P03.05 Deep spatial profiling of the immune landscape of MSI and MSS colorectal tumors

IntroductionIn colorectal cancer (CRC) there have been many recent advances in immune related biomarkers that are both prognostic and predictive of response to immunotherapy. Microsatellite instability (MSI)/mismatch repair deficiency dMMR is present in 15–20% of CRCs and correlates with increased immunogenic mutations that often augment lymphocyte infiltration into the tumor microenvironment (TME). Additionally, location of tumor infiltrating T cells in two areas of the TME, the tumor center (CT) and invasive margin (IM) has also been shown to be prognostic and predictive of response to immunotherapy. Here we use multiplexed protein and RNA digital spatial profiling to elicit the immune landscape of MSI-MSS characterized CRC tumors.MethodsForty-eight CRC tumors were analyzed for gene expression using the NanoString® nCounter® PanCancer IO 360™ Research Use Only (RUO) Gene Expression Panel and assessed for 48 cell typing and biological signatures, including MMR loss/MSI predictor and the Tumor Inflammation Signature (TIS). A subset of 18 CRC tumors (6 MSI-TIS-hi, 6 MSS-TIS-hi, 6 MSS-TIS-lo) was selected for analysis with the RUO GeoMx™ Digital Spatial Profiler (DSP) using 40 antibodies (human IO protein panel), or 84 RNA probes (human IO RNA panel). Selection of regions of interest (ROIs) in two locations, CT and IM were guided by staining with fluorescent markers (CD45, CD3, pan-CK, DNA). 300–600 µM diameter circle ROIs were selected, and in some cases segmented by pan-CK+/pan-CK-. For 2 immune hot samples contour profiling at the IM into stromal and tumor regions was performed using 1400+ RNA probes with NGS readout.SummaryUsing whole tissue gene expression analysis, we determined the TIS and IO 360 signature scores for 48 CRC tumors using PanCancer IO 360 assay. 18 tumors within this cohort were selected based on TIS status to further dissect the location-dependent immune contexture of the TME. Protein DSP confirmed loss of dMMR markers (MSH2/MLH1) and identified an increased amount of potentially suppressive macrophages (CD163+PD-L1+) in MSI-TIS-hi versus MSS-TIS-hi tumors. Segmentation of ROIs based on tumor versus stroma (pan-CK±) identified samples with high proportions of tumor-invading TILs. Two MSI-TIS-hi profiled using probes against 1400+ mRNA targets confirmed protein results (CD163 in IM) and identified tumor-related signatures corresponding to the inside of the tumor (Cytokeratins, HER2/ERBB2, MET).ConclusionsHere we show the use of novel high-plex spatial profiling to profile location and pathways in the TME of MSI and MSS CRC tumors. These findings elicit unique biology related to the location and signaling of immune cells, which have the potential to unveil targets for therapeutic combinations.Disclosure InformationS.E. Church: A. Employment (full or part-time); Significant; NanoString Technologies. J. Reeves: A. Employment (full or part-time); Significant; NanoString Technologies. D.R. Zollinger: A. Employment (full or part-time); Significant; NanoString Technologies. J. McKay-Fleisch: A. Employment (full or part-time); Significant; NanoString Technologies. A.J. Bahrami: A. Employment (full or part-time); Significant; NanoString Technologies. M. Holpert: A. Employment (full or part-time); Significant; NanoString Technologies. A.M. White: A. Employment (full or part-time); Significant; NanoString Technologies. M.D. Bailey: A. Employment (full or part-time); Significant; NanoString Technologies. C.R. Merritt: A. Employment (full or part-time); Significant; NanoString Technologies. M. Hoang: A. Employment (full or part-time); Significant; NanoString Technologies. S. Warren: A. Employment (full or part-time); Significant; NanoString Technologies. J.M. Beechem: A. Employment (full or part-time); Significant; NanoString Technologies.

