Methods and applications for single-cell and spatial multi-omics

Nature reviews. Genetics

2023 Mar 02

Vandereyken, K;Sifrim, A;Thienpont, B;Voet, T;
PMID: 36864178 | DOI: 10.1038/s41576-023-00580-2

The joint analysis of the genome, epigenome, transcriptome, proteome and/or metabolome from single cells is transforming our understanding of cell biology in health and disease. In less than a decade, the field has seen tremendous technological revolutions that enable crucial new insights into the interplay between intracellular and intercellular molecular mechanisms that govern development, physiology and pathogenesis. In this Review, we highlight advances in the fast-developing field of single-cell and spatial multi-omics technologies (also known as multimodal omics approaches), and the computational strategies needed to integrate information across these molecular layers. We demonstrate their impact on fundamental cell biology and translational research, discuss current challenges and provide an outlook to the future.

Single-cell proteomics enabled by next-generation sequencing or mass spectrometry

Nature methods

2023 Mar 01

Bennett, HM;Stephenson, W;Rose, CM;Darmanis, S;
PMID: 36864196 | DOI: 10.1038/s41592-023-01791-5

In the last decade, single-cell RNA sequencing routinely performed on large numbers of single cells has greatly advanced our understanding of the underlying heterogeneity of complex biological systems. Technological advances have also enabled protein measurements, further contributing to the elucidation of cell types and states present in complex tissues. Recently, there have been independent advances in mass spectrometric techniques bringing us one step closer to characterizing single-cell proteomes. Here we discuss the challenges of detecting proteins in single cells by both mass spectrometry and sequencing-based methods. We review the state of the art for these techniques and propose that there is a space for technological advancements and complementary approaches that maximize the advantages of both classes of technologies.

Spatial profiling technologies illuminate the tumor microenvironment

Cancer cell

2023 Feb 13

Elhanani, O;Ben-Uri, R;Keren, L;
PMID: 36800999 | DOI: 10.1016/j.ccell.2023.01.010

The tumor microenvironment (TME) is composed of many different cellular and acellular components that together drive tumor growth, invasion, metastasis, and response to therapies. Increasing realization of the significance of the TME in cancer biology has shifted cancer research from a cancer-centric model to one that considers the TME as a whole. Recent technological advancements in spatial profiling methodologies provide a systematic view and illuminate the physical localization of the components of the TME. In this review, we provide an overview of major spatial profiling technologies. We present the types of information that can be extracted from these data and describe their applications, findings and challenges in cancer research. Finally, we provide a future perspective of how spatial profiling could be integrated into cancer research to improve patient diagnosis, prognosis, stratification to treatment and development of novel therapeutics.

Alternative Splicing

Methods in Molecular Biology

2022 Jul 27

Scheiffele, P;Mauger, O;
| DOI: 10.1007/978-1-0716-2521-7

This detailed volume collects commonly used and cutting-edge methods to analyze alternative splicing, a key step in gene regulation. After an introduction of the alternative splicing mechanism and its targeting for therapeutic strategies, the book continues with techniques for analyzing alternative splicing profiles in complex biological systems, visualizing and localizing alternative spliced transcripts with cellular and sub-cellular resolution, probing regulators of alternative splicing, as well as assessing the functional consequences of alternative splicing. Written for the highly successful _Methods in Molecular Biology_ series, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, reproducible protocols, and tips on troubleshooting and avoiding known pitfalls.

Best practice standards for circular RNA research

Nature methods

2022 May 26

Nielsen, AF;Bindereif, A;Bozzoni, I;Hanan, M;Hansen, TB;Irimia, M;Kadener, S;Kristensen, LS;Legnini, I;Morlando, M;Jarlstad Olesen, MT;Pasterkamp, RJ;Preibisch, S;Rajewsky, N;Suenkel, C;Kjems, J;
PMID: 35618955 | DOI: 10.1038/s41592-022-01487-2

Circular RNAs (circRNAs) are formed in all domains of life and via different mechanisms. There has been an explosion in the number of circRNA papers in recent years; however, as a relatively young field, circRNA biology has an urgent need for common experimental standards for isolating, analyzing, expressing and depleting circRNAs. Here we propose a set of guidelines for circRNA studies based on the authors' experience. This Perspective will specifically address the major class of circRNAs in Eukarya that are generated by a spliceosome-catalyzed back-splicing event. We hope that the implementation of best practice principles for circRNA research will help move the field forward and allow a better functional understanding of this fascinating group of RNAs.

