RNA profiling of human dorsal root ganglia reveals sex-differences in mechanisms promoting neuropathic pain

Brain : a journal of neurology

2022 Jul 22

Ray, PR;Shiers, S;Caruso, JP;Tavares-Ferreira, D;Sankaranarayanan, I;Uhelski, ML;Li, Y;North, RY;Tatsui, C;Dussor, G;Burton, MD;Dougherty, PM;Price, TJ;
PMID: 35867896 | DOI: 10.1093/brain/awac266

Neuropathic pain is a leading cause of high impact pain, is often disabling and is poorly managed by current therapeutics. Here we focused on a unique group of neuropathic pain patients undergoing thoracic vertebrectomy where the dorsal root ganglia is removed as part of the surgery allowing for molecular characterization and identification of mechanistic drivers of neuropathic pain independently of preclinical models. Our goal was to quantify whole transcriptome RNA abundances using RNA-seq in pain-associated human dorsal root ganglia from these patients, allowing comprehensive identification of molecular changes in these samples by contrasting them with non-pain associated dorsal root ganglia. We sequenced 70 human dorsal root ganglia, and among these 50 met inclusion criteria for sufficient neuronal mRNA signal for downstream analysis. Our expression analysis revealed profound sex differences in differentially expressed genes including increase of IL1B, TNF, CXCL14, and OSM in male and including CCL1, CCL21, PENK and TLR3 in female dorsal root ganglia associated with neuropathic pain. Co-expression modules revealed enrichment in members of JUN-FOS signalling in males, and centromere protein coding genes in females. Neuro-immune signalling pathways revealed distinct cytokine signalling pathways associated with neuropathic pain in males (OSM, LIF, SOCS1) and females (CCL1, CCL19, CCL21). We validated cellular expression profiles of a subset of these findings using RNAscope in situ hybridization. Our findings give direct support for sex differences in underlying mechanisms of neuropathic pain in patient populations.

Dysregulation of CXCL14 promotes malignant phenotypes of esophageal squamous carcinoma cells via regulating SRC and EGFR signaling

Biochemical and biophysical research communications

2022 Apr 04

Guo, J;Chang, C;Yang, LY;Cai, HQ;Chen, DX;Zhang, Y;Cai, Y;Wang, JJ;Wei, WQ;Hao, JJ;Wang, MR;
PMID: 35421632 | DOI: 10.1016/j.bbrc.2022.03.144

The present study was to identify abnormal methylation genes implicated in esophageal squamous cell carcinoma (ESCC). Genomic methylation alterations in ESCC tissues were analyzed using laser-microdissection and whole-genome bisulfite sequencing. CXCL14 promoter was frequently hypermethylated in ESCC tissues. The correlation of CXCL14 hypermethylation status and the mRNA and protein expression levels were validated using nested methylation-specific PCR (nMS-PCR), RNAscope in situ hybridization (RISH) and Western blot. RISH results showed completely negative CXCL14 expression in 34.3% (34/99) ESCC, compared with those in the basal layer cells of normal epithelia. Low expression of CXCL14 was more present in patients with lower differentiation. The anticancer role of CXCL14 has been commonly associated with immune regulation in the literature. Here, we observed by functional analysis that CXCL14 can also act as a tumor suppressor in ESCC cells. 5-Aza-dC treatment suppressed CXCL14 methylation and up-regulated the expression of CXCL14. Ectopic expression of CXCL14 suppressed the proliferation, invasion, tumor growth, and lung metastasis of ESCC cells. Both ectopic expression and induction of CXCL14 with 5-Aza-dC inhibited the activity of SRC, MEK1/2 and STAT3 in ESCC cells, while activated EGFR. Importantly, a combination of CXCL14 expression and SRC or EGFR inhibitor dramatically repressed the proliferation of ESCC cells and the growth of xenografts. Our findings revealed a direct tumor suppressor role of CXCL14, but not through the immune system. The data suggest that for ESCC patients with low level CXCL14, increasing CXCL14 expression combined with inhibition of SRC or EGFR might be a promising therapeutic strategy.

Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type

Nat Neurosci.

