NanoString Gene Expression Analysis Research Articles

CSIG-21. THE ROLE OF GUT MICROBIOTA IN GLIOBLASTOMA: INSIGHTS FROM MICROBIOME-GENE EXPRESSION INTERACTIONS

Abstract Glioblastoma, the most aggressive form of primary brain tumor, remains a formidable clinical challenge due to its invasiveness and resistance to therapy. Emerging evidence suggests that the gut microbiome plays a crucial role in modulating cancer progression through complex interactions with host physiology. In this study, we investigated the influence of gut microbiota and tumor gene expression by analyzing stool and tissue samples from glioblastoma patients. Utilizing 16S rRNA sequencing of stool samples and NanoString gene expression analysis of tumor tissue samples, we explored the relationship between gut microbiome taxa and gene expression levels in tissue. Pathway analysis using KEGG and Gene Ontology databases was employed to elucidate the functional implications of these interactions. Our findings reveal contrasting effects of specific gut microbiota taxa and gene expression, with Fusobacterium demonstrating a potential oncogenic role, while Bifidobacteriaceae and Akkermansiaceae display potential tumor-suppressive effects. Fusobacterium is associated with the upregulation of key oncogenes and pathways promoting cancer cell survival, growth, and metabolism, whereas Bifidobacteriaceae and Akkermansiaceae are linked to the downregulation of genes involved in pathways promoting cancer cell survival and proliferation, such as MAPK signaling and stress response pathways, potentially inhibiting cancer cell growth. These results highlight the intricate interplay between gut microbiota composition and cancer biology and underscore the potential of gut microbiota modulation as a therapeutic strategy in glioblastoma management.

Gene and Protein Expression of MAGE and Associated Immune Landscape Elements in Non-Small-Cell Lung Carcinoma and Urothelial Carcinomas.

Melanoma-associated antigen-A (MAGE-A) is expressed in multiple cancers with restricted expression in normal tissue. We sought to assess the MAGE-A3/A6 expression profile as well as immune landscape in urothelial (UC) and non-small cell lung carcinoma (NSCLC). We also assessed co-expression of immune-associated markers, including programmed cell death ligand 1 (PD-L1) in tumor and/or immune cells, and assessed the effect of checkpoint inhibitor treatment on these markers in the context of urothelial carcinoma. We used formalin-fixed paraffin-embedded (FFPE) tissue sections from a variety of tumor types were screened by IHC for MAGE-A and PD-L1 expression. Gene expression analyses by RNA sequencing were performed on RNA extracted from serial tissue sections. UC tumor samples from patients treated with checkpoint inhibitors were assessed by IHC and NanoString gene expression analysis for MAGE-A and immune marker expression before and after treatment. Overall, 84 samples (57%) had any detectable MAGE-A expression. Detectable MAGE-A expression was present at similar frequencies in both tumor tissue types, with 41 (50%) NSCLC and 43 (64%) UC. MAGE-A expression was not significantly changed before and after checkpoint inhibitor therapy by both IHC and NanoString mRNA sequencing. Other immune markers were similarly unchanged post immune checkpoint inhibitor therapy. Stable expression of MAGE-A3/A6 pre and post checkpoint inhibitor treatment indicates that archival specimens harvested after checkpoint therapy are applicable to screening potential candidates for MAGE therapies.

Upregulation of Neuroinflammation-Associated Genes in the Brain of SARS-CoV-2-Infected Mice.

Neurological manifestations are a significant complication of coronavirus disease 2019 (COVID-19), but the underlying mechanisms are yet to be understood. Recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced neuroinvasion and encephalitis were observed in K18-hACE2 mice, leading to mortality. Our goal in this study was to gain insights into the molecular pathogenesis of neurological manifestations in this mouse model. To analyze differentially expressed genes (DEGs) in the brains of mice following SARS-CoV-2 infection, we performed NanoString gene expression analysis using three individual animal samples at 1, 3, and 6 days post-infection. We identified the DEGs by comparing them to animals that were not infected with the virus. We found that genes upregulated at day 6 post-infection were mainly associated with Toll-like receptor (TLR) signaling, RIG-I-like receptor (RLR) signaling, and cell death pathways. However, downregulated genes were associated with neurodegeneration and synaptic signaling pathways. In correlation with gene expression profiles, a multiplexed immunoassay showed the upregulation of multiple cytokines and chemokines involved in inflammation and cell death in SARS-CoV-2-infected brains. Furthermore, the pathway analysis of DEGs indicated a possible link between TLR2-mediated signaling pathways and neuroinflammation, as well as pyroptosis and necroptosis in the brain. In conclusion, our work demonstrates neuroinflammation-associated gene expression profiles, which can provide key insight into the severe disease observed in COVID-19 patients.