Single-cell atlases: shared and tissue-specific cell types across human organs

Nature reviews. Genetics

2022 Feb 25

Elmentaite, R;Domínguez Conde, C;Yang, L;Teichmann, SA;
PMID: 35217821 | DOI: 10.1038/s41576-022-00449-w

The development of single-cell and spatial transcriptomics methods was instrumental in the conception of the Human Cell Atlas initiative, which aims to generate an integrated map of all cells across the human body. These technology advances are bringing increasing depth and resolution to maps of human organs and tissues, as well as our understanding of individual human cell types. Commonalities as well as tissue-specific features of primary and supportive cell types across human organs are beginning to emerge from these human tissue maps. In this Review, we highlight key biological insights obtained from cross-tissue studies into epithelial, fibroblast, vascular and immune cells based on single-cell gene expression data in humans and contrast it with mechanisms reported in mice.

Experimental challenges to modeling prostate cancer heterogeneity

Cancer letters

2021 Oct 21

Flores-Téllez, TDNJ;Baena, E;
PMID: 34688843 | DOI: 10.1016/j.canlet.2021.10.012

Tumor heterogeneity plays a key role in prostate cancer prognosis, therapy selection, relapse, and acquisition of treatment resistance. Prostate cancer presents a heterogeneous diversity at inter- and intra-tumor and inter-patient levels which are influenced by multiple intrinsic and/or extrinsic factors. Recent studies have started to characterize the complexity of prostate tumors and these different tiers of heterogeneity. In this review, we discuss the most common factors that contribute to tumoral diversity. Moreover, we focus on the description of the in vitro and in vivo approaches, as well as high-throughput technologies, that help to model intra-tumoral diversity. Further understanding tumor heterogeneities and the challenges they present will guide enhanced patient risk stratification, aid the design of more precise therapies, and ultimately help beat this chameleon-like disease.

Macrophage/microglia-derived IL-1β induces glioblastoma growth via the STAT3/NF-κB pathway

Human cell

2021 Sep 30

Kai, K;Komohara, Y;Esumi, S;Fujiwara, Y;Yamamoto, T;Uekawa, K;Ohta, K;Takezaki, T;Kuroda, J;Shinojima, N;Hamasaki, T;Mukasa, A;
PMID: 34591282 | DOI: 10.1007/s13577-021-00619-8

Glioblastoma is a glioma characterized by highly malignant features. Numerous studies conducted on the relationship between glioblastoma and the microenvironment have indicated the significance of tumor-associated macrophages/microglia (TAMs) in glioblastoma progression. Since interleukin (IL)-1β secreted by TAMs has been suggested to promote glioblastoma growth, we attempted to elucidate the detailed mechanisms of IL-1β in glioblastoma growth in this study. A phospho-receptor tyrosine kinase array and RNA-sequencing studies indicated that IL-1β induced the activation of signal transducer and activator of transcription-3 and nuclear factor-kappa B signaling. Glioblastoma cells stimulated by IL-1β induced the production of IL-6 and CXCL8, which synergistically promoted glioblastoma growth via signal transducer and activator of transcription-3 and nuclear factor-kappa B signaling. By immunohistochemistry, IL-1β expression was seen on TAMs, especially in perinecrotic areas. These results suggest that IL-1β might be a useful target molecule for anti-glioblastoma therapy.

An mPOA-ARC AgRP Pathway Modulates Cold-Evoked Eating Behavior

SSRN Electronic Journal

2021 Feb 09

Yang, S;Tan, Y;Wu, X;Wang, J;Sun, J;Gan, L;Shen, B;Huang, J;
| DOI: 10.2139/ssrn.3780283

Behavioral thermoregulation involves enhanced appetite under decreased temperature. The neural circuitry mediating the crosstalk between behavioral thermoregulation and energy homeostasis remains to be elucidated. We find that the hypothalamic orexigenic agouti-related neuropeptide (AgRP) neurons in the arcuate nucleus (ARC) are profoundly activated by cold exposure. Inhibition of AgRP neurons attenuates the cold-evoked eating behavior. The calcium signals in AgRP neurons display an immediate-response pattern in response to cold stimulation. We find that the upstream cold-responsive neurons in the medial preoptic area (mPOA) make excitatory synaptic connections onto AgRP neurons. These findings reveal an mPOA-ARC neural pathway that modulates the cold-evoked eating behavior.

The origin of the expressed retrotransposed gene ACTBL2 and its influence on human melanoma cells\' motility and focal adhesion formation

Scientific reports

2021 Feb 08

Malek, N;Michrowska, A;Mazurkiewicz, E;Mrówczyńska, E;Mackiewicz, P;Mazur, AJ;
PMID: 33558623 | DOI: 10.1038/s41598-021-82074-x

We have recently found that β-actin-like protein 2 (actbl2) forms complexes with gelsolin in human melanoma cells and can polymerize. Phylogenetic and bioinformatic analyses showed that actbl2 has a common origin with two non-muscle actins, which share a separate history from the muscle actins. The actin groups' divergence started at the beginning of vertebrate evolution, and actbl2 actins are characterized by the largest number of non-conserved amino acid substitutions of all actins. We also discovered that ACTBL2 is expressed at a very low level in several melanoma cell lines, but a small subset of cells exhibited a high ACTBL2 expression. We found that clones with knocked-out ACTBL2 (CR-ACTBL2) or overexpressing actbl2 (OE-ACTBL2) differ from control cells in the invasion, focal adhesion formation, and actin polymerization ratio, as well as in the formation of lamellipodia and stress fibers. Thus, we postulate that actbl2 is the seventh actin isoform and is essential for cell motility.

Identification of novel neurocircuitry through which leptin targets multiple inputs to the dopamine system to reduce food reward seeking

Biological Psychiatry

2021 Jan 01

Omrani, A;de Vrind, V;Lodder, B;Stoltenborg, I;Kooij, K;Wolterink-Donselaar, I;Luijendijk-Berg, M;Garner, K;van ’t Sant, L;Rozeboom, A;Dickson, S;Meye, F;Adan, R;
| DOI: 10.1016/j.biopsych.2021.02.017

A large number of dopamine neurons projecting to the NAc are innervated by local VTA LepR-expressing gamma-aminobutyric acid (GABA) neurons. Leptin enhances the activity of these GABA neurons and thereby inhibits NAc-projecting dopamine neurons. In addition, we find that lateral hypothalamic (LH) LepR-expressing neurons projecting to the VTA are inhibited by leptin and that these neurons modulate dopamine neurons indirectly via inhibition of VTA GABA neurons. In accordance with such a disinhibitory function, optogenetically stimulating LH LepR projections to the VTA potently activates dopamine neurons in vivo. Moreover, we found that chemogenetic activation of LH LepR neurons increases the motivation to obtain a food reward only when mice are in positive energy balance.

Oxycodone withdrawal induces HDAC1/HDAC2-dependent transcriptional maladaptations in the reward pathway in a mouse model of peripheral nerve injury

Nature neuroscience

2023 Jun 08

Pryce, KD;Serafini, RA;Ramakrishnan, A;Nicolais, A;Giosan, IM;Polizu, C;Torres-Berrío, A;Vuppala, S;Kronman, H;Ruiz, A;Gaspari, S;Peña, CJ;Sakloth, F;Mitsi, V;van Duzer, J;Mazitschek, R;Jarpe, M;Shen, L;Nestler, EJ;Zachariou, V;
PMID: 37291337 | DOI: 10.1038/s41593-023-01350-3

The development of physical dependence and addiction disorders due to misuse of opioid analgesics is a major concern with pain therapeutics. We developed a mouse model of oxycodone exposure and subsequent withdrawal in the presence or absence of chronic neuropathic pain. Oxycodone withdrawal alone triggered robust gene expression adaptations in the nucleus accumbens, medial prefrontal cortex and ventral tegmental area, with numerous genes and pathways selectively affected by oxycodone withdrawal in mice with peripheral nerve injury. Pathway analysis predicted that histone deacetylase (HDAC) 1 is a top upstream regulator in opioid withdrawal in nucleus accumbens and medial prefrontal cortex. The novel HDAC1/HDAC2 inhibitor, Regenacy Brain Class I HDAC Inhibitor (RBC1HI), attenuated behavioral manifestations of oxycodone withdrawal, especially in mice with neuropathic pain. These findings suggest that inhibition of HDAC1/HDAC2 may provide an avenue for patients with chronic pain who are dependent on opioids to transition to non-opioid analgesics.

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	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

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