Bulk RNA-seq Analysis Research Articles

SPI1 Is the Master Regulator of the Small Cell Variant of Anaplastic Large Cell Lymphoma Controlled By Methylation of SPI1 Gene Promoter Region

Introduction The small cell variant of anaplastic large cell lymphoma (SC-ALCL) is a subtype of anaplastic lymphoma kinase (ALK)-positive ALCL characterized by chemoresistance and poor prognosis, necessitating novel treatment strategies. Recent advances in targeted therapies have resulted in significant responses in various chemoresistant hematological malignancies. Therefore, understanding the underlying oncogenic mechanisms and identifying potential therapeutic targets are critical for overcoming SC-ALCL. Immunohistochemically, SC-ALCL comprises two distinct tumor cell populations: small tumor cells demonstrating negative to weak ALK and CD30 protein expression, and large tumor cells with robust expression of these proteins. We previously reported that small tumor cells display particular resistance to chemotherapy; however, the mechanisms underlying chemoresistance and the molecular characteristics of small tumor cells remain elusive. This study aimed to elucidate the molecular attributes of small tumor cells and pave the way for the development of innovative treatment approaches. Methods We investigated the characteristics of small tumor cells using patient blood samples diagnosed with SC-ALCL leukemic presentation and publicly available RNA-seq data from eight ALK-positive ALCL cell lines. Small and large tumor cells were separated from blood samples based on CD30 expression using CD30 - a phycoerythrin antibody and a magnetic bead separation kit. The presence of the NPM-ALK fusion gene was confirmed in both populations by PCR analysis. Bulk RNA-seq analysis was performed on four samples: small and large tumor cells at the onset and at the first (mainly occupied by small tumor cells) and second (mainly occupied by large tumor cells) relapses. Gene set enrichment analysis was used to identify gene expression patterns in small tumor cells. Signature genes of small tumor cells were identified by filtering with the following thresholds: false discovery rate-adjusted P-value <0.05, and fold-change >2 compared to large tumor cells. The transcription factors regulating the signature genes of small tumor cells were predicted using the g:Profiler software. Result Bulk RNA sequencing analysis of the four blood samples revealed that the SPI1 gene was significantly upregulated in small tumor cells compared to that in large tumor cells. SPI1 has emerged as the highest-scoring transcription factor, regulating the expression of 570 genes that characterize small tumor cells. Approximately 70% of these signature genes overlapped with the SPI1 target genes. Furthermore, gene set enrichment analysis revealed that small tumor cells display increased anti-apoptotic gene activity and decreased cell proliferation gene activity. These observations were validated by integrated gene expression analysis using publicly available RNA-seq data from eight ALK-positive ALCL cell lines (Figure 1). The study found high expression of SPI1 and SPI1 targeted genes in small tumor cells, accompanied by low expression of anti-apoptotic genes in ALK-positive ALCL cell lines, which were more analogous to large tumor cells than small tumor cells. Single-cell RNA-seq analysis validated the high expression of the SPI1 gene in small tumor cells. To explore the mechanism regulating SPI1 gene expression, we examined DNA methylation patterns in the SPI1 promoter region using conventional bisulfite sequencing. Conventional bisulfite sequencing revealed a hypomethylated SPI1 promoter region in small tumor cells and a hypermethylated region in large tumor cells, potentially accounting for the differential SPI1 expression. Conclusion This study highlights SPI1gene as the master regulator determining the unique characteristics of small tumor cells in SC-ALCL. Furthermore, the regulation of SPI1 gene expression appeared to be mediated by DNA methylation patterns within the SPI1 promoter region. These findings pave the way for innovative treatment strategies for SC-ALCL such as SPI1 targeting therapy and methylation-based therapies, offering promising avenues for treating SC-ALCL.

North American Adult T-Cell Leukemia/Lymphoma Has Frequent Mutations in CCR4 and Responds in Vitro to a Small Molecule CCR4 Antagonist, CCR4-351