Download Full-text

Polycombs and microRNA-223 regulate human granulopoiesis by transcriptional control of target gene expression

Blood ◽

10.1182/blood-2011-08-371344 ◽

2012 ◽

Vol 119 (17) ◽

pp. 4034-4046 ◽

Cited By ~ 101

Author(s):

Giuseppe Zardo ◽

Alberto Ciolfi ◽

Laura Vian ◽

Linda M. Starnes ◽

Monia Billi ◽

...

Keyword(s):

Gene Expression ◽

Dna Sequences ◽

Transcriptional Control ◽

Transcriptional Level ◽

Seed Region ◽

Mrna Targets ◽

Epigenetic Events ◽

Evolutionarily Conserved ◽

Lineage Decision ◽

Chromatin Remodeling Complexes

Abstract Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin “bivalent domains,” hypermethylation, recruitment of polycomb (PcG)–RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.

Download Full-text

Label-free proteomic analysis reveals large dynamic changes to the cellular proteome upon expression of the miRNA-23a-27a-24-2 microRNA cluster

Biochemistry and Cell Biology ◽

10.1139/bcb-2019-0014 ◽

2020 ◽

Vol 98 (1) ◽

pp. 61-69 ◽

Cited By ~ 1

Author(s):

Ramanaguru S. Piragasam ◽

S. Faraz Hussain ◽

Steven G. Chaulk ◽

Zaeem A. Siddiqi ◽

Richard P. Fahlman

Keyword(s):

Gene Expression ◽

Proteomic Analysis ◽

Small Rnas ◽

Regulatory Networks ◽

Mirna Cluster ◽

Label Free ◽

Dynamic Changes ◽

Mrna Targets ◽

Order Of Magnitude ◽

Non Coding Rnas

In deciphering the regulatory networks of gene expression controlled by the small non-coding RNAs known as microRNAs (miRNAs), a major challenge has been with the identification of the true mRNA targets by these RNAs within the context of the enormous numbers of predicted targets for each of these small RNAs. To facilitate the system-wide identification of miRNA targets, a variety of system wide methods, such as proteomics, have been implemented. Here we describe the utilization of quantitative label-free proteomics and bioinformatics to identify the most significant changes to the proteome upon expression of the miR-23a-27a-24-2 miRNA cluster. In light of recent work leading to the hypothesis that only the most pronounced regulatory events by miRNAs may be physiologically relevant, our data reveal that label-free analysis circumvents the limitations of proteomic labeling techniques that limit the maximum differences that can be quantified. The result of our analysis identifies a series of novel candidate targets that are reduced in abundance by more than an order of magnitude upon the expression of the miR-23a-27a-24-2 cluster.

Download Full-text

Multiplexed, Targeted Gene Expression Profiling and Genetic Analysis on Electronic Microarrays

Clinical Chemistry ◽

10.1093/clinchem/48.11.1873 ◽

2002 ◽

Vol 48 (11) ◽

pp. 1873-1882 ◽

Cited By ~ 18

Author(s):

Elaine M Weidenhammer ◽

Brenda F Kahl ◽

Ling Wang ◽

Larry Wang ◽

Melanie Duhon ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Target Genes ◽