2018 Aug 27

"Boldog E, Bakken TE, Hodge RD, Novotny M, Aevermann BD, Baka J, Bordé S, Close JL, Diez-Fuertes F, Ding SL, Faragó N, Kocsis AK, Kovács B, Maltzer Z, McCorrison JM, Miller JA, Molnár G, Oláh G, Ozsvár A, Rózsa M, Shehata SI, Smith KA, Sunkin SM, Tran D
PMID: 30150662 | DOI: 10.1038/s41593-018-0205-2

We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries

Proceedings of the National Academy of Sciences of the United States of America

2021 May 18

Meinsohn, MC;Saatcioglu, HD;Wei, L;Li, Y;Horn, H;Chauvin, M;Kano, M;Nguyen, NMP;Nagykery, N;Kashiwagi, A;Samore, WR;Wang, D;Oliva, E;Gao, G;Morris, ME;Donahoe, PK;Pépin, D;
PMID: 33980714 | DOI: 10.1073/pnas.2100920118

Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

Expression of the chemokine CXCL14 in the tumour stroma is an independent marker of survival in breast cancer.

Br J Cancer.

2016 May 26

Sjöberg E, Augsten M, Bergh J, Jirström K, Östman A.
PMID: 27115465 | DOI: 10.1038/bjc.2016.104.

Abstract

BACKGROUND:

Expression of the chemokine CXCL14 has previously been shown to be elevated in the tumour stroma of, for example, prostate and breast cancer. Cancer-associated fibroblast-derived CXCL14 enhances tumour growth in mouse models of prostate and breast cancer. However, the prognostic significance of compartment-specific expression of CXCL14 has not been studied.

METHODS:

CXCL14 mRNA expression was analysed in a breast cancer tissue microarray (TMA) of formalin-fixed, paraffin-embedded tumours by the RNAscope 2.0 Assay. Epithelial and stromal expression was analysed separately and correlated with clinicopathological characteristics and survival.

RESULTS:

CXCL14 was variably and independently expressed in malignant and stromal cells of breast cancer. Total and stromal expression of CXCL14 did not associate with clinicopathological parameters. Epithelial CXCL14 expression was significantly associated with oestrogen receptor α (ERα)-positive tumours and lower proliferation status. Total CXCL14 expression correlated significantly with shorter breast cancer-specific and recurrence-free survival. High stromal, but not epithelial, CXCL14 expression was significantly associated with shorter survival in univariable and multivariable analyses. Moreover, the correlation between stromal CXCL14 expression and survival was more prominent in ER negative, triple negative and basal-like breast cancers.

CONCLUSIONS:

The identification of prognostic significance of stromal CXCL14 in breast cancer demonstrates novel clinical relevance of a stroma-derived secreted factor and illustrates the importance of tumour compartment-specific analyses. On the basis of the prognostic signals from difficult-to-treat subgroups, CXCL14 should also be considered as a candidate drug target.

CXCL14 promotes a robust brain tumor-associated immune response in glioma

Clinical cancer research : an official journal of the American Association for Cancer Research

2022 May 05

Kumar, A;Mohamed, E;Tong, S;Chen, K;Mukherjee, J;Lim, Y;Wong, CM;Boosalis, Z;Shai, A;Pieper, RO;Gupta, N;Perry, A;Bollen, AW;Molinaro, AM;Solomon, DA;Shieh, JTC;Phillips, JJ;
PMID: 35511927 | DOI: 10.1158/1078-0432.CCR-21-2830