Expression of LC3A, LC3B and p62/SQSTM1 autophagy proteins in hepatocellular carcinoma (HCC) tissues and the predicted microRNAs involved in the autophagy-related pathway.

Autophagy plays multifaceted roles in regulating hepatocellular carcinoma (HCC) and the mechanisms involved are under-explored. Regulatory microRNAs (miRNAs) have been reported to target autophagy proteins but their roles in HCC is not well studied. Using HCC patient tissues, this study aims to investigate the association of autophagy with several clinicopathological parameters as well as identifying the autophagy-related miRNAs and the possible pathways. Autophagy level in the HCC patient-derived cancer and non-cancer tissues was determined by immunohistochemistry (IHC) targeting SQSTM1, LC3A and LC3B proteins. Significance tests of clinicopathological variables were tested using the Fisher's exact or Chi-square tests. Gene and miRNA expression assays were carried out and analyzed using Nanostring platform and software followed by validation of other online bioinformatics tools, namely String and miRabel. Autophagy expression was significantly higher in cancerous tissues compared to adjacent non-cancer tissues. High LC3B expression was associated with advanced tumor histology grade and tumor location. Nanostring gene expression analysis revealed that SQSTM1, PARP1 and ATG9A genes were upregulated in HCC tissues compared to non-cancer tissues while SIRT1 gene was downregulated. These genes are closely related to an autophagy pathway in HCC. Further, using miRabel tool, three downregulated miRNAs (hsa-miR-16b-5p, hsa-miR-34a-5p, and hsa-miR-660-5p) and one upregulated miRNA (hsa-miR-539-5p) were found to closely interact with the abovementioned autophagy-related genes. We then mapped out the possible pathway involving the genes and miRNAs in HCC tissues. We conclude that autophagy events are more active in HCC tissues compared to the adjacent non-cancer tissues. We also reported the possible role of several miRNAs in regulating autophagy-related genes in the autophagy pathway in HCC. This may contribute to the development of potential therapeutic targets for improving HCC therapy. Future investigations are warranted to validate the target genes reported in this study using a larger sample size and more targeted molecular technique.

Combining VPS34 inhibitors with STING agonists enhances type I interferon signaling and anti-tumor efficacy.

An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway.

STING signalling compensates for low tumour mutation burden to drive anti-tumour immunity

While mutation-derived neoantigens are well recognized in generating anti-tumour T cell response, increasing evidences highlight the complex association between tumour mutation burden (TMB) and tumour infiltrating lymphocytes (TILs). The exploration of non-TMB determinants of active immune response could improve the prognosis prediction and provide guidance for current immunotherapy. The transcriptomic and whole exome sequence data in The Cancer Genome Atlas were used to examine the relationship between TMB and exhausted CD8+ T cells (Tex), as an indicator of tumour antigen-specific T cells across nine major cancer types. Computational clustering analysis was performed on 4510 tumours to identify different immune profiles. NanoString gene expression analysis and single cell RNA-seq analysis using fresh human breast cancer were performed for finding validation. TMB was found to be poorly correlated with active immune response in various cancer types. Patient clustering analysis revealed a group of tumours with abundant Tex but low TMB. In those tumours, we observed significantly higher expression of the stimulator of interferon genes (STING) signalling. Dendritic cells, particularly those of BATF3+ lineage, were also found to be essential for accumulation of Tex within tumours. Mechanistically, loss of genomic and cellular integrity, marked by decreased DNA damage repair, defective replication stress response, and increased apoptosis were shown to drive STING activation. These results highlight that TMB alone does not fully predict tumour immune profiles, with STING signalling compensating for low TMB in non-hypermutated tumours to enhance anti-tumour immunity. Translating these results, STING agonists may benefit patients with non-hypermutated tumours. STING activation may serve as an additional biomarker to predict response to immune checkpoint blockades alongside TMB. Our research also unravelled the interplay between genomic instability and STING activation, informing potential combined chemotherapy targeting the axis of genomic integrity and immunotherapy. City of Hope Christopher Family Endowed Innovation Fund for Alzheimer's Disease and Breast Cancer Research in honor of Vineta Christopher; Breast Cancer Alliance Early Career Investigator Award; National Cancer Institute of the National Institutes of Health under award number R01CA256989 and R01CA240392.