Adult T-cell leukemia/lymphoma (ATLL) is a rare and aggressive disease of malignant CD4+ T cells that develops in human T-lymphotropic virus-1 (HTLV-1) carriers. Patients diagnosed with ATLL have the worst survival among all peripheral T-cell lymphomas. There is an urgent need for in-depth understanding of its pathogenesis and development of new treatment strategies. We and others have shown that ATLLs diagnosed in the Japanese (J-ATLL) and North American (NA-ATLL) patients have differences in clinical characteristics and immune-genetic landscape. Among the most frequently mutated genes in J-ATLL cohorts is CCR4, a chemokine receptor that is targeted by C-terminal truncation mutations in one third of the cases. These mutations result in a more stabilized surface CCR4 and have been reported to cause elevated Akt activation following chemokine stimulation. Since our previous targeted exon sequencing study did not include CCR4, this work was initiated to assess CCR4 mutation frequency in NA-ATLL cases and to evaluate the preclinical activity of a small molecule CCR4 antagonist. This is because, although an anti-CCR4 mAb, mogamulizumab, has been approved in Japan to treat ATLL, this therapy failed in a Phase 2 trial among patients outside of Japan. Thus, mogamulizumab is not available as a routine treatment for NA-ATLL patients. We compiled CCR4 gene status data from bulk RNA-seq (n=10) and whole genome sequencing (WGS) analysis (n = 10). Out of a total of 17 NA-ATLL cases, 8 (47.1%) carried CCR4 gene alterations that included in-frame and frameshift indels, as well as nonsense mutations targeted to the C-terminus cytoplasmic tail and also the trans-membrane domain. In subtype analysis, 6 out 14 acute cases (42.9%) and 2 out of 3 (66.7%) of chronic cases carried at least one copy of mutated CCR4 gene, indicating that the mutation frequency in NA-ATLL is possibly even higher than that reported for the Japanese cohorts ( Figure 1). We then evaluated cell surface CCR4 expression using flow cytometry. The primary ATLL cells (CD4 +CD7 -CD8 -) from all 7 diagnostic samples uniformly express markedly increased levels of CCR4 relative to healthy control CD4 T cells. Interestingly, CCR4 expression among 8 ATLL cell lines is highly variable, implying that in vivo, tumor microenvironment factors may also contribute to sustained CCR4 upregulation in addition to the influence from the HTLV-1 oncoprotein, HBZ. FLX475 is an orally active small molecule CCR4 antagonist that is currently in a Phase 1/2 study (NCT03674567) in several types of advanced cancer including EBV+ NK/T cell lymphoma. So far, favorable clinical antitumor activity has been observed that includes complete responses with FLX475 monotherapy and encouraging combination activity (Lin C.-C. et al ESMO-IO 2022 Dec; #187P). Using several CCR4-positive cell lines, we examined in vitro anti-ATLL activity of CCR4-351, a CCR4 antagonist that is a highly related analog of FLX475. CCR4 is the exclusive homing receptor for two chemokine molecules, CCL17 and CCL22. In a transwell assay, both chemokines can elicit strong chemotaxis activity in a J-ATLL cell line ED40515(+). However, only CCL22 but not CCL17 can induce significant chemotaxis in two NA-ATLL cell lines, ATL#13 and ATL#29. The exact cause of this difference between these cell lines awaits future studies. In these transwell-based assays, CCR4-351 exhibited potent chemotaxis inhibitory activity with IC 50 values ranging from 7-60 nM ( Figure 2). In addition, when the cell lines were cultured under growth factor-deprivation conditions, CCR4-351 treatment caused a mild reduction in total cell viability, suggesting an additional anti-ATLL mechanism of action. In summary, in this first study of the CCR4 pathway status in NA-ATLL, we found that nearly half of the cases in our ATLL cohort carried a mutated CCR4 gene. Contrary to primary ATLL cells, ATLL cell lines exhibit a wide variation in CCR4 expression. CCL22 but not CCL17 induced strong chemotaxis behavior in NA-ATLL cell lines, which was potently inhibited by a small molecule CCR4 antagonist, CCR4-351. Since extramedullary presentation is frequently seen in NA-ATLL and central nervous system involvement is an adverse prognostic feature, inhibiting chemotaxis with a CCR4 antagonist such as FLX475 may be an effective therapeutic approach.

Hematopoietic Stem and Progenitor Cells Acquire an Inflammatory Memory of the Acute Myeloid Leukemia Niche

Recent studies suggest that acute myeloid leukemia (AML) generates inflammation in the bone marrow (BM) niche to an extent that compromises the function of healthy hematopoietic stem and progenitor cells (HSPC) and impacts patient prognosis. AML xenograft studies have identified several pro-inflammatory mediators (IL-6, IL-1b) that are secreted into the BM niche by leukemic blasts. Prior work by our laboratory also showed contributions involving other AML-secreted factors, in regulating HSPCs in the leukemic niche. HSPC are cellular components of the innate immune system, but the full extent of their inflammatory adaptation by AML is unknown. To overcome some of the experimental constraints inherent in the xenograft approach, we first established non-conditioned congenic grafts of C1498 murine AML cells in C57B/6 recipients. Assessment of the BM revealed that, even at low AML burden, these animals contained a range of elevated pro-inflammatory factors. Single cell RNA-Seq studies of healthy HSPCs, obtained concurrently at sacrifice confirmed an inflammatory transcriptional signature in HSC and MPP populations, with GSEA enrichment for interferon-alpha and -gamma signaling pathways. These observations led us to hypothesize that AML-experienced (inflammation-primed) healthy HSPCs may acquire a durable long-term inflammatory memory that shapes their response to subsequent stimulation during relapse or infection. To test this hypothesis, we adoptively transferred FACS-sorted healthy AML-experienced HSPCs from C1498-engrafted mice (HSPC AML) into secondary recipients. HSPCs transplanted from PBS-injected recipients (HSPC PBS) serve as controls. To assess whether HSPC AML would respond to immune challenge more aggressively compared to HSPC PBS, we exposed these animals with a single-injection of lipopolysaccharide (LPS), followed by tissue harvest and analysis after 24 hours. Bulk RNA-Seq analysis of HSPC AML from the LPS-challenged recipients showed amplified GSEA signatures for tumor necrosis factor- and NF-kB signaling pathways compared to HSPC PBS. In addition, LPS-challenged HSPC AML showed amplified expression by several krüppel-like factors (KLF) genes. Analysis for transposase-accessible chromatin loci (ATAC-Seq), concurrently obtained from LPS-challenged HSPC AML echoed the enrichment of KLF motifs as well. Collectively, the C1498 model suggests that initial AML exposure programs HSPC AML for an amplified inflammatory and differentiation program. To further ascertain disease relevance and reproducibility we turned to a doxycycline-inducible (i)MLL-AF9 AML model. Given the low leukemic burden in the BM of C1498-grafted animals, we wanted to test the hypothesis that secreted factors accounted, at least in part, for the inflammatory memory. Indeed, injection of AML secreted factors from iMLL-AF9 models revealed a strong inflammatory signature in bulk RNA-Seq studies. To determine if innate immune priming in HSPC can be directly mediated by AML crosstalk, we exposed ex vivo-expanded HSPCs to purified secreted factors (SF AML) from (i)MLL-AF9 AML blasts, followed by a secondary inflammatory challenge with LPS seven days later. Gene expression analysis of SF AML-experienced LPS-challenged HSPCs confirmed myeloid-skewed progenitor differentiation and increased expression of inflammatory targets. Together, our data show for the first time that phenotypically normal, AML-experienced HSPCs carry a durable inflammatory legacy whereby primary exposure to AML secreted factors alters their response to secondary stimuli. Our experiments phenocopy observations of trained immunity in other experimental systems, and suggest the acquisition of an innate immune memory in the AML niche. Immune priming may impact the role of these HSPC in the context of inflammation associated with aging, or clones with predisposing molecular lesions such as TET2 or DNMT3A.