The immunosuppressive tumor microenvironment present in the majority of diffuse glioma limits therapeutic response to immunotherapy. As the determinants of the glioma-associated immune response are relatively poorly understood, the study of glioma with more robust tumor-associated immune responses may be particularly useful to identify novel immunomodulatory factors that can promote T cell effector function in glioma.We used multiplex immune-profiling, proteomic profiling, and gene expression analysis to define the tumor-associated immune response in two molecular subtypes of glioma and identify factors that may modulate this response. We then used patient-derived glioma cultures and an immunocompetent murine model for malignant glioma to analyze the ability of tumor-intrinsic factors to promote a CD8+ T cell response.As compared with IDH-mutant astrocytoma, MAPK-activated pleomorphic xanthoastrocytoma (PXA) harbored increased numbers of activated cytotoxic CD8+ T cells and Iba1+ microglia/macrophages, increased MHC class I expression, enrichment of genes associated with antigen presentation and processing, and increased tumor cell secretion of the chemokine CXCL14. CXCL14 promoted activated CD8+ T cell chemotaxis in vitro, recruited tumor-infiltrating CD8+ T cells in vivo, and prolonged overall survival in a cytotoxic T cell-dependent manner. The immunomodulatory molecule B7-H3 was also highly expressed in PXA.We identify the MAPK-activated lower grade astrocytoma PXA, as having an immune-rich tumor microenvironment and suggest this tumor may be particularly vulnerable to immunotherapeutic modulation. We also identify CXCL14 as an important determinant of the glioma-associated immune microenvironment, sufficient to promote an anti-tumor CD8+ T cell response.

The small and large intestine contain related mesenchymal subsets that derive from embryonic Gli1+ precursors

Nature communications

2023 Apr 21

Pærregaard, SI;Wulff, L;Schussek, S;Niss, K;Mörbe, U;Jendholm, J;Wendland, K;Andrusaite, AT;Brulois, KF;Nibbs, RJB;Sitnik, K;Mowat, AM;Butcher, EC;Brunak, S;Agace, WW;
PMID: 37085516 | DOI: 10.1038/s41467-023-37952-5

The intestinal lamina propria contains a diverse network of fibroblasts that provide key support functions to cells within their local environment. Despite this, our understanding of the diversity, location and ontogeny of fibroblasts within and along the length of the intestine remains incomplete. Here we show that the small and large intestinal lamina propria contain similar fibroblast subsets that locate in specific anatomical niches. Nevertheless, we find that the transcriptional profile of similar fibroblast subsets differs markedly between the small intestine and colon suggesting region specific functions. We perform in vivo transplantation and lineage-tracing experiments to demonstrate that adult intestinal fibroblast subsets, smooth muscle cells and pericytes derive from Gli1-expressing precursors present in embryonic day 12.5 intestine. Trajectory analysis of single cell RNA-seq datasets of E12.5 and adult mesenchymal cells suggest that adult smooth muscle cells and fibroblasts derive from distinct embryonic intermediates and that adult fibroblast subsets develop in a linear trajectory from CD81+ fibroblasts. Finally, we provide evidence that colonic subepithelial PDGFRαhi fibroblasts comprise several functionally distinct populations that originate from an Fgfr2-expressing fibroblast intermediate. Our results provide insights into intestinal stromal cell diversity, location, function, and ontogeny, with implications for intestinal development and homeostasis.

Neuronal cell types, projections, and spatial organization of the central amygdala

iScience

2022 Dec 22

O'Leary, TP;Kendrick, RM;Bristow, BN;Sullivan, KE;Wang, L;Clements, J;Lemire, AL;Cembrowski, MS;
PMID: 36425768 | DOI: 10.1016/j.isci.2022.105497

The central amygdala (CEA) has been richly studied for interpreting function and behavior according to specific cell types and circuits. Such work has typically defined molecular cell types by classical inhibitory marker genes; consequently, whether marker-gene-defined cell types exhaustively cover the CEA and co-vary with connectivity remains unresolved. Here, we combined single-cell RNA sequencing, multiplexed fluorescent in situ hybridization, immunohistochemistry, and long-range projection mapping to derive a "bottom-up" understanding of CEA cell types. In doing so, we identify two major cell types, encompassing one-third of all CEA neurons, that have gone unresolved in previous studies. In spatially mapping these novel types, we identify a non-canonical CEA subdomain associated with Nr2f2 expression and uncover an Isl1-expressing medial cell type that accounts for many long-range CEA projections. Our results reveal new CEA organizational principles across cell types and spatial scales and provide a framework for future work examining cell-type-specific behavior and function.

Sharp cell-type-identity changes differentiate the retrosplenial cortex from the neocortex

Cell reports

2023 Mar 28

Sullivan, KE;Kraus, L;Kapustina, M;Wang, L;Stach, TR;Lemire, AL;Clements, J;Cembrowski, MS;
PMID: 36881508 | DOI: 10.1016/j.celrep.2023.112206

The laminae of the neocortex are fundamental processing layers of the mammalian brain. Notably, such laminae are believed to be relatively stereotyped across short spatial scales such that shared laminae between nearby brain regions exhibit similar constituent cells. Here, we consider a potential exception to this rule by studying the retrosplenial cortex (RSC), a brain region known for sharp cytoarchitectonic differences across its granular-dysgranular border. Using a variety of transcriptomics techniques, we identify, spatially map, and interpret the excitatory cell-type landscape of the mouse RSC. In doing so, we uncover that RSC gene expression and cell types change sharply at the granular-dysgranular border. Additionally, supposedly homologous laminae between the RSC and the neocortex are effectively wholly distinct in their cell-type composition. In collection, the RSC exhibits a variety of intrinsic cell-type specializations and embodies an organizational principle wherein cell-type identities can vary sharply within and between brain regions.

Single-cell RNA-sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification

Nature communications

2022 Jul 05

Petitpré, C;Faure, L;Uhl, P;Fontanet, P;Filova, I;Pavlinkova, G;Adameyko, I;Hadjab, S;Lallemend, F;
PMID: 35790771 | DOI: 10.1038/s41467-022-31580-1

Different types of spiral ganglion neurons (SGNs) are essential for auditory perception by transmitting complex auditory information from hair cells (HCs) to the brain. Here, we use deep, single cell transcriptomics to study the molecular mechanisms that govern their identity and organization in mice. We identify a core set of temporally patterned genes and gene regulatory networks that may contribute to the diversification of SGNs through sequential binary decisions and demonstrate a role for NEUROD1 in driving specification of a Ic-SGN phenotype. We also find that each trajectory of the decision tree is defined by initial co-expression of alternative subtype molecular controls followed by gradual shifts toward cell fate resolution. Finally, analysis of both developing SGN and HC types reveals cell-cell signaling potentially playing a role in the differentiation of SGNs. Our results indicate that SGN identities are drafted prior to birth and reveal molecular principles that shape their differentiation and will facilitate studies of their development, physiology, and dysfunction.

Spatially organized multicellular immune hubs in human colorectal cancer

Cell

2021 Aug 24

Pelka, K;Hofree, M;Chen, JH;Sarkizova, S;Pirl, JD;Jorgji, V;Bejnood, A;Dionne, D;Ge, WH;Xu, KH;Chao, SX;Zollinger, DR;Lieb, DJ;Reeves, JW;Fuhrman, CA;Hoang, ML;Delorey, T;Nguyen, LT;Waldman, J;Klapholz, M;Wakiro, I;Cohen, O;Albers, J;Smillie, CS;Cuoco, MS;Wu, J;Su, MJ;Yeung, J;Vijaykumar, B;Magnuson, AM;Asinovski, N;Moll, T;Goder-Reiser, MN;Applebaum, AS;Brais, LK;DelloStritto, LK;Denning, SL;Phillips, ST;Hill, EK;Meehan, JK;Frederick, DT;Sharova, T;Kanodia, A;Todres, EZ;Jané-Valbuena, J;Biton, M;Izar, B;Lambden, CD;Clancy, TE;Bleday, R;Melnitchouk, N;Irani, J;Kunitake, H;Berger, DL;Srivastava, A;Hornick, JL;Ogino, S;Rotem, A;Vigneau, S;Johnson, BE;Corcoran, RB;Sharpe, AH;Kuchroo, VK;Ng, K;Giannakis, M;Nieman, LT;Boland, GM;Aguirre, AJ;Anderson, AC;Rozenblatt-Rosen, O;Regev, A;Hacohen, N;
PMID: 34450029 | DOI: 10.1016/j.cell.2021.08.003

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

Search form

Probes for INS

Content for comparison

Gene

Product

Research area

Category

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSIONS:

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China