Intraductal xenografts show lobular carcinoma cells rely on their own extracellular matrix and LOXL1

EMBO molecular medicine

2021 Feb 22

Sflomos, G;Battista, L;Aouad, P;De Martino, F;Scabia, V;Stravodimou, A;Ayyanan, A;Ifticene-Treboux, A;RLS, ;Bucher, P;Fiche, M;Ambrosini, G;Brisken, C;
PMID: 33616307 | DOI: 10.15252/emmm.202013180

Invasive lobular carcinoma (ILC) is the most frequent special histological subtype of breast cancer, typically characterized by loss of E-cadherin. It has clinical features distinct from other estrogen receptor-positive (ER+ ) breast cancers but the molecular mechanisms underlying its characteristic biology are poorly understood because we lack experimental models to study them. Here, we recapitulate the human disease, including its metastatic pattern, by grafting ILC-derived breast cancer cell lines, SUM-44 PE and MDA-MB-134-VI cells, into the mouse milk ducts. Using patient-derived intraductal xenografts from lobular and non-lobular ER+ HER2- tumors to compare global gene expression, we identify extracellular matrix modulation as a lobular carcinoma cell-intrinsic trait. Analysis of TCGA patient datasets shows matrisome signature is enriched in lobular carcinomas with overexpression of elastin, collagens, and the collagen modifying enzyme LOXL1. Treatment with the pan LOX inhibitor BAPN and silencing of LOXL1 expression decrease tumor growth, invasion, and metastasis by disrupting ECM structure resulting in decreased ER signaling. We conclude that LOXL1 inhibition is a promising therapeutic strategy for ILC.

COCO/DAND5 inhibits developmental and pathological ocular angiogenesis

EMBO molecular medicine

2021 Feb 15

Popovic, N;Hooker, E;Barabino, A;Flamier, A;Provost, F;Buscarlet, M;Bernier, G;Larrivée, B;
PMID: 33587337 | DOI: 10.15252/emmm.202012005

Neovascularization contributes to multiple visual disorders including age-related macular degeneration (AMD) and retinopathy of prematurity. Current therapies for treating ocular angiogenesis are centered on the inhibition of vascular endothelial growth factor (VEGF). While clinically effective, some AMD patients are refractory or develop resistance to anti-VEGF therapies and concerns of increased risks of developing geographic atrophy following long-term treatment have been raised. Identification of alternative pathways to inhibit pathological angiogenesis is thus important. We have identified a novel inhibitor of angiogenesis, COCO, a member of the Cerberus-related DAN protein family. We demonstrate that COCO inhibits sprouting, migration and cellular proliferation of cultured endothelial cells. Intravitreal injections of COCO inhibited retinal vascularization during development and in models of retinopathy of prematurity. COCO equally abrogated angiogenesis in models of choroidal neovascularization. Mechanistically, COCO inhibited TGFβ and BMP pathways and altered energy metabolism and redox balance of endothelial cells. Together, these data show that COCO is an inhibitor of retinal and choroidal angiogenesis, possibly representing a therapeutic option for the treatment of neovascular ocular diseases.

A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

Nature communications

2021 Feb 11

Zhu, Y;Feng, F;Hu, G;Wang, Y;Yu, Y;Zhu, Y;Xu, W;Cai, X;Sun, Z;Han, W;Ye, R;Qu, D;Ding, Q;Huang, X;Chen, H;Xu, W;Xie, Y;Cai, Q;Yuan, Z;Zhang, R;
PMID: 33574281 | DOI: 10.1038/s41467-021-21213-4

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

Decoding neuronal composition and ontogeny of individual hypothalamic nuclei

Neuron

2021 Feb 06

Ma, T;Wong, SZH;Lee, B;Ming, GL;Song, H;
PMID: 33600763 | DOI: 10.1016/j.neuron.2021.01.026

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

Lung Mast Cells Have a High Constitutive Expression of Carboxypeptidase A3 mRNA That Is Independent from Granule-Stored CPA3

Cells

2021 Feb 03

Siddhuraj, P;Clausson, CM;Sanden, C;Alyamani, M;Kadivar, M;Marsal, J;Wallengren, J;Bjermer, L;Erjefält, JS;
PMID: 33546258 | DOI: 10.3390/cells10020309