A chemosensory-like histidine kinase is dispensable for chemotaxis in vitro but regulates the virulence of Borrelia burgdorferi through modulating the stability of RpoS.

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1β, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.

Inhibition of the urea cycle by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin increases serum ammonia levels in mice

The aryl hydrocarbon receptor is a ligand-activated transcription factor known for mediating the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. TCDD induces nonalcoholic fatty liver disease (NAFLD)-like pathologies including simple steatosis that can progress to steatohepatitis with fibrosis and bile duct proliferation in male mice. Dose-dependent progression of steatosis to steatohepatitis with fibrosis by TCDD has been associated with metabolic reprogramming, including the disruption of amino acid metabolism. Here, we used targeted metabolomic analysis to reveal dose-dependent changes in the level of ten serum and eleven hepatic amino acids in mice upon treatment with TCDD. Bulk RNA-seq and protein analysis showed TCDD repressed CPS1, OTS, ASS1, ASL, and GLUL, all of which are associated with the urea cycle and glutamine biosynthesis. Urea and glutamine are end products of the detoxification and excretion of ammonia, a toxic byproduct of amino acid catabolism. Furthermore, we found that the catalytic activity of OTC, a rate-limiting step in the urea cycle was also dose dependently repressed. These results are consistent with an increase in circulating ammonia. Collectively, the repression of the urea and glutamate-glutamine cycles increased circulating ammonia levels and the toxicity of TCDD.

Sfrp1 as a Pivotal Paracrine Factor in the Trained Pericardial Stem Cells that Foster Reparative Activity.

Tissue damage often induces local inflammation that in turn dictates a series of subsequential responses, such as stem cell activation and growth, to maintain tissue homeostasis. The aim of the study is to testify the possibility of using inflammation-trained stem cells as optimal donor cells to augment the efficacy of cell therapy. The pericardial stem/stromal cells derived from the animals after myocardial infarction (MI-pSC) showed an enhanced myogenic potential and augmented reparative activity after transplantation in the injured hearts, as compared to the Sham-pSC. Bulk RNA-Seq analysis revealed significant upregulation of a panel of myogenic and trophic genes in the MI-pSC and, notably, Sfrp1 as an important anti-apoptotic factor induced robustly in the MI-pSC. Injection of the MI-pSC yielded measurable numbers of surviving cardiomyocytes (Tunel and Casp-3 negative) within the infarct area, but the effects were significantly diminished by siRNA-based silence of Sfrp1 gene in the pSC. Primed Sham-pSC with pericardial fluid from MI rats mimicked the upregulation of Sfrp1 and enhanced myogenic potential and reparative activity of pSC. Taken together, our results illustrated the inflammation-trained pSC favor a reparative activity through upregulation of Sfrp1 gene that confers anti-apoptotic activity in the injured cardiomyocytes. Therefore, the active form of stem cells may be used as a cardiac protective agent to boost therapeutical potential of stem cells.