T Cell/Natural Killer (NK) Cell Transcriptional Profiles Are Associated with Response to Gilteritinib in FLT3-Mutated Acute Myeloid Leukemia

S.R. & A.C. equally contributed Background: Despite significative improvements in the management of FLT3-mutated AML and its prognosis since FLT3 inhibitors ( FLT3i) adoption, a significant proportion of patients results refractory or ultimately relapses after this targeted treatment. Gilteritinib is an oral and selective FLT3i with single-agent activity as salvage therapy in Relapsed/Refractory patients (R/R). The investigation of resistance mechanism to Gilteritinib is crucial to guide next therapeutic choices and improve drug effectiveness. Thus far, correlative studies have elucidated mainly intrinsic and mutational resistance mechanisms. However, less is known regarding potential extrinsic mechanisms and about the role of the immune system in influencing this FLT3i activity. Herein, we aimed at investigating intrinsic as well as novel extrinsic (immunological) mechanisms of resistance to Gilteritinib in routine clinical practice. Methods: A cohort of FLT3-mut AML patients treated with Gilteritinib at Hematology Institute of Bologna was selected. DNA/RNA were extracted from bone marrow (BM) samples pre-therapy and at first response assessment and screened by a custom NGS panel covering the full-coding sequence of 28 genes and a NanoString gene-expression analysis using the PanCancer IO 360 Panel. Data were collected in accordance with GCP and Helsinki declaration. NanoString data were acquired and analyzed using the nCounter FLEX Analysis System and the nSolver advanced analysis module (version 2.0.115). DESeq2 multi-factor design was employed to account for potential confounders. A public sc-RNA-seq dataset of newly diagnosed AMLs (Dufva et al. Cancer Cell 2020) was used to map DEGs between Responders and Non-Responders to Gilteritinib onto reference AML BM samples. Single-cell gene expression scores were computed using the UCell package in R. Results: Overall, 20 FLT3-mutated R/R AML patients receiving Gilteritinib as single-agent therapy were enrolled in this study. The median age at Gilteritinib therapy was 62 (range 33 - 77) years and 13/20 (65%) patients were male. Seventeen out of 20 (85 %) patients harbored a FLT3-ITD mutation, 2/20 (10 %) a TKD mutation and 1 (5 %) patient both mutations. Eleven out of 20 (55 %) patients had a concomitant NPM1 mutation. Patients were dichotomized into Responders (R) (n=9) and Non-Responders (NR) (n = 11) according to BM response. Intrinsic mechanisms of resistance were evaluated by NGS analyses on 13 patients (R=7 and NR=6). Firstly, FLT3 full-coding sequencing did not detect any clearly pathogenetic Non-Canonical (NC) mutation: M737I, M837I, P890L and P534L were four detected NC variants with a possible role in FLT3i sensitivity, however their low VAF (1-2 %) suggests a “passenger” role in this cohort. Secondly, evaluating variations in VAF of non- FLT3 variants detected pre- and post-therapy we observed a new variant onset in 4/6 (66.7 %) Gilteritinib NR patients, affecting genes involved in RAS pathway in all cases (1 NRAS, 2 KRAS and 1 PTPN11). Significant differences in immune transcriptional profiles were observed when comparing R and NR patients (R = 9 and NR = 11), including higher expression of genes reflecting T/NK function ( CD8A, GZMK, KLRK1, GNLY) and, more specifically, T-cell activity ( PRF1, EOMES, ZAP70, TNFSF8) in responders. Conversely, oncogenes involved in MAPK/Akt ( PROM1, ANGPT1, ANGPT2) or cell-cycle regulators ( CDKN2A, CCNA1) were overexpressed in NR patients (Fig. 1a). We next selected the top 30 DEGs between R and NR and we averaged gene expression across all BM samples. Gene set enrichment analysis (GSEA) revealed that approximately 25% of the significantly enriched pathways when using the top 30 DEGs as input were related to T-cell function / TCR signaling. By projecting the top 30 DEGs genes onto a sc-RNA-seq map of the AML tumor microenvironment, we found that they were largely expressed by CD8 + T EM, CD8 + T CM and NK cells (Fig. 1b). Summary/Conclusion: This study confirmed evolutionary trajectories often involving RAS pathway mutations as an intrinsic mechanism of resistance to Gilteritinib therapy. Of note, differences in immune transcriptomic profiles, involving mainly CD8+ T-Cells and NK-Cells, were associated with response, pointing to a previously unappreciated role of the immune system in mediating response to Gilteritinib.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr-/- mice.

Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. We generated myeloid-specific Stat4ΔLysMLdlr-/-, neutrophil-specific Stat4ΔS100A8Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4ΔLysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed Stat4ΔLysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

Abstract 4843: Characterization of T-cell metabolic defects in chronic lymphocytic leukemia preclinical model