Mrna Targets ◽

Amplification Method ◽

Polymorphism Detection ◽

Targeted Gene Expression ◽

Electronic Microarray ◽

Electronic Field

Abstract Background: Electronic microarrays comprise independent microelectrode test sites that can be electronically biased positive or negative, or left neutral, to move and concentrate charged molecules such as DNA and RNA to one or more test sites. We developed a protocol for multiplexed gene expression profiling of mRNA targets that uses electronic field-facilitated hybridization on electronic microarrays. Methods: A multiplexed, T7 RNA polymerase-mediated amplification method was used for expression profiling of target mRNAs from total cellular RNA; targets were detected by hybridization to sequence-specific capture oligonucleotides on electronic microarrays. Activation of individual test sites on the electronic microarray was used to target hybridization to designated subsets of sites and allow comparisons of target concentrations in different samples. We used multiplexed amplification and electronic field-facilitated hybridization to analyze expression of a model set of 10 target genes in the U937 cell line during lipopolysaccharide-mediated differentiation. Performance of multiple genetic analyses (single-nucleotide polymorphism detection, gene expression profiling, and splicing isoform detection) on a single electronic microarray was demonstrated using the ApoE and ApoER2 genes as a model system. Results: Targets were detected after a 2-min hybridization reaction. With noncomplementary capture probes, no signal was detectable. Twofold changes in target concentration were detectable throughout the (∼64-fold) range of concentrations tested. Levels of 10 targets were analyzed side by side across seven time points. By confining electronic activation to subsets of test sites, polymorphism detection, expression profiling, and splicing isoform analysis were performed on a single electronic microarray. Conclusions: Microelectronic array technology provides specific target detection and quantification with advantages over currently available methodologies for targeted gene expression profiling and combinatorial genomics testing.

Download Full-text

CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins

eLife ◽

10.7554/elife.16072 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 13

Author(s):

Lizhen Chen ◽

Zhijie Liu ◽

Bing Zhou ◽

Chaoliang Wei ◽

Yu Zhou ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Alternative Splicing ◽

Binding Proteins ◽

Rna Binding ◽

Axon Regeneration ◽

Rna Binding Proteins ◽

Mrna Isoforms ◽

C Elegans ◽

Mrna Targets

Axon injury triggers dramatic changes in gene expression. While transcriptional regulation of injury-induced gene expression is widely studied, less is known about the roles of RNA binding proteins (RBPs) in post-transcriptional regulation during axon regeneration. In C. elegans the CELF (CUGBP and Etr-3 Like Factor) family RBP UNC-75 is required for axon regeneration. Using crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq) we identify a set of genes involved in synaptic transmission as mRNA targets of UNC-75. In particular, we show that UNC-75 regulates alternative splicing of two mRNA isoforms of the SNARE Syntaxin/unc-64. In C. elegans mutants lacking unc-75 or its targets, regenerating axons form growth cones, yet are deficient in extension. Extending these findings to mammalian axon regeneration, we show that mouse Celf2 expression is upregulated after peripheral nerve injury and that Celf2 mutant mice are defective in axon regeneration. Further, mRNAs for several Syntaxins show CELF2 dependent regulation. Our data delineate a post-transcriptional regulatory pathway with a conserved role in regenerative axon extension.

Download Full-text

MicroRNA in Platelet Production and Activation

Blood ◽

10.1182/blood.v116.21.sci-39.sci-39 ◽

2010 ◽

Vol 116 (21) ◽

pp. SCI-39-SCI-39

Author(s):

Paul F. Bray

Keyword(s):

