Leveraging single cell sequencing to unravel intra-tumour heterogeneity and tumour evolution in human cancers

The Journal of pathology

2022 Apr 19

Bowes, A;Tarabichi, M;Pillay, N;Van Loo, P;
PMID: 35438189 | DOI: 10.1002/path.5914

Intra-tumour heterogeneity and tumour evolution are well-documented phenomena in human cancers. While the advent of next-generation sequencing technologies has facilitated the large-scale capture of genomic data, the field of single cell genomics is nascent but rapidly advancing and generating many new insights into the complex molecular mechanisms of tumour biology. In this review, we provide an overview of current single cell DNA sequencing technologies, exploring how recent methodological advancements have enumerated new insights into intra-tumour heterogeneity and tumour evolution. Areas highlighted include the potential power of single cell genome sequencing studies to explore evolutionary dynamics contributing to tumourigenesis through to progression, metastasis and therapy resistance. We also explore the use of in-situ sequencing technologies to study intra-tumour heterogeneity in a spatial context, as well as examining the use of single cell genomics to perform lineage tracing in both normal and malignant tissues. Finally, we consider the use of multi-modal single cell sequencing technologies. Taken together, it is hoped that these many facets of single cell genome sequencing will improve our understanding of tumourigenesis, progression and lethality in cancer leading to the development of novel therapies. This article is protected by

CircRNA circCOL1A1 Acts as a Sponge of miR-30a-5p to Promote Vascular Smooth Cell Phenotype Switch through Regulation of Smad1 Expression

Thrombosis and haemostasis

2022 Dec 03

Ye, M;Ni, Q;Wang, H;Wang, Y;Yao, Y;Li, Y;Wang, W;Yang, S;Chen, J;Lv, L;Zhao, Y;Xue, G;Guo, X;Zhang, L;
PMID: 36462769 | DOI: 10.1055/s-0042-1757875

Phenotypic switch of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of atherosclerosis. The mRNA expression of the synthetic biomarker Collagen Type I Alpha 1 Chain (COL1A1) gene is upregulated during the switch of VSMCs from the contractile to the synthetic phenotype. The association of noncoding circular RNAs transcribed by the COL1A1 gene with VSMC phenotype alteration and atherogenesis remains unclear. Here we reported a COL1A1 circular RNA (circCOL1A1) which is specifically expressed in VSMCs and is upregulated during phenotype alteration of VSMCs. CircCOL1A1 is also detectable in the serum or plasma. Healthy vascular tissues have a low expression of CircCOL1A1, while it is upregulated in atherosclerosis patients. Through ex vivo and in vitro assays, we found that circCOL1A1 can promote VSMC phenotype switch. Mechanistic analysis showed that circCOL1A1 may exert its function as a competing endogenous RNA of miR-30a-5p. Upregulation of circCOL1A1 ameliorates the inhibitory effect of miR-30a-5p on its target SMAD1, which leads to suppression of transforming growth factor-β (TGF-β) signaling. Our findings demonstrate that circCOL1A1 promotes the phenotype switch of VSMCs through the miR-30a-5p/SMAD1/TGF-β axis and it may serve as a novel marker of atherogenesis or as a therapeutic target for atherosclerosis.Thieme. All rights reserved.

CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs

Cancers

2022 Jan 15

Koppula, A;Abdelgawad, A;Guarnerio, J;Batish, M;Parashar, V;
PMID: 35053590 | DOI: 10.3390/cancers14020428

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method 'circular fluorescent in situ hybridization' (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.

Visualization of Defined Gene Sequences in Single Nuclei by DNA In Situ Hybridization (DISH)

Neuromethods

2022 Jun 01

Park, C;Kaeser, G;Chun, J;
| DOI: 10.1007/978-1-0716-2357-2_13

Gains and/or losses of large genomic loci such as full or partial aneuploidies/aneusomies can be routinely identified in single cells using fluorescence in situ hybridization (FISH); however, standard FISH typically cannot resolve single genes or gene variations. Here we provide a protocol for DNA in situ hybridization (DISH) that is capable of identifying single gene loci and gene variants within the nucleus of single cells. DISH was developed to enable detection of newly identified mosaic structural variants resembling complementary DNAs (cDNAs) that were termed genomic cDNAs or gencDNAs. gencDNAs are intron-less gene copies with expansive sequence diversity, even within cells from a single individual, and are proposed to be formed through somatic gene recombination (SGR). gencDNAs were first discovered for the human amyloid precursor protein (_APP_) gene through increased copy numbers and forms in Alzheimer’s disease (AD) brains where both full-length annotated splice-isoforms and novel shortened _APP_ sequences containing intraexonic junctions (IEJs), and single nucleotide variants (SNVs) were observed within genomic DNA. Modification of a commercially available RNA ISH technology, BaseScope , particularly through the use of _sense_-strand probes that were combined with distinct tissue preparative steps for nuclear probe access, enabled DISH detection of gencDNAs, as well as detection of germline control sequences. Protocol details include considerations for probe design, use on human brain cell nuclei (generalizable to other species), and appropriate positive and negative controls.

The Alternative Matrisome: alternative splicing of ECM proteins in development, homeostasis and tumor progression

Matrix biology : journal of the International Society for Matrix Biology

2022 May 07

Rekad, Z;Izzi, V;Lamba, R;Ciais, D;Van Obberghen-Schilling, E;
PMID: 35537652 | DOI: 10.1016/j.matbio.2022.05.003

The extracellular matrix (ECM) is a fundamental component of the tissue of multicellular organisms that is comprised of an intricate network of multidomain proteins and associated factors, collectively known as the matrisome. The ECM creates a biophysical environment that regulates essential cellular processes such as adhesion, proliferation and migration and impacts cell fate decisions. The composition of the ECM varies across organs, developmental stages and diseases. Interestingly, most ECM genes generate transcripts that undergo extensive alternative splicing events, producing multiple protein variants from one gene thus enhancing ECM complexity and impacting matrix architecture. Extensive studies over the past several decades have linked ECM remodeling and expression of alternatively spliced ECM isoforms to cancer, and reprogramming of the alternative splicing patterns in cells has recently been proposed as a new hallmark of tumor progression. Indeed, tumor-associated alternative splicing occurs in both malignant and non-malignant cells of the tumor environment and growing evidence suggests that expression of specific ECM splicing variants could be a key step for stromal activation. In this review, we present a general overview of alternative splicing mechanisms, featuring examples of ECM components. The importance of ECM variant expression during essential physiological processes, such as tissue organization and embryonic development is discussed as well as the dysregulation of alternative splicing in cancer. The overall aim of this review is to address the complexity of the ECM by highlighting the importance of the yet-to-be-fully-characterized "alternative" matrisome in physiological and pathological states such as cancer.

Elevated Placental microRNA-155 Is a Biomarker of a Preeclamptic Subtype

Hypertension (Dallas, Tex. : 1979)

2022 Dec 15

Wang, Z;Liu, D;Dai, Y;Li, R;Zheng, Y;Zhao, G;Wang, J;Diao, Z;Cao, C;Lv, H;Gu, N;Zhou, H;Ding, H;Li, J;Zhu, X;Duan, H;Shen, L;Zhang, Q;Chen, J;Hu, H;Wang, X;Zheng, M;Zhou, Y;Hu, Y;
PMID: 36519433 | DOI: 10.1161/HYPERTENSIONAHA.122.19914

Preeclampsia is a complicated syndrome with marked heterogeneity. The biomarker-based classification for this syndrome is more constructive to the targeted prevention and treatment of preeclampsia. It has been reported that preeclamptic patients had elevated miR-155 in placentas or circulation. Here, we investigated the characteristics of patients with high placental miR-155 (pl-miR-155).Based on the 95th percentile (P95) of pl-miR-155 in controls, preeclamptic patients were divided into high miR-155 group (≥P95) and normal miR-155 group (<P95). The changes of placental pathology, clinical manifestations, and placental transcriptome of preeclamptic patients were clustered by t-distributed stochastic neighbor embedding and hierarchical clustering analysis. The placental restricted miR-155 overexpression mouse model was constructed, and the phenotype, placental pathology, and transcriptome were evaluated. Furthermore, the therapeutic potential of antagonist of miR-155 was explored by administrating with antagomir-155.About one-third of preeclamptic patients had high pl-miR-155 expression, which was positively correlated with circulating miR-155 levels. These patients could be clustered as 1 group, according to clinical manifestation, placental pathology, or transcriptomes by t-distributed stochastic neighbor embedding and hierarchical clustering analysis. Further, the pregnant mice with placental restricted miR-155 overexpression could simulate the changes of clinical signs, pathology, and transcriptome of placentas in patients with high pl-miR-155. AntagomiR-155 treatment relieved the preeclampsia-like phenotype and improved the placental vascular development in mice.There is at least 1 type of preeclampsia with upregulated miR-155 presenting more severe clinical manifestations. MiR-155 may be a potential therapeutic target in patients with high pl-miR-155.

Targeting Alternative Splicing for Therapeutic Interventions

Methods in molecular biology (Clifton, N.J.)

2022 Jul 27

Centa, JL;Hastings, ML;
PMID: 35895256 | DOI: 10.1007/978-1-0716-2521-7_2

Targeting of pre-mRNA splicing has yielded a rich variety of strategies for altering gene expression as a treatment for disease. The search for therapeutics that can modulate splicing has been dominated by antisense oligonucleotides (ASOs) and small molecule compounds, with each platform achieving remarkably effective results in the clinic. The success of RNA-targeting drugs has led to the exploration of new strategies to expand the repertoire of this type of therapeutic. Here, we discuss some of the more common causes of faulty gene expression and provide examples of approaches that have been developed to target and correct these defects for therapeutic value.

Identification of germ cell-specific Mga variant mRNA that promotes meiosis via impediment of a non-canonical PRC1

Scientific reports

2021 May 06

Kitamura, Y;Uranishi, K;Hirasaki, M;Nishimoto, M;Suzuki, A;Okuda, A;
PMID: 33958653 | DOI: 10.1038/s41598-021-89123-5

A non-canonical PRC1 (PRC1.6) prevents precocious meiotic onset. Germ cells alleviate its negative effect by reducing their amount of MAX, a component of PRC1.6, as a prerequisite for their bona fide meiosis. Here, we found that germ cells produced Mga variant mRNA bearing a premature termination codon (PTC) during meiosis as an additional mechanism to impede the function of PRC1.6. The variant mRNA encodes an anomalous MGA protein that lacks the bHLHZ domain and thus functions as a dominant negative regulator of PRC1.6. Notwithstanding the presence of PTC, the Mga variant mRNA are rather stably present in spermatocytes and spermatids due to their intrinsic inefficient background of nonsense-mediated mRNA decay. Thus, our data indicate that meiosis is controlled in a multi-layered manner in which both MAX and MGA, which constitute the core of PRC1.6, are at least used as targets to deteriorate the integrity of the complex to ensure progression of meiosis.

RAC1B modulates intestinal tumourigenesis via modulation of WNT and EGFR signalling pathways

Nature communications

2021 Apr 20

Gudiño, V;Pohl, SÖ;Billard, CV;Cammareri, P;Bolado, A;Aitken, S;Stevenson, D;Hall, AE;Agostino, M;Cassidy, J;Nixon, C;von Kriegsheim, A;Freile, P;Popplewell, L;Dickson, G;Murphy, L;Wheeler, A;Dunlop, M;Din, F;Strathdee, D;Sansom, OJ;Myant, KB;
PMID: 33879799 | DOI: 10.1038/s41467-021-22531-3

Current therapeutic options for treating colorectal cancer have little clinical efficacy and acquired resistance during treatment is common, even following patient stratification. Understanding the mechanisms that promote therapy resistance may lead to the development of novel therapeutic options that complement existing treatments and improve patient outcome. Here, we identify RAC1B as an important mediator of colorectal tumourigenesis and a potential target for enhancing the efficacy of EGFR inhibitor treatment. We find that high RAC1B expression in human colorectal cancer is associated with aggressive disease and poor prognosis and deletion of Rac1b in a mouse colorectal cancer model reduces tumourigenesis. We demonstrate that RAC1B interacts with, and is required for efficient activation of the EGFR signalling pathway. Moreover, RAC1B inhibition sensitises cetuximab resistant human tumour organoids to the effects of EGFR inhibition, outlining a potential therapeutic target for improving the clinical efficacy of EGFR inhibitors in colorectal cancer.

A critical role of the bone marrow envelope in human bone remodeling

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

2023 Apr 10

Andersen, TL;Jensen, PR;Sikjaer, TT;Rejnmark, L;Ejersted, C;Delaisse, JM;
PMID: 37038371 | DOI: 10.1002/jbmr.4815

Proper bone remodeling depends not only on a team of bone-resorbing osteoclasts and bone-forming osteoblasts. It also depends on the site-specific delivery of a large amount of osteoblast lineage cells to the bone remodeling site. How this delivery occurs is poorly known. Here, we got insight into this mechanism by analyzing the distribution of markers of osteoblastogenesis on bone surfaces and in their bone marrow neighborhood in human cancellous bone. We found a CD271-positive/PDGFβ-R-positive cell layer surrounding the bone marrow, that provides osteoblastogenic potential along all bone surfaces whether quiescent or remodeling. This bone marrow envelope cell layer takes the appearance of a canopy above remodeling sites, where it then also shows an upregulation of the proliferation marker Ki67, SMA, Tenascin-C, fibronectin and MMP13. This indicates that the canopy is a region of the bone marrow envelope where early markers of osteoblastogenesis are activated concurrently with initiation of bone remodeling. Important, the high proliferation index in the canopy is not associated with increasing cell densities at the canopy level, but well at the bone surface level, thereby supporting delivery of cells from the canopy to the bone surface. This delivery route explains why lack of canopies was previously found to coincide with lack of bone formation, and fits current knowledge on the canopies as a target for regulators of bone remodeling. We conclude that the coordination of bone marrow envelope activities and bone surface activities allows integrating osteoblastogenesis and bone remodeling into a same functional unit, and propose that the bone marrow envelope is critical for preserving bone health.This article is protected by

The UPF3B Splice Variant Modulated by HnRNPR Promotes Invasion and Metastasis of Hepatocellular Carcinoma via Inhibiting the CDH1/Hippo Pathway

papers.ssrn.com

2023 Mar 29

Wang, H;Luo, W;Zhang, H;Cheng, J;Li, H;yang, W;Li, W;Wang, J;Yang, X;Zhang, T;Han, D;Wang, Q;Liu, Y;Wang, J;Qian, D;Liu, L;
| DOI: 10.2139/ssrn.4401714

Preprints with The Lancet is part of SSRN´s First Look, a place where journals identify content of interest prior to publication. Authors have opted in at submission to The Lancet family of journals to post their preprints on Preprints with The Lancet. The usual SSRN checks and a Lancet-specific check for appropriateness and transparency have been applied. Preprints available here are not Lancet publications or necessarily under review with a Lancet journal. These preprints are early stage research papers that have not been peer-reviewed. The findings should not be used for clinical or public health decision making and should not be presented to a lay audience without highlighting that they are preliminary and have not been peer-reviewed. For more information on this collaboration, see the comments published in The Lancet about the trial [https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(18)31125-5/fulltext] period, and our decision to make this a permanent [https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)31950-4/fulltext] offering, or visit The Lancet´s FAQ [https://www.thelancet.com/preprints] page, and for any feedback please contact preprints@lancet.com.

First passage time analysis of spatial mutation patterns reveals sub-clonal evolutionary dynamics in colorectal cancer

PLoS computational biology

2023 Mar 01

Haughey, MJ;Bassolas, A;Sousa, S;Baker, AM;Graham, TA;Nicosia, V;Huang, W;
PMID: 36913406 | DOI: 10.1371/journal.pcbi.1010952

The signature of early cancer dynamics on the spatial arrangement of tumour cells is poorly understood, and yet could encode information about how sub-clones grew within the expanding tumour. Novel methods of quantifying spatial tumour data at the cellular scale are required to link evolutionary dynamics to the resulting spatial architecture of the tumour. Here, we propose a framework using first passage times of random walks to quantify the complex spatial patterns of tumour cell population mixing. First, using a simple model of cell mixing we demonstrate how first passage time statistics can distinguish between different pattern structures. We then apply our method to simulated patterns of mutated and non-mutated tumour cell population mixing, generated using an agent-based model of expanding tumours, to explore how first passage times reflect mutant cell replicative advantage, time of emergence and strength of cell pushing. Finally, we explore applications to experimentally measured human colorectal cancer, and estimate parameters of early sub-clonal dynamics using our spatial computational model. We infer a wide range of sub-clonal dynamics, with mutant cell division rates varying between 1 and 4 times the rate of non-mutated cells across our sample set. Some mutated sub-clones emerged after as few as 100 non-mutant cell divisions, and others only after 50,000 divisions. The majority were consistent with boundary driven growth or short-range cell pushing. By analysing multiple sub-sampled regions in a small number of samples, we explore how the distribution of inferred dynamics could inform about the initial mutational event. Our results demonstrate the efficacy of first passage time analysis as a new methodology in spatial analysis of solid tumour tissue, and suggest that patterns of sub-clonal mixing can provide insights into early cancer dynamics.

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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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