Abstract Chronic lymphocytic leukemia (CLL) is the most predominant leukemia affecting elderly population in western countries. T-cell dysfunction is a CLL hallmark contributing to the low success rates of adoptive cell therapy in patients. Generally, exhausted states of tumor infiltrating lymphocytes are associated with defects in metabolic and mitochondrial adaptation, preventing normal cytotoxic functions. A handful of recent reports have demonstrated metabolic defects in CLL patients’ T-cells. Nevertheless, the field is still lacking sufficient studies to validate these metabolic changes in the preclinical Eµ-TCL1 murine model which is the most established model for CLL. Our aim is to uncover T-cell mitochondrial and metabolic defects in the Eµ-TCL1 splenic microenvironment. In this study, CLL was induced in mice via the adoptive transfer of leukemic splenocytes from transgenic Eµ-TCL1 mice into wild type (WT) recipients. Multiparameter flow cytometry was used to investigate mitochondrial properties and cellular glucose uptake of splenic CLL CD8+ T-cells. Moreover, we measured the expression levels of PGC-1α, a master regulator of mitochondrial function, and exhaustion-related markers in CD8+ T-cells. T-cell mitochondrial function was measured using mitochondrial stress test following in vitro CD3/CD28 stimulation for 72 hours. Nanostring gene expression analysis was done to compare metabolic gene expression profiles of CD8+ T-cells from either WT or Eµ-TCL1 mice. Probing Eµ-TCL1 splenocytes with mitotracker green and tetramethylrhodamine methyl ester revealed accumulation of depolarized mitochondria in CD8+ T-cells compared to WT counterparts. Our results also showed significant changes in mitochondrial and cellular reactive oxygen species levels as indicated by mitosox and H2DCFDA staining, respectively. Additionally, marked reduction of glucose uptake by Eµ-TCL1 CD8+ T-cells was noted following incubation with 2-NBDG glucose analog. Observed mitochondrial abnormalities in the Eµ-TCL1 CD8+ T-cells were associated with a significant downregulation of PGC-1α expression. Flow cytometry analysis demonstrated a significant increase in the expression of exhaustion markers in Eµ-TCL1 CD8+ T-cells and reduction in self-renewal marker, TCF-1. Interestingly, the abnormal mitochondrial properties were directly correlated with PD-1 expression in Eµ-TCL1 T-cells. Nanostring significance score analysis of Eµ-TCL1 versus WT CD8+ T-cell RNA revealed upregulation of glycolysis, mitochondrial respiration, T-cell receptor and costimulatory pathways. On the other hand, there was a downregulation in autophagy, AMPK and amino acid transporter pathways. In conclusion, our work characterizes for the first time the metabolic and mitochondrial defects of CD8+ T-cells from Eµ-TCL1 murine model and outlines the correlation of these defects with T-cell exhaustion in CLL. Citation Format: Wael Gamal, Melanie Mediavilla-Varela, Kamira Maharaj, Angimar Uriepero, Vishaal Kunta, Eva Sahakian, Javier Pinilla-Ibarz. Characterization of T-cell metabolic defects in chronic lymphocytic leukemia preclinical model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4843.

P047 Osteopontin in colitis-associated carcinoma (CAC)

Abstract Background Inflammatory bowel disease (IBD) patients pose a lifelong risk of developing colitis-associated carcinoma (CAC). Current understanding and mechanisms of CAC development stem from murine CAC models while studies on human CAC carcinogenesis are scarce. Thus, we aimed to understand CAC carcinogenesis, using human samples from patients suffering from Crohn′s disease (CD), CD-associated CAC (CD-CAC), ulcerative colitis (UC), and UC-associated CAC (UC-CAC) and further validated the findings in vitro. Methods We obtained FFPE specimens from 40 patients that suffered from either one of the IBDs or CACs and furthermore inflammation-free healthy controls (10 patients per group). These samples were micro-dissected, followed by RNA isolation and Nanostring gene expression analysis. Our data revealed a strong upregulation of SPP1 (Secreted Phosphoprotein-1) gene encoding for osteopontin (OPN) upon comparing heathy controls to IBD and to CAC. We then used in vitro-models to elucidate the mechanism of OPN-induced carcinogenesis. We stimulated intestinal epithelial cell lines HT-29/B6 and T84 cells with rhOPN (recombinant human OPN), following bulk RNAseq. We validated the findings using Seahorse assay for analysis of mitochondrial respiration (determining oxygen consumption rate, OCR). Western phospho-blots were done to unravel signal transduction events. Results Nanostring gene expression data revealed the following: (i) SPP1 gene encoding for OPN and epithelial-to-mesenchymal (EMT) transcription factors fibronectin-1 (FN1) and ZEB1 were strongly upregulated in UC-CAC and CD-CAC patients when compared to UC and CD. (ii) IL17-pathway genes were significantly downregulated in UC-CAC patients. (iii) Tight junction and polarity genes were altered in both UC-CAC and CD-CAC patients. rhOPN stimulation in vitro to HT29/B6 and T84 cells excluded OPN as the trigger of EMT but uncovered upregulated mitochondrial respiratory chain genes as a potential trigger for tumour cell proliferation. Validation of RNAseq findings by Seahorse assay revealed that after sequential injections of oligomycin, FCCP, rotenone and antimycin A both, HT-29/B6 and T84 cells, had higher basal and maximal OCRs in response to rhOPN (Fig. 1). T84, but not HT-29/B6 cells also showed an upregulated ATP production in response to rhOPN. Conclusion SPP1 expression correlates with CAC carcinogenesis. Putative underlying pathways rather include modulation of mitochondrial respiratory chain activity by differential gene expression than induction of EMT.

CSF1 expression associated with M2 macrophages and decreased survival in colorectal liver metastases

Background: Over half of patients with colorectal cancer develop liver metastases. A deeper understanding of the colorectal liver metastasis (CRLM)-specific tumor immune microenvironment is needed to improve therapeutic options. Within the tumor immune microenvironment, macrophages play a key role in tumor invasion and metastasis. Specifically, the M2 macrophage phenotype is known to be immunosuppressive, pro-tumorigenic, and is induced by CSF1 signaling. Using NanoString gene expression analysis on resected CRLM specimens, we previously identified that CSF1 was expressed at higher levels in patients with resected CRLM who had poor overall survival compared to those with better survival. Given these findings at the gene expression level, we hypothesized that tumors of patients with poor survival would have a higher prevalence of M2 macrophages. The aim of this study was to assess the M2 macrophage frequency in the tumor immune microenvironment of patients who underwent resection of CRLM.

Multi‐omics analyses reveal unexpected effects of PLCg2 deficiency in the brain

AbstractBackground Plcg2 polymorphisms are associated with both reduced (P522R) and increased (M28L) risk of Alzheimer’s disease (AD). The normal function of phospholipase C‐gamma‐2 (PLCg2) protein is to catalyze the hydrolysis of the membrane phosphatidylinositol‐4,5‐bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol trisphosphate (IP3), which subsequently feed into downstream signaling pathways involving protein kinase C (PKC) activation, lipid homeostasis, and calcium regulation. In the brain, Plcg2 is primarily expressed by microglia. The Plcg2 P522R protective variant is a functional hypermorph, while it is predicted that M28L is a loss‐of‐function variant. The specific consequences of PLCg2 loss of function in the brain are unknown. We predicted that reduction of PLCg2 would lead to disrupted PIP2 and DAG metabolism, and that these changes would be associated with decreased PKC and calcium signaling resulting in changes in microglia phenotype.MethodsAt 3 months of age, PLCg2 knockout (KO), PLCg2 heterozygous, and wild‐type (WT) littermate mice were euthanized. We analyzed their brains using, shotgun lipidomics, proteomics, gene expression analysis by a Nanostring Glia Profiling panel, and immunohistochemistry (IHC).ResultsLipidomic analyses revealed expected but sex‐specific changes in total brain PIP2 and DAG content in KOs compared to WTs. Surprisingly, significant decreases in myelin‐specific lipids (e.g., cerebrosides) were identified in both sexes. Unexpectedly, Nanostring gene expression analysis revealed sex‐specific changes in myelin‐related genes, as well as subtle effects on microglia phenotype in KOs compared to WTs, suggestive of reduced microglia . Surprisingly, exploratory proteomics analyses revealed subtle changes in KO females compared to WTs, primarily related to amino acid metabolism. IHC for microglial markers confirmed subtle differences in microglia (Iba1 density and area) in KOs compared to WTs.ConclusionsOur data demonstrate that loss of PLCg2 in the brain generally leads to subtle differences in microglia phenotype in the absence of inflammatory stimuli (suggestive of reduced microglial reactivity) and a surprising potential role for PLCg2 in myelin and amino acid homeostasis that requires further investigation. Overall, our data indicate that loss of PLCg2 function has subtle effects on brain homeostasis that may underlie enhanced vulnerability to AD pathology via microglia and myelin dysfunction.

464 CCR8 is a new therapeutic target in cutaneous T-cell lymphomas

Cutaneous T-cell lymphomas (CTCL) are rare non-Hodgkin lymphomas of skin-tropic T cells. Advanced-stage CTCL are frequently refractory to treatment and early relapses are common. There is a clear unmet need for new treatments with long-term efficacy in CTCL. CCR8 is a chemokine receptor expressed by skin resident memory T cells which are suspected to be the tumor cells of origin in mycosis fungoides. CCR8 is also strongly expressed by tumor-infiltrating Treg involved in immune escape. We combined blood flow cytometry in 13 patients with CTCL and healthy controls, in vitro stimulation of CTCL patients cells, Nanostring gene expression analysis, and single cell RNA sequencing analysis on human CTCL skin to study CCR8 expression and function in CTCL.

ENTPD1/CD39 as a predictive marker of treatment response to gemogenovatucel-T as maintenance therapy in newly diagnosed ovarian cancer

BackgroundBroadened use of predictive molecular and phenotypic profiling amongst oncologists has facilitated optimal integration of targeted- and immuno-therapeutics into clinical care. However, the use of predictive immunomarkers in ovarian cancer (OC) has not consistently translated into clinical benefit. Vigil (gemogenovatucel-T) is a novel plasmid engineered autologous tumor cell immunotherapy designed to knock down the tumor suppressor cytokines, TGFβ1 and TGFβ2, augment local immune function via increased GMCSF expression and enhance presentation of clonal neoantigen epitopes.MethodsAll patients enrolled in the VITAL trial (NCT02346747) of maintenance Vigil vs. placebo as front-line therapy with homologous recombination proficient (HRP) stage IIIB-IV newly diagnosed ovarian cancer underwent NanoString gene expression analysis. Tissue was obtained from surgically resected ovarian tumor tissue following surgical debulking. A statistical algorithm was used to analyze the NanoString gene expression data.ResultsUsing the NanoString Statistical Algorithm (NSA), we identify high expression of ENTPD1/CD39 (which functions as the rate-limiting step in the production of the immune suppressor adenosine from ATP to ADP) as a presumptive predictor of response to Vigil versus placebo regardless of HRP status on the basis of relapse free survival (median not achieved vs 8.1 months, p = 0.00007) and overall survival (median not achieved vs 41.4 months, p = 0.013) extension.ConclusionNSA should be considered for application to investigational targeted therapies in order to identify populations most likely to benefit from treatment, in preparation for efficacy conclusive trials.

Characterization of Immunopathology and Small Airway Remodeling in Constrictive Bronchiolitis.

Rationale: Constrictive bronchiolitis (ConB) is a relatively rare and understudied form of lung disease whose underlying immunopathology remains incompletely defined. Objectives: Our objectives were to quantify specific pathological features that differentiate ConB from other diseases that affect the small airways and to investigate the underlying immune and inflammatory phenotype present in ConB. Methods: We performed a comparative histomorphometric analysis of small airways in lung biopsy samples collected from 50 soldiers with postdeployment ConB, 8 patients with sporadic ConB, 55 patients with chronic obstructive pulmonary disease, and 25 nondiseased control subjects. We measured immune and inflammatory gene expression in lung tissue using the NanoString nCounter Immunology Panel from six control subjects, six soldiers with ConB, and six patients with sporadic ConB. Measurements and Main Results: Compared with control subjects, we found shared pathological changes in small airways from soldiers with postdeployment ConB and patients with sporadic ConB, including increased thickness of the smooth muscle layer, increased collagen deposition in the subepithelium, and lymphocyte infiltration. Using principal-component analysis, we showed that ConB pathology was clearly separable both from control lungs and from small airway disease associated with chronic obstructive pulmonary disease. NanoString gene expression analysis from lung tissue revealed T-cell activation in both groups of patients with ConB with upregulation of proinflammatory pathways, including cytokine-cytokine receptor interactions, NF-κB (nuclear factor-κB) signaling, TLR (Toll-like receptor) signaling, T-cell receptor signaling, and antigen processing and presentation. Conclusions: These findings indicate shared immunopathology among different forms of ConB and suggest that an ongoing T-helper cell type 1-type adaptive immune response underlies airway wall remodeling in ConB.

Defective fractalkine-CX3CR1 signaling aggravates neuroinflammation and affects recovery from cuprizone-induced demyelination.

Microglia have been implicated in multiple sclerosis (MS) pathogenesis. The fractalkine receptor CX3CR1 limits the activation of pathogenic microglia and the human polymorphic CX3CR1I249/M280 (hCX3CR1I249/M280 ) variant increases disease progression in models of MS. However, the role of hCX3CR1I249/M280 variant on microglial activation and central nervous system repair mechanisms remains unknown. Therefore, using transgenic mice expressing the hCX3CR1I249/M280 variant, we aimed to determine the contribution of defective CX3CR1 signaling to neuroinflammation and remyelination in the cuprizone model of focal demyelination. Here, we report that mice expressing hCX3CR1I249/M280 exhibit marked demyelination and microgliosis following acute cuprizone treatment. Nanostring gene expression analysis in demyelinated lesions showed that hCX3CR1I249/M280 but not CX3CR1-deficient mice up-regulated the cuprizone-induced gene profile linked to inflammatory, oxidative stress, and phagocytic pathways. Although CX3CR1-deficient (CX3CR1-KO) and fractalkine-deficient (FKN-KO) mice displayed a comparable demyelination and microglial activation phenotype to hCX3CR1I249/M280 mice, only CX3CR1-deficient and CX3CR1-WT mice showed significant myelin recovery 1 week from cuprizone withdrawal. Confocal microscopy showed that hCX3CR1I249/M280 variant inhibits the generation of cells involved in myelin repair. Our results show that defective fractalkine signaling contributes to regional differences in demyelination, and suggest that the CX3CR1 pathway activity may be a key mechanism for limiting toxic gene responses in neuroinflammation. Cover Image for this issue:https://doi.org/10.1111/jnc.15416.

Easy NanoString Gene Expression Analysis with the NanoTube

The NanoString nCounter analysis system has become a popular platform for the quantification of RNA expression, and numerous statistical and bioinformatic methods have been developed to analyze the resulting data. However, many of the current methods and tools are unavailable to those without expert knowledge in computer programming. We have developed the NanoTube R package to make this analysis easier for anyone with basic R skills, as well as a simple web application (developed using R‐Shiny) for those with no programming skills. This includes all of the steps required in nCounter data analysis, from data processing and quality control to differential expression, gene set analysis, and data visualization. In addition to manufacturer‐recommended data analysis steps, functions for interoperability with other R packages for NanoString data are provided for those who prefer different analysis methods. And because this library is open‐source, it can be expanded by others with additional analysis and visualization needs.We demonstrate the software by applying it to a publically available data set comparing the gene expression of patients with autoimmune retinopathy against healthy controls (GEO data series GSE117751). In addition to confirming the enrichment of multiple immune pathways identified in the previous analysis of the data, the EGFR signaling pathway is identified as a potential pathway of interest in autoimmune retinopathy patients. Use of this software will help researchers understand their gene expression data more quickly, and can also allow for the rapid reanalysis of public NanoString data sets to uncover new biological discoveries. The NanoTube R package and user’s guide are available on the Bioconductor software repository (https://www.bioconductor.org/packages/devel/bioc/html/NanoTube.html), while the beta version of the web application can be downloaded from GitHub (https://github.com/calebclass/Shiny‐NanoTube).

A Model to Detect Significant Prostate Cancer Integrating Urinary Peptide and Extracellular Vesicle RNA Data.

Simple SummaryProstate cancer is one of the leading causes of cancer-related death in men in the world, but a large proportion of men that are diagnosed with prostate cancer do not have a form of the disease that will cause them long term harm. Therefore, there is a need to accurately predict the aggressiveness of the disease without taking an invasive biopsy. In this study, we develop a test that can predict whether a patient has prostate cancer and how aggressive that cancer is. This test combines clinical measurements, levels of four genes collected from a fraction of the urine, and levels of six peptides found in urine. We found that this test, deemed ‘ExoSpec’, has the potential to improve the pathway for men with a clinical suspicion of prostate cancer and could reduce the requirement for biopsies by 30%.There is a clinical need to improve assessment of biopsy-naïve patients for the presence of clinically significant prostate cancer (PCa). In this study, we investigated whether the robust integration of expression data from urinary extracellular vesicle RNA (EV-RNA) with urine proteomic metabolites can accurately predict PCa biopsy outcome. Urine samples collected within the Movember GAP1 Urine Biomarker study (n = 192) were analysed by both mass spectrometry-based urine-proteomics and NanoString gene-expression analysis (167 gene-probes). Cross-validated LASSO penalised regression and Random Forests identified a combination of clinical and urinary biomarkers for predictive modelling of significant disease (Gleason Score (Gs) ≥ 3 + 4). Four predictive models were developed: ‘MassSpec’ (CE-MS proteomics), ‘EV-RNA’, and ‘SoC’ (standard of care) clinical data models, alongside a fully integrated omics-model, deemed ‘ExoSpec’. ExoSpec (incorporating four gene transcripts, six peptides, and two clinical variables) is the best model for predicting Gs ≥ 3 + 4 at initial biopsy (AUC = 0.83, 95% CI: 0.77–0.88) and is superior to a standard of care (SoC) model utilising clinical data alone (AUC = 0.71, p < 0.001, 1000 resamples). As the ExoSpec Risk Score increases, the likelihood of higher-grade PCa on biopsy is significantly greater (OR = 2.8, 95% CI: 2.1–3.7). The decision curve analyses reveals that ExoSpec provides a net benefit over SoC and could reduce unnecessary biopsies by 30%.

Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice.

Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice.Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1+/- mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI.Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1+/- mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1+/- mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1+/- mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage.Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI.

Ibrutinib Inhibits cGVHD Pathogenic Pre-Germinal Center B-Cells and Follicular Helper Cells While Preserving Immune Memory and Th1 T-Cells

Background: Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Treatment relies upon corticosteroids and immunosuppressive agents with limited efficacy and considerable side-effects. Evolving research points to the complex involvement of immune T, B, and myeloid cells in the pathogenesis of cGVHD. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) which inhibits the activation of B-cells, specific T-cell subsets, and certain myeloid cells that are implicated in cGVHD. Substantial clinical benefit was demonstrated in an open-label phase 1b/2 study of ibrutinib in patients with cGVHD that had failed frontline therapy (Miklos et al ASH 2016). Based upon this data, ibrutinib is indicated by the US FDA for the treatment of adult patients with cGVHD after failure of one or more lines of systemic therapy.Methods: Blood samples were collected before and multiple timepoints after starting ibrutinib 420mg daily therapy from 42 subjects with cGVHD that had failed frontline therapy (Miklos et al ASH 2016). Correlative analyses included Nanostring gene expression profiling, Luminex and ELISA based plasma testing, CyTOF phospho-immunophenotyping, and ex-vivo T-cell stimulation assays.Results: Initial data from 8 patients using targeted, Nanostring gene expression analysis showed ibrutinib reduced inflammatory genes including NFkB-1 and chemokines CXCL10, CCL7 and CCL3 (2.6, 2.3, 25, and 1.8-fold decrease over first 3 months, respectively). Luminex plasma quantification supported gene expression profiling results, and unveiled several additional chemotactic, inflammatory, and fibrotic factors which were down modulated by ibrutinib therapy including CCL22, CXCL9, CXCL10, IL-8, sCD25, CCL4, and ST2 (69-80% of patients showed decreasing trend). 42-parameter CyTOF single cell analysis revealed a 10-fold reduction in absolute numbers of cGVHD implicated pre-germinal center B-cells (CD19+, CD27+, CD38+, IgD+), and notably diminished Tfh, Th17, and total B cells (2.6, 1.5, and 1.4-fold decrease over first 90 days, respectively) following ibrutinib therapy. Relative numbers of CD4 and CD8 T-cells, class-switched B-cells, and CD14+ monocytes remained unchanged, and iNKT, Th1, and Treg cells increased incrementally (1.8, 1.2, and 1.1-fold at day 90, respectively). IgG plasma levels persisted while IgM significantly decreased corroborating an ibrutinib germinal center effect that did not deplete long-lived plasma cells. Furthermore, T cell reactivity against influenza increased, and antibodies against EBV and tetanus remained unchanged. Strikingly, single cell phosphorylation analysis showed BTK and ITK signaling was attenuated following ibrutinib treatment in defined B and T cell subsets (Figure 1). Specifically, PLCγ1/2 activation was simultaneously diminished in the pre-germinal center and plasmablast B-cell subsets.Conclusions: Our examination of the biologic and cellular mechanisms which support the clinical results of ibrutinib in the setting of cGVHD demonstrate that ibrutinib selectively inhibits cGVHD pathogenic pre-germinal center B-cells and Tfh cells, while preserving immune memory and Th1 T-cells. These data are especially salient given the clinical efficacy of ibrutinib in cGVHD, and the meticulous nature of the correlative studies designed to elucidate the multifactorial mechanism of action in this life-threatening disease. [Display omitted] DisclosuresSahaf:Pharmacyclics: Research Funding; Stanford: Patents & Royalties. Tebaykin:Stanford University: Patents & Royalties. Hopper:Pharmacyclics: Employment. Cheung:Pharmacyclics: Employment; AbbVie: Equity Ownership. Bittencourt:Pharmacyclics: Employment, Research Funding; AbbVie: Equity Ownership, Research Funding. Cutler:Astellas: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Pfizer: Consultancy. Arora:Takeda Oncology: Consultancy. Waller:Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Cerus: Equity Ownership; Helocyte: Consultancy; National Institutes of Health: Research Funding; Katz Foundation: Research Funding; AMGEN: Consultancy; PRA: Consultancy; Coulter Foundation: Research Funding; Celldex: Consultancy; Chimerix: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding. Jagasia:Janssen: Consultancy, Research Funding; Mallinckrodt: Consultancy; Therakos: Consultancy, Research Funding. Flowers:Pharmacyclics: Consultancy. Logan:Novartis: Consultancy, Research Funding; Incyte: Consultancy; Kite: Research Funding; Astellas: Research Funding; Jazz: Consultancy; Amgen: Consultancy; Pharmacyclics: Research Funding; Shire: Consultancy. Jaglowski:Pharmacyclics: Consultancy, Research Funding. Lih:AbbVie: Equity Ownership; Pharmacyclics: Employment. Solman:Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, Expenses; AbbVie: Equity Ownership. Lal:Gilead Sciences: Employment; AbbVie: Equity Ownership; The Permanente Medical Group (TPMG): Employment; Pharmacyclics LLC, an AbbVie Company: Employment. Styles:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Hill:Amgen: Employment, Equity Ownership, Patents & Royalties; Pharmacyclics: Employment; AbbVie: Equity Ownership; Miravant Medical: Patents & Royalties. James:AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment. Bendall:Fluidigm Sciences: Consultancy, Other: Travel, Accomodations, Expenses; Bluebird Bio: Other: Travel, Accomodations, Expenses, Research Funding; Ionpath, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Boehringer-ingelheim: Consultancy, Other: Travel, Accomodations, Expenses; Pharmacyclics: Research Funding. Dubovsky:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership.