathways that exhibited significant changes. These findings suggest that DEGs and the TNF signaling pathway may play crucial roles in chemotherapy resistance in peripheral T-cell lymphoma.ConclusionOur study revealed that the expression of specific genes, including TNFRSF1B, TRADD2, and MAP3K7, may play an important role in chemotherapy resistance in peripheral T-cell lymphoma. Moreover, we identified the downregulation of the TNF signaling pathway, a crucial pathway involved in cell survival, death, and differentiation, as a potential contributor to the development of chemotherapy resistance in peripheral T-cell lymphoma. These findings provide valuable insights into the molecular mechanisms underlying chemotherapy resistance and highlight potential targets for overcoming treatment resistance in this challenging disease.