Transcriptomic Analysis of Clonal Hematopoiesis in Solid Tumors Reveals Driver Mutation-Specific Patterns of Immune Dysregulation

Introduction: Clonal hematopoiesis (CH) is a pre-malignant state wherein somatic mutations confer a proliferative advantage to hematopoietic stem cells, driving inflammatory changes that increase the risk of age-related diseases and mortality. CH is common in individuals with solid cancers and associated with poorer overall survival due to increased tumor progression, raising the possibility that CH alters the tumor-immune microenvironment (TIME). This study defines the inflammatory program of tumor-infiltrating leukocytes to help elucidate the role of CH in the TIME. Methods: Somatic mutations compatible with CH were detected in the whole exome sequences of 6,537 cancer patients with 16 major cancer types from The Cancer Genome Atlas (TCGA) project using Mutect2. CH variant curation followed a pre-defined list of putative variants in 63 genes known to drive hematologic clonality. Calls were filtered to remove sequencing artifacts, germline variants, and small clones with variant allele fraction (VAF) < 2%. Bulk RNA-seq analysis was conducted on data from the colon adenocarcinoma (n=240) and lung adenocarcinoma (n=402) groups. Sequencing reads were pseudo-aligned and quantified with kallisto, then differential expression analysis was performed with DESeq2, controlled for cancer type, tumor biopsy site, and patient sex. Differentially expressed genes were subject to gene set enrichment analysis (GSEA) of gene ontology (GO) terms using clusterProfiler. Results: The prevalence of CH in this cohort was 8.8% (n=576/6,537) and its presence was strongly associated with age (p=1.0x10 -25). The highest rate of CH was found in cutaneous melanoma (23.5%, n=110/468) and the lowest rate was in esophageal carcinoma (2.4%, n=3/126). Most patients with CH had just 1 variant (82.8%, n=477/576), but some patients exhibited 2 variants (10.1%, n=58/576), and a small group of patients carried 3 or more variants. A total of 772 CH variants were detected in the cohort, and as expected their distribution across the CH driver genes was not uniform: DNMT3A mutations were most common (n=193), followed by TET2 (n=59), and ASXL1 (n=47; Fig 1). KMT2C mutations were also present in this cohort (n=41) at far higher rates than reported in the general population, providing possible evidence that KMT2C-mutant CH is associated with cancer or its treatments. Across the group of colon and lung cancer patients subject to RNA-seq analysis, a total of 220 genes (111 upregulated, 109 downregulated) were found to be differentially expressed by CH status at p adj < 0.05. GSEA of GO terms revealed significant activation of gene sets involved in innate and adaptive immune processes with upregulation of both positive and negative regulatory systems, suggesting more intricate mechanisms of immune dysfunction as opposed to generalized hyper-inflammatory activity in the TIME (Fig. 2). GSEA also revealed enrichment of non-immune pathways tied to tumorigenesis. Among the significantly dysregulated genes are the neutrophil activators PPBP and CXCL6, monocyte/macrophage markers LILRA2, CPM, CHI3L1, and CD163L1, and the natural killer cell receptors KLRC2/3. Strikingly, these inflammatory changes were absent in patients with DNMT3A, TET2, or ASXL1 (DTA) CH, and were instead driven by non-DTA CH mutations in a VAF-dependent manner. Investigation of tumor transcriptomes within each cancer type revealed that CH in colon cancer patients has reciprocal effects on inflammation and immunity - DTA CH in this cancer demonstrated suppression of immune-associated gene sets while non-DTA CH was more consistent with the general trend of immune enrichment and activation. Conclusions: This work demonstrates that while general inflammatory changes are evident at the tumor level with CH, there is a critical need to recognize the nuances of CH-associated immune mechanisms in cancer, which appear to vary by cancer type and CH driver. Ongoing efforts will continue to characterize the TIME of the other TCGA cancers through transcriptomic analysis and in situ histological imaging. This study also provides some early clinical evidence for the selection of KMT2C variants in cancer patients, though more work is needed to understand their clonal dynamics and clinical implications. In light of these findings, future studies will pave the way for CH as a biomarker in cancer medicine and a cornerstone of precision immuno-oncology.

Identification of the immune-associated characteristics and predictive biomarkers of keratoconus based on single-cell RNA-sequencing and bulk RNA-sequencing.

Whether keratoconus (KC) is an inflammatory disease is currently debated. Hence, we aimed to investigate the immune-related features of KC based on single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) data. scRNA-seq data were obtained from the Genome Sequence Archive (GSA), bulk RNA-seq data were obtained from the Gene Expression Omnibus (GEO), and immune-associated genes(IAGs) were obtained from the ImmPort database. Cell clusters of KC were annotated, and different cell clusters were then selected. The IAG score of each cell was calculated using the AUCell package. Three bulk RNA-seq datasets were merged and used to identify the differentially expressed genes (DEGs), biological functions, and immune characteristics. Weighted gene coexpression network analysis (WGCNA) was used to select the IAG score-related hub genes. Based on scRNA-seq and bulk RNA-seq analyses, three machine learning algorithms, including random forest (RF), support vector machine (SVM), and least absolute shrinkage and selection operator (LASSO) regression analysis, were used to identify potential prognostic markers for KC. A predictive nomogram was developed based on prognostic markers. Six cell clusters were identified in KC, and decreased corneal stromal cell-5 (CSC-5) and increased CSC-6 were found in KC. CSC and immune cell clusters had the highest IAG scores. The bulk RNA-seq analysis identified 1362 DEGs (553 upregulated and 809 downregulated) in KC. We found different immune cell populations and differentially expressed cytokines in KC. More than three key IAG score-related modules and 367 genes were identified. By integrating the scRNA-seq and bulk RNA-seq analyses, 250 IAGs were selected and then incorporated into three machine learning models, and 10 IAGs (CEP112, FYN, IFITM1, IGFBP5, LPIN2, MAP1B, RNASE1, RUNX3, SMIM10, and SRGN) were identified as potential prognostic genes that were significantly associated with cytokine and matrix metalloproteinase(MMP)1-14 expression. Finally, a predictive nomogram was constructed and validated. Taken together, our results identified CSCs and immune cell clusters that may play a key role during KC progression by regulating immunological features and maintaining cell stability.

The slan antigen identifies the prototypical non-classical CD16+-monocytes in human blood.

Peripheral monocytes in humans are conventionally divided into classical (CL, CD14++CD16-), intermediate (INT, CD14++CD16+) and non-classical (NC, CD14dim/-CD16++) cells, based on their expression levels of CD14 and CD16. A major fraction of the NC-monocytes has been shown to express the 6-sulfo LacNAc (slan) antigen, but whether these slan+/NC-monocytes represent the prototypical non-classical monocytes or whether they are simply a sub-fraction with identical features as the remainder of NC monocytes is still unclear. We analyzed transcriptome (by bulk and single cell RNA-seq), proteome, cell surface markers and production of discrete cytokines by peripheral slan+/NC- and slan-/NC-monocytes, in comparison to total NC-, CL- and INT- monocytes. By bulk RNA-seq and proteomic analysis, we found that slan+/NC-monocytes express higher levels of genes and proteins specific of NC-monocytes than slan-/NC-monocytes do. Unsupervised clustering of scRNA-seq data generated one cluster of NC- and one of INT-monocytes, where all slan+/NC-monocytes were allocated to the NC-monocyte cluster, while slan-/NC-monocytes were found, in part (13.4%), within the INT-monocyte cluster. In addition, total NC- and slan-/NC-monocytes, but not slan+/NC-monocytes, were found by both bulk RNA-seq and scRNA-seq to contain a small percentage of natural killer cells. In addition to comparatively characterize total NC-, slan-/NC- and slan+/NC-monocyte transcriptomes and proteomes, our data prove that slan+/NC-, but not slan-/NC-, monocytes are more representative of prototypical NC-monocytes.

Deciphering intratumoral heterogeneity of hepatocellular carcinoma with microvascular invasion with radiogenomic analysis

Background and aimsThe recurrence and metastasis of hepatocellular carcinoma (HCC) are mainly caused by microvascular invasion (MVI). Our study aimed to uncover the cellular atlas of MVI+ HCC and investigate the underlying immune infiltration patterns with radiomics features.MethodsThree MVI positive HCC and three MVI negative HCC samples were collected for single-cell RNA-seq analysis. 26 MVI positive HCC and 30 MVI negative HCC tissues were underwent bulk RNA-seq analysis. For radiomics analysis, radiomics features score (Radscore) were built using preoperative contrast MRI for MVI prediction and overall survival prediction. We deciphered the metabolism profiles of MVI+ HCC using scMetabolism and scFEA. The correlation of Radscore with the level of APOE+ macrophages and iCAFs was identified. Whole Exome Sequencing (WES) was applied to distinguish intrahepatic metastasis (IM) and multicentric occurrence (MO). Transcriptome profiles were compared between IM and MO.ResultsElevated levels of APOE+ macrophages and iCAFs were detected in MVI+ HCC. There was a strong correlation between the infiltration of APOE+ macrophages and iCAFs, as confirmed by immunofluorescent staining. MVI positive tumors exhibited increased lipid metabolism, which was attributed to the increased presence of APOE+ macrophages. APOE+ macrophages and iCAFs were also found in high levels in IM, as opposed to MO. The difference of infiltration level and Radscore between two nodules in IM was relatively small. Furthermore, we developed Radscore for predicting MVI and HCC prognostication that were also able to predict the level of infiltration of APOE+ macrophages and iCAFs.ConclusionThis study demonstrated the interactions of cell subpopulations and distinct metabolism profiles in MVI+ HCC. Besides, MVI prediction Radscore and MVI prognostic Radscore were highly correlated with the infiltration of APOE+ macrophages and iCAFs, which helped to understand the biological significance of radiomics and optimize treatment strategy for MVI+ HCC.

THU551 Glycoprotein-NMB (GPNMB) Is A Potential Lymphangioleiomyomatosis (LAM) Therapeutic Target And Biomarker

Abstract Disclosure: E. Gibbons: None. S.R. Hammes: None. Lymphangioleiomyomatosis (LAM) is a rare, progressive, destructive cystic lung disease affecting almost exclusively female-sexed individuals. Mutations in the tuberous sclerosis (TSC1 or TSC2) genes constitutively activate the mTORC1 pathway, promoting hyperproliferation of smooth muscle cells, creating small tumors throughout the lungs. These tumors, through largely unknown mechanisms, result in the formation of large cysts that replace normal alveolar space. Inhibition of mTORC1 with Sirolimus slows but does not stop LAM progression; therefore, there is a need for new LAM treatment options and biomarkers. Due to LAM’s estrogen sensitivity, female specificity, and metastatic nature, we previously proposed LAM cells originate from the uterine myometrium. We therefore designed a uterine-specific TSC2-null mouse model where all the mice generate uterine tumors characteristic of LAM and half develop lung metastases, suggesting that indeed LAM tumors may be derived from the uterus. Using bulk RNASeq analysis of uterine tissue from our mouse model, when focusing on genes regulated by estrogen and TSC2, we discovered increased expression of melanocytic markers, including Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB), a known oncogenic protein in other cancers. GPNMB was not only highly expressed in our mouse model; it was also expressed intracellularly and on the cell surface of TSC2-null cell lines and in LAM tumor cells from patient lungs and uteri. Notably, the extracellular domain of GPNMB was released from the surface of TSC2-null cell lines, suggesting that the ectodomain may also be released from TSC2-null cells in LAM patients. Here we show that, GPNMB protein and mRNA expression increased when the cells were serum starved, mimicking conditions of a tumor microenvironment. This increase was dependent on mTORC1 signaling and mRNA transcription. Knocking down GPNMB expression by siRNA had minimal effects on TSC2-null cells pro-tumorigenic functions in vitro aside from decreasing invasion. However, knocking out GPNMB in TSC2-null cells using CRISPR/Cas9 significantly decreased mouse xenograft tumor growth. Additionally, treating mice with a GPNMB antibody drug conjugate abrogated xenograft growth, highlighting GPNMB’s potential as a therapeutic target. Also, GPNMB’s ectodomain was released from the cell surface of TSC2-null cells by the proteases Adam10 and 17. Of note, the ectodomain is implicated in mediating GPNMB’s effects on tumor growth, suggesting that Adam10/17 inhibition may mitigate TSC2-null tumor growth. Importantly, the GPNMB ectodomain is a potential serum biomarker of LAM, as there was a significant increase in GPNMB ectodomain levels in patient serum as compared with healthy controls. Furthermore, serum GPNMB levels decreased in most patients after Sirolimus treatment, implicating GPNMB as a potential marker for responsiveness to treatment. Presentation: Thursday, June 15, 2023

Divergent tumor and immune cell reprogramming underlying immunotherapy response and immune-related adverse events in lung squamous cell carcinoma

BackgroundLung squamous cell carcinoma (LUSC) remains a leading cause of cancer-related deaths with few therapeutic strategies. Immune checkpoint inhibitors (ICIs) have demonstrated promising efficacy in patients with LUSC. However, ICIs...

Deciphering Treg cell roles in esophageal squamous cell carcinoma: a comprehensive prognostic and immunotherapeutic analysis.

Background: Esophageal squamous cell carcinoma (ESCC) is a prevalent and aggressive form of cancer that poses significant challenges in terms of prognosis and treatment. Regulatory T cells (Treg cells) have gained attention due to their influential role in immune modulation within the tumor microenvironment (TME). Understanding the intricate interactions between Treg cells and the tumor microenvironment is essential for unraveling the mechanisms underlying ESCC progression and for developing effective prognostic models and immunotherapeutic strategies. Methods: A combination of single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq analysis was utilized to explore the role of Treg cells within the TME of ESCC. The accuracy and applicability of the prognostic model were assessed through multi-dimensional evaluations, encompassing an examination of the model's performance across various dimensions, such as the mutation landscape, clinical relevance, enrichment analysis, and potential implications for immunotherapy strategies. Results: The pivotal role of the macrophage migration inhibitory factor (MIF) signaling pathway within the ESCC TME was investigated, with a focus on its impact on Treg cells and other subpopulations. Through comprehensive integration of bulk sequencing data, a Treg-associated signature (TAS) was constructed, revealing that ESCC patients with elevated TAS (referred to as high-TAS individuals) experienced significantly improved prognoses. Heightened immune infiltration and increased expression of immune checkpoint markers were observed in high-TAS specimens. The model's validity was established through the IMvigor210 dataset, demonstrating its robustness in predicting prognosis and responsiveness to immunotherapy. Heightened therapeutic benefits were observed in immune-based interventions for high-TAS ESCC patients. Noteworthy differences in pathway enrichment patterns emerged between high and low-TAS cohorts, highlighting potential avenues for therapeutic exploration. Furthermore, the clinical relevance of key model genes was substantiated by analyzing clinical samples from ten paired tumor and adjacent tissues, revealing differential expression levels. Conclusion: The study established a TAS that enables accurate prediction of patient prognosis and responsiveness to immunotherapy. This achievement holds significant implications for the clinical management of ESCC, offering valuable insights for informed therapeutic interventions.

Myeloid cell-specific deletion of epidermal growth factor receptor aggravates acute cardiac injury.

Myeloid cells, including macrophages, play important roles as first responders to cardiac injury and stress. Epidermal growth factor receptor (EGFR) has been identified as a mediator of macrophage responsiveness to select diseases, though its impact on cardiac function or remodeling following acute ischemic injury is unknown. We aimed to define the role of myeloid cell-specific EGFR in the regulation of cardiac function and remodeling following acute myocardial infarction (MI)-induced injury. Floxed EGFR mice were bred with homozygous LysM-Cre (LMC) transgenic mice to yield myeloid-specific EGFR knockout (mKO) mice. Via echocardiography, immunohistochemistry, RNA sequencing and flow cytometry, the impact of myeloid cell-specific EGFR deletion on cardiac structure and function was assessed at baseline and following injury. Compared with LMC controls, myeloid cell-specific EGFR deletion led to an increase in cardiomyocyte hypertrophy at baseline. Bulk RNASeq analysis of isolated cardiac Cd11b+ myeloid cells revealed substantial changes in mKO cell transcripts at baseline, particularly in relation to predicted decreases in neovascularization. In response to myocardial infarction, mKO mice experienced a hastened decline in cardiac function with isolated cardiac Cd11b+ myeloid cells expressing decreased levels of the pro-reparative mediators Vegfa and Il10, which coincided with enhanced cardiac hypertrophy and decreased capillary density. Overall, loss of EGFR qualitatively alters cardiac resident macrophages that promotes a low level of basal stress and a more rapid decrease in cardiac function along with worsened repair following acute ischemic injury.

Deletion of Ascl1 in pancreatic β-cells improves insulin secretion, promotes parasympathetic innervation, and attenuates dedifferentiation during metabolic stress

ObjectiveASCL1, a pioneer transcription factor, is essential for neural cell differentiation and function. Previous studies have shown that Ascl1 expression is increased in pancreatic β-cells lacking functional KATP channels or after feeding of a high fat diet (HFD) suggesting that it may contribute to the metabolic stress response of β-cells. MethodsWe generated β-cell-specific Ascl1 knockout mice (Ascl1βKO) and assessed their glucose homeostasis, islet morphology and gene expression after feeding either a normal diet or HFD for 12 weeks, or in combination with a genetic disruption of Abcc8, an essential KATP channel component. ResultsAscl1 expression is increased in response to both a HFD and membrane depolarization and requires CREB-dependent Ca2+ signaling. No differences in glucose homeostasis or islet morphology were observed in Ascl1βKO mice fed a normal diet or in the absence of KATP channels. However, male Ascl1βKO mice fed a HFD exhibited decreased blood glucose levels, improved glucose tolerance, and increased β-cell proliferation. Bulk RNA-seq analysis of islets from Ascl1βKO mice from three studied conditions showed alterations in genes associated with the secretory function. HFD-fed Ascl1βKO mice showed the most extensive changes with increased expression of genes necessary for glucose sensing, insulin secretion and β-cell proliferation, and a decrease in genes associated with β-cell dysfunction, inflammation and dedifferentiation. HFD-fed Ascl1βKO mice also displayed increased expression of parasympathetic neural markers and cholinergic receptors that was accompanied by increased insulin secretion in response to acetylcholine and an increase in islet innervation. ConclusionsAscl1 expression is induced by stimuli that cause Ca2+-signaling to the nucleus and contributes in a multifactorial manner to the loss of β-cell function by promoting the expression of genes associated with cellular dedifferentiation, attenuating β-cells proliferation, suppressing acetylcholine sensitivity, and repressing parasympathetic innervation of islets. Thus, the removal of Ascl1 from β-cells improves their function in response to metabolic stress.

Clinical-mediated discovery of pyroptosis in CD8+ T cell and NK cell reveals melanoma heterogeneity by single-cell and bulk sequence

Histologically, melanoma tissues had fewer positive cells percentage of pyroptosis-related genes (PRGs), GZMA, GSDMB, NLRP1, IL18, and CHMP4A in epidermal than in normal skin. Pyroptosis, a new frontier in cancer, affects the tumor microenvironment and tumor immunotherapy. Nevertheless, the role of pyroptosis remains controversial, which reason is partly due to the heterogeneity of the cellular composition in melanoma. In this study, we present a comprehensive analysis of the single-cell transcriptome landscape of pyroptosis in melanoma specimens. Our findings reveal dysregulation in the expression of PRGs, particularly in immune cells, such as CD8+ cells (representing CD8+ T cells) and CD57+ cells (representing NK cells). Additionally, the immunohistochemical and multiplex immunofluorescence staining experiments results further confirmed GZMA+ cells and GSDMB+ cells were predominantly expressed in immune cells, especially in CD8 + T cells and NK cells. Melanoma specimens secreted a minimal presence of GZMA+ merged CD8+ T cells (0.11%) and GSDMB+ merged CD57+ cells (0.08%), compared to the control groups exhibiting proportions of 4.02% and 0.62%, respectively. The aforementioned findings indicate that a reduced presence of immune cells within tumors may play a role in diminishing the ability of pyroptosis, consequently posing a potential risk to the anti-melanoma properties. To quantify clinical relevance, we constructed a prognostic risk model and an individualized nomogram (C-index=0.58, P = 0.002), suggesting a potential role of PRGs in malignant melanoma prevention. In conclusion, our integrated single-cell and bulk RNA-seq analysis identified immune cell clusters and immune gene modules with experiment validation, contributing to our better understanding of pyroptosis in melanoma.

Ethanol Causes Cell Death and Neuronal Differentiation Defect During Initial Neurogenesis of the Neural Retina by Disrupting Calcium Signaling in Human Retinal Organoids.

Fetal Alcohol Syndrome (FAS) affects a significant proportion, exceeding 90%, of afflicted children, leading to severe ocular aberrations such as microphthalmia and optic nerve hypoplasia. During the early stages of pregnancy, the commencement of neural retina neurogenesis represents a critical period for human eye development, concurrently exposing the developing retinal structures to the highest risk of prenatal ethanol exposure due to a lack of awareness. Despite the paramount importance of this period, the precise influence and underlying mechanisms of short-term ethanol exposure on the developmental process of the human neural retina have remained largely elusive. In this study, we utilize the human embryonic stem cells derived retinal organoids (hROs) to recapitulate the initial retinal neurogenesis and find that 1% (v/v) ethanol slows the growth of hROs by inducing robust cell death and retinal ganglion cell differentiation defect. Bulk RNA-seq analysis and two-photon microscope live calcium imaging reveal altered calcium signaling dynamics derived from ethanol-induced down-regulation of RYR1 and CACNA1S. Moreover, the calcium-binding protein RET, one of the downstream effector genes of the calcium signaling pathway, synergistically integrates ethanol and calcium signals to abort neuron differentiation and cause cell death. To sum up, our study illustrates the effect and molecular mechanism of ethanol on the initial neurogenesis of the human embryonic neural retina, providing a novel interpretation of the ocular phenotype of FAS and potentially informing preventative measures for susceptible populations.

Prognosis and therapeutic benefits prediction based on NK cell marker genes through single-cell RNA-seq with integrated bulk RNA-seq analysis for hepatocellular carcinoma.

Tumor-infiltrating immune cells greatly participate in regulating tumorigenesis and metastasis of hepatocellular carcinoma (HCC). Natural killer cell, as an important role of innate immunity, plays an indispensable role in antitumor immunity and regulate tumor development. In this study, we firstly identified 251 NK cell marker genes of HCC based on single-cell RNA sequencing data. Subsequently, an NK cell marker genes-related prognostic signature (NKPS) was developed in the cancer genome atlas (TCGA) cohort for risk stratification and prognosis prediction. The predictive value of the NKPS in prognosis was well validated in different clinical subgroups and three external datasets (ICGC-LIHC cohort, GSE14520 cohort and Guilin cohort). Moreover, multivariate analysis revealed the independent prognostic value of NKPS for OS in HCC. Further functional analysis indicated the NKPS was associated with basic cellular processes, that may contribute to the development and progression of HCC. Thereafter, immune characteristics as well as the therapeutic benefits in NKPS risk score-defined subgroups were analyzed. Patients with low-risk score exhibited immune-active status, manifested as higher immune scores, more infiltration of CD8+ T cells and macrophage M1, and higher T-cell receptor (TCR) richness and diversity. Remarkably, the NKPS was negatively correlated with immunotherapy response-related signatures. In addition, the low-risk group exhibited significantly improved therapeutic benefits, either from immunotherapy or traditional chemotherapy and target therapy. Overall, the NKPS showed an excellent predictive value for prognosis and therapeutic responses for HCC, which might also provide novel insights into better HCC management strategies.

Robustness of single-cell RNA-seq for identifying differentially expressed genes

BackgroundA common feature of single-cell RNA-seq (scRNA-seq) data is that the number of cells in a cell cluster may vary widely, ranging from a few dozen to several thousand. It is not clear whether scRNA-seq data from a small number of cells allow robust identification of differentially expressed genes (DEGs) with various characteristics.ResultsWe addressed this question by performing scRNA-seq and poly(A)-dependent bulk RNA-seq in comparable aliquots of human induced pluripotent stem cells-derived, purified vascular endothelial and smooth muscle cells. We found that scRNA-seq data needed to have 2,000 or more cells in a cluster to identify the majority of DEGs that would show modest differences in a bulk RNA-seq analysis. On the other hand, clusters with as few as 50–100 cells may be sufficient for identifying the majority of DEGs that would have extremely small p values or transcript abundance greater than a few hundred transcripts per million in a bulk RNA-seq analysis.ConclusionFindings of the current study provide a quantitative reference for designing studies that aim for identifying DEGs for specific cell clusters using scRNA-seq data and for interpreting results of such studies.

Differential gene expression in the calvarial and cortical bone of juvenile female mice.

Both the calvarial and the cortical bones develop through intramembranous ossification, yet they have very different structures and functions. The calvaria enables the rapid while protected growth of the brain, whereas the cortical bone takes part in locomotion. Both types of bones undergo extensive modeling during embryonic and post-natal growth, while bone remodeling is the most dominant process in adults. Their shared formation mechanism and their highly distinct functions raise the fundamental question of how similar or diverse the molecular pathways that act in each bone type are. To answer this question, we aimed to compare the transcriptomes of calvaria and cortices from 21-day old mice by bulk RNA-Seq analysis. The results revealed clear differences in expression levels of genes related to bone pathologies, craniosynostosis, mechanical loading and bone-relevant signaling pathways like WNT and IHH, emphasizing the functional differences between these bones. We further discussed the less expected candidate genes and gene sets in the context of bone. Finally, we compared differences between juvenile and mature bone, highlighting commonalities and dissimilarities of gene expression between calvaria and cortices during post-natal bone growth and adult bone remodeling. Altogether, this study revealed significant differences between the transcriptome of calvaria and cortical bones in juvenile female mice, highlighting the most important pathway mediators for the development and function of two different bone types that originate both through intramembranous ossification.