The mast cell granule metalloprotease CPA3 is proposed to have important tissue homeostatic functions. However, the basal CPA3 mRNA and protein expression among mast cell populations has remained poorly investigated. Using a novel histology-based methodology that yields quantitative data on mRNA and protein expression at a single-cell level, the present study maps CPA3 mRNA and protein throughout the MCT and MCTC populations in healthy skin, gut and lung tissues. MCTC cells had both a higher frequency of CPA3 protein-containing cells and a higher protein-staining intensity than the MCT population. Among the tissues, skin MCs had highest CPA3 protein intensity. The expression pattern at the mRNA level was reversed. Lung mast cells had the highest mean CPA3 mRNA staining. Intriguingly, the large alveolar MCT population, that lack CPA3 protein, had uniquely high CPA3 mRNA intensity. A broader multi-tissue RNA analysis confirmed the uniquely high CPA3 mRNA quantities in the lung and corroborated the dissociation between chymase and CPA3 at the mRNA level. Taken together, our novel data suggest a hitherto underestimated contribution of mucosal-like MCT to baseline CPA3 mRNA production. The functional consequence of this high constitutive expression now reveals an important area for further research.

Single-cell atlas of developing murine adrenal gland reveals relation of Schwann cell precursor signature to neuroblastoma phenotype

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

2021 Feb 02

Hanemaaijer, ES;Margaritis, T;Sanders, K;Bos, FL;Candelli, T;Al-Saati, H;van Noesel, MM;Meyer-Wentrup, FAG;van de Wetering, M;Holstege, FCP;Clevers, H;
PMID: 33500353 | DOI: 10.1073/pnas.2022350118

Neuroblastoma is the most common extracranial solid tumor and accounts for ∼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor ("SCP") and the "neuroblast" cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.

Noncanonical TGFβ Signaling Promotes Specialized Neuroretina Tip-Cell Sprouting and Blood-Retina Barrier Formation

SSRN Electronic Journal

2021 Jan 28

Zarkada, G;Howard, J;Xiao, X;Park, H;Bizou, M;Leclerc, S;Künzel, S;Cagnone, G;Joyal, J;Andelfinger, G;Eichmann, A;Dubrac, A;
| DOI: 10.2139/ssrn.3770937

Endothelial tip cells guiding tissue vascularization are primary targets for angiogenic therapies. Whether tip cells require differential signals to develop their complex branching patterns remained unknown. Here we show that tip cells invading the neuroretina (D-tip cells) are distinct from tip cells guiding the superficial plexus (S-tip cells). D-tip cells have a unique transcriptional signature, display blood-retina barrier properties and TGFβ signaling requirements. Endothelial deletion of TGFβ receptor I (ALK5) inhibits D-tip cell differentiation and diving vessels. ALK5 endothelial deficiency results in a dysfunctional S-like tip cell molecular signature, aberrant contractile pericytes, and hemorrhagic vascular malformations, while SMAD mutants do not develop this phenotype. Oxygen-induced retinopathy retinas exhibit S-like tip cells with increased Alk5 signaling, and _Alk5_ deletion impedes retina revascularization. Our data reveal stage-specific tip cell heterogeneity as a requirement for retinal vascular development and suggest that noncanonical-TGFβ signaling could improve retinal revascularization and neural function in ischemic retinopathy.

A spatially resolved brain region- and cell type-specific isoform atlas of the postnatal mouse brain

Nature communications

2021 Jan 19

Joglekar, A;Prjibelski, A;Mahfouz, A;Collier, P;Lin, S;Schlusche, AK;Marrocco, J;Williams, SR;Haase, B;Hayes, A;Chew, JG;Weisenfeld, NI;Wong, MY;Stein, AN;Hardwick, SA;Hunt, T;Wang, Q;Dieterich, C;Bent, Z;Fedrigo, O;Sloan, SA;Risso, D;Jarvis, ED;Flicek, P;Luo, W;Pitt, GS;Frankish, A;Smit, AB;Ross, ME;Tilgner, HU;
PMID: 33469025 | DOI: 10.1038/s41467-020-20343-5

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 ( www.isoformAtlas.com ). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity.

Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia

Nature cell biology

2021 Jan 18

Chumduri, C;Gurumurthy, RK;Berger, H;Dietrich, O;Kumar, N;Koster, S;Brinkmann, V;Hoffmann, K;Drabkina, M;Arampatzi, P;Son, D;Klemm, U;Mollenkopf, HJ;Herbst, H;Mangler, M;Vogel, J;Saliba, AE;Meyer, TF;
PMID: 33462395 | DOI: 10.1038/s41556-020-00619-0

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

Hypoxia-induced suppression of alternative splicing of MBD2 promotes breast cancer metastasis via activation of FZD1

Cancer research

2021 Jan 05

Liu, Z;Sun, L;Cai, Y;Shen, S;Zhang, T;Wang, N;Wu, G;Ma, W;Li, ST;Suo, C;Hao, Y;Jia, WD;Semenza, GL;Gao, P;Zhang, H;
PMID: 33402389 | DOI: 10.1158/0008-5472.CAN-20-2876

Metastasis is responsible for the majority of breast cancer (BrCa) deaths; however, the mechanisms underlying metastasis in this disease remain largely elusive. Here we report that under hypoxic conditions, alternative splicing of MBD2 is suppressed, favoring the production of MBD2a which facilitates BrCa metastasis. Specifically, MBD2a promoted, whereas its lesser known short form MBD2c suppressed metastasis. Activation of HIF-1 under hypoxia facilitated MBD2a production via repression of SRSF2-mediated alternative splicing. As a result, elevated MBD2a outcompeted MBD2c for binding to promoter CpG islands to activate expression of FZD1, thereby promoting EMT and metastasis. Strikingly, clinical data reveals significantly correlated expression of MBD2a and MBD2c with the invasiveness of malignancy, indicating opposing roles for MBD2 splicing variants in regulating human BrCa metastasis. Collectively, our findings establish a novel link between MBD2 switching and tumor metastasis and provide a promising therapeutic strategy and predictive biomarkers for hypoxia-driven BrCa metastasis.

Neurod4 converts endogenous neural stem cells to neurons with synaptic formation after spinal cord injury

iScience

2021 Jan 01

Fukuoka, T;Kato, A;Hirano, M;Ohka, F;Aoki, K;Awaya, T;Adilijiang, A;Sachi, M;Tanahashi, K;Yamaguchi, J;Motomura, K;Shimizu, H;Nagashima, Y;Ando, R;Wakabayashi, T;Lee-Liu, D;Larrain, J;Nishimura, Y;Natsume, A;
| DOI: 10.1016/j.isci.2021.102074

The transcriptome analysis of injured Xenopus laevis tadpole and mice suggested that Neurod4L.S., a basic-helix-loop-helix transcription factor, was the most promising transcription factor to exert neuroregeneration after spinal cord injury (SCI) in mammals. We generated a pseudotyped retroviral vector with the neurotropic lymphocytic choriomeningitis virus (LCMV) envelope to deliver murine Neurod4 to mice undergoing SCI. SCI induced ependymal cells to neural stem cells (NSCs) in the central canal. The LCMV envelope-based pseudotypedvector preferentially introduced Neurod4 into activated NSCs, which converted to neurons with axonal regrowth and suppressed the scar-forming glial lineage. Neurod4-induced inhibitory neurons predominantly projected to the subsynaptic domains of motor neurons at the epicenter, and Neurod4-induced excitatory neurons predominantly projected to subsynaptic domains of motor neurons caudal to the injury site suggesting the formation of functional synapses. Thus, Neurod4 is a potential therapeutic factor that can improve anatomical and functional recovery after SCI.

Infectious titres of human papillomaviruses (HPVs) in patient lesions, methodological considerations in evaluating HPV infectivity and implications for the efficacy of high-level disinfectants

EBioMedicine

2021 Jan 01

Ozbun, MA;Bondu, V;Patterson, NA;Sterk, RT;Waxman, AG;Bennett, EC;McKee, R;Sharma, A;Yarwood, J;Rogers, M;Eichenbaum, G;
PMID: 33422988 | DOI: 10.1016/j.ebiom.2020.103165

Recent publications from a single research group have suggested that aldehyde-based high-level disinfectants (HLDs), such as ortho-phthalaldehyde (OPA), are not effective at inactivating HPVs and that therefore, patients may be at risk of HPV infection from medical devices. These results could have significant public health consequences and therefore necessitated evaluation of their reproducibility and clinical relevance. We developed methods and used standardised controls to: (1) quantify the infectious levels of clinically-sourced HPVs from patient lesions and compare them to laboratory-derived HPVs, (2) evaluate experimental factors that should be controlled to ensure consistent and reproducible infectivity measurements of different HPV genotypes, and (3) determine the efficacy of select HLDs. A novel focus forming unit (FFU) infectivity assay demonstrated that exfoliates from patient anogenital lesions and respiratory papillomas yielded infectious HPV burdens up to 2.7 × 103 FFU; therefore, using 2.2 × 102 to 1.0 × 104 FFU of laboratory-derived HPVs in disinfection assays provides a relevant range for clinical exposures. RNase and neutralising antibody sensitivities were used to ensure valid infectivity measures of tissue-derived and recombinant HPV preparations. HPV infectivity was demonstrated over a dynamic range of 4-5 log10; and disinfection with OPA and hypochlorite was achieved over 3 to >4 log10 with multiple genotypes of tissue-derived and recombinant HPV isolates. This work, along with a companion publication from an independent lab in this issue, address a major public health question by showing that HPVs are susceptible to HLDs. Advanced Sterilization Products; US NIH (R01CA207368, U19AI084081, P30CA118100).

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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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