Gene Expression ◽

Zinc Finger Protein ◽

Conflicts Of Interest ◽

Platelet Reactivity ◽

Functional Characterization ◽

Fine Tuning ◽

Interindividual Variation ◽

Erythroid Progenitor ◽

Mrna Targets ◽

Potential Biomarker

Abstract Abstract SCI-39 Alterations in gene expression are at the heart of both megakaryocytopoiesis and interindividual variation in platelet reactivity. Over the past decade there has been an increasing awareness of the important role played by microRNAs (miRNAs) in these diverse cell biologic and physiologic processes. MiRNAs are noncoding RNAs that target complementary sequences in mRNAs, leading to mRNA degradation or inhibition of translation. More than 1000 miRNAs have been identified, which are estimated to regulate 30%-90% of all coding genes. Expression of miRNAs is cell and developmental stage specific. MiRNAs regulate hematopoietic lineage commitment, as well as B lymphopoiesis, granulopoiesis, erythropoiesis and monocytopoiesis. Recent data also demonstrate a clear role for miRNAs in megakaryocytopoiesis (Meg-poiesis). The molecular genetic basis for the transition from one stage of Meg-poiesis to another requires fine-tuning of the various control elements, and miRNAs act as “rheostats” in this process. MiRNA-150 has a strong effect on Meg-erythroid progenitor cells, inducing commitment to the Meg lineage at the expense of erythropoiesis. MiRNA-150 directly down-regulates MYB, a transcription factor important in thrombopoiesis. Over-expressed miRNA-146a knocks down CXCR4 (receptor for SDF-1), reducing Meg proliferation and maturation. MiR-146a gene expression is negatively regulated by the promyelocytic zinc finger protein, PLZF. Thus, miRNAs act as intermediaries of transcription factors that control Meg-poiesis. An increasing number of genetic diseases are being described due to mutations in miRNA genes or their mRNA targets. Deletion of miR-145 and miR-146a on 5q is sufficient to cause the 5q- phenotype; miR-125b-2 up-regulates Meg-poiesis and may be involved in megakaryoblastic leukemia. Platelets appear to be a particularly rich source of miRNAs, many of which are expressed at very high levels. Notably, platelets contain Dicer, TRBP2 and Ago2, and are capable of synthesizing miRNAs from pre-miRNAs. MiRNAs regulate Bcl-xL and Bak, raising the possibility that platelet miRNAs affect platelet lifespan. Because miRNAs are very stable, they also represent a potential biomarker, and we have found sets of miRNAs that appear to classify platelet reactivity to epinephrine. Using bioinformatic approaches, we have identified miRNA-mRNA pairs differentially expressed in platelets with differing phenotypes. This approach permits functional characterization of novel platelet mRNAs, and elucidation of a potential genetic mechanism for adjusting megakaryocyte/platelet mRNA expression. Thus, information extracted from these RNA networks continue to provide insights into systems biology of higher organisms. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

High multiplex, digital spatial profiling of proteins and RNA in fixed tissue using genomic detection methods

10.1101/559021 ◽

2019 ◽

Cited By ~ 10

Author(s):

Christopher R. Merritt ◽

Giang T. Ong ◽

Sarah Church ◽

Kristi Barker ◽

Gary Geiss ◽

...

Keyword(s):

Immune Cell ◽

Tissue Sample ◽

Detection Methods ◽

Fixed Tissue ◽

Affinity Reagents ◽

Spatial Profiling ◽

Rna Probes ◽

Oligonucleotide Detection ◽

Modern Genomic ◽

Non Destructive

ABSTRACTWe have developed Digital Spatial Profiling (DSP), a non-destructive method for high-plex spatial profiling of proteins and RNA, using oligonucleotide detection technologies with unlimited multiplexing capability. The key breakthroughs underlying DSP are threefold: (1) multiplexed readout of proteins/RNA using oligo-tags; (2) oligo-tags attached to affinity reagents (antibodies/RNA probes) through a photocleavable (PC) linker; (3) photocleaving light projected onto the tissue sample to release PC-oligos in any spatial pattern. Here we show precise analyte reproducibility, validation, and cellular resolution using DSP. We also demonstrate biological proof-of-concept using lymphoid, colorectal tumor, and autoimmune tissue as models to profile immune cell populations, stroma, and cancer cells to identify factors specific for the diseased microenvironment. DSP utilizes the unlimited multiplexing capability of modern genomic approaches, while simultaneously providing spatial context of protein and RNA to examine biological questions based on analyte location and distribution.

Download Full-text

Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets

10.1101/007898 ◽

2014 ◽

Author(s):

Patrick Danaher ◽

Robin Lynn White ◽

Erin Hanson ◽

Jack Ballantyne

Keyword(s):

Gene Expression ◽

Body Fluid ◽

Body Fluids ◽

Menstrual Blood ◽

Body Fluid Identification ◽

Fluid Identification ◽

Mrna Profiling ◽

Mrna Targets ◽

Vaginal Secretions ◽

Specific Mrna

A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString® probes to 23 tissue/body fluid specific mRNA targets. The body fluids/tissues targeted were peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We tested and compared a simple 5 minute room temperature cellular lysis protocol against standard RNA isolation from same source material as a means to facilitate ease-of-use in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source blood, semen, vaginal secretions, menstrual blood and skin samples all demonstrated the expected tissue specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. Two of the five mixtures were called perfectly using the assay algorithm, and one of the component fluids was identified in the each of the 'false negative' mixtures. Crucially, our algorithm produced zero false positive fluid identifications across this study's 98 samples. Further optimization of the biomarker 'Codeset' will be required before it can be used in casework, particularly with respect to increasing the signal to noise ratio of the saliva biomarkers. With suitable modifications, this simplified protocol with minimal hands on requirement should facilitate routine use of mRNA profiling in casework laboratories.

Download Full-text

Organelle Visualization With Multicolored Fluorescent Markers in Bamboo

Frontiers in Plant Science ◽

10.3389/fpls.2021.658836 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mengdi Zhang ◽

Shuai Hu ◽

Fang Yi ◽

Yanli Gao ◽

Dongmei Zhu ◽

...

Keyword(s):

Gene Expression ◽

Subcellular Localization ◽

Molecular Mechanisms ◽

Expression System ◽

Transient Gene Expression ◽

Moso Bamboo ◽

Functional Study ◽

Efficient System ◽

Gene Expression System ◽

Fluorescent Markers

Bamboo is an important model plant to study the molecular mechanisms of rapid shoot growth and flowering once in a lifetime. However, bamboo research about protein functional characterization is largely lagged behind, mainly due to the lack of gene transformation platforms. In this study, a protoplast transient gene expression system in moso bamboo has been first established. Using this reliable and efficient system, we have generated a set of multicolored fluorescent markers based on the targeting sequences from endogenous proteins, which have been validated by their comparative localization with Arabidopsis organelle markers, in a combination with pharmaceutical treatments. Moreover, we further demonstrated the power of this multicolor marker set for rapid, combinatorial analysis of the subcellular localization of uncharacterized proteins, which may play potential functions in moso bamboo flowering and fast growth of shoots. Finally, this protoplast transient gene expression system has been elucidated for functional analysis in protein–protein interaction by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. Taken together, in combination with the set of moso bamboo organelle markers, the protoplast transient gene expression system could be used for subcellular localization and functional study of unknown proteins in bamboo and will definitely promote rapid progress in diverse areas of research in bamboo plants.

Download Full-text

An unsupervised feature extraction and selection strategy for identifying epithelial-mesenchymal transition state metrics in breast cancer and melanoma

10.1101/865139 ◽

2019 ◽

Cited By ~ 2

Author(s):

David J. Klinke ◽

Arezo Torang

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Tumor Microenvironment ◽

Epithelial Mesenchymal Transition ◽

Cell Phenotype ◽

Holistic View ◽

Mesenchymal Transition ◽

Feature Extraction And Selection ◽

Cellular Context ◽

Immune Contexture

Digital cytometry is opening up new avenues to better understand the heterogeneous cell types present within the tumor microenvironment. While the focus is towards elucidating immune and stromal cells as clinical correlates, there is still a need to better understand how a change in tumor cell phenotype, such as the epithelial-mesenchymal transition, influences the immune contexture. To complement existing digital cytometry methods, our objective was to develop an unsupervised gene signature capturing a change in differentiation state that is tailored to the specific cellular context of breast cancer and melanoma, as a illustrative example. Towards this aim, we used principal component analysis coupled with resampling to develop unsupervised gene expression-based state metrics specific for the cellular context that characterize the state of cellular differentiation within an epithelial to mesenchymal-like state space and independently correlate with metastatic potential. First developed using cell line data, the orthogonal state metrics were refined to exclude the contributions of normal fibroblasts and to provide tissue-level state estimates based on bulk tissue RNA-seq measures. The resulting gene expression-based metrics for differentiation state aim to inform a more holistic view of how the malignant cell phenotype influences the immune contexture within the tumor microenvironment.

Download